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"v1_Abstract": "Castration-resistant prostate cancer (CRPC) expresses high levels of the anti-apoptotic proteins Bcl-2, Bcl-xL and Mcl-1, resulting in resistance to apoptosis and association with poor prognosis. Docetaxel, an antimitotic drug that is the first-line treatment strategy for CRPC, is known to provide a small survival benefit. However, docetaxel chemotherapy alone is not enough to counteract the high levels of Bcl-2/Bcl-xL/Mcl-1 present in CRPC. ABT-737 is a small molecule that binds to Bcl-2/Bcl-xL (but not Mcl-1) with high affinity and disrupts their interaction with pro-apoptotic Bax/Bak, thus enhancing apoptosis. Our results indicate that ABT-737 can sensitize androgen-dependent LNCaP and CRPC PC3 cells to docetaxeland to the novel antimitotic ENMD-1198-mediated caspase-dependent apoptosis. CRPC DU145 cells, however, are more resistant to ABT-737 because they are Bax null and not because they express the highest levels of anti-apoptotic Mcl-1 (associated with ABT-737 resistance). Knockdown of Bax or Bak in LNCaP indicates that ABT-737-induced antimitotic enhancement of apoptosis is more dependent on the levels of Bax than Bak. Furthermore, we find that the ability of docetaxel to increase cyclin B1/Cdk1-mediated phosphorylation of Bcl-2/Bcl-xL and decrease Mcl-1 is required for ABT-737 to enhance apoptosis in PC3 cells, as determined by addition of Cdk1 inhibitor purvalanol A and expression of shRNA specific for cyclin B1. Overall, our data suggests that the high levels of anti-apoptotic proteins in Bax-expressing CRPC cells can be overcome by targeting Bcl-2/Bcl-xL with ABT-737 and Mcl-1 with antimitotics.",
"v2_Abstract": "Castration-resistant prostate cancer (CRPC) expresses high levels of the anti-apoptotic proteins Bcl-2, Bcl-xL and Mcl-1, resulting in resistance to apoptosis and association with poor prognosis. Docetaxel, an antimitotic drug that is the first-line treatment strategy for CRPC, is known to provide a small survival benefit. However, docetaxel chemotherapy alone is not enough to counteract the high levels of Bcl-2/Bcl-xL/Mcl-1 present in CRPC. ABT-737 is a small molecule that binds to Bcl-2/Bcl-xL (but not Mcl-1) with high affinity and disrupts their interaction with pro-apoptotic Bax/Bak, thus enhancing apoptosis. Our results indicate that ABT-737 can sensitize androgen-dependent LNCaP and CRPC PC3 cells to docetaxeland to the novel antimitotic ENMD-1198-mediated caspase-dependent apoptosis. CRPC DU145 cells, however, are more resistant to ABT-737 because they are Bax null and not because they express the highest levels of anti-apoptotic Mcl-1 (associated with ABT-737 resistance). Knockdown of Bax or Bak in LNCaP indicates that ABT-737-induced antimitotic enhancement of apoptosis is more dependent on the levels of Bax than Bak. Furthermore, we find that the ability of docetaxel to increase cyclin B1/Cdk1-mediated phosphorylation of Bcl-2/Bcl-xL and decrease Mcl-1 is required for ABT-737 to enhance apoptosis in PC3 cells, as determined by addition of Cdk1 inhibitor purvalanol A and expression of shRNA specific for cyclin B1. Overall, our data suggests that the high levels of anti-apoptotic proteins in Bax-expressing CRPC cells can be overcome by targeting Bcl-2/Bcl-xL with ABT-737 and Mcl-1 with antimitotics.",
"v1_text": "materials and methods : Reagents ABT-737 was obtained from Abbott Laboratories (Abbott Park, IL, USA), Doc from Sanofi-Aventis (Bridgewater, NJ, USA), and ENMD-1198 from EntreMed, Inc (Rockville, MD, USA). Q-VD pan-caspase inhibitor was purchased from R&D Systems (Minneapolis, MN, USA); purvalanol A from A.G. Scientific (San Diego, CA, USA); Trypan blue (0.4%) from Invitrogen (Grand Island, NY, USA); and Coomassie blue from EMD Chemicals (Billerica, MA, USA). All other reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA). Cell culture Human PCa cell lines LNCaP, DU145, and PC3 were obtained from the American Type Culture Collection (Manassas, VA, USA) (van Bokhoven et al., 2003) and used within 6 months of resuscitation of original cultures. All cells were maintained in RPMI 1640 medium (Invitrogen) with 5% fetal bovine serum (Hyclone, Waltham, MA), 100 U/ml penicillin, 100 \u03bcg/ml streptomycin, and 0.25 \u03bcg/ml amphotericin (Invitrogen). Unlike LNCaP, LN-AI cells are able to grow for long-term in RPMI 1640 with 5% charcoal-stripped fetal bovine serum (Hyclone) and are referred to as LN-AI/CSS (Gomez, de las Pozas & Perez-Stable, 2006). 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 PeerJ reviewing PDF | (v2013:06:610:1:1:NEW 5 Aug 2013) R ev ie w in g M an us cr ip t Western blot analysis Preparation of total protein lysates and western blot analysis was done as previously described (Gomez, de las Pozas & Perez-Stable, 2006). The following antibodies were used: Bcl-2 (N-19), Bax (N-20), Mcl-1 (S-19), cyclin B1 (GNS1), AIF (E-1), and horseradish peroxidase-conjugated secondary antibody from Santa Cruz Biotechnology (Santa Cruz, CA, USA); Bak (NT) from EMD Millipore (Billerica, MA, USA); Bcl-xL (#610211), cytochrome c (7H8.2C12), Smac (#612245) from BD Biosciences (San Diego, CA, USA); cleaved PARP (9541), phospho-(Ser70) Bcl-2 (2827), CoxIV (#4844), Bid (#2002) from Cell Signaling Technology (Danvers, MA, USA); phospho-(Ser62) Bcl-xl (30655) from Abcam (Cambridge, MA, USA); and Noxa (114C307.1) from Novus Biologicals (Littleton, CO, USA). After immunodetection, our preference for loading controls was for staining of total proteins transferred to the membrane with Coomassie blue because drug treatments often affect the levels of typical housekeeping proteins such as actin or tubulin. ABT-737 cell viability assay LNCaP, DU145, and PC-3 cells were seeded in 96-well plates. The next day, fresh media containing ABT-737 (1, 2.5, 5, 10 \u00b5M), or control (0.1% DMSO) were added and cells incubated for three days. The CellTiter Aqueous cell proliferation colorimetric method from Promega (Madison, WI, USA) was used to determine cell viability, as per manufacturer\u2019s instructions. Cell viability was normalized against the vehicle control and the data expressed as a percentage of control from three independent experiments done in triplicate. Drug treatments PCa cells were cultured in media containing Doc (1nM), 1198 (1 \u00b5M), ABT-737 (1 \u00b5M), Doc or 1198 + ABT-737, Q-VD (10 \u00b5M), purvalanol A (5 \u00b5M), Doc or 1198 + Q-VD or purvalanol A, or 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 PeerJ reviewing PDF | (v2013:06:610:1:1:NEW 5 Aug 2013) R ev ie w in g M an us cr ip t DMSO (0.1%) control for varying times (24\u221272 h). In all the experiments, floating and trypsinized attached cells were pooled for further analysis. Trypan blue exclusion assay Treated and control PCa cells were harvested, resuspended in PBS, diluted 1:1 in 0.4% trypan blue, dead blue and live non-blue cells immediately counted using a hemacytometer, and the % dead blue cells determined from at least three independent experiments done in duplicate. Annexin-FITC/propidium iodide (PI) flow cytometry Treated and control PCa cells were resuspended in binding buffer followed by the addition of annexin V-FITC and PI (Annexin V Kit sc-4252 AK, Santa Cruz Biotechnology). After 20 min., cells were analyzed by flow cytometry using a Coulter XL flow cytometer and the percentage of annexin+ cells determined using WinMDI version 2.8 from two independent experiments done in triplicate. Mitochondrial protein release assay Treated and control PCa cells were resuspended in a buffer containing 100-200 \u00b5M digitonin, 20 mM Hepes, pH 7.5, 10 mM KCl, 1.5 mM MgCl, 1 mM EGTA, 1 mM EDTA, 1 mM DTT, 250 mM sucrose, and protease inhibitors (Roche, Nutley, NJ) at 50 \u00b5l/1 x106 cells. After 5 min on ice, cells were centrifuged at 14k rpm 5 min and the supernatant used for western blot analysis. Digitonin is a detergent that preferentially permeabilizes plasma membrane compared to mitochondrial membrane (Gottlieb & Granville, 2002). Retrovirus transduction of DU145 and LNCaP with Bax hBax C3-EGFP (Addgene, Cambridge, MA, USA; plasmid 19741) (Nechushtan et al., 1999) was digested with HindIII, blunt-ended with Klenow DNA polymerase, digested with EcoRI, and the 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 PeerJ reviewing PDF | (v2013:06:610:1:1:NEW 5 Aug 2013) R ev ie w in g M an us cr ip t 0.6 kb Bax insert ligated into pBABE puro plasmid (BamH1-blunt/EcoRI) using DNA ligase (New England Biolabs, Ipswich, MA, USA). Retrovirus production and infection were done by transfecting HEK293T cells (American Type Culture Collection) with pBABE/Bax or pBABE/EV (empty vector), pUMVC3, and pCVM-VSV-G with FuGene HD (Roche), addition of filtered (0.45 \u00b5M) media after 48 h to DU145 and LNCaP cells, and selection with puromycin (Invitrogen; 2 \u00b5g/ml) for 1 week. Cell death in DU145/Bax, DU145/EV, LNCaP/Bax, and LNCaP/EV cells treated with DMSO control were similar to parental cells (not shown). Lentiviral transduction of LNCaP, DU145, and PC3 with shRNA The shRNA design, lentivirus production, and infection were done as previously described (Stewart et al, 2003). The following DNA oligonucleotides (Operon, Huntsville, AL, USA) targeting Mcl-1, Bax, Bak, and cyclin B1 were cloned into pLKO.1 lentivirus vector: shMcl-1 (M2): CCGGGCTGGAGA TTATCTCTCGGTACTCGAGTACCGAGAGATAATCTCCAGCTTTTTG; shMcl-1 (M3): CCGGGCTAAACACTTGAAGACCATACTCGAGTATGGTCTTCAAGTGTTTAGC TTTTTG; shBax-1: CCGGGCCGGAACTGATCAGAACCATCTCGAGATGGTTCTGATCAGTTCCGG CTTTTTG (PC3); shBax-2: CCGGGCCTCAGGATGCGTCCACCAACTCGAGTTGGTGGACG CATCCTGAGGCTTTTTG (LNCaP); shBak-1: CCGGTGGTACGAAGATTCTTCAAATCTC GAGATTTGAAGAATCTTCGTACCATTTTTG (LNCaP); shBak-3: CCGGATGAGTACTTCA CCAAGATTGCTCGAGCAATCTTGGTGAAGTACTCATTTTTTG (PC3); shCyclin B1-2: CCGG GCCAAATACCTGATGGAACTACTCGAGTAGTTCCATCAGGTATTTGGCTTTTTG; and shCyclin B1-3: CCGGGCCATCCTAATTGACTGGCTACTCGAGTAGCCAGTCAATTAGGATG GCTTTTTG. The control shRNA was targeted against green fluorescent protein (GFP). For Mcl-1 knockdown in DU145/Bax and PC3/shCyclin B1 cells (puromycin resistant), shMcl-1 and shGFP oligonucleotides 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 PeerJ reviewing PDF | (v2013:06:610:1:1:NEW 5 Aug 2013) R ev ie w in g M an us cr ip t were cloned into pLKO.1/hygromycin plasmid and transduced cells selected with 400 \u00b5g/ml hygromycin (Invitrogen) for two weeks. Cell death in DU145/shMcl-1, DU145/shGFP, DU145/Bax/shMcl-1, DU145/Bax/shGFP, LNCaP/shBax, LNCaP/shBak, LNCaP/shGFP, PC3/shBax, PC3/shBak, PC3/shCyclin B1, PC3/shGFP, PC3/shCyclin B1/shMcl-1, and PC3/shCyclin B1/shGFP cells treated with DMSO control were similar to parental cells (not shown). Statistical analysis Statistical differences between drug-treated and control PCa cells were determined by two-tailed Student\u2019s t-test (unequal variance) with P<0.05 considered significant. results : CRPC cells express high anti-apoptotic Bcl-2/Bcl-xL/Mcl-1 and low or null pro-apoptotic Bax/Bak Since ABT-737 is a Bcl-2/Bcl-xL antagonist that should promote the pro-apoptotic function of Bax/Bak, we compared the protein levels of Bcl-2, Bcl-xL, Bax, and Bak in LNCaP, DU145, and PC3 cells. LNCaP cells are androgen-dependent, contain wild-type p53, and exhibit higher sensitivity to antimitotic-mediated apoptosis relative to DU145 and PC3, which are castration-resistant and p53 mutated or null (van Bokhoven et al., 2003; Reiner et al., 2009). As expected, DU145 and PC3 expressed higher Bcl-2/Bcl-xL when compared to LNCaP cells (Fig. 1A). Expression of Mcl-1, an anti-apoptotic member of the Bcl-2 family that is not targeted by ABT-737 and is associated with chemoresistance to ABT-737 treatment (van Delft et al., 2006; Chen et al, 2007; Lestini et al., 2009; Hauck et al., 2009; Yecies et al., 2010), was highest in DU145 compared to PC3 and LNCaP cells. The protein levels of pro-apoptotic Bax and Bak were lower in CRPC cells compared to LNCaP; Bax is null in DU145 (Tang et al., 1998). These results suggest that the high anti-apoptotic Bcl-2/Bcl-xL and low pro-apoptotic Bax/Bak protein environment present in CRPC cells may benefit from ABT-737 treatment in order to enhance apoptotic cell death. However, DU145 cells were more resistant to ABT-737 as a single agent when compared to LNCaP and PC3 cells (Fig. 1B). ABT-737 enhances Doc/1198-mediated apoptosis in LNCaP and PC3 but not in DU145 cells CRPC cells such as DU145 and PC3 are more resistant to Doc treatment compared to androgen-dependent cells such as LNCaP and combinations with other drugs or agents are required to increase therapeutic efficacy. Our results showed that the combination of 1 nM Doc or 1 \u00b5M 1198 with a sub-cytotoxic dose of ABT-737 (1 \u00b5M) significantly increased cell death and cleaved-PARP (measure of caspase activity) compared to 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 PeerJ reviewing PDF | (v2013:06:610:1:1:NEW 5 Aug 2013) R ev ie w in g M an us cr ip t the single agents in LNCaP and PC3 but not in DU145 cells (Fig. 2A, B; Supplemental Fig. S1A). Similar results were obtained in LNCaP-AI/CSS, a CRPC variant of LNCaP that is more chemoresistant (Supplemental Fig. S1B). The pan-caspase inhibitor Q-VD (10 \u00b5M) blocked Doc + ABT-737-mediated cell death and cleaved-PARP, indicating that increased caspase activity was required (Fig. 2A, B). ABT-737 targets the mitochondria to initiate the intrinsic pathway of apoptosis by increasing the release of mitochondrial proteins such as cytochrome c, which in turn activates the caspase cascade (Chipuk et al., 2010). Our results indicated that ABT-737 enhanced Doc-mediated release of cytochrome c, Smac (blocks inhibitor of apoptosis [IAP] family; LaCasse et al., 2008), and apoptosis-inducing factor (AIF; translocates to nucleus to increase DNA fragmentation; Susin et al., 1999) from the mitochondria in LNCaP and PC3 but not in DU145 cells (Fig. 2C; Supplemental Fig. S2). In addition, there was less cytoplasmic Bax protein in Doc + ABT-737 treated LNCaP and PC3 cells, likely as a result of greater translocation of Bax to the mitochondria. Thus, ABT-737 enhancement of Doc-mediated pro-apoptotic protein release from the mitochondria correlates with increased apoptotic cell death in LNCaP and PC3 but not in DU145. Bax expression but not Mcl-1 suppression sensitizes DU145 to ABT-737 enhancement of Doc/1198-mediated apoptosis We investigated the mechanisms why DU145 cells are more resistant to ABT-737. One possibility stems from the Bax null status of DU145 cells, which would indicate that ABT-737 mediates its cytotoxicity via the Bax pathway. This is supported by a previous finding indicating that transient transfection of Bax into DU145 cells increases sensitivity to ABT-737 + TRAIL (Song, Kandasamy & Kraft, 2008). Another possibility is that ABT-737 resistance of DU145 cells arises from elevated Mcl-1, which unlike Bcl-2/Bcl-xL does not interact with ABT-737 and may therefore block the ability of ABT-737 to increase apoptosis (van Delft et al., 2006; Chen et al, 2007; Lestini et al., 2009; Hauck et al., 2009; Yecies et al., 2010). To address these possibilities, we isolated DU145 cells stably expressing Bax and DU145 cells with stable Mcl-1 knockdown. Our results showed that ABT-737 significantly enhanced Doc/1198-mediated cell death in DU145/Bax cells above Doc/1198 treatment alone (Fig. 3A, B). Similar results were obtained in LNCaP/Bax cells (Supplemental Fig S3). In contrast, knockdown of Mcl-1 in DU145 or DU145/Bax cells did not significantly enhance Doc/1198 + ABT-737-mediated apoptotic cell death (Fig. 3C; Supplemental Fig. S4). These results indicate that DU145 cells are more resistant to ABT-737 enhancement of antimitotic-mediated apoptosis because they are Bax null and not because they express high Mcl-1. In contrast, knockdown of Mcl-1 in LNCaP and PC3 cells increased cell death and cl-PARP, confirming the importance of Mcl-1 in resistance to Doc/1198 + ABT-737 treatment (Supplemental Fig. S5). ABT-737-mediated enhancement of Doc/1198-induced apoptosis in LNCaP is more dependent on Bax than Bak 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 PeerJ reviewing PDF | (v2013:06:610:1:1:NEW 5 Aug 2013) R ev ie w in g M an us cr ip t ABT-737 inhibits the interactions of Bcl-2/Bax and Bcl-xL/Bak, thus allowing Bax and Bak to induce MOMP and apoptosis (Oltersdorf et al., 2005; Tagscherer et al., 2008). To further investigate the relative importance of Bax versus Bak in mediating ABT-737 enhancement of Doc/1198-mediated cell death in PCa cells, we isolated LNCaP and PC3 cells stably expressing either shBax or shBak. Results showed that knockdown of Bax lowered Doc/1198 + ABT-737-mediated cell death and cleaved-PARP in both LNCaP and PC3 cells compared to the negative control shGFP cells (Fig. 4A). Knockdown of Bak also lowered Doc/1198 + ABT-737-induced cell death and cleaved-PARP in PC3 cells but had no significant effects in LNCaP cells (Fig. 4B). These results suggest that ABT-737 enhancement of Doc/1198-mediated apoptosis in LNCaP cells is more dependent on Bax than to Bak but PC3 cells are dependent on both Bax and Bak. Doc counteracts the ABT-737-mediated increase in Mcl-1 We determined if Doc and Doc + ABT-737 had any effects on the protein levels of Bcl-2 family members in LNCaP and PC3 cells. Interestingly, Doc decreased and ABT-737 increased Mcl-1 but the combination of Doc + ABT-737 lowered Mcl-1 (Fig. 5). The mechanism why ABT-737 alone increases Mcl-1 protein is not known but may reflect the observation that acquired resistance to ABT-737 involves increased Mcl-1 (Yecies et al., 2010). There were no clear differences in the protein levels of Bcl-2, Bcl-xL, Bax, Bak, Bid, and Noxa with the exception in LNCaP where there was less Bcl-2 (Doc, 24 h) and Bak (Doc+ ABT-737, 48 h). These results suggest that the Doc + ABT-737 enhancement of apoptosis may depend upon the ability of Doc to counteract the ABT-737-mediated increase in Mcl-1. ABT-737-mediated enhancement of Doc-induced apoptosis is dependent on cyclinB1/Cdk1-mediated phosphorylation of Bcl-2/Bcl-xL and decrease of Mcl-1 Small molecule inhibitors of Cdk1 prevent Doc-mediated increase in cyclin B1/Cdk1 activity and blocks induction of apoptosis in CRPC cells (Perez-Stable, 2006; Gomez, de las Pozas & Perez-Stable, 2006). We investigated whether cyclin B1/Cdk1-mediated increase in Bcl-2/Bcl-xL phosphorylation and decrease in Mcl-1 is important for the ABT-737 enhancement of Doc-induced apoptosis. Treatment (Doc alone and Doc + ABT-737) of LNCaP and PC3 but not DU145 cells increased phospho (P)-Bcl-2, whereas the levels of P-Bcl-xL was similar in all three 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 PeerJ reviewing PDF | (v2013:06:610:1:1:NEW 5 Aug 2013) R ev ie w in g M an us cr ip t cell lines (Supplemental Fig. S6). Addition of 5 \u00b5M purvalanol A, a specific inhibitor of cyclin B1/Cdk1 activity (Gray et al., 1998) blocked Doc + ABT-737 cell death and the increase in P-Bcl-2/P-Bcl-xL in PC3 cells (Fig. 6A). However, purvalanol A increased Mcl-1 protein in Doc + ABT-737 treated PC3 cells, suggesting that this may also play an important role in blocking apoptotic cell death (Fig. 6A). To further determine whether the Doc-mediated increase in cyclin B1/Cdk1 activity, P-Bcl-2/P-Bcl-xL, and decrease in Mcl-1 is important for ABT-737 + Doc cell death, we isolated PC3 cells stably expressing shCyclin B1. Results showed that knockdown of cyclin B1 lowered Doc + ABT-737 cell death, cleaved-PARP, and P-Bcl-2/P-Bcl-xL in PC3/shCyclin B1 cells compared to shGFP control cells (Fig. 6B). However, given that there is less cyclin B1 to mediate Cdk1 degradation of Mcl-1 by Doc, it is not surprising that Mcl-1 levels are slightly higher in Doc + ABT-737 treated PC3/shCyclin B1 compared to shGFP cells (Fig. 6B). We then isolated PC3/shCyclin B1 cells stably expressing shMcl-1 and the results showed that Doc + ABT-737 increased cell death (67%) nearly to the levels of PC3/shMcl-1 cells (83%) (Fig. 7; Supplemental Fig. S5B). Overall, these results suggest that the ABT-737 enhancement of Doc-mediated apoptosis is more dependent on the ability of cyclin B1/Cdk1 to increase the degradation of Mcl-1 than to phosphorylate Bcl-2/Bcl-xL. figure legends : Figure 1 Bcl-2 family protein levels and sensitivity to ABT-737 in PCa cells. (A) Western blot analysis showing that the levels of anti-apoptotic Bcl-2 and Bcl-xL proteins are higher in DU145 (D) and PC3 (P) CRPC cells compared to androgen-dependent LNCaP (L) cells, whereas anti-apoptotic Mcl-1 is highest in DU145. The levels of pro-apoptotic Bax and Bak are higher in LNCaP compared to DU145 (Bax null) and PC3. After detection, Coomassie blue stain of total protein transferred to the membrane is the loading control. (B) Cell viability assay showing that LNCaP and PC3 are similarly sensitive to various concentrations of ABT-737 (1-5 \u00b5M; three days), whereas DU145 is more resistant. 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 PeerJ reviewing PDF | (v2013:06:610:1:1:NEW 5 Aug 2013) R ev ie w in g M an us cr ip t Figure 2 ABT-737 enhances Doc-mediated apoptotic cell death in LNCaP and PC3 but not in DU145 PCa cells. (A) Trypan blue exclusion assay showing that the combination of 1 nM Doc + 1 \u00b5M ABT-737 increases total cell death in LNCaP and PC3 but not in DU145 compared to Doc or ABT-737 alone (*, P<2x10-4). LNCaP were treated for 48 h and DU145 and PC3 for 72 h. Pan-caspase inhibitor Q-VD (10 \u00b5M) blocks the Doc + ABT-737 increase in cell death. Western blot analysis showing that Doc + ABT-737 increases cleaved-PARP (cP) levels in LNCaP and PC3 but not in DU145 compared to Doc or ABT-737 alone. Q-VD blocks the Doc + ABT-737 increase in cP in all cells. (B) Flow cytometric analysis showing higher annexin-FITC stained LNCaP and PC3 but not DU145 cells treated with Doc + ABT-737 compared to Doc or ABT-737 alone (*, P<2x10-5). Q-VD blocks the Doc + ABT-737 increase in annexin + cells in LNCaP and PC3. (C) Mitochondrial protein release assay and western blot showing increased cytochrome c, Smac, AIF and decreased Bax in LNCaP cells treated with Doc + ABT-737 compared to Doc or ABT-737 alone, and control. In DU145 cells, cytochrome c, Smac, and AIF mitochondrial release were similar in Doc + ABT-737 as in Doc alone. Cox IV protein is negative indicating no mitochondrial contamination whereas actin is the positive control. +C for both LNCaP and DU145 is lysate prepared from LNCaP cells using the standard method for total proteins. Figure 3 DU145 cells are resistant to ABT-737 because they are Bax null. (A) Trypan blue exclusion assay showing that Doc + ABT-737 (DA) or 1198 + ABT-737 (98A) increases cell death at 72 h in DU145/Bax compared to Doc (D) or 1198 (98) alone (*, P<0.008), whereas there is no effect in DU145/EV (empty vector) control cells. Western blot analysis showing that DA or 98A increases cP in DU145/Bax cells compared to D or 98 alone, whereas there is no increase of cP in DU/EV cells. Bax is expressed in DU145/Bax but not in DU145/EV cells. (B) Annexin-FITC/PI flow cytometric analysis showing higher annexin+ and PI+ cells in DA treated 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 PeerJ reviewing PDF | (v2013:06:610:1:1:NEW 5 Aug 2013) R ev ie w in g M an us cr ip t DU145/Bax compared to DU145/EV cells (*, P<1x10-6). (C) Trypan blue exclusion assay showing that DA or 98A does not significantly increase cell death in DU145/shMcl-1 and DU145/shGFP control cells compared to D or 98 alone. Western blot analysis showing no difference in the cP levels in DU145/shMcl-1 and DU145/shGFP control cells treated with D, DA, 98, or 98A. Mcl-1 is expressed much higher in DU145/shGFP compared to DU145/shMcl-1 cells. Figure 4 Bax suppression has a greater effect on ABT-737-mediated Doc/1198-induced apoptotic cell death than Bak suppression in LNCaP cells. (A) Trypan blue exclusion assay showing significantly less cell death in LNCaP/shBax and PC3/shBax cells treated with DA or 98A compared to shGFP (G) control cells (*, P<0.02). Western blot analysis showing less cP and Bax in LNCaP/shBax and PC3/shBax cells treated with DA or 98A compared to control cells. (B) Trypan blue exclusion assay showing less cell death in PC3/shBak (*, P<6x10-4) but not in LNCaP/shBak cells treated with DA or 98A compared to control cells. Western blot analysis showing less cP in PC3/shBak cells treated with D or 98 + A but little difference in LNCaP/shBak cells compared to control cells. Bak is lower in LNCaP/shBak and PC3/shBak cells compared to control cells. Figure 5 Doc counteracts the ABT-737-mediated increase in Mcl-1 protein. Western blot showing that treatment of LNCaP and PC3 cells with 1 \u00b5M ABT-737 (A) increases Mcl-1 but treatment with 1 nM Doc (D) decreases Mcl-1. Combination of Doc + ABT-737 (DA) decreases Mcl-1. In LNCaP, there is less Bcl-2 (D, 24 h) and Bak (DA, 48 h) but few differences in Bcl-xL, Bax, Bid, and Noxa. In PC-3, there are few differences Bcl-2, Bcl-xL, Bax, Bak, Bid, and Noxa. Figure 6 Inhibition of cyclinB1/Cdk1-mediated phosphorylation of Bcl-2/Bcl-xL blocks ABT-737 enhancement of Doc induced apoptosis in PC3 cells. (A) Trypan blue exclusion assay showing that 5 \u00b5M purvalanol A (P) lowers DA cell death in PC3 cells (*, P<9x10-8). 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 PeerJ reviewing PDF | (v2013:06:610:1:1:NEW 5 Aug 2013) R ev ie w in g M an us cr ip t Western blot analysis showing that P blocks the DA increase in cl-PARP, P-Bcl-2 and P-Bcl-xL, whereas there is no change in the total levels of Bcl-2 and Bcl-xL; P treatment increases Mcl-1 in DA treated cells. Vertical line in cl-PARP indicates sample from same blot not in sequence. (B) Trypan blue exclusion assay showing less cell death in DA treated PC3/shCyclin B1-2 and -3 cells compared to PC3/shGFP control cells (*, P<4x10-5). Western blot analysis showing less cyclin B1, cP, P-Bcl-2, P-Bcl-xL, and slightly greater Mcl-1 in PC3/shCyclin B1-2 and -3 cells treated with DA compared to PC3/shGFP control cells. Figure 7 ABT-737 enhancement of Doc-mediated apoptosis is more dependent on the ability of cyclin B1/Cdk1 to decrease Mcl-1 than to phosphorylate Bcl-2/Bcl-xL. Trypan blue exclusion assay showing greater cell death in DA treated (48 h) PC3/shCyclin B1/shMcl-1 (B1-2/M3, B1-3/M3) cells compared to control PC3/shCyclin B1/shGFP (B1-2/G, B1-3/G) and PC3/shGFP (G) cells (*, P< 0.002). Western blot analysis showing increased cl-PARP and decreased Mcl-1 in PC3/shCyclin B1/shMcl-1 compared control shGFP cells after DA treatment (24 h). There is less cyclin B1, P-Bcl-1, and P-Bcl-xL in PC3/shCyclin B1/shGFP or shMcl-1 compared to PC3/shGFP cells, whereas there are no changes in total Bcl-1 or Bcl-xL. Figure 8 Schematic of how ABT-737 can sensitize Bax+ CRPC cells to Doc. Treatment of CRPC cells such as PC3 with Doc deregulates cyclin B1/Cdk1 activity and increases phosphorylation of Bcl-2/Bcl-xL and degradation of Mcl-1. However, the high levels of Bcl-2/Bcl-xL/Mcl-1 in CRPC cells presents a block to apoptosis. Addition of ABT-737 disrupts the anti-apoptotic activity of Bcl-2/Bcl-xL and more effectively allows Doc treatment to bypass the block and increase apoptosis. Cyclin B1/Cdk1 hyperactivity caused by Doc treatment also lowers Mcl-1, an ABT-737 resistance factor, to further increase apoptosis. 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 PeerJ reviewing PDF | (v2013:06:610:1:1:NEW 5 Aug 2013) R ev ie w in g M an us cr ip t Figure 1 \"Bcl-2 family protein levels and sensitivity to ABT-737 in PCa cells\" \"(A) Western blot analysis showing that the levels of anti-apoptotic Bcl-2 and Bcl-xL proteins are higher in DU145 (D) and PC3 (P) CRPC cells compared toandrogen-dependent LNCaP (L) cells, whereas anti-apoptotic Mcl-1 is highest in DU145. The levels of pro-apoptotic Bax and Bak are higher in LNCaP compared to DU145 (Bax null) and PC3. After detection, Coomassie blue stain of total protein transferred to the membrane is the loading control. (B) Cell viabilityassay showing that LNCaP and PC3 are similarly sensitive to various concentrations of ABT-737 (1-5 m M; three days), whereas DU145 is more resistant.\" PeerJ reviewing PDF | (v2013:06:610:1:1:NEW 5 Aug 2013) R ev ie w in g M an us cr ip t Figure 2 ABT-737 enhances Doc-mediated apoptotic cell death in LNCaP and PC3 but not in DU145 PCa cells (A) Trypan blue exclusion assay showing that the combination of 1 nM Doc + 1 uM ABT-737 increases total cell death in LNCaP and PC3 but not in DU145 compared to Doc or ABT-737 alone (*, P<2x10 -4 ). LNCaP were treated for 48 h and DU145 and PC3 for 72 h. Pan-caspase inhibitor Q-VD (10 uM) blocks the Doc + ABT-737 increase in cell death. Western blot analysis showing that Doc + ABT-737 increases cleaved-PARP (cP) levels in LNCaP and PC3 but not in DU145 compared to Doc or ABT-737 alone. Q-VD blocks the Doc + ABT-737 increase in cP in all cells. (B) Flow cytometric analysis showing higher annexin-FITC stained LNCaP and PC3 but not DU145 cells treated with Doc + ABT-737 compared to Doc or ABT-737 alone (*, P<2 x10 -5 ) . Q-VD blocks the Doc + ABT-737 increase in annexin + cells in LNCaP and PC3. (C) Mitochondrial protein release assay and western blot showing increased cytochrome c, Smac, AIF and decreased Bax in LNCaP cells treated with Doc + ABT-737 compared to Doc or ABT-737 alone, and control. In DU145 cells, cytochrome c, Smac, and AIF mitochondrial release were similar in Doc + ABT-737 as in Doc alone. Cox IV protein is negative indicating no mitochondrial contamination whereas actin is the positive control. +C for both LNCaP and DU145 is lysate prepared from LNCaP cells using the standard method for total proteins. PeerJ reviewing PDF | (v2013:06:610:1:1:NEW 5 Aug 2013) R ev ie w in g M an us cr ip t PeerJ reviewing PDF | (v2013:06:610:1:1:NEW 5 Aug 2013) R ev ie w in g M an us cr ip t Figure 3 DU145 cells are resistant to ABT-737 because they are Bax null (A) Trypan blue exclusion assay showing that Doc + ABT-737 (DA) or 1198 + ABT-737 (98A) increases cell death at 72 h in DU145/Bax compared to Doc (D) or 1198 (98) alone (*, P<0.008), whereas there is no effect in DU145/EV (empty vector) control cells. Western blot analysis showing that DA or 98A increases cP in DU145/Bax cells compared to D or 98 alone, whereas there is no increase of cP in DU/EV cells. Bax is expressed in DU145/Bax but not in DU145/EV cells. (B) Annexin-FITC/PI flow cytometric analysis showing higher annexin+ and PI+ cells in DA treated DU145/Bax compared to DU145/EV cells (*, P<1x10 -6 ). (C) Trypan blue exclusion assay showing that DA or 98A does not significantly increase cell death in DU145/shMcl-1 and DU145/shGFP control cells compared to D or 98 alone. Western blot analysis showing no difference in the cP levels in DU145/shMcl-1 and DU145/shGFP control cells treated with D, DA, 98, or 98A. Mcl-1 is expressed much higher in DU145/shGFP compared to DU145/shMcl-1 cells. PeerJ reviewing PDF | (v2013:06:610:1:1:NEW 5 Aug 2013) R ev ie w in g M an us cr ip t Figure 4 Bax suppression has a greater effect on ABT-737-mediated Doc/1198-induced apoptotic cell death than Bak suppression in LNCaP cells (A) Trypan blue exclusion assay showing significantly less cell death in LNCaP/shBax and PC3/shBax cells treated with DA or 98A compared to shGFP (G) control cells (*, P<0.02). Western blot analysis showing less cP and Bax in LNCaP/shBax and PC3/shBax cells treated with DA or 98A compared to control cells. (B) Trypan blue exclusion assay showing less cell death in PC3/shBak (*, P<6x10 -4 ) but not in LNCaP/shBak cells treated with DA or 98A compared to control cells. Western blot analysis showing less cP in PC3/shBak cells treated with D or 98 + A but little difference in LNCaP/shBak cells compared to control cells. Bak is lower in LNCaP/shBak and PC3/shBak cells compared to control cells. PeerJ reviewing PDF | (v2013:06:610:1:1:NEW 5 Aug 2013) R ev ie w in g M an us cr ip t Figure 5 Doc counteracts the ABT-737-mediated increase in Mcl-1 protein Western blot showing that treatment of LNCaP and PC3 cells with 1 uM ABT-737 (A) increases Mcl-1 but treatment with 1 nM Doc (D) decreases Mcl-1. Combination of Doc + ABT-737 (DA) decreases Mcl-1. In LNCaP, there is less Bcl-2 (D, 24 h) and Bak (DA, 48 h) but few differences in Bcl-xL, Bax, Bid, and Noxa. In PC-3, there are few differences Bcl-2, Bcl-xL, Bax, Bak, Bid, and Noxa. PeerJ reviewing PDF | (v2013:06:610:1:1:NEW 5 Aug 2013) R ev ie w in g M an us cr ip t Figure 6 Inhibition of cyclinB1/Cdk1-mediated phosphorylation of Bcl-2/Bcl-xL blocks ABT-737 enhancement of Doc induced apoptosis in PC3 cells (A) Trypan blue exclusion assay showing that 5 uM purvalanol A (P) lowers DA cell death in PC3 cells (*, P<9x10 -8 ). Western blot analysis showing that P blocks the DA increase in cl-PARP, P-Bcl-2 and P-Bcl-xL, whereas there is no change in the total levels of Bcl-2 and Bcl-xL; P treatment increases Mcl-1 in DA treated cells. Vertical line in cl-PARP indicates sample from same blot not in sequence. (B) Trypan blue exclusion assay showing less cell death in DA treated PC3/shCyclin B1-2 and -3 cells compared to PC3/shGFP control cells (*, P<4x10 -5 ). Western blot analysis showing less cyclin B1, cP, P-Bcl-2, P-Bcl-xL, and slightly greater Mcl-1 in PC3/shCyclin B1-2 and -3 cells treated with DA compared to PC3/shGFP control cells. PeerJ reviewing PDF | (v2013:06:610:1:1:NEW 5 Aug 2013) R ev ie w in g M an us cr ip t Figure 7 ABT-737 enhancement of Doc-mediated apoptosis is more dependent on the ability of cyclin B1/Cdk1 to decrease Mcl-1 than to phosphorylate Bcl-2/Bcl-xL Trypan blue exclusion assay showing greater cell death in DA treated (48 h) PC3/shCyclin B1/shMcl-1 (B1-2/M3, B1-3/M3) cells compared to control PC3/shCyclin B1/shGFP (B1-2/G, B1-3/G) and PC3/shGFP (G) cells (*, P< 0.002). Western blot analysis showing increased cl-PARP and decreased Mcl-1 in PC3/shCyclin B1/shMcl-1 compared control shGFP cells after DA treatment (24 h). There is less cyclin B1, P-Bcl-1, and P-Bcl-xL in PC3/shCyclin B1/shGFP or shMcl-1 compared to PC3/shGFP cells, whereas there are no changes in total Bcl-1 or Bcl-xL. PeerJ reviewing PDF | (v2013:06:610:1:1:NEW 5 Aug 2013) R ev ie w in g M an us cr ip t Figure 8 Schematic of how ABT-737 can sensitize Bax+ CRPC cells to Doc Treatment of CRPC cells such as PC3 with Doc deregulates cyclin B1/Cdk1 activity and increases phosphorylation of Bcl-2/Bcl-xL and degradation of Mcl-1. However, the high levels of Bcl-2/Bcl-xL/Mcl-1 in CRPC cells presents a block to apoptosis. Addition of ABT-737 disrupts the anti-apoptotic activity of Bcl-2/Bcl-xL and more effectively allows Doc treatment to bypass the block and increase apoptosis. Cyclin B1/Cdk1 hyperactivity caused by Doc treatment also lowers Mcl-1, an ABT-737 resistance factor, to further increase apoptosis. PeerJ reviewing PDF | (v2013:06:610:1:1:NEW 5 Aug 2013) R ev ie w in g M an us cr ip t acknowledgements : We thank Dr. Pryamvada Rai for critical reading of the manuscript and helpful suggestions; Deanna Palenzuela for technical assistance; Ron Hamelik for assistance with flow cytometry; and Drs. Bernard Roos and Guy Howard for support. discussion : Progression of PCa to CRPC is often associated with overexpression of the anti-apoptotic proteins Bcl-2, Bcl-xL, and Mcl-1, resulting in resistance to apoptosis and poor prognosis (Karnak & Xu, 2011). Doc is an antimitotic drug approved for the treatment of CRPC but the high levels of Bcl-2/Bcl-xL/Mcl-1 confers a block, resulting in less apoptosis and reduced efficacy. Here we report that the Bcl-2/Bcl-xL small molecule antagonist ABT-737 can overcome this block and increase Doc and 1198 (new antimitotic)-induced apoptosis in CRPC PC3 and LN-AI/CSS cells. However, another CRPC cell line, DU145, is more resistant to ABT-737 due to the lack of the pro-apoptotic Bax protein and the combination with Doc or 1198 did not further increase apoptosis. Our results also indicate that the ABT-737 enhancement of Doc-mediated 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 PeerJ reviewing PDF | (v2013:06:610:1:1:NEW 5 Aug 2013) R ev ie w in g M an us cr ip t apoptosis in LNCaP and PC3 cells is dependent upon the ability of Doc to constitutively activate cyclin B1/Cdk1 activity and hyperphosphorylate Bcl-2/Bcl-xL and decrease Mcl-1. Overall, these results provide mechanistic insight into how ABT-737 can sensitize Bax expressing CRPC cells to Doc/1198 treatment by overcoming the block in apoptosis due to the high levels of Bcl-2/Bcl-xL/Mcl-1 (Fig. 8). There is strong evidence that Bcl-2/Bcl-xL overexpression is important for CRPC progression (Miyake, Monia & Gleave, 2000). Our results indicate that the commonly utilized CRPC cell lines DU145 and PC3 express higher levels of Bcl2/Bcl-xL and lower levels of Bax/Bak when compared to androgen-dependent LNCaP cells (Fig. 1A). Several reports indicate that the efficacy of ABT-737 positively correlates with Bcl-2 levels, i.e., the higher the Bcl-2 the better the response to ABT-737 alone or in combination with other chemotherapeutic agents (Del Gaizo Moore et al., 2008; Hann et al., 2008; Mason et al., 2009; Oakes et al., 2012). This correlation does not appear to apply to PCa cell lines as LNCaP has the lowest levels of Bcl-2 yet are as sensitive to ABT-737 as PC3 cells with the highest level of Bcl-2 (Fig. 1). Numerous reports indicate that Mcl-1 overexpression in a variety of cancers can mediate resistance to ABT-737 (van Delft et al., 2006; Chen et al, 2007; Lestini et al., 2009; Hauck et al., 2009; Yecies et al., 2010). However, our results indicate that DU145 cells are more resistant to ABT-737 as a single agent and in combination with Doc/1198 due to the lack of Bax expression and not because they express the highest levels of Mcl-1. In contrast, knockdown of Mcl-1 in Bax expressing LNCaP and PC3 cells enhances sensitivity to Doc/1198 + ABT-737. It is possible that in PCa cells, the complete loss of Bax (as in DU145) is a more dominant mechanism for ABT-737 resistance compared to overexpression of Mcl-1. In PC3, however, the Bax levels are much lower than in LNCaP, yet both cells respond similarly to ABT-737, suggesting that only a low amount of Bax protein is required. In PCa clinical biopsies, loss of Bax immunostaining relative to normal non-cancer prostate epithelium is a useful biomarker for categorizing patient risk and response to 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 PeerJ reviewing PDF | (v2013:06:610:1:1:NEW 5 Aug 2013) R ev ie w in g M an us cr ip t radiation therapy (Pollack et al., 2003; Khor et al., 2007). Therefore, determining the percentage of Bax negative cells in human PCa specimens may provide a useful biomarker for identifying patients that should respond to ABT-737 treatment. During a normal mitotic cell cycle phase, cyclin B1/Cdk1 transiently phosphorylates numerous substrates, including the anti-apoptotic proteins Bcl-2, Bcl-xL, and Mcl-1. Antimitotic drugs such as Doc or 1198 can prevent the degradation of cyclin B1, resulting in constitutively active Cdk1 activity and hyperphosphorylation of Bcl-2/Bcl-xL/Mcl-1. Our results suggest that cyclinB1/Cdk1-mediated phosphorylation of Bcl-2/Bcl-xL is important for the enhancement of apoptosis by the Doc + ABT-737 combination in LNCaP and PC3 cells. This supports previous results that the antimitotic drug vinblastine promotes apoptosis in HeLa cervical carcinoma cells by a similar mechanism (Terrano, Upreti & Chambers, 2010). More recently, the sensitivity of cancer cells to antimitotic drugs has been shown to have a dependence on the ability of cyclin B1/Cdk1 to phosphorylate and increase the degradation of Mcl-1 in order to enhance apoptosis (Harley et al., 2010; Wertz et al., 2011). Our results suggest that the antimitotic-mediated increase in cyclin B1/Cdk1 activity and reduction of Mcl-1 protein counteracts the increase of Mcl-1 by ABT-737, resulting in enhanced apoptosis. Antimitotic drugs or radiation treatment also enhance cyclin B1/Cdk1 phosphorylation of numerous other substrates that are implicated in either increasing or decreasing apoptosis (O'Connor et al., 2000; Konishi et al., 2002; Berndtsson et al., 2005; Allan & Clarke, 2009; Andersen et al., 2009; Nantajit et al., 2010). Overall, our results suggest that the cyclin B1/Cdk1-mediated hyperphosphorylation of Bcl-2, Bcl-xL, and Mcl-1 is a major mechanism linking mitotic arrest to the induction of apoptosis in PCa cells. Although our results do not address whether the Doc/1198 + ABT-737 combination will be effective in vivo, there is evidence in lung, leukemia, prostate, and breast cancer to indicate that the antimitotic + ABT-737 combination should prove to be effective in animal models of PCa (Oakes et al., 2012; Shoemaker et al., 2006; Kang et al., 2007; Bray et al., 2009). Navitoclax, 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 PeerJ reviewing PDF | (v2013:06:610:1:1:NEW 5 Aug 2013) R ev ie w in g M an us cr ip t previously known as ABT-263, is an orally bioavailable analog of ABT-737 with identical function that is currently in Phase II trials for refractory lymphoid malignancies and solid tumors and appears to be a promising agent for use in combination with Doc (Tse et al., 2008; Shi et al., 2011). Our results provide a strong mechanistic rationale for combining targeted chemotherapy against Bcl2/Bcl-xL, as with navitoclax, with the currently approved drug for CRPC Doc, which targets the destruction of Mcl-1, a navitoclax resistance factor. With the addition of cabazitaxel to help in the treatment of patients that develop Doc-resistance, it is also likely that the combination of cabazitaxel and navitoclax will further improve overall survival (de Bono et al., 2010). abt-737, a small molecule bcl-2/bcl-xl antagonist, increases antimitotic-mediated apoptosis in human prostate cancer cells : Castration-resistant prostate cancer (CRPC) expresses high levels of the anti-apoptotic proteins Bcl-2, Bcl-xL and Mcl-1, resulting in resistance to apoptosis and association with poor prognosis. Docetaxel, an antimitotic drug that is the first-line treatment strategy for CRPC, is known to provide a small survival benefit. However, docetaxel chemotherapy alone is not enough to counteract the high levels of Bcl-2/Bcl-xL/Mcl-1 present in CRPC. ABT-737 is a small molecule that binds to Bcl-2/Bcl-xL (but not Mcl-1) with high affinity and disrupts their interaction with pro-apoptotic Bax/Bak, thus enhancing apoptosis. Our results indicate that ABT-737 can sensitize androgen-dependent LNCaP and CRPC PC3 cells to docetaxel- and to the novel antimitotic ENMD-1198-mediated caspase-dependent apoptosis. CRPC DU145 cells, however, are more resistant to ABT-737 because they are Bax null and not because they express the highest levels of anti-apoptotic Mcl-1 (associated with ABT-737 resistance). Knockdown of Bax or Bak in LNCaP indicates that ABT-737-induced antimitotic enhancement of apoptosis is more dependent on the levels of Bax than Bak. Furthermore, we find that the ability of docetaxel to increase cyclin B1/Cdk1-mediated phosphorylation of Bcl-2/Bcl-xL and decrease Mcl-1 is required for ABT-737 to enhance apoptosis in PC3 cells, as determined by addition of Cdk1 inhibitor purvalanol A and expression of shRNA specific for cyclin B1. Overall, our data suggests that the high levels of anti-apoptotic proteins in Bax-expressing CRPC cells can be overcome by targeting Bcl-2/Bcl-xL with ABT-737 and Mcl-1 with antimitotics. PeerJ reviewing PDF | (v2013:06:610:1:1:NEW 5 Aug 2013) R ev ie w in g M an us cr ip t Ricardo Parrondo1, Alicia de las Pozas1, Teresita Reiner1, and Carlos Perez-Stable1, 2,3 1Geriatric Research, Education, and Clinical Center and Research Service, Bruce W. Carter Veterans Affairs Medical Center, Miami, FL, USA 33125 2Division of Gerontology & Geriatric Medicine, Department of Medicine, University of Miami Miller School of Medicine, Miami FL, USA 33136 3Sylvester Comprehensive Cancer Center, University of Miami Miller School of Medicine, Miami FL, USA 33136 Corresponding author: Carlos Perez-Stable, Bruce W. Carter Veterans Affairs Medical Center, GRECC (11-GRC), 1201 NW 16 Street, Miami, FL 33125 USA. Phone: (305) 575-7000, extension 4391. E-mail: cperez@med.miami.edu INTRODUCTION 1 2 3 4 5 6 7 8 9 10 11 PeerJ reviewing PDF | (v2013:06:610:1:1:NEW 5 Aug 2013) R ev ie w in g M an us cr ip t Prostate cancer (PCa) is a leading cause of cancer-related death in men and remains incurable in the metastatic setting. Despite the initial response to androgen deprivation, PCa gradually progresses to castration-resistant prostate cancer (CRPC) (Hadaschik & Gleave, 2007; Attar, Takimoto & Gottardis, 2009). Docetaxel (Doc) is an FDA approved first-line treatment for patients with CRPC but confers only a small survival benefit (Tannock et al., 2004). Once CRPC patients fail Doc chemotherapy, only the Doc derivative cabazitaxel confers a slightly longer overall survival (de Bone et al., 2010). To further improve overall survival of CRPC patients, a better mechanistic understanding of Doc-induced CRPC cell death is required to develop more effective combinatorial treatments. The anti-proliferative activity of Doc results from its ability to bind microtubules and disrupt mitosis (Jordan & Wilson, 2004). Doc activates the mitotic checkpoint and blocks the degradation of cyclin B1, leading to a prolonged activation of cyclin-dependant kinase 1 (Cdk1) and increased mitotic arrest, followed by induction of mitotic catastrophe or apoptosis and also lysosome-dependent cell death (Castedo et al., 2004; Mediavilla-Varela et al., 2009). We have previously shown that small molecule inhibitors of Cdk1 can prevent Doc-mediated increase in cyclin B1/Cdk1 activity and block induction of apoptosis in CRPC cells (Perez-Stable, 2006; Gomez, de las Pozas & Perez-Stable, 2006). This finding indicates that prolonged cyclin B1/Cdk1 activity phosphorylates apoptotic signaling targets that can subsequently lead to apoptosis, although the precise mechanisms have been difficult to determine. However, it is likely that the mechanism involves substrates phosphorylated by cyclin B1/Cdk1. The mitotic response to Doc shows little variation between cell types, whereas the ability to subsequently undergo apoptosis shows large variations (Shi, Orth & Mitchison, 2008; Gascoigne & Taylor, 2008). This suggests that sensitivity to Doc depends to a greater extent on cell type-specific apoptotic signaling mechanisms rather than on pathways that mediate mitotic arrest. Bcl-2, Bcl-xL, and Mcl-1 are anti-apoptotic proteins of the Bcl-2 family that are highly expressed 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 PeerJ reviewing PDF | (v2013:06:610:1:1:NEW 5 Aug 2013) R ev ie w in g M an us cr ip t in CRPC, resulting in resistance to apoptosis and association with poor prognosis (Karnak & Xu, 2011). Bcl-2, Bcl-xL, and Mcl-1 protect cells from apoptosis by binding to Bax and Bak, pro-apoptotic members of the Bcl-2 family, thereby preventing their homodimerization. Bax and Bak homodimers promote apoptosis by forming pores in the mitochondria, leading to mitochondrial outer membrane permeabilization (MOMP), cytochrome c release, and the activation of the caspase cascade (Chipuk et al., 2010). Interestingly, microtubule inhibitors such as Doc induce Bcl-2 and Bcl-xL phosphorylation, thus antagonizing their anti-apoptotic function (Haldar, Jena & Croce, 1995; Poruchynsky et al., 1998). Furthermore, phospho-defective Bcl-2 and Bcl-xL mutants block the pro-apoptotic effects of microtubule inhibitors, reinforcing the notion that mitotic phosphorylation of Bcl-2 and Bcl-xL inhibits their anti-apoptotic function (Haldar, Basu & Croce, 1998; Basu & Haldar,2003; Upreti, et al., 2008; Terrano, Upreti & Chambers, 2010). Recent biochemical data now shows that cyclin B1/Cdk1 is the kinase that phosphorylates Bcl-2 and Bcl-xL during prolonged mitosis after treatment with the microtubule inhibitor vinblastine (Terrano, Upreti & Chambers, 2010). In addition, cyclin B1/Cdk1 can also phosphorylate Mcl-1 to increase its degradation during prolonged mitotic arrest (Harley et al., 2010; Wertz et al., 2011). During a normal mitotic cell cycle phase, cyclin B1/Cdk1 only transiently phosphorylates Bcl-2, Bcl-xL, and Mcl-1, thus limiting the pro-cell death effect. Therefore, it is likely that during Doc-mediated mitotic arrest, prolonged cyclin B1/Cdk1 activity hyperphosphorylates Bcl-2, Bcl-xL, and Mcl-1to block their anti-apoptotic function, which is likely important for increasing cell death. It is known that Doc chemotherapy alone is not enough to overcome the high levels of Bcl-2, Bcl-xL, and Mcl-1 present in patients with CRPC (Karnak & Xu, 2011). A chemical library identified ABT-737 as a small molecule that binds Bcl-2/Bcl-xL (but not Mcl-1) with high affinity to disrupt their interaction with Bax/Bak and enhance the apoptotic signals, especially when combined with other chemotherapeutic drugs (Oltersdorf et al., 2005; Tagscherer et al., 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 PeerJ reviewing PDF | (v2013:06:610:1:1:NEW 5 Aug 2013) R ev ie w in g M an us cr ip t 2008). The purpose of the present study is to determine whether ABT-737 combined with Doc or a novel antimitotic ENMD-1198 (a more stable and potent derivative of 2-methoxyestradiol with clinical promise [LaVallee et al., 2008; Zhou et al., 2011]) can overcome the high levels of Bcl-2/Bcl-xL/Mcl-1 in CRPC cells and enhance apoptotic cell death. Our results show that ABT-737 enhances Doc and 1198-mediated caspase-dependent apoptosis in some PCa cells and that this enhancement is dependent on expression of Bax and on cyclin B1/Cdk1-mediated phosphorylation of Bcl-2/Bcl-xL and decrease in Mcl-1.",
"v2_text": "abt-737, a small molecule bcl-2/bcl-xl antagonist, increases antimitotic-mediated apoptosis in human prostate cancer cells : Castration-resistant prostate cancer (CRPC) expresses high levels of the anti-apoptotic proteins Bcl-2, Bcl-xL and Mcl-1, resulting in resistance to apoptosis and association with poor prognosis. Docetaxel, an antimitotic drug that is the first-line treatment strategy for CRPC, is known to provide a small survival benefit. However, docetaxel chemotherapy alone is not enough to counteract the high levels of Bcl-2/Bcl-xL/Mcl-1 present in CRPC. ABT-737 is a small molecule that binds to Bcl-2/Bcl-xL (but not Mcl-1) with high affinity and disrupts their interaction with pro-apoptotic Bax/Bak, thus enhancing apoptosis. Our results indicate that ABT-737 can sensitize androgen-dependent LNCaP and CRPC PC3 cells to docetaxel- and to the novel antimitotic ENMD-1198-mediated caspase-dependent apoptosis. CRPC DU145 cells, however, are more resistant to ABT-737 because they are Bax null and not because they express the highest levels of anti-apoptotic Mcl-1 (associated with ABT-737 resistance). Knockdown of Bax or Bak in LNCaP indicates that ABT-737-induced antimitotic enhancement of apoptosis is more dependent on the levels of Bax than Bak. Furthermore, we find that the ability of docetaxel to increase cyclin B1/Cdk1-mediated phosphorylation of Bcl-2/Bcl-xL and decrease Mcl-1 is required for ABT-737 to enhance apoptosis in PC3 cells, as determined by addition of Cdk1 inhibitor purvalanol A and expression of shRNA specific for cyclin B1. Overall, our data suggests that the high levels of anti-apoptotic proteins in Bax-expressing CRPC cells can be overcome by targeting Bcl-2/Bcl-xL with ABT-737 and Mcl-1 with antimitotics. PeerJ reviewing PDF | (v2013:06:610:0:1:NEW 25 Jun 2013) R ev ie w in g M an us cr ip t Ricardo Parrondo1, Alicia de las Pozas1, Teresita Reiner1, and Carlos Perez-Stable1, 2,3 1 2 1Geriatric Research, Education, and Clinical Center and Research Service, Bruce W. Carter 3 Veterans Affairs Medical Center, Miami, FL, USA 33125 4 2Division of Gerontology & Geriatric Medicine, Department of Medicine, University of Miami 5 Miller School of Medicine, Miami FL, USA 33136 6 3Sylvester Comprehensive Cancer Center, University of Miami Miller School of Medicine, 7 Miami FL, USA 33136 8 9 10 Corresponding author: Carlos Perez-Stable, Bruce W. Carter Veterans Affairs Medical Center, 11 GRECC (11-GRC), 1201 NW 16 Street, Miami, FL 33125 USA. Phone: (305) 575-7000, 12 extension 4391. E-mail: cperez@med.miami.edu 13 14 15 16 17 18 19 20 21 22 PeerJ reviewing PDF | (v2013:06:610:0:1:NEW 25 Jun 2013) R ev ie w in g M an us cr ip t 2 INTRODUCTION 23 Prostate cancer (PC) is a leading cause of cancer-related death in men and remains incurable in 24 the metastatic setting. Despite the initial response to androgen deprivation, PC gradually 25 progresses to castration-resistant prostate cancer (CRPC) (Hadaschik & Gleave, 2007; 26 Attar, Takimoto & Gottardis, 2009). Docetaxel (Doc) is an FDA approved first-line treatment 27 for patients with CRPC but confers only a small survival benefit (Tannock et al., 2004). Once 28 CRPC patients fail Doc chemotherapy, only the Doc derivative cabazitaxel confers a slightly 29 longer overall survival (de Bone et al., 2010). To further improve overall survival of CRPC 30 patients, a better mechanistic understanding of Doc-induced CRPC cell death is required to 31 develop more effective combinatorial treatments. 32 The anti-proliferative activity of Doc results from its ability to bind microtubules and 33 disrupt mitosis (Jordan & Wilson, 2004). Doc activates the mitotic checkpoint and blocks the 34 degradation of cyclin B1, leading to a prolonged activation of cyclin-dependant kinase 1 (Cdk1) 35 and increased mitotic arrest, followed by induction of mitotic catastrophe or apoptosis (Castedo 36 et al., 2004). We have previously shown that small molecule inhibitors of Cdk1 can prevent 37 Doc-mediated increase in cyclin B1/Cdk1 activity and block induction of apoptosis in CRPC 38 cells (Perez-Stable, 2006; Gomez, de las Pozas & Perez-Stable, 2006). This finding indicates 39 that prolonged cyclin B1/Cdk1 activity phosphorylates apoptotic signaling targets that can 40 subsequently lead to apoptosis, although the precise mechanisms have been difficult to 41 determine. However, it is likely that the mechanism involves substrates phosphorylated by cyclin 42 B1/Cdk1. 43 The mitotic response to Doc shows little variation between cell types, whereas the ability to 44 subsequently undergo apoptosis shows large variations (Shi, Orth & Mitchison, 2008; Gascoigne 45 PeerJ reviewing PDF | (v2013:06:610:0:1:NEW 25 Jun 2013) R ev ie w in g M an us cr ip t 3 & Taylor, 2008). This suggests that sensitivity to Doc depends to a greater extent on cell type-46 specific apoptotic signaling mechanisms rather than on pathways that mediate mitotic arrest. 47 Bcl-2, Bcl-xL, and Mcl-1 are anti-apoptotic proteins of the Bcl-2 family that are highly 48 expressed in CRPC, resulting in resistance to apoptosis and association with poor prognosis 49 (Karnak & Xu, 2011). Bcl-2, Bcl-xL, and Mcl-1 protect cells from apoptosis by binding to Bax 50 and Bak, pro-apoptotic members of the Bcl-2 family, thereby preventing their homodimerization. 51 Bax and Bak homodimers promote apoptosis by forming pores in the mitochondria, leading to 52 mitochondrial outer membrane permeabilization (MOMP), cytochrome c release, and the 53 activation of the caspase cascade (Chipuk et al., 2010). 54 Interestingly, microtubule inhibitors such as Doc induce Bcl-2 and Bcl-xL phosphorylation, 55 thus antagonizing their anti-apoptotic function (Haldar, Jena & Croce, 1995; Poruchynsky et al., 56 1998). Furthermore, phospho-defective Bcl-2 and Bcl-xL mutants block the pro-apoptotic 57 effects of microtubule inhibitors, reinforcing the notion that mitotic phosphorylation of Bcl-2 and 58 Bcl-xL inhibits their anti-apoptotic function (Haldar, Basu & Croce, 1998; Basu & Haldar,2003; 59 Upreti, et al., 2008; Terrano, Upreti & Chambers, 2010). Recent biochemical data now shows 60 that cyclin B1/Cdk1 is the kinase that phosphorylates Bcl-2 and Bcl-xL during prolonged mitosis 61 after treatment with the microtubule inhibitor vinblastine (Terrano, Upreti & Chambers, 2010). 62 In addition, cyclin B1/Cdk1 can also phosphorylate Mcl-1 to increase its degradation during 63 prolonged mitotic arrest (Harley et al., 2010; Wertz et al., 2011). Therefore, it is likely that 64 during Doc-mediated mitotic arrest, prolonged cyclin B1/Cdk1 activity hyperphosphorylates Bcl-65 2, Bcl-xL, and Mcl-1to block their anti-apoptotic function. 66 It is known that Doc chemotherapy alone is not enough to overcome the high levels of Bcl-67 2, Bcl-xL, and Mcl-1 present in patients with CRPC (Karnak & Xu, 2011). A chemical library 68 PeerJ reviewing PDF | (v2013:06:610:0:1:NEW 25 Jun 2013) R ev ie w in g M an us cr ip t 4 identified ABT-737 as a small molecule that binds Bcl-2/Bcl-xL (but not Mcl-1) with high 69 affinity to disrupt their interaction with Bax/Bak and enhance the apoptotic signals, especially 70 when combined with other chemotherapeutic drugs (Oltersdorf et al., 2005; Tagscherer et al., 71 2008). The purpose of the present study is to determine whether ABT-737 combined with Doc 72 or a novel antimitotic ENMD-1198 (a more stable and potent derivative of 2-methoxyestradiol 73 with clinical promise [LaVallee et al., 2008; Zhou et al., 2011]) can overcome the high levels of 74 Bcl-2/Bcl-xL/Mcl-1 in CRPC cells and enhance apoptotic cell death. Our results show that 75 ABT-737 enhances Doc and 1198-mediated caspase-dependent apoptosis in some PC cells and 76 that this enhancement is dependent on expression of Bax and on cyclin B1/Cdk1-mediated 77 phosphorylation of Bcl-2/Bcl-xL and decrease in Mcl-1. 78 79 MATERIALS AND METHODS 80 Reagents 81 ABT-737 was obtained from Abbott Laboratories (Abbott Park, IL, USA), Doc from Sanofi-82 Aventis (Bridgewater, NJ, USA), and ENMD-1198 from EntreMed, Inc (Rockville, MD, USA). 83 Q-VD pan-caspase inhibitor was purchased from R&D Systems (Minneapolis, MN, USA); 84 purvalanol A from A.G. Scientific (San Diego, CA, USA); Trypan blue (0.4%) from Invitrogen 85 (Grand Island, NY, USA); and Coomassie blue from EMD Chemicals (Billerica, MA, USA). All 86 other reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA). 87 Cell culture 88 Human PC cell lines LNCaP, DU145, and PC3 were obtained from the American Type Culture 89 Collection (Manassas, VA, USA) (van Bokhoven et al., 2003) and used within 6 months of 90 resuscitation of original cultures. All cells were maintained in RPMI 1640 medium (Invitrogen) 91 PeerJ reviewing PDF | (v2013:06:610:0:1:NEW 25 Jun 2013) R ev ie w in g M an us cr ip t 5 with 5% fetal bovine serum (Hyclone, Waltham, MA), 100 U/ml penicillin, 100 \u03bcg/ml 92 streptomycin, and 0.25 \u03bcg/ml amphotericin (Invitrogen). Unlike LNCaP, LN-AI cells are able to 93 grow for long-term in RPMI 1640 with 5% charcoal-stripped fetal bovine serum (Hyclone) and 94 are referred to as LN-AI/CSS (Gomez, de las Pozas & Perez-Stable, 2006). 95 Western blot analysis 96 Preparation of total protein lysates and western blot analysis was done as previously described 97 (Gomez, de las Pozas & Perez-Stable, 2006). The following antibodies were used: Bcl-2 (N-19), 98 Bax (N-20), Mcl-1 (S-19), cyclin B1 (GNS1), AIF (E-1), and horseradish peroxidase-conjugated 99 secondary antibody from Santa Cruz Biotechnology (Santa Cruz, CA, USA); Bak (NT) from 100 EMD Millipore (Billerica, MA, USA); Bcl-xL (#610211), cytochrome c (7H8.2C12), Smac 101 (#612245) from BD Biosciences (San Diego, CA, USA); cleaved PARP (9541), phospho-(Ser70) 102 Bcl-2 (2827), CoxIV (#4844), Bid (#2002) from Cell Signaling Technology (Danvers, MA, 103 USA); phospho-(Ser62) Bcl-xl (30655) from Abcam (Cambridge, MA, USA); and Noxa 104 (114C307.1) from Novus Biologicals (Littleton, CO, USA). Our preference for loading controls 105 was for staining of total protein with Coomassie blue because drug treatments often affect the 106 levels of typical housekeeping proteins such as actin or tubulin. 107 ABT-737 cell viability assay 108 LNCaP, DU145, and PC-3 cells were seeded in 96-well plates. The next day, fresh media 109 containing ABT-737 (1, 2.5, 5, 10 \u00b5M), or control (0.1% DMSO) were added and cells incubated 110 for three days. The CellTiter Aqueous cell proliferation colorimetric method from Promega 111 (Madison, WI, USA) was used to determine cell viability, as per manufacturer\u2019s instructions. 112 Cell viability was normalized against the vehicle control and the data expressed as a percentage 113 of control from three independent experiments done in triplicate. 114 PeerJ reviewing PDF | (v2013:06:610:0:1:NEW 25 Jun 2013) R ev ie w in g M an us cr ip t 6 Drug treatments 115 PC cells were cultured in media containing Doc (1nM), 1198 (1 \u00b5M), ABT-737 (1 \u00b5M), Doc or 116 1198 + ABT-737, Q-VD (10 \u00b5M), purvalanol A (5 \u00b5M), Doc or 1198 + Q-VD or purvalanol A, 117 or DMSO (0.1%) control for varying times (24\u221272 h). In all the experiments, floating and 118 trypsinized attached cells were pooled for further analysis. 119 Trypan blue exclusion assay 120 Treated and control PC cells were harvested, resuspended in PBS, diluted 1:1 in 0.4% trypan 121 blue, dead blue and live non-blue cells immediately counted using a hemacytometer, and the % 122 dead blue cells determined from at least three independent experiments done in duplicate. 123 Annexin-FITC/propidium iodide (PI) flow cytometry 124 Treated and control PC cells were resuspended in binding buffer followed by the addition of 125 annexin V-FITC and PI (Annexin V Kit sc-4252 AK, Santa Cruz Biotechnology). After 20 min., 126 cells were analyzed by flow cytometry using a Coulter XL flow cytometer and the percentage of 127 annexin+ cells determined using WinMDI version 2.8 from two independent experiments done 128 in triplicate. 129 Mitochondrial protein release assay 130 Treated and control PC cells were resuspended in a buffer containing 100-200 \u00b5M digitonin, 20 131 mM Hepes, pH 7.5, 10 mM KCl, 1.5 mM MgCl, 1 mM EGTA, 1 mM EDTA, 1 mM DTT, 250 132 mM sucrose, and protease inhibitors (Roche, Nutley, NJ) at 50 \u00b5l/1 x106 cells. After 5 min on 133 ice, cells were centrifuged at 14k rpm 5 min and the supernatant used for western blot analysis. 134 Digitonin is a detergent that preferentially permeabilizes plasma membrane compared to 135 mitochondrial membrane (Gottlieb & Granville, 2002). 136 PeerJ reviewing PDF | (v2013:06:610:0:1:NEW 25 Jun 2013) R ev ie w in g M an us cr ip t 7 Retrovirus transduction of DU145 and LNCaP with Bax 137 hBax C3-EGFP (Addgene, Cambridge, MA, USA; plasmid 19741) (Nechushtan et al., 1999) 138 was digested with HindIII, blunt-ended with Klenow DNA polymerase, digested with EcoRI, 139 and the 0.6 kb Bax insert ligated into pBABE puro plasmid (BamH1-blunt/EcoRI) using DNA 140 ligase (New England Biolabs, Ipswich, MA, USA). Retrovirus production and infection were 141 done by transfecting HEK293T cells (American Type Culture Collection) with pBABE/Bax or 142 pBABE/EV (empty vector), pUMVC3, and pCVM-VSV-G with FuGene HD (Roche), addition 143 of filtered (0.45 \u00b5M) media after 48 h to DU145 and LNCaP cells, and selection with puromycin 144 (Invitrogen; 2 \u00b5g/ml) for 1 week. Cell death in DU145/Bax, DU145/EV, LNCaP/Bax, and 145 LNCaP/EV cells treated with DMSO control were similar to parental cells (not shown). 146 Lentiviral transduction of LNCaP, DU145, and PC3 with shRNA 147 The shRNA design, lentivirus production, and infection were done as previously described (Stewart et 148 al, 2003). The following DNA oligonucleotides (Operon, Huntsville, AL, USA) targeting Mcl-1, Bax, 149 Bak, and cyclin B1 were cloned into pLKO.1 lentivirus vector: shMcl-1 (M2): CCGGGCTGGAGA 150 TTATCTCTCGGTACTCGAGTACCGAGAGATAATCTCCAGCTTTTTG; shMcl-1 (M3): 151 CCGGGCTAAACACTTGAAGACCATACTCGAGTATGGTCTTCAAGTGTTTAGC TTTTTG; 152 shBax-1: CCGGGCCGGAACTGATCAGAACCATCTCGAGATGGTTCTGATCAGTTCCGG 153 CTTTTTG (PC3); shBax-2: CCGGGCCTCAGGATGCGTCCACCAACTCGAGTTGGTGGACG 154 CATCCTGAGGCTTTTTG (LNCaP); shBak-1: CCGGTGGTACGAAGATTCTTCAAATCTC 155 GAGATTTGAAGAATCTTCGTACCATTTTTG (LNCaP); shBak-3: CCGGATGAGTACTTCA 156 CCAAGATTGCTCGAGCAATCTTGGTGAAGTACTCATTTTTTG (PC3); shCyclin B1-2: CCGG 157 GCCAAATACCTGATGGAACTACTCGAGTAGTTCCATCAGGTATTTGGCTTTTTG; and 158 shCyclin B1-3: CCGGGCCATCCTAATTGACTGGCTACTCGAGTAGCCAGTCAATTAGGATG 159 PeerJ reviewing PDF | (v2013:06:610:0:1:NEW 25 Jun 2013) R ev ie w in g M an us cr ip t 8 GCTTTTTG. The control shRNA was targeted against green fluorescent protein (GFP). For Mcl-1 160 knockdown in DU145/Bax and PC3/shCyclin B1 cells (puromycin resistant), shMcl-1 and shGFP 161 oligonucleotides were cloned into pLKO.1/hygromycin plasmid and transduced cells selected with 400 162 \u00b5g/ml hygromycin (Invitrogen) for two weeks. Cell death in DU145/shMcl-1, DU145/shGFP, 163 DU145/Bax/shMcl-1, DU145/Bax/shGFP, LNCaP/shBax, LNCaP/shBak, LNCaP/shGFP, PC3/shBax, 164 PC3/shBak, PC3/shCyclin B1, PC3/shGFP, PC3/shCyclin B1/shMcl-1, and PC3/shCyclin B1/shGFP 165 cells treated with DMSO control were similar to parental cells (not shown). 166 Statistical analysis 167 Statistical differences between drug-treated and control PC cells were determined by two-tailed 168 Student\u2019s t-test (unequal variance) with P<0.05 considered significant. 169 170 RESULTS 171 CRPC cells express high anti-apoptotic Bcl-2/Bcl-xL/Mcl-1 and low or null pro-apoptotic 172 Bax/Bak 173 Since ABT-737 is a Bcl-2/Bcl-xL antagonist that should promote the pro-apoptotic function of 174 Bax/Bak, we compared the protein levels of Bcl-2, Bcl-xL, Bax, and Bak in LNCaP, DU145, and 175 PC3 cells. LNCaP cells are androgen-dependent, contain wild-type p53, and exhibit higher 176 sensitivity to antimitotic-mediated apoptosis relative to DU145 and PC3, which are castration-177 resistant and p53 mutated or null (van Bokhoven et al., 2003; Reiner et al., 2009). As expected, 178 DU145 and PC3 expressed higher Bcl-2/Bcl-xL when compared to LNCaP cells (Fig. 1A). 179 Expression of Mcl-1, an anti-apoptotic member of the Bcl-2 family that is not targeted by ABT-180 737 and is associated with chemoresistance to ABT-737 treatment (van Delft et al., 2006; Chen 181 et al, 2007; Lestini et al., 2009; Hauck et al., 2009; Yecies et al., 2010), was highest in DU145 182 PeerJ reviewing PDF | (v2013:06:610:0:1:NEW 25 Jun 2013) R ev ie w in g M an us cr ip t 9 compared to PC3 and LNCaP cells. The protein levels of pro-apoptotic Bax and Bak were lower 183 in CRPC cells compared to LNCaP; Bax is null in DU145 (Tang et al., 1998). These results 184 suggest that the high anti-apoptotic Bcl-2/Bcl-xL and low pro-apoptotic Bax/Bak protein 185 environment present in CRPC cells may benefit from ABT-737 treatment in order to enhance 186 apoptotic cell death. However, DU145 cells were more resistant to ABT-737 as a single agent 187 when compared to LNCaP and PC3 cells (Fig. 1B). 188 ABT-737 enhances Doc/1198-mediated apoptosis in LNCaP and PC3 but not in DU145 cells 189 CRPC cells such as DU145 and PC3 are more resistant to Doc treatment compared to androgen-190 dependent cells such as LNCaP and combinations with other drugs or agents are required to 191 increase therapeutic efficacy. Our results showed that the combination of 1 nM Doc or 1 \u00b5M 192 1198 with a sub-cytotoxic dose of ABT-737 (1 \u00b5M) significantly increased cell death and 193 cleaved-PARP (measure of caspase activity) compared to the single agents in LNCaP and PC3 194 but not in DU145 cells (Fig. 2A, B; Supplemental Fig. S1A). Similar results were obtained in 195 LNCaP-AI/CSS, a CRPC variant of LNCaP that is more chemoresistant (Supplemental Fig. 196 S1B). The pan-caspase inhibitor Q-VD (10 \u00b5M) blocked Doc + ABT-737-mediated cell death 197 and cleaved-PARP, indicating that increased caspase activity was required (Fig. 2A, B). 198 ABT-737 targets the mitochondria to initiate the intrinsic pathway of apoptosis by 199 increasing the release of mitochondrial proteins such as cytochrome c, which in turn activates the 200 caspase cascade (Chipuk et al., 2010). Our results indicated that ABT-737 enhanced Doc-201 mediated release of cytochrome c, Smac (blocks inhibitor of apoptosis [IAP] family; LaCasse et 202 al., 2008), and apoptosis-inducing factor (AIF; translocates to nucleus to increase DNA 203 fragmentation; Susin et al., 1999) from the mitochondria in LNCaP and PC3 but not in DU145 204 cells (Fig. 2C; Supplemental Fig. S2). In addition, there was less cytoplasmic Bax protein in 205 PeerJ reviewing PDF | (v2013:06:610:0:1:NEW 25 Jun 2013) R ev ie w in g M an us cr ip t 10 Doc + ABT-737 treated LNCaP and PC3 cells, likely as a result of greater translocation of Bax 206 to the mitochondria. Thus, ABT-737 enhancement of Doc-mediated pro-apoptotic protein release 207 from the mitochondria correlates with increased apoptotic cell death in LNCaP and PC3 but not 208 in DU145. 209 Bax expression but not Mcl-1 suppression sensitizes DU145 to ABT-737 enhancement of 210 Doc/1198-mediated apoptosis 211 We investigated the mechanisms why DU145 cells are more resistant to ABT-737. One 212 possibility stems from the Bax null status of DU145 cells, which would indicate that ABT-737 213 mediates its cytotoxicity via the Bax pathway. This is supported by a previous finding indicating 214 that transient transfection of Bax into DU145 cells increases sensitivity to ABT-737 + TRAIL 215 (Song, Kandasamy & Kraft, 2008). Another possibility is that ABT-737 resistance of DU145 216 cells arises from elevated Mcl-1, which unlike Bcl-2/Bcl-xL does not interact with ABT-737 and 217 may therefore block the ability of ABT-737 to increase apoptosis (van Delft et al., 2006; Chen et 218 al, 2007; Lestini et al., 2009; Hauck et al., 2009; Yecies et al., 2010). To address these 219 possibilities, we isolated DU145 cells stably expressing Bax and DU145 cells with stable Mcl-1 220 knockdown. Our results showed that ABT-737 significantly enhanced Doc/1198-mediated cell 221 death in DU145/Bax cells above Doc/1198 treatment alone (Fig. 3A, B). Similar results were 222 obtained in LNCaP/Bax cells (Supplemental Fig S3). In contrast, knockdown of Mcl-1 in 223 DU145 or DU145/Bax cells did not significantly enhance Doc/1198 + ABT-737-mediated 224 apoptotic cell death (Fig. 3C; Supplemental Fig. S4). These results indicate that DU145 cells 225 are more resistant to ABT-737 enhancement of antimitotic-mediated apoptosis because they are 226 Bax null and not because they express high Mcl-1. In contrast, knockdown of Mcl-1 in LNCaP 227 PeerJ reviewing PDF | (v2013:06:610:0:1:NEW 25 Jun 2013) R ev ie w in g M an us cr ip t 11 and PC3 cells increased cell death and cl-PARP, confirming the importance of Mcl-1 in 228 resistance to Doc/1198 + ABT-737 treatment (Supplemental Fig. S5). 229 ABT-737-mediated enhancement of Doc/1198-induced apoptosis in LNCaP is more 230 dependent on Bax than Bak 231 ABT-737 inhibits the interactions of Bcl-2/Bax and Bcl-xL/Bak, thus allowing Bax and Bak to 232 induce MOMP and apoptosis (Oltersdorf et al., 2005; Tagscherer et al., 2008). To further 233 investigate the relative importance of Bax versus Bak in mediating ABT-737 enhancement of 234 Doc/1198-mediated cell death in PC cells, we isolated LNCaP and PC3 cells stably expressing 235 either shBax or shBak. Results showed that knockdown of Bax lowered Doc/1198 + ABT-737-236 mediated cell death and cleaved-PARP in both LNCaP and PC3 cells compared to the negative 237 control shGFP cells (Fig. 4A). Knockdown of Bak also lowered Doc/1198 + ABT-737-induced 238 cell death and cleaved-PARP in PC3 cells but had no significant effects in LNCaP cells (Fig. 239 4B). These results suggest that ABT-737 enhancement of Doc/1198-mediated apoptosis in 240 LNCaP cells is more dependent on Bax than to Bak but PC3 cells are dependent on both Bax and 241 Bak. 242 Doc counteracts the ABT-737-mediated increase in Mcl-1 243 We determined if Doc and Doc + ABT-737 had any effects on the protein levels of Bcl-2 family 244 members in LNCaP and PC3 cells. Interestingly, Doc decreased and ABT-737 increased Mcl-1 245 but the combination of Doc + ABT-737 lowered Mcl-1 (Fig. 5). The mechanism why ABT-737 246 alone increases Mcl-1 protein is not known but may reflect the observation that acquired 247 resistance to ABT-737 involves increased Mcl-1 (Yecies et al., 2010). There were no clear 248 differences in the protein levels of Bcl-2, Bcl-xL, Bax, Bak, Bid, and Noxa with the exception in 249 LNCaP where there was less Bcl-2 (Doc, 24 h) and Bak (Doc+ ABT-737, 48 h). These results 250 PeerJ reviewing PDF | (v2013:06:610:0:1:NEW 25 Jun 2013) R ev ie w in g M an us cr ip t 12 suggest that the Doc + ABT-737 enhancement of apoptosis may depend upon the ability of Doc 251 to counteract the ABT-737-mediated increase in Mcl-1. 252 ABT-737-mediated enhancement of Doc-induced apoptosis is dependent on cyclinB1/Cdk1-253 mediated phosphorylation of Bcl-2/Bcl-xL and decrease of Mcl-1 254 Small molecule inhibitors of Cdk1 prevent Doc-mediated increase in cyclin B1/Cdk1 activity 255 and blocks induction of apoptosis in CRPC cells (Perez-Stable, 2006; Gomez, de las Pozas & 256 Perez-Stable, 2006). We investigated whether cyclin B1/Cdk1-mediated increase in Bcl-2/Bcl-257 xL phosphorylation and decrease in Mcl-1 is important for the ABT-737 enhancement of Doc-258 induced apoptosis. Treatment (Doc alone and Doc + ABT-737) of LNCaP and PC3 but not 259 DU145 cells increased phospho (P)-Bcl-2, whereas the levels of P-Bcl-xL was similar in all three 260 cell lines (Supplemental Fig. S6). Addition of 5 \u00b5M purvalanol A, a specific inhibitor of cyclin 261 B1/Cdk1 activity (Gray et al., 1998) blocked Doc + ABT-737 cell death and the increase in P-262 Bcl-2/P-Bcl-xL in PC3 cells (Fig. 6A). However, purvalanol A increased Mcl-1 protein in Doc 263 + ABT-737 treated PC3 cells, suggesting that this may also play an important role in blocking 264 apoptotic cell death (Fig. 6A). 265 To further determine whether the Doc-mediated increase in cyclin B1/Cdk1 activity, P-Bcl-266 2/P-Bcl-xL, and decrease in Mcl-1 is important for ABT-737 + Doc cell death, we isolated PC3 267 cells stably expressing shCyclin B1. Results showed that knockdown of cyclin B1 lowered Doc 268 + ABT-737 cell death, cleaved-PARP, and P-Bcl-2/P-Bcl-xL in PC3/shCyclin B1 cells compared 269 to shGFP control cells (Fig. 6B). However, given that there is less cyclin B1 to mediate Cdk1 270 degradation of Mcl-1 by Doc, it is not surprising that Mcl-1 levels are slightly higher in Doc + 271 ABT-737 treated PC3/shCyclin B1 compared to shGFP cells (Fig. 6B). We then isolated 272 PC3/shCyclin B1 cells stably expressing shMcl-1 and the results showed that Doc + ABT-737 273 PeerJ reviewing PDF | (v2013:06:610:0:1:NEW 25 Jun 2013) R ev ie w in g M an us cr ip t 13 increased cell death (67%) nearly to the levels of PC3/shMcl-1 cells (83%) (Fig. 7; 274 Supplemental Fig. S5B). Overall, these results suggest that the ABT-737 enhancement of Doc-275 mediated apoptosis is more dependent on the ability of cyclin B1/Cdk1 to increase the 276 degradation of Mcl-1 than to phosphorylate Bcl-2/Bcl-xL. 277 278 DISCUSSION 279 Progression of PC to CRPC is often associated with overexpression of the anti-apoptotic proteins 280 Bcl-2, Bcl-xL, and Mcl-1, resulting in resistance to apoptosis and poor prognosis (Karnak & Xu, 281 2011). Doc is an antimitotic drug approved for the treatment of CRPC but the high levels of Bcl-282 2/Bcl-xL/Mcl-1 confers a block, resulting in less apoptosis and reduced efficacy. Here we report 283 that the Bcl-2/Bcl-xL small molecule antagonist ABT-737 can overcome this block and increase 284 Doc and 1198 (new antimitotic)-induced apoptosis in CRPC PC3 and LN-AI/CSS cells. 285 However, another CRPC cell line, DU145, is more resistant to ABT-737 due to the lack of the 286 pro-apoptotic Bax protein and the combination with Doc or 1198 did not further increase 287 apoptosis. Our results also indicate that the ABT-737 enhancement of Doc-mediated apoptosis in 288 LNCaP and PC3 cells is dependent upon the ability of Doc to constitutively activate cyclin 289 B1/Cdk1 activity and hyperphosphorylate Bcl-2/Bcl-xL and decrease Mcl-1. Overall, these 290 results provide mechanistic insight into how ABT-737 can sensitize Bax expressing CRPC cells 291 to Doc/1198 treatment by overcoming the block in apoptosis due to the high levels of Bcl-2/Bcl-292 xL/Mcl-1 (Fig. 8). 293 There is strong evidence that Bcl-2/Bcl-xL overexpression is important for CRPC 294 progression (Miyake, Monia & Gleave, 2000). Our results indicate that the commonly utilized 295 CRPC cell lines DU145 and PC3 express higher levels of Bcl2/Bcl-xL and lower levels of 296 PeerJ reviewing PDF | (v2013:06:610:0:1:NEW 25 Jun 2013) R ev ie w in g M an us cr ip t 14 Bax/Bak when compared to androgen-dependent LNCaP cells (Fig. 1A). Several reports 297 indicate that the efficacy of ABT-737 positively correlates with Bcl-2 levels, i.e., the higher the 298 Bcl-2 the better the response to ABT-737 alone or in combination with other chemotherapeutic 299 agents (Del Gaizo Moore et al., 2008; Hann et al., 2008; Mason et al., 2009; Oakes et al., 2012). 300 This correlation does not appear to apply to PC cell lines as LNCaP has the lowest levels of Bcl-301 2 yet are as sensitive to ABT-737 as PC3 cells with the highest level of Bcl-2 (Fig. 1). 302 Numerous reports indicate that Mcl-1 overexpression in a variety of cancers can mediate 303 resistance to ABT-737 (van Delft et al., 2006; Chen et al, 2007; Lestini et al., 2009; Hauck et al., 304 2009; Yecies et al., 2010). However, our results indicate that DU145 cells are more resistant to 305 ABT-737 as a single agent and in combination with Doc/1198 due to the lack of Bax expression 306 and not because they express the highest levels of Mcl-1. In contrast, knockdown of Mcl-1 in 307 Bax expressing LNCaP and PC3 cells enhances sensitivity to Doc/1198 + ABT-737. It is 308 possible that in PC cells, the complete loss of Bax (as in DU145) is a more dominant mechanism 309 for ABT-737 resistance compared to overexpression of Mcl-1. In PC3, however, the Bax levels 310 are much lower than in LNCaP, yet both cells respond similarly to ABT-737, suggesting that 311 only a low amount of Bax protein is required. In PC clinical biopsies, loss of Bax 312 immunostaining relative to normal non-cancer prostate epithelium is a useful biomarker for 313 categorizing patient risk and response to radiation therapy (Pollack et al., 2003; Khor et al., 314 2007). Therefore, determining the percentage of Bax negative cells in human PC specimens may 315 provide a useful biomarker for identifying patients that should respond to ABT-737 treatment. 316 During a normal mitotic cell cycle phase, cyclin B1/Cdk1 transiently phosphorylates 317 numerous substrates, including the anti-apoptotic proteins Bcl-2, Bcl-xL, and Mcl-1. Antimitotic 318 drugs such as Doc or 1198 can prevent the degradation of cyclin B1, resulting in constitutively 319 PeerJ reviewing PDF | (v2013:06:610:0:1:NEW 25 Jun 2013) R ev ie w in g M an us cr ip t 15 active Cdk1 activity and hyperphosphorylation of Bcl-2/Bcl-xL/Mcl-1. Our results suggest that 320 cyclinB1/Cdk1-mediated phosphorylation of Bcl-2/Bcl-xL is important for the enhancement of 321 apoptosis by the Doc + ABT-737 combination in LNCaP and PC3 cells. This supports previous 322 results that the antimitotic drug vinblastine promotes apoptosis in HeLa cervical carcinoma cells 323 by a similar mechanism (Terrano, Upreti & Chambers, 2010). More recently, the sensitivity of 324 cancer cells to antimitotic drugs has been shown to have a dependence on the ability of cyclin 325 B1/Cdk1 to phosphorylate and increase the degradation of Mcl-1 in order to enhance apoptosis 326 (Harley et al., 2010; Wertz et al., 2011). Our results suggest that the antimitotic-mediated 327 increase in cyclin B1/Cdk1 activity and reduction of Mcl-1 protein counteracts the increase of 328 Mcl-1 by ABT-737, resulting in enhanced apoptosis. Antimitotic drugs or radiation treatment 329 also enhance cyclin B1/Cdk1 phosphorylation of numerous other substrates that are implicated in 330 either increasing or decreasing apoptosis (O'Connor et al., 2000; Konishi et al., 2002; 331 Berndtsson et al., 2005; Allan & Clarke, 2009; Andersen et al., 2009; Nantajit et al., 2010). 332 Overall, our results suggest that the cyclin B1/Cdk1-mediated hyperphosphorylation of Bcl-2, 333 Bcl-xL, and Mcl-1 is a major mechanism linking mitotic arrest to the induction of apoptosis in 334 PC cells. 335 Although our results do not address whether the Doc/1198 + ABT-737 combination will be 336 effective in vivo, there is evidence in lung, leukemia, prostate, and breast cancer to indicate that 337 the antimitotic + ABT-737 combination should prove to be effective in animal models of PC 338 (Oakes et al., 2012; Shoemaker et al., 2006; Kang et al., 2007; Bray et al., 2009). Navitoclax, 339 previously known as ABT-263, is an orally bioavailable analog of ABT-737 with identical 340 function that is currently in Phase II trials for refractory lymphoid malignancies and solid tumors 341 and appears to be a promising agent for use in combination with Doc (Tse et al., 2008; Shi et al., 342 PeerJ reviewing PDF | (v2013:06:610:0:1:NEW 25 Jun 2013) R ev ie w in g M an us cr ip t 16 2011). Our results provide a strong mechanistic rationale for combining targeted chemotherapy 343 against Bcl2/Bcl-xL, as with navitoclax, with the currently approved drug for CRPC Doc, which 344 targets the destruction of Mcl-1, a navitoclax resistance factor. With the addition of cabazitaxel 345 to help in the treatment of patients that develop Doc-resistance, it is also likely that the 346 combination of cabazitaxel and navitoclax will further improve overall survival (de Bono et al., 347 2010). 348 349 ACKNOWLEDGEMENTS 350 We thank Dr. Pryamvada Rai for critical reading of the manuscript and helpful suggestions; 351 Deanna Palenzuela for technical assistance; Ron Hamelik for assistance with flow cytometry; 352 and Drs. Bernard Roos and Guy Howard for support. 353 354 REFERENCES 355 Allan LA, Clarke PR. 2007. Phosphorylation of caspase-9 by CDK1/cyclin B1 protects mitotic 356 cells against apoptosis. Molecular Cell 26:301\u2013310. 357 Andersen JL, Johnson CE, Freel CD, Parrish AB, Day JL, Buchakjian MR, Nutt LK, 358 Thompson JW, Moseley MA, Kornbluth S. 2009. Restraint of apoptosis during mitosis 359 through interdomain phosphorylation of caspase-2. EMBO Journal 28:3216\u20133227. 360 Attar RM, Takimoto CH, Gottardis MM. 2009. Castration-resistant prostate cancer: locking 361 up the molecular escape routes. Clinical Cancer Research 15:3251\u20133255. 362 Basu A, Haldar S. 2003. Identification of a novel Bcl-xL phosphorylation site regulating the 363 sensitivity of taxol- or 2-methoxyestradiol-induced apoptosis. FEBS Letters 538:41\u201347. 364 PeerJ reviewing PDF | (v2013:06:610:0:1:NEW 25 Jun 2013) R ev ie w in g M an us cr ip t 17 Berndtsson M, Konishi Y, Bonni A, Hagg M, Shoshan M, Linder S, Havelka AM. 2005. 365 Phosphorylation of BAD at Ser-128 during mitosis and paclitaxel-induced apoptosis. FEBS 366 Letters 579:3090\u20133094. 367 Bray K, Chen HY, Karp CM, May M, Ganesan S, Karantza-Wadsworth V, DiPaola RS, 368 White E. 2009. Bcl-2 modulation to activate apoptosis in prostate cancer. Molecular Cancer 369 Research 7:1487\u20131496. 370 Castedo M, Perfettini JL, Roumier T, Andreau K, Medema R, Kroemer G. 2004. Cell death 371 by mitotic catastrophe: a molecular definition. Oncogene 23:2825\u20132837. 372 Chen S, Dai Y, Harada H, Dent P, Grant S. 2007. Mcl-1 down-regulation potentiates ABT-373 737 lethality by cooperatively inducing Bak activation and Bax translocation. Cancer Research 374 67:782\u2013791. 375 Chipuk JE, Moldoveanu T, Llambi F, Parsons MJ, Green DR. 2010. The BCL-2 family 376 reunion. Molecular Cell 37:299\u2013310. 377 de Bono JS, Oudard S, Ozguroglu M, Hansen S, Machiels JP, Kocak I, Gravis G, Bodrogi 378 I, Mackenzie MJ, Shen L, Roessner M, Gupta S, Sartor AO; TROPIC Investigators. 2010. 379 Prednisone plus cabazitaxel or mitoxantrone for metastatic castration-resistant prostate cancer 380 progressing after docetaxel treatment: a randomised open-label trial. Lancet 376:1147\u20131154. 381 Del Gaizo Moore V, Schlis KD, Sallan SE, Armstrong SA, Letai A. 2008. BCL-2 382 dependence and ABT-737 sensitivity in acute lymphoblastic leukemia. Blood 111:2300\u20132309. 383 Gascoigne KE, Taylor SS. 2008. Cancer cells display profound intra- and interline variation 384 following prolonged exposure to antimitotic drugs. Cancer Cell 14:111\u2013122. 385 PeerJ reviewing PDF | (v2013:06:610:0:1:NEW 25 Jun 2013) R ev ie w in g M an us cr ip t 18 Gomez LA, de las Pozas A, Perez-Stable C. 2006. Sequential combination of flavopiridol and 386 docetaxel reduces the levels of XIAP and AKT proteins and stimulates apoptosis in human 387 LNCaP prostate cancer cells. Molecular Cancer Therapeutics 5:1216\u20131226. 388 Gottlieb RA, Granville DJ. 2002. Analyzing mitochondrial changes during apoptosis. Methods 389 26:341\u2013347. 390 Gray NS, Wodicka L, Thunnissen AM, Norman TC, Kwon S, Espinoza FH, Morgan DO, 391 Barnes G, LeClerc S, Meijer L, Kim SH, Lockhart DJ, Schultz PG. 1998. Exploiting 392 chemical libraries, structure, and genomics in the search for kinase inhibitors. Science 281: 533\u2013393 538. 394 Hadaschik BA, Gleave ME. 2007. Therapeutic options for hormone-refractory prostate cancer 395 in 2007. Urologic Oncology 25:413\u2013419. 396 Haldar S, Basu A, Croce CM. 1998. Serine-70 is one of the critical sites for drug-induced Bcl2 397 phosphorylation in cancer cells. Cancer Research 58:1609\u20131615. 398 Haldar S, Jena N, Croce CM. 1995. Inactivation of Bcl-2 by phosphorylation. Proceedings of 399 the National Academy of Sciences, USA 92: 4507\u20134511. 400 Hann CL, Daniel VC, Sugar EA, Dobromilskaya I, Murphy SC, Cope L, Lin X, Hierman 401 JS, Wilburn DL, Watkins DN, Rudin CM. 2008. Therapeutic efficacy of ABT-737, a selective 402 inhibitor of BCL-2, in small cell lung cancer. Cancer Research 68:2321\u20132328. 403 Harley ME, Allan LA, Sanderson HS, Clarke PR. 2010. Phosphorylation of Mcl-1 by CDK1-404 cyclin B1 initiates its Cdc20-dependent destruction during mitotic arrest. EMBO Journal 29: 405 2407\u20132420. 406 PeerJ reviewing PDF | (v2013:06:610:0:1:NEW 25 Jun 2013) R ev ie w in g M an us cr ip t 19 Hauck P, Chao BH, Litz J, Krystal GW. 2009. Alterations in the Noxa/Mcl-1 axis determine 407 sensitivity of small cell lung cancer to the BH3 mimetic ABT-737. Molecular Cancer 408 Therapeutics 8:883\u2013892. 409 Jordan MA, Wilson L. 2004. Microtubules as a target for anticancer drugs. Nature Reviews 410 Cancer 4:253\u2013265. 411 Kang MH, Kang YH, Szymanska B, Wilczynska-Kalak U, Sheard MA, Harned TM, Lock 412 RB, Reynolds CP. 2007. Activity of vincristine, L-ASP, and dexamethasone against acute 413 lymphoblastic leukemia is enhanced by the BH3-mimetic ABT-737 in vitro and in vivo. Blood 414 110:2057\u20132066. 415 Karnak D, Xu L. 2010. Chemosensitization of prostate cancer by modulating Bcl-2 family 416 proteins. Current Drug Targets 11:699\u2013707. 417 Khor LY, Moughan J, Al-Saleem T, Hammond EH, Venkatesan V, Rosenthal SA, Ritter 418 MA, Sandler HM, Hanks GE, Shipley WU, Pollack A. 2007. Bcl-2 and Bax expression 419 predict prostate cancer outcome in men treated with androgen deprivation and radiotherapy on 420 radiation therapy oncology group protocol 92-02. Clinical Cancer Research 13:3585\u20133590. 421 Konishi Y, Lehtinen M, Donovan N, Bonni A. 2002. Cdc2 phosphorylation of BAD links the 422 cell cycle to the cell death machinery. Molecular Cell 9:1005\u20131016. 423 LaCasse EC, Mahoney DJ, Cheung HH, Plenchette S, Baird S, Korneluk RG. 2008. IAP-424 targeted therapies for cancer. Oncogene 27:6252\u20136275. 425 LaVallee TM, Burke PA, Swartz GM, Hamel E, Agoston GE, Shah J, Suwandi L, Hanson 426 AD, Fogler WE, Sidor CF, Treston AM. 2008. Significant antitumor activity in vivo following 427 PeerJ reviewing PDF | (v2013:06:610:0:1:NEW 25 Jun 2013) R ev ie w in g M an us cr ip t 20 treatment with the microtubule agent ENMD-1198. Molecular Cancer Therapeutics 7:1472\u2013428 1482. 429 Lestini BJ, Goldsmith KC, Fluchel MN, Liu X, Chen NL, Goyal B, Pawel BR, Hogarty MD. 430 2009. Mcl1 downregulation sensitizes neuroblastoma to cytotoxic chemotherapy and small 431 molecule Bcl2-family antagonists. Cancer Biology & Therapy 8:1587\u20131595. 432 Mason KD, Khaw SL, Rayeroux KC, Chew E, Lee EF, Fairlie WD, Grigg AP, Seymour JF, 433 Szer J, Huang DC, Roberts AW. 2009. The BH3 mimetic compound, ABT-737, synergizes 434 with a range of cytotoxic chemotherapy agents in chronic lymphocytic leukemia. Leukemia 435 23:2034\u20132041. 436 Miyake H, Monia BP, Gleave ME. 2000. Inhibition of progression to androgen-independence 437 by combined adjuvant treatment with antisense BCL-XL and antisense Bcl-2 oligonucleotides 438 plus taxol after castration in the Shionogi tumor model. International Journal of Cancer 86:855\u2013439 862. 440 Nantajit D, Fan M, Duru N, Wen Y, Reed JC, Li JJ. 2010. Cyclin B1/Cdk1 phosphorylation 441 of mitochondrial p53 induces anti-apoptotic response. PLoS One 5:e12341. 442 Nechushtan A, Smith CL, Hsu YT, Youle RJ. 1999. Conformation of the Bax C-terminus 443 regulates subcellular location and cell death. EMBO Journal 18:2330\u20132341. 444 Oakes SR, Vaillant F, Lim E, Lee L, Breslin K, Feleppa F, Deb S, Ritchie ME, Takano E, 445 Ward T, Fox SB, Generali D, Smyth GK, Strasser A, Huang DC, Visvader JE, Lindeman GJ. 446 2012. Sensitization of BCL-2-expressing breast tumors to chemotherapy by the BH3 mimetic ABT-447 737. Proceedings of the National Academy of Sciences USA 109:2766\u20132771. 448 PeerJ reviewing PDF | (v2013:06:610:0:1:NEW 25 Jun 2013) R ev ie w in g M an us cr ip t 21 O'Connor DS, Grossman D, Plescia J, Li F, Zhang H, Villa A, Tognin S, Marchisio PC, 449 Altieri DC. 2000. Regulation of apoptosis at cell division by p34cdc2 phosphorylation of survivin. 450 Proceedings of the National Academy of Sciences USA 97:13103\u201313107. 451 Oltersdorf T, Elmore SW, Shoemaker AR, Armstrong RC, Augeri DJ, Belli BA, Bruncko 452 M, Deckwerth TL, Dinges J, Hajduk PJ, Joseph MK, Kitada S, Korsmeyer SJ, Kunzer AR, 453 Letai A, Li C, Mitten MJ, Nettesheim DG, Ng S, Nimmer PM, O'Connor JM, Oleksijew A, 454 Petros AM, Reed JC, Shen W, Tahir SK, Thompson CB, Tomaselli KJ, Wang B, Wendt 455 MD, Zhang H, Fesik SW, Rosenberg SH. 2005. An inhibitor of Bcl-2 family proteins induces 456 regression of solid tumours. Nature 435:677\u2013681. 457 Perez-Stable C. 2006. 2-Methoxyestradiol and paclitaxel have similar effects on the cell cycle 458 and induction of apoptosis in prostate cancer cells. Cancer Letters 231:49\u221264. 459 Pollack A, Cowen D, Troncoso P, Zagars GK, von Eschenbach AC, Meistrich ML, 460 McDonnell T. 2003. Molecular markers of outcome after radiotherapy in patients with prostate 461 carcinoma: Ki-67, bcl-2, bax, and bcl-x. Cancer 97:1630\u20131638. 462 Poruchynsky MS, Wang EE, Rudin CM, Blagosklonny MV, Fojo T. 1998. Bcl-xL is 463 phosphorylated in malignant cells following microtubule disruption. Cancer Research 58:3331\u2013464 3338. 465 Reiner T, de las Pozas A, Gomez LA, Perez-Stable C. 2009. Low dose combination of 2-466 methoxyestradiol and docetaxel can block prostate cancer cells in mitosis and induce apoptosis. 467 Cancer Letters 276:21\u201331. 468 Shi J, Orth JD, Mitchison T. 2008. Cell type variation in responses to antimitotic drugs that 469 target microtubules and kinesin-5. Cancer Research 68:3269\u20133276. 470 PeerJ reviewing PDF | (v2013:06:610:0:1:NEW 25 Jun 2013) R ev ie w in g M an us cr ip t 22 Shi J, Zhou Y, Huang HC, Mitchison TJ. 2011. Navitoclax (ABT-263) accelerates apoptosis 471 during drug-induced mitotic arrest by antagonizing Bcl-xL. Cancer Research 71:4518\u20134526. 472 Shoemaker AR, Oleksijew A, Bauch J, Belli BA, Borre T, Bruncko M, Deckwirth T, Frost 473 DJ, Jarvis K, Joseph MK, Marsh K, McClellan W, Nellans H, Ng S, Nimmer P, O'Connor 474 JM, Oltersdorf T, Qing W, Shen W, Stavropoulos J, Tahir SK, Wang B, Warner R, Zhang 475 H, Fesik SW, Rosenberg SH, Elmore SW. 2006. A small-molecule inhibitor of Bcl-XL 476 potentiates the activity of cytotoxic drugs in vitro and in vivo. Cancer Research 66:8731\u20138739. 477 Song JH, Kandasamy K, Kraft AS. 2008. ABT-737 induces expression of the death receptor 5 478 and sensitizes human cancer cells to TRAIL-induced apoptosis. Journal of Biological Chemistry 479 283:25003\u201325013. 480 Stewart SA, Dykxhoorn DM, Palliser D, Mizuno H, Yu EY, An DS, Sabatini DM, Chen IS, 481 Hahn WC, Sharp PA, Weinberg RA, Novina CD. 2003. Lentivirus-delivered stable gene 482 silencing by RNAi in primary cells. RNA 9:493\u2013501. 483 Susin SA, Lorenzo HK, Zamzami N, Marzo I, Snow BE, Brothers GM, Mangion J, Jacotot 484 E, Costantini P, Loeffler M, Larochette N, Goodlett DR, Aebersold R, Siderovski DP, 485 Penninger JM, Kroemer G. 1999. Molecular characterization of mitochondrial apoptosis-486 inducing factor. Nature 397:441\u2013446. 487 Tagscherer KE, Fassl A, Campos B, Farhadi M, Kraemer A, B\u00f6ck BC, Macher-488 Goeppinger S, Radlwimmer B, Wiestler OD, Herold-Mende C, Roth W. 2008. Apoptosis-489 based treatment of glioblastomas with ABT-737, a novel small molecule inhibitor of Bcl-2 490 family proteins. Oncogene 27:6646\u20136656. 491 PeerJ reviewing PDF | (v2013:06:610:0:1:NEW 25 Jun 2013) R ev ie w in g M an us cr ip t 23 Tang DG, Li L, Chopra DP, Porter AT. 1998. Extended survivability of prostate cancer cells 492 in the absence of trophic factors: increased proliferation, evasion of apoptosis, and the role of 493 apoptosis proteins. Cancer Research 58:3466\u20133479. 494 Tannock IF, de Wit R, Berry WR, Horti J, Pluzanska A, Chi KN, Oudard S, Th\u00e9odore C, 495 James ND, Turesson I, Rosenthal MA, Eisenberger MA; TAX 327 Investigators. 2004. 496 Docetaxel plus prednisone or mitoxantrone plus prednisone for advanced prostate cancer. New 497 England Journal of Medicine 351:1502\u20131512. 498 Terrano DT, Upreti M, Chambers TC. 2010. Cyclin-dependent kinase 1-mediated Bcl-xL/Bcl-499 2 phosphorylation acts as a functional link coupling mitotic arrest and apoptosis. Molecular and 500 Cellular Biology 30:640\u2013656. 501 Tse C, Shoemaker AR, Adickes J, Anderson MG, Chen J, Jin S, Johnson EF, Marsh KC, 502 Mitten MJ, Nimmer P, Roberts L, Tahir SK, Xiao Y, Yang X, Zhang H, Fesik S, Rosenberg 503 SH, Elmore SW. 2008. ABT-263: a potent and orally bioavailable Bcl-2 family inhibitor. 504 Cancer Research 68:3421\u20133428. 505 Upreti M, Galitovskaya EN, Chu R, Tackett AJ, Terrano DT, Granell S, Chambers TC. 506 2008. Identification of the major phosphorylation site in Bcl-xL induced by microtubule 507 inhibitors and analysis of its functional significance. Journal of Biological Chemistry 508 283:35517\u201335525. 509 van Bokhoven A, Varella-Garcia M, Korch C, Johannes WU, Smith EE, Miller HL, 510 Nordeen SK, Miller GJ, Lucia MS. 2003. Molecular characterization of human prostate 511 carcinoma cell lines. Prostate 57:205\u2013225. 512 PeerJ reviewing PDF | (v2013:06:610:0:1:NEW 25 Jun 2013) R ev ie w in g M an us cr ip t van Delft MF, Wei AH, Mason KD, Vandenberg CJ, Chen L, Czabotar PE, Willis SN, Scott 513 CL, Day CL, Cory S, Adams JM, Roberts AW, Huang DC. 2006. The BH3 mimetic ABT-514 737 targets selective Bcl-2 proteins and efficiently induces apoptosis via Bak/Bax if Mcl-1 is 515 neutralized. Cancer Cell 10:389\u2013399. 516 Wertz IE, Kusam S, Lam C, Okamoto T, Sandoval W, Anderson DJ, Helgason E, Ernst 517 JA, Eby M, Liu J, Belmont LD, Kaminker JS, O'Rourke KM, Pujara K, Kohli PB, 518 Johnson AR, Chiu ML, Lill JR, Jackson PK, Fairbrother WJ, Seshagiri S, Ludlam MJ, 519 Leong KG, Dueber EC, Maecker H, Huang DC, Dixit VM. 2011. Sensitivity to antitubulin 520 chemotherapeutics is regulated by MCL1 and FBW7. Nature 471:110-114. 521 Yecies D, Carlson NE, Deng J, Letai A. 2010. Acquired resistance to ABT-737 in lymphoma 522 cells that up-regulate MCL-1 and BFL-1. Blood 115:3304\u20133313. 523 Zhou Q, Gustafson D, Nallapareddy S, Diab S, Leong S, Lewis K, Gore L, Messersmith 524 WA, Treston AM, Eckhardt SG, Sidor C, Camidge DR. 2011. A phase I dose-escalation, 525 safety and pharmacokinetic study of the 2-methoxyestradiol analog ENMD-1198 administered 526 orally to patients with advanced cancer. Investigational New Drugs 29:340\u2013346. 527 528 529 FIGURE LEGENDS 530 R ev ie w in g M an us cr ip t LNCaP compared to DU145 (Bax null) and PC3. Coomassie blue stain of total protein is the 535 loading control. (B) Cell viability assay showing that LNCaP and PC3 are similarly sensitive to 536 various concentrations of ABT-737 (1-5 \u00b5M; three days), whereas DU145 is more resistant. 537 Figure 2 ABT-737 enhances Doc-mediated apoptotic cell death in LNCaP and PC3 but not 538 in DU145 PC cells. (A) Trypan blue exclusion assay showing that the combination of 1 nM Doc 539 + 1 \u00b5M ABT-737 increases total cell death in LNCaP and PC3 but not in DU145 compared to 540 Doc or ABT-737 alone (*, P<2x10-4). LNCaP were treated for 48 h and DU145 and PC3 for 72 541 h. Pan-caspase inhibitor Q-VD (10 \u00b5M) blocks the Doc + ABT-737 increase in cell death. 542 Western blot analysis showing that Doc + ABT-737 increases cleaved-PARP (cP) levels in 543 LNCaP and PC3 but not in DU145 compared to Doc or ABT-737 alone. Q-VD blocks the Doc + 544 ABT-737 increase in cP in all cells. (B) Flow cytometric analysis showing higher annexin-FITC 545 stained LNCaP and PC3 but not DU145 cells treated with Doc + ABT-737 compared to Doc or 546 ABT-737 alone (*, P<2x10-5). Q-VD blocks the Doc + ABT-737 increase in annexin + cells in 547 LNCaP and PC3. (C) Mitochondrial protein release assay and western blot showing increased 548 cytochrome c, Smac, AIF and decreased Bax in LNCaP cells treated with Doc + ABT-737 549 compared to Doc or ABT-737 alone, and control. In DU145 cells, cytochrome c, Smac, and AIF 550 mitochondrial release were similar in Doc + ABT-737 as in Doc alone. Cox IV protein is 551 negative indicating no mitochondrial contamination whereas actin is the positive control. +C for 552 both LNCaP and DU145 is lysate prepared from LNCaP cells using the standard method for total 553 proteins. 554 R ev ie w in g M an us cr ip t death at 72 h in DU145/Bax compared to Doc (D) or 1198 (98) alone (*, P<0.008), whereas 557 there is no effect in DU145/EV (empty vector) control cells. Western blot analysis showing that 558 DA or 98A increases cP in DU145/Bax cells compared to D or 98 alone, whereas there is no 559 increase of cP in DU/EV cells. Bax is expressed in DU145/Bax but not in DU145/EV cells. (B) 560 Annexin-FITC/PI flow cytometric analysis showing higher annexin+ and PI+ cells in DA treated 561 DU145/Bax compared to DU145/EV cells (*, P<1x10-6). (C) Trypan blue exclusion assay 562 showing that DA or 98A does not significantly increase cell death in DU145/shMcl-1 and 563 DU145/shGFP control cells compared to D or 98 alone. Western blot analysis showing no 564 difference in the cP levels in DU145/shMcl-1 and DU145/shGFP control cells treated with D, 565 DA, 98, or 98A. Mcl-1 is expressed much higher in DU145/shGFP compared to DU145/shMcl-1 566 cells. 567 Figure 4 Bax suppression has a greater effect on ABT-737-mediated Doc/1198-induced 568 apoptotic cell death than Bak suppression in LNCaP cells. (A) Trypan blue exclusion assay 569 showing significantly less cell death in LNCaP/shBax and PC3/shBax cells treated with DA or 570 98A compared to shGFP (G) control cells (*, P<0.02). Western blot analysis showing less cP 571 and Bax in LNCaP/shBax and PC3/shBax cells treated with DA or 98A compared to control 572 cells. (B) Trypan blue exclusion assay showing less cell death in PC3/shBak (*, P<6x10-4) but 573 not in LNCaP/shBak cells treated with DA or 98A compared to control cells. Western blot 574 analysis showing less cP in PC3/shBak cells treated with D or 98 + A but little difference in 575 LNCaP/shBak cells compared to control cells. Bak is lower in LNCaP/shBak and PC3/shBak 576 cells compared to control cells. 577 R ev ie w in g M an us cr ip t treatment with 1 nM Doc (D) decreases Mcl-1. Combination of Doc + ABT-737 (DA) decreases 580 Mcl-1. In LNCaP, there is less Bcl-2 (D, 24 h) and Bak (DA, 48 h) but few differences in Bcl-581 xL, Bax, Bid, and Noxa. In PC-3, there are few differences Bcl-2, Bcl-xL, Bax, Bak, Bid, and 582 Noxa. 583 Figure 6 Inhibition of cyclinB1/Cdk1-mediated phosphorylation of Bcl-2/Bcl-xL blocks 584 ABT-737 enhancement of Doc induced apoptosis in PC3 cells. (A) Trypan blue exclusion 585 assay showing that 5 \u00b5M purvalanol A (P) lowers DA cell death in PC3 cells (*, P<9x10-8). 586 Western blot analysis showing that P blocks the DA increase in cl-PARP, P-Bcl-2 and P-Bcl-xL, 587 whereas there is no change in the total levels of Bcl-2 and Bcl-xL; P treatment increases Mcl-1 in 588 DA treated cells. Vertical line in cl-PARP indicates sample from same blot not in sequence. (B) 589 Trypan blue exclusion assay showing less cell death in DA treated PC3/shCyclin B1-2 and -3 590 cells compared to PC3/shGFP control cells (*, P<4x10-5). Western blot analysis showing less 591 cyclin B1, cP, P-Bcl-2, P-Bcl-xL, and slightly greater Mcl-1 in PC3/shCyclin B1-2 and -3 cells 592 treated with DA compared to PC3/shGFP control cells. 593 R ev ie w in g M an us cr ip t 28 Figure 8 Schematic of how ABT-737 can sensitize Bax+ CRPC cells to Doc. Treatment of 602 CRPC cells such as PC3 with Doc deregulates cyclin B1/Cdk1 activity and increases 603 phosphorylation of Bcl-2/Bcl-xL and degradation of Mcl-1. However, the high levels of Bcl-604 2/Bcl-xL/Mcl-1 in CRPC cells presents a block to apoptosis. Addition of ABT-737 disrupts the 605 anti-apoptotic activity of Bcl-2/Bcl-xL and more effectively allows Doc treatment to bypass the 606 block and increase apoptosis. Cyclin B1/Cdk1 hyperactivity caused by Doc treatment also lowers 607 Mcl-1, an ABT-737 resistance factor, to further increase apoptosis. 608 PeerJ reviewing PDF | (v2013:06:610:0:1:NEW 25 Jun 2013) R ev ie w in g M an us cr ip t Figure 1 Bcl-2 family protein levels and sensitivity to ABT-737 in PC cells (A) Western blot analysis showing that the levels of anti-apoptotic Bcl-2 and Bcl-xL proteins are higher in DU145 (DU) and PC3 (PC) CRPC cells compared to androgen-dependent LNCaP cells, whereas anti-apoptotic Mcl-1 is highest in DU145. The levels of pro-apoptotic Bax and Bak are higher in LNCaP compared to DU145 (Bax null) and PC3. Coomassie blue stain of total protein is the loading control. (B) Cell viability assay showing that LNCaP and PC3 are similarly sensitive to various concentrations of ABT-737 (1-5 uM; three days), whereas DU145 is more resistant. PeerJ reviewing PDF | (v2013:06:610:0:1:NEW 25 Jun 2013) R ev ie w in g M an us cr ip t Figure 2 ABT-737 enhances Doc-mediated apoptotic cell death in LNCaP and PC3 but not in DU145 PC cells (A) Trypan blue exclusion assay showing that the combination of 1 nM Doc + 1 uM ABT-737 increases total cell death in LNCaP and PC3 but not in DU145 compared to Doc or ABT-737 alone (*, P<2x10 -4 ). LNCaP were treated for 48 h and DU145 and PC3 for 72 h. Pan-caspase inhibitor Q-VD (10 uM) blocks the Doc + ABT-737 increase in cell death. Western blot analysis showing that Doc + ABT-737 increases cleaved-PARP (cP) levels in LNCaP and PC3 but not in DU145 compared to Doc or ABT-737 alone. Q-VD blocks the Doc + ABT-737 increase in cP in all cells. (B) Flow cytometric analysis showing higher annexin-FITC stained LNCaP and PC3 but not DU145 cells treated with Doc + ABT-737 compared to Doc or ABT-737 alone (*, P<2 x10 -5 ) . Q-VD blocks the Doc + ABT-737 increase in annexin + cells in LNCaP and PC3. (C) Mitochondrial protein release assay and western blot showing increased cytochrome c, Smac, AIF and decreased Bax in LNCaP cells treated with Doc + ABT-737 compared to Doc or ABT-737 alone, and control. In DU145 cells, cytochrome c, Smac, and AIF mitochondrial release were similar in Doc + ABT-737 as in Doc alone. Cox IV protein is negative indicating no mitochondrial contamination whereas actin is the positive control. +C for both LNCaP and DU145 is lysate prepared from LNCaP cells using the standard method for total proteins. PeerJ reviewing PDF | (v2013:06:610:0:1:NEW 25 Jun 2013) R ev ie w in g M an us cr ip t PeerJ reviewing PDF | (v2013:06:610:0:1:NEW 25 Jun 2013) R ev ie w in g M an us cr ip t Figure 3 DU145 cells are resistant to ABT-737 because they are Bax null (A) Trypan blue exclusion assay showing that Doc + ABT-737 (DA) or 1198 + ABT-737 (98A) increases cell death at 72 h in DU145/Bax compared to Doc (D) or 1198 (98) alone (*, P<0.008), whereas there is no effect in DU145/EV (empty vector) control cells. Western blot analysis showing that DA or 98A increases cP in DU145/Bax cells compared to D or 98 alone, whereas there is no increase of cP in DU/EV cells. Bax is expressed in DU145/Bax but not in DU145/EV cells. (B) Annexin-FITC/PI flow cytometric analysis showing higher annexin+ and PI+ cells in DA treated DU145/Bax compared to DU145/EV cells (*, P<1x10 -6 ). (C) Trypan blue exclusion assay showing that DA or 98A does not significantly increase cell death in DU145/shMcl-1 and DU145/shGFP control cells compared to D or 98 alone. Western blot analysis showing no difference in the cP levels in DU145/shMcl-1 and DU145/shGFP control cells treated with D, DA, 98, or 98A. Mcl-1 is expressed much higher in DU145/shGFP compared to DU145/shMcl-1 cells. PeerJ reviewing PDF | (v2013:06:610:0:1:NEW 25 Jun 2013) R ev ie w in g M an us cr ip t Figure 4 Bax suppression has a greater effect on ABT-737-mediated Doc/1198-induced apoptotic cell death than Bak suppression in LNCaP cells (A) Trypan blue exclusion assay showing significantly less cell death in LNCaP/shBax and PC3/shBax cells treated with DA or 98A compared to shGFP (G) control cells (*, P<0.02). Western blot analysis showing less cP and Bax in LNCaP/shBax and PC3/shBax cells treated with DA or 98A compared to control cells. (B) Trypan blue exclusion assay showing less cell death in PC3/shBak (*, P<6x10 -4 ) but not in LNCaP/shBak cells treated with DA or 98A compared to control cells. Western blot analysis showing less cP in PC3/shBak cells treated with D or 98 + A but little difference in LNCaP/shBak cells compared to control cells. Bak is lower in LNCaP/shBak and PC3/shBak cells compared to control cells. PeerJ reviewing PDF | (v2013:06:610:0:1:NEW 25 Jun 2013) R ev ie w in g M an us cr ip t Figure 5 Doc counteracts the ABT-737-mediated increase in Mcl-1 protein Western blot showing that treatment of LNCaP and PC3 cells with 1 uM ABT-737 (A) increases Mcl-1 but treatment with 1 nM Doc (D) decreases Mcl-1. Combination of Doc + ABT-737 (DA) decreases Mcl-1. In LNCaP, there is less Bcl-2 (D, 24 h) and Bak (DA, 48 h) but few differences in Bcl-xL, Bax, Bid, and Noxa. In PC-3, there are few differences Bcl-2, Bcl-xL, Bax, Bak, Bid, and Noxa. PeerJ reviewing PDF | (v2013:06:610:0:1:NEW 25 Jun 2013) R ev ie w in g M an us cr ip t Figure 6 Inhibition of cyclinB1/Cdk1-mediated phosphorylation of Bcl-2/Bcl-xL blocks ABT-737 enhancement of Doc induced apoptosis in PC3 cells (A) Trypan blue exclusion assay showing that 5 uM purvalanol A (P) lowers DA cell death in PC3 cells (*, P<9x10 -8 ). Western blot analysis showing that P blocks the DA increase in cl-PARP, P-Bcl-2 and P-Bcl-xL, whereas there is no change in the total levels of Bcl-2 and Bcl-xL; P treatment increases Mcl-1 in DA treated cells. Vertical line in cl-PARP indicates sample from same blot not in sequence. (B) Trypan blue exclusion assay showing less cell death in DA treated PC3/shCyclin B1-2 and -3 cells compared to PC3/shGFP control cells (*, P<4x10 -5 ). Western blot analysis showing less cyclin B1, cP, P-Bcl-2, P-Bcl-xL, and slightly greater Mcl-1 in PC3/shCyclin B1-2 and -3 cells treated with DA compared to PC3/shGFP control cells. PeerJ reviewing PDF | (v2013:06:610:0:1:NEW 25 Jun 2013) R ev ie w in g M an us cr ip t Figure 7 ABT-737 enhancement of Doc-mediated apoptosis is more dependent on the ability of cyclin B1/Cdk1 to decrease Mcl-1 than to phosphorylate Bcl-2/Bcl-xL Trypan blue exclusion assay showing greater cell death in DA treated (48 h) PC3/shCyclin B1/shMcl-1 (B1-2/M3, B1-3/M3) cells compared to control PC3/shCyclin B1/shGFP (B1-2/G, B1-3/G) and PC3/shGFP (G) cells (*, P< 0.002). Western blot analysis showing increased cl-PARP and decreased Mcl-1 in PC3/shCyclin B1/shMcl-1 compared control shGFP cells after DA treatment (24 h). There is less cyclin B1, P-Bcl-1, and P-Bcl-xL in PC3/shCyclin B1/shGFP or shMcl-1 compared to PC3/shGFP cells, whereas there are no changes in total Bcl-1 or Bcl-xL. PeerJ reviewing PDF | (v2013:06:610:0:1:NEW 25 Jun 2013) R ev ie w in g M an us cr ip t Figure 8 Schematic of how ABT-737 can sensitize Bax+ CRPC cells to Doc Treatment of CRPC cells such as PC3 with Doc deregulates cyclin B1/Cdk1 activity and increases phosphorylation of Bcl-2/Bcl-xL and degradation of Mcl-1. However, the high levels of Bcl-2/Bcl-xL/Mcl-1 in CRPC cells presents a block to apoptosis. Addition of ABT-737 disrupts the anti-apoptotic activity of Bcl-2/Bcl-xL and more effectively allows Doc treatment to bypass the block and increase apoptosis. Cyclin B1/Cdk1 hyperactivity caused by Doc treatment also lowers Mcl-1, an ABT-737 resistance factor, to further increase apoptosis. PeerJ reviewing PDF | (v2013:06:610:0:1:NEW 25 Jun 2013) R ev ie w in g M an us cr ip t",
"url": "https://peerj.com/articles/145/reviews/",
"review_1": "Vladimir Uversky \u00b7 Aug 6, 2013 \u00b7 Academic Editor\nACCEPT\nThank you for addressing all the critical points of the reviewers and for revising the manuscript accordingly.",
"review_2": "Vladimir Uversky \u00b7 Jul 31, 2013 \u00b7 Academic Editor\nMINOR REVISIONS\nPlease address critical points raised by the first reviewer.",
"review_3": "Reviewer 1 \u00b7 Jul 31, 2013\nBasic reporting\nNo Comments\nExperimental design\nNo Comments\nValidity of the findings\nNo Comments\nAdditional comments\nThis manuscript describes in silico screening of CalB mutants for enzymatic amidase activity. Then the computational results are benchmarked against experimental studies. The computational approach gives reasonable results in general -- the authors demonstrate that 15 out of 22 mutants are correctly predicted in qualitative activity. The manuscript is scholarly and well written. The methods are complete and clear. The data are new and appear to be true. I recommend publishing with minor revision.\nI have a few minor suggestions/questions.\n1. In table 1, two columns are both labeled with \u201cCat.\u201d The authors need to make more detailed labels to distinguish (one from Exp. and the other is from Calc.). Also, the authors need to include the cutoff of 12.5 kcal/mol in the table legends otherwise it is difficult for readers to correlate calculated barriers with calculated cat.\n2. In pg. 8, the authors state that the cutoff is done purely for a post hoc comparison of experimental and computed data. And they used 12.5 kcal/mol as cutoff. The authors need to expand this section to give detailed descriptions about how they reach the value of 12.5 in their study.\n3. For results and discussion part, the two subtitles are \u201cSet S\u201d and \u201cSet L\u201d. It might be useful to guide readers if the authors can use more detailed subtitles which can generally summarize their results.\n4. In the last part of results and discussion, the authors state that I189G as a single mutant is counterproductive but lowers the barrier of G39A-A141Q-L278A. This would suggest there are couplings between I189 and G39/A141/L278. While it is difficult to figure out such kind of couplings, one simple question is, is I189 close to G39/A141/L278 in the structure.\nCite this review as\nAnonymous Reviewer (2013) Peer Review #1 of \"In silico screening of 393 mutants facilitates enzyme engineering of amidase activity in CalB (v0.1)\". PeerJ https://doi.org/10.7287/peerj.145v0.1/reviews/1",
"review_4": "Reviewer 2 \u00b7 Jul 16, 2013\nBasic reporting\nMeets all necessary criteria\nExperimental design\nExcellent\nValidity of the findings\nExcellent\nAdditional comments\nThis is an excellent paper, nicely demonstrating the utility of a practical computational modelling approach to the prediction of enzyme activity. The method uses semiempirical quantum chemical methods to model amidase reactivity in a lipase. This is thorough work of high quality. The paper is well written and the results are analysed and presented in appropriate detail. The results will be of wide interest. This is a demonstration of a method that will find real industrial application, as well as in other contexts. The paper is suitable for publication essentially as is. A minor point is that some of the references might be lacking information (e.g. refs 27 and 28 do not look complete as written). Also, the authors might want to comment a little more on how amidase activity is achieved. There is wide debate about amidase versus esterase activity, and, while this is not the focus here, comparison with enzymes such as fatty acid amide hydrolase, which has been the subject of much modelling work, could be useful (e.g. a little more discussion on what specific features may provide amidase activity and how the designed amidases compare to natural amidaases). This could assist in future rational design (as well as in testing the quantum chemical methods by comparisons on related reactions.\nCite this review as\nAnonymous Reviewer (2013) Peer Review #2 of \"In silico screening of 393 mutants facilitates enzyme engineering of amidase activity in CalB (v0.1)\". PeerJ https://doi.org/10.7287/peerj.145v0.1/reviews/2",
"pdf_1": "https://peerj.com/articles/145v0.2/submission",
"pdf_2": "https://peerj.com/articles/145v0.1/submission",
"all_reviews": "Review 1: Vladimir Uversky \u00b7 Aug 6, 2013 \u00b7 Academic Editor\nACCEPT\nThank you for addressing all the critical points of the reviewers and for revising the manuscript accordingly.\nReview 2: Vladimir Uversky \u00b7 Jul 31, 2013 \u00b7 Academic Editor\nMINOR REVISIONS\nPlease address critical points raised by the first reviewer.\nReview 3: Reviewer 1 \u00b7 Jul 31, 2013\nBasic reporting\nNo Comments\nExperimental design\nNo Comments\nValidity of the findings\nNo Comments\nAdditional comments\nThis manuscript describes in silico screening of CalB mutants for enzymatic amidase activity. Then the computational results are benchmarked against experimental studies. The computational approach gives reasonable results in general -- the authors demonstrate that 15 out of 22 mutants are correctly predicted in qualitative activity. The manuscript is scholarly and well written. The methods are complete and clear. The data are new and appear to be true. I recommend publishing with minor revision.\nI have a few minor suggestions/questions.\n1. In table 1, two columns are both labeled with \u201cCat.\u201d The authors need to make more detailed labels to distinguish (one from Exp. and the other is from Calc.). Also, the authors need to include the cutoff of 12.5 kcal/mol in the table legends otherwise it is difficult for readers to correlate calculated barriers with calculated cat.\n2. In pg. 8, the authors state that the cutoff is done purely for a post hoc comparison of experimental and computed data. And they used 12.5 kcal/mol as cutoff. The authors need to expand this section to give detailed descriptions about how they reach the value of 12.5 in their study.\n3. For results and discussion part, the two subtitles are \u201cSet S\u201d and \u201cSet L\u201d. It might be useful to guide readers if the authors can use more detailed subtitles which can generally summarize their results.\n4. In the last part of results and discussion, the authors state that I189G as a single mutant is counterproductive but lowers the barrier of G39A-A141Q-L278A. This would suggest there are couplings between I189 and G39/A141/L278. While it is difficult to figure out such kind of couplings, one simple question is, is I189 close to G39/A141/L278 in the structure.\nCite this review as\nAnonymous Reviewer (2013) Peer Review #1 of \"In silico screening of 393 mutants facilitates enzyme engineering of amidase activity in CalB (v0.1)\". PeerJ https://doi.org/10.7287/peerj.145v0.1/reviews/1\nReview 4: Reviewer 2 \u00b7 Jul 16, 2013\nBasic reporting\nMeets all necessary criteria\nExperimental design\nExcellent\nValidity of the findings\nExcellent\nAdditional comments\nThis is an excellent paper, nicely demonstrating the utility of a practical computational modelling approach to the prediction of enzyme activity. The method uses semiempirical quantum chemical methods to model amidase reactivity in a lipase. This is thorough work of high quality. The paper is well written and the results are analysed and presented in appropriate detail. The results will be of wide interest. This is a demonstration of a method that will find real industrial application, as well as in other contexts. The paper is suitable for publication essentially as is. A minor point is that some of the references might be lacking information (e.g. refs 27 and 28 do not look complete as written). Also, the authors might want to comment a little more on how amidase activity is achieved. There is wide debate about amidase versus esterase activity, and, while this is not the focus here, comparison with enzymes such as fatty acid amide hydrolase, which has been the subject of much modelling work, could be useful (e.g. a little more discussion on what specific features may provide amidase activity and how the designed amidases compare to natural amidaases). This could assist in future rational design (as well as in testing the quantum chemical methods by comparisons on related reactions.\nCite this review as\nAnonymous Reviewer (2013) Peer Review #2 of \"In silico screening of 393 mutants facilitates enzyme engineering of amidase activity in CalB (v0.1)\". PeerJ https://doi.org/10.7287/peerj.145v0.1/reviews/2\nReview 5: nan\nReview 6: \n",
"LLM_extracted_review": "['1. Thank you for addressing all the critical points of the reviewers and for revising the manuscript accordingly.\\n\\n2. Please address critical points raised by the first reviewer.\\n\\n3. The manuscript describes in silico screening of CalB mutants for enzymatic amidase activity. The computational approach gives reasonable results in general -- the authors demonstrate that 15 out of 22 mutants are correctly predicted in qualitative activity. The manuscript is scholarly and well written. The methods are complete and clear. The data are new and appear to be true. I recommend publishing with minor revision. \\n - In table 1, two columns are both labeled with \u201cCat.\u201d The authors need to make more detailed labels to distinguish (one from Exp. and the other is from Calc.). Also, the authors need to include the cutoff of 12.5 kcal/mol in the table legends otherwise it is difficult for readers to correlate calculated barriers with calculated cat.\\n - In pg. 8, the authors state that the cutoff is done purely for a post hoc comparison of experimental and computed data. They need to expand this section to give detailed descriptions about how they reach the value of 12.5 in their study.\\n - For results and discussion part, the two subtitles are \u201cSet S\u201d and \u201cSet L\u201d. It might be useful to guide readers if the authors can use more detailed subtitles which can generally summarize their results.\\n - In the last part of results and discussion, the authors state that I189G as a single mutant is counterproductive but lowers the barrier of G39A-A141Q-L278A. This would suggest there are couplings between I189 and G39/A141/L278. A simple question is, is I189 close to G39/A141/L278 in the structure?\\n\\n4. This is an excellent paper, nicely demonstrating the utility of a practical computational modelling approach to the prediction of enzyme activity. The method uses semiempirical quantum chemical methods to model amidase reactivity in a lipase. This is thorough work of high quality. The paper is well written and the results are analysed and presented in appropriate detail. The results will be of wide interest. \\n - A minor point is that some of the references might be lacking information (e.g. refs 27 and 28 do not look complete as written).\\n - The authors might want to comment a little more on how amidase activity is achieved. There is wide debate about amidase versus esterase activity, and while this is not the focus here, comparison with enzymes such as fatty acid amide hydrolase could be useful. A little more discussion on what specific features may provide amidase activity and how the designed amidases compare to natural amidaases could assist in future rational design and testing the quantum chemical methods by comparisons on related reactions.']"
} |