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Anti-androgen receptor activity of apoptotic CK2 inhibitor CX4945 in human prostate cancer LNCap cells
Bioorganic & Medicinal Chemistry Letters 22 (2012) 5470–5474
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Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Anti-androgen receptor activity of apoptotic CK2 inhibitor CX4945 in human prostate cancer LNCap cells Byung Jun Ryu a, , Seung-hwa Baek a, , Jiyeon Kim a, Su Jung Bae a, Sung-Youn Chang b, Jung-Nyoung Heo b, Hyuk Lee b, Sang Yeol Lee c, Seong Hwan Kim a,d,⇑ a
Laboratory of Translational Therapeutics, Pharmacology Research Center, Yuseong-gu, Daejeon 305-600, Republic of Korea Medicinal Chemistry Center, Bio-Organic Science Division, Korea Research Institute of Chemical Technology, PO Box 107, Yuseong-gu, Daejeon 305-600, Republic of Korea c Department of Life Science, Gachon University, Sungnam 461-701, Republic of Korea d Department of Pharmaceutical and Medical Chemistry, Korea University of Science and Technology, Daejeon 305-350, Republic of Korea b
a r t i c l e
i n f o
Article history: Received 2 March 2012 Revised 26 June 2012 Accepted 9 July 2012 Available online 14 July 2012 Keywords: CK2 CX4945 Androgen receptor Apoptosis Prostate cancer
a b s t r a c t Androgen receptor (AR) is crucial for transcriptional signaling in prostate cancers. The anti-cancer activity of protein kinase CK2 (formerly called casein kinase 2)-specific small molecule inhibitors have been reported in several cancers including prostate cancers. The orally available CX4945, a potent and selective small molecule inhibitor of CK2, has advanced into human clinical trials and has exhibited strong antitumor activity. The inhibition of CK2 leads to a down-regulation of the AR-dependent transcription, but the functional relevance of CX4945 to AR-dependent transcription in AR-positive LNCap cells has not been studied yet. Our observation of inhibitory effects of CX4945 on the expression or phosphorylation levels of CK2a, Akt and anti-apoptotic molecules including IAP family members agreed with a previous study showing the effect of CK2 inhibition in cancer cells. This study also provides novel information on the impact of CX4945 in the inhibition of AR-dependent transcriptional activation in LNCap cells via its down-regulation. Pharmacologic inhibition experiment revealed that CX4945 could exhibit its anti-cancer activity in LNCap cells via the independent inhibitions of AR and Akt-survivin signalings. Ó 2012 Elsevier Ltd. All rights reserved.
Androgen receptor (AR) is crucial for transcriptional signaling in prostate cancer, one of the most commonly diagnosed cancers in elderly men.1 Testosterone and its reduced metabolite dihydrotestosterone (DHT) are the two major growth factors of prostate cells. DHT, as the principal androgen, mainly regulates intraprostatic androgen-mediated cellular events. In the absence of the ligand binding, the cytoplasmic AR is held inactive, but upon androgen binding, the androgen–AR complex enters the nucleus and then triggers the transcription of androgen-regulated genes.2 Typical androgen-regulated genes such as prostate-specific antigen (PSA) and transmembrane serine protease 2 (TMPRSS2) are induced by DHT or synthetic androgen in AR-positive human prostate cancer LNCap cells.3 Since androgenic stimulation in prostate cancer is the major cause of its progression, the suppression of plasma testosterone by medical or surgical castration or the interruption of androgenic stimulation by AR antagonists or inhibitors of AR signaling leads to symptom reduction in men with advanced or metastatic prostate cancers.
⇑ Corresponding author. Tel.: +82 42 860 7687.
E-mail address: [email protected] (S.H. Kim). These authors contributed equally to this study.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmcl.2012.07.031
Protein kinase CK2 (formerly called casein kinase 2) is a ubiquitous serine/threonine kinase that controls a broad spectrum of cellular processes through phosphorylation of thousands of protein substrates involved in the regulation of the cell cycle, proliferation, and apoptosis.4 Constitutively active CK2 is elevated in several types of cancers where it protects cells from apoptosis, and its over-expression results in tumorigenesis in cellular and animal models.5,6 Since the inhibition of CK2 has been shown to induce apoptotic cell death of tumor cells, it has been considered as a promising druggable target for cancer therapy. The inhibition of CK2 activity or its expression decreases cellular proliferation as well as induces apoptosis in cancer cells including prostate cancers.4d,7 Decreased CK2 activity either by a specific antisense oligonucleotide or by a selective small molecule inhibitor of CK2 has been shown to reduce prostate cancer cell growth and inhibit prostate tumor growth in xenograft models.7,8 Recently, orally available CX4945 (Fig. 1A), a potent and selective small molecule inhibitor of CK2, was advanced into human clinical trials. This compound exhibited a dose-dependent anti-tumor activity in AR-independent PC-3 cells-inoculated xenograft model with the reduction in the phosphorylation of the biomarker p21 at T145.8c The apoptotic susceptibility of AR-sensitive prostate cancer LNCap cells to the knock-down of CK2 was similar to that of
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Figure 1. CX4945 induces the death of LNCap cells with caspase-3 activation. The effects of CX4945 (its structure in A) on the viability of LNCap cells (B) and RWPE-1 cells (C), the induction of caspase-3 activity (D), and the induction of annexin V-positive apoptotic cells (E) were evaluated. ⁄⁄, P <0.01; ⁄⁄⁄, P <0.001.
AR-refractory prostate cancer PC-3 cells, suggesting the AR-independent anti-prostate cancer activity of CK2.8a However, several studies have shown that the inhibition of CK2 by its specific inhibitors provoked different responses in LNCap cells and in PC-3 cells.8b,9 In contrast to PC-3 cells, LNCap cells responded to CK2 inhibition with apoptosis, suggesting that the mechanism of action on the apoptotic activity of CK2 inhibition might differ in the two types of cells. This suggestion in conjunction with the result showing that the inhibition of CK2 led to a down-regulation of the ARdependent transcription led us to investigate the functional relevance of CX4945 to AR-dependent transcription in LNCap cells.10 Herein, we have evaluated the anti-AR activity of CX4945 in LNCap cells. CX4945 was synthesized as described in a previous study and according to the protocol used in Cylene Pharmaceuticals where CX4945 was developed, cells were treated with CX4945 for 4 days and then cell viability was measured.8d,11,12 The IC50 value of CX4945 in LNCap cells was 4.59 lM (Fig. 1B), comparable to the IC50 values in several cancer cells (around 1.71–20.01 lM) reported by Cylene Pharmaceuticals. In normal prostate RWPE-1 cells, CX4945 did not affect the cell viability when compared to its activity in LNCap cells (Fig. 1C); at 10 lM, CX4945 time-dependently inhibited the viability of LNCap cells, but RWPE-1 cells survived 94% even incubated with CX4945 for 3 days.12 Since LNCap cells have been shown to respond to CK2 inhibition with apoptosis, we extended our study to examine the effect of CX4945 on the induction of caspase-3 activity.8b,9,12 Consistent with previous reports, CX4945 at levels greater than 10 lM significantly increased caspase-3 activity (Fig. 1D), suggesting that the cytotoxicity of CX4945 could be due to its potential to trigger apoptotic cell death.11 Additionally, the previous study showed that all normal cell lines failed to show a detectable change of caspase 3/7 activity as high as 100 lM of CX4945.11 The effect of CX4945 on the induction of apoptotic death in LNCap cells was also confirmed by staining annexin V that is used as a probe to detect
cells expressing phosphatidylserine (PS).13 When cells undergo apoptosis, PS becomes exposed on the surface of cells. As shown in Fig. 1E, the annexin V-positive LNCap cells were found when treated with CX4945. The apoptotic activity of CX4945 was further evaluated by measuring the CX4945-induced aberrant expressions of CK2a, CX4945related biomarkers and apoptosis-related molecules.14 These measurements could be helpful to explain the anti-apoptotic activity of CX4945 in LNCap cells. Since 72% of LNCap cells survived when treated with 10 lM CX4946 for 1 day (data not shown), CX4945 was used at concentration 10 lM or below in the following experiments. An evaluation of the effect of CX4945 on the expression of its target CK2 revealed that CX4945 reduced the protein level of CK2a in both the cytosolic and nuclear fractions (Fig. 2A). The immunohistochemical expression of nuclear CK2a was greater in tumor tissues than in adenomas and normal colorectal tissues and furthermore, a strong association between the nuclear localization of CK2a and poor prognostic factors in human prostate adenocarcinomas has been also reported.15,16 Considering these results suggesting the importance of nuclear CK2a levels in cancers, CX4945, with the potential to reduce the nuclear level of CK2a, might be an optimum drug candidate for treating cancers. The down-regulation of CK2 expression has been shown to induce apoptosis in several cancer cells including prostate cancers.4d,17 We then evaluated the effect of CX4945 on the phosphorylated levels of p21 and Akt. The phosphorylated forms of both are considered as biomarkers indicating the anti-cancer activity of CX4945 since it has been reported to inhibit the phosphorylations of p21 (at threonine 145) and Akt (at the canonical serine 473) in xenograft models.8c,11 Here, in LNCap cells, CX4945 strongly inhibited the phosphorylations of p21 and Akt (Fig. 2A). Akt signaling is required for the survival of cancers and has been shown to regulate the expression of survivin. Additionally, CK2 has been shown to functionally interact with Akt to promote the
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of CK2 in relation to the pathobiology of androgen-dependent and -independent prostate cancer has been identified with the inhibition of CK2 leading to a down-regulation of the AR-dependent transcription in LNCap cells.10,23 Thus, we investigated the functional involvement of CX4945 in AR signaling to better understand the underlying mechanism responsible for CX4945-mediated apoptosis in AR-positive prostate cancers. We conducted Western blot and high-content screening (HCS) analyses to elucidate the functional involvement of CX4945 in AR signaling.13,24 Western blot analysis revealed that CX4945 completely inhibited the nuclear translocation of AR via its down-regulation (Fig. 3A). The inhibitory effect of CX4945 on the nuclear translocation of AR was also confirmed by HCS analysis (Fig. 3B and C); AR translocation was significantly induced by DHT treatment, while its induction was inhibited by CX4945 in a dose dependent fashion. Even in the absence of DHT, CX4945 significantly inhibited the nuclear translocation of AR in LNCap cells. Additionally, consistent with the outcome of the studies in Fig. 3A, the reduced level of AR translocation by CX4945 was less than its basal level, suggesting that CX4945 could inhibit the nuclear translocation of AR via its down-regulation.
Figure 2. Aberrant levels of CX4945-responsive biomarkers in LNCap cells. (A) The effects of CX4945 on the phosphorylation of p21 and Akt, and the expression of CHOP and survivin were evaluated by Western blot analysis. (B) The effect of Akt inhibitor LY294002 on the expressions of survivin and AR was evaluated by Western blot analysis.
expression of survivin and enhance cell survival.18 We observed the down-regulation of survivin by CX4945 (Fig. 2A). Also, the levels of the anti-apoptotic X-linked inhibitor of apoptosis protein (XIAP) were reduced by CX4945. Survivin and XIAP are members of the class of inhibitors of apoptosis proteins (IAPs) that are elevated in cancers and have been proposed to block caspase activity.19 The inhibition or down-regulation of CK2 has been shown to reduce the expression of IAP proteins, suggesting that CK2 could sensitize cells toward induction of apoptosis by attenuating IAPs expression.20 Another IAP, cIAP-1, but not cIAP-2, was shown to be reduced by TBB in ALVA-41 prostate cancer cells, but neither of these IAPs was affected by CX4945 in LNCap cells (data not shown).20 The Akt-dependent expression of survivin was further supported by a pharmacologic study; Akt inhibitor LY294002 blocked the phosphorylation of Akt and the expression of survivin in LNCap cells (Fig. 2B). CK2 inhibition in LNCap cells has been shown to induce apoptosis via the endoplasmic reticulum (ER) stress response.8b To determine whether CX4945 could trigger the ER stress response, we measured the level of transcription factor C/EBP-homologous protein (CHOP), a protein known to be involved in the ER stress response and to induce apoptosis.21 CX4945 strongly induced CHOP protein expression (Fig. 2A). Since CHOP has been involved in ER stress-induced apoptosis by enhancing DR5 expression in human carcinoma cells, we also investigated the effect of CX4945 on DR5 expression.22 CX4945 had no effect on DR5 expression (Fig. 2A), suggesting that CX4945-induced CHOP might regulate the transcriptional levels of genes other than DR5. The development and progression of prostate cancer are dependent on AR signaling. Since androgens are the primary regulators of prostate cancer cell growth and proliferation, androgen ablation by orchiectomy or reduction of androgen levels by administration of anti-androgens that act as AR antagonists or inhibitors of AR signaling have been common therapeutic approaches for treating patients with prostate cancers. In fact, significant functional activity
Figure 3. Anti-AR activity of CX4945 in LNCap cells. The effect of CX4945 on the nuclear translocation of AR was evaluated by (A) Western blot analysis and (B) HCS analysis. (C) Fluorescence intensity of AR-GFP was measured with Cellomics ArrayScan. Control vs. treated group: ⁄, P <0.05; ⁄⁄, P <0.01; ⁄⁄⁄, P <0.001. DHTtreated group versus DHT plus CX4945-treatd group:#, P <0.05; ##, P <0.01; ###, P <0.001.
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Figure 4. Effect of CX4945 on AR activity. The effects of CX4945 on AR activity were evaluated by (A) AR reporter luciferase activity assay and (B) by measuring the ARresponsive gene expression based on real-time PCR. Control versus treated group: ⁄⁄, P <0.01; ⁄⁄⁄, P <0.001. DHT-treated group versus DHT plus CX4945-treatd group: #, P <0.05; ##, P <0.01; ###, P <0.001.
Since the inhibitory effect of CX4945 on the nuclear translocation of AR could strongly affect its role in transcriptional regulation, the effect of CX4945 on AR activity was further evaluated by AR reporter luciferase activity assay and also by quantifying the mRNA expression levels of the most typical androgen-regulated genes such as PSA and TMPRSS2 by the real-time PCR.25,26 In the absence of DHT, CX4945 significantly inhibited AR reporter luciferase activity in a dose-dependent manner and it also significantly inhibited DHT-mediated activation of AR (Fig. 4A). Considering that CX4945 did not significantly affect cell viability, the CX4945-mediated reduction of AR activity was not due to its cytotoxicity. Additionally, real-time PCR analysis revealed that the mRNA levels of PSA and TMPRSS2 were significantly reduced by 10 lM CX4945 in the absence of DHT. Consistent with DHT-mediated activation of AR, the expressions of both genes were strongly induced by DHT, but those inductions were inhibited by CX4945; importantly, DHT-induced mRNA expression of TMPRSS2 was significantly and dose-dependently inhibited by CX4945 (Fig. 4B). DHT-induced mRNA of PSA was significantly inhibited by CX4945 only at 3 lM. DHT is a major mediator of androgen induction of PSA gene expression in LNCap cells, but several signaling pathways including CK2 signaling and their cross-talk with AR signaling have been shown to be involved in the regulation of PSA gene expression.27 This might be the reason that CX4945 could not strongly inhibit the DHT-induced PSA mRNA expression. Apparently, the activation of protein kinase C or the inhibition of HER-2 signaling led to impairment of PSA expression in LNCap cells, and furthermore, the cross talk between androgen and Wnt signaling pathways has been reported by showing that inhibitor of b-catenin and T-cell factor (ICAT) enhanced expression of endogenous PSA. ICAT is a b-catenin binding protein that inhibits the canonical Wnt/b-catenin signaling pathway. In summary, our observations on the inhibitory effects of CX4945 on the expression or phosphorylation levels of CK2a, Akt, and anti-apoptotic molecules including IAP family members agree with a previous study showing the effect of CK2 inhibition in cancer cells. Our results provide novel information on the impact of CX4945 in the inhibition of AR-dependent transcriptional activation in LNCap cells, suggesting that the apoptotic activity of CX4945 could be attributed to its potential to inhibit transcriptional activity of AR via down-regulation of its expression in LNCap cells. Also, considering that LY294002 decreased the expression of survivin, but not of AR (Fig. 2B), the anti-cancer properties of CX4945 in LNCap cells may function by the independent inhibition of AR and Akt-survivin signalings. The further development of CK2 inhibition by specific inhibitors like CX4945 might lead to a
frontline strategy for the clinical treatment of patients with prostate cancers. Acknowledgments This work was supported by KRICT’s project, SI-1205 funded by the Ministry of Knowledge Economy, Republic of Korea. AR translocation was the result of a study on the ‘Joint Research Support Project’, supported by University of Science and Technology (UST). References and notes 1. Jemal, A.; Siegel, R.; Ward, E.; Hao, Y.; Xu, J.; Murray, T.; Thun, M. J. CA Cancer J. Clin. 2008, 58, 71. 2. (a) Evans, R. M. Science 1988, 240, 889; (b) Chatterjee, B. Mol. Cell Biochem. 2003, 253, 89; (c) Vis, A. N.; Schröder, F. H. BJU Int. 2009, 104, 438. 3. (a) Zhu, Y. S.; Cai, L. Q.; You, X.; Cordero, J. J.; Huang, Y.; Imperato-McGinley, J. J. Androl. 2003, 24, 681; (b) Barbier, O.; Bélanger, A. Best Pract. Res. Clin. Endocrinol. Metab. 2008, 22, 259; (c) Tran, C.; Ouk, S.; Clegg, N. J.; Chen, Y.; Watson, P. A.; Arora, V.; Wongvipat, J.; Smith-Jones, P. M.; Yoo, D.; Kwon, A.; Wasielewska, T.; Welsbie, D.; Chen, C. D.; Higano, C. S.; Beer, T. M.; Hung, D. T.; Scher, H. I.; Jung, M. E.; Sawyers, C. L. Science 2009, 324, 787. 4. (a) Allende, J. E.; Allende, C. C. FASEB J. 1995, 9, 313; (b) Litchfield, D. W. Biochem. J. 2003, 369, 1; (c) Meggio, F.; Pinna, L. A. FASEB J. 2003, 17, 349; (d) Wang, G.; Unger, G.; Ahmad, K. A.; Slaton, J. W.; Ahmed, K. Mol. Cell Biochem. 2005, 274, 77; (e) Homma, M. K.; Homma, Y. Mol Cell Biochem. 2008, 316, 49. 5. (a) Prowald, K.; Fischer, H.; Issinger, O. G. FEBS Lett. 1984, 176, 479; (b) Guerra, B.; Issinger, O. G. Electrophoresis 1999, 20, 391; (c) Tawfic, S.; Yu, S.; Wang, H.; Faust, R.; Davis, A.; Ahmed, K. Histol. Histopathol. 2001, 16, 573. 6. (a) Channavajhala, P.; Seldin, D. C. Oncogene 2002, 21, 5280; (b) LandesmanBollag, E.; Song, D. H.; Romieu-Mourez, R.; Sussman, D. J.; Cardiff, R. D.; Sonenshein, G. E.; Seldin, D. C. Mol. Cell Biochem. 2001, 227, 153. 7. Slaton, J. W.; Unger, G. M.; Sloper, D. T.; Davis, A. T.; Ahmed, K. Mol. Cancer Res. 2004, 2, 712. 8. (a) Wang, H.; Davis, A.; Yu, S.; Ahmed, K. Mol. Cell Biochem. 2001, 227, 167; (b) Hessenauer, A.; Schneider, C. C.; Götz, C.; Montenarh, M. Cell Signal. 2011, 23, 145; (c) Pierre, F.; Chua, P. C.; O’Brien, S. E.; Siddiqui-Jain, A.; Bourbon, P.; Haddach, M.; Michaux, J.; Nagasawa, J.; Schwaebe, M. K.; Stefan, E.; Vialettes, A.; Whitten, J. P.; Chen, T. K.; Darjania, L.; Stansfield, R.; Bliesath, J.; Drygin, D.; Ho, C.; Omori, M.; Proffitt, C.; Streiner, N.; Rice, W. G.; Ryckman, D. M.; Anderes, K. Mol. Cell Biochem. 2011, 356, 37; (d) Pierre, F.; Chua, P. C.; O’Brien, S. E.; Siddiqui-Jain, A.; Bourbon, P.; Haddach, M.; Michaux, J.; Nagasawa, J.; Schwaebe, M. K.; Stefan, E.; Vialettes, A.; Whitten, J. P.; Chen, T. K.; Darjania, L.; Stansfield, R.; Anderes, K.; Bliesath, J.; Drygin, D.; Ho, C.; Omori, M.; Proffitt, C.; Streiner, N.; Trent, K.; Rice, W. G.; Ryckman, D. M. J. Med. Chem. 2011, 54, 635. 9. Hessenauer, A.; Montenarh, M.; Götz, C. Int. J. Oncol. 2003, 22, 1263. 10. Götz, C.; Bachmann, C.; Montenarh, M. Prostate 2007, 67, 125. 11. Siddiqui-Jain, A.; Drygin, D.; Streiner, N.; Chua, P.; Pierre, F.; O’Brien, S. E.; Bliesath, J.; Omori, M.; Huser, N.; Ho, C.; Proffitt, C.; Schwaebe, M. K.; Ryckman, D. M.; Rice, W. G.; Anderes, K. Cancer Res. 2010, 70, 10288. 12. (a) Cell culture - Human prostate cancer LNCap cells were cultured in RPMI1640 (Hyclone, UT) supplemented with 10% fetal bovine serum (FBS, Hyclone), 100 U/ml of penicillin and 100 lg/ml of streptomycin in humidified atmosphere of 5% CO2 at 37 °C. The culture medium was changed every 3 days. Human normal prostate RWPE-1 cells were cultured in Keratinocyte-Serum Free Medium (Invitrogen) with 0.05 mg/ml of bovine pituitary extract and
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15. 16. 17. 18.
19.
20. 21. 22. 23. 24.
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5 ng/ml of human recombinant epidermal growth factor; (b) Cell viability assay - LNCap cells were seeded in a 96-well plate at 3 103 cells/well, cultured for 24 h and then incubated with CX4945 for 4 days. Then, cell viability was measured in triplicates by the Cell Counting Kit-8 (Dojindo Molecular Technologies, ML) according to the manufacturer’s protocol. Absorbance was measured by using Wallac EnVision microplate reader (PerkinElmer, Finland). RWPE-1 cells were seeded in a 96-well plate at 1 104 cells/well, cultured for 24 h and then incubated with CX4945 for 1-3 days. Then, cell viability was measured in triplicates by CCK-8. (c) Caspase-3 activity assay – LNCap cells were seeded in a 96-well plate at 1 104 cells/well, cultured for 24 h and then incubated with CX4945 for 1 or 2 days. Then, caspase-3 activity was measured in triplicates by Caspase-3 activity assay kit (Promega, WI) according to the manufacturer’s protocol. Significance was determined by Student’s t-test and differences were considered significant when P < 0.05. Immunocytochemistry - LNCaP cells (5 104 cells) were plated on the 1% gelatin coated coverslips and treated with CX-4945 (30 lM) for 72 hrs. After incubation, cells were rinsed and stained with Annexin V staining kit (Clonetech, CA, USA) according to the manufacturer’s protocol. All cells were then fixed with 2% formaldehyde and the annexin V-positive cells were visualized under the fluorescent microscope. Nuclei were stained with 10 lg/ ml DAPI. Scale bars represent 200 lm. Western blot analysis - Western blot was performed as described in a previous study; Hwang, M. K.; Min, Y. K.; Kim, S. H. Biochem. Cell Biol. 2009, 87, 919. Briefly, cells were homogenized and centrifuged at 10,000 g for 15 min. The supernatant was used as the cytoplasmic protein fraction and nuclear proteins were extracted using NucBuster Protein Extraction kit (Novagen, Germanay). Denatured proteins were separated on the gels and transferred onto PVDF (Millipore, CA). After incubation with antibody, membranes were developed with Pierce SuperSignal West Femto Maximum Sensitivity Substrate (Fisher Scientific, PA) using the LAS-3000 luminescent image analyzer (Fuji Photo Film Co., Ltd., Japan). Antibodies against p21, Akt, p-Akt, CHOP, DR5, XIAP and survivin were purchased from Cell Signaling Technology Inc. (MA). Antibodies against AR, actin, histone H3, p-p21, cIAP-1, cIAP-2 and secondary antibodies were purchased from Santa Cruz Biotechnology, Inc. (CA). Antibody against CK2a was purchased from Millipore (CA). Zou, J.; Luo, H.; Zeng, Q.; Dong, Z.; Wu, D.; Liu, L. J. Transl. Med. 2011, 9, 97. Laramas, M.; Pasquier, D.; Filhol, O.; Ringeisen, F.; Descotes, J. L.; Cochet, C. Eur. J. Cancer. 2007, 43, 928. Ahmad, K. A.; Wang, G.; Slaton, J.; Unger, G.; Ahmed, K. Anticancer Drugs. 2005, 1037, 16. Ponce, D. P.; Yefi, R.; Cabello, P.; Maturana, J. L.; Niechi, I.; Silva, E.; Galindo, M.; Antonelli, M.; Marcelain, K.; Armisen, R.; Tapia, J. C. Mol. Cell Biochem. 2011, 356, 127. (a) Bortner, C. D.; Cidlowski, J. A. Annu. Rev. Pharmacol. Toxicol. 2002, 42, 259; (b) McEleny, K. R.; Watson, R. W.; Coffey, R. N.; O’Neill, A. J.; Fitzpatrick, J. M. Prostate 2002, 51, 133; (c) Krajewska, M.; Krajewski, S.; Banares, S.; Huang, X.; Turner, B.; Bubendorf, L.; Kallioniemi, O. P.; Shabaik, A.; Vitiello, A.; Peehl, D.; Gao, G. J.; Reed, J. C. Clin. Cancer Res. 2003, 9, 4914. Wang, G.; Ahmad, K. A.; Ahmed, K. Cancer Res. 2006, 66, 2242. Maytin, E. V.; Ubeda, M.; Lin, J. C.; Habener, J. F. Exp. Cell Res. 2001, 267, 193. Yamaguchi, H.; Wang, H. G. J. Biol. Chem. 2004, 279, 45495. Wang, G.; Ahmad, K. A.; Unger, G.; Slaton, J. W.; Ahmed, K. J. Cell Biochem. 2006, 99, 382. HCS analysis—LNCap cells were seeded in a black clear 96-well plate (Greiner Bio One, NC) at 2.5104 cells/well in RPMI1640 with 10% charcoal-stripped,
dextran-treated FBS (CD-FBS, Sigma, MO). After 1 day incubation, media were changed to RPMI1640 with 0.1% CD-FBS and cells were cultured for 16 hr. Cells were pre-treated with CX4945 for 9 hr before incubating with 10 nM DHT (Tokyo Chemical Industry, Japan) for 3 h and then, cells were washed twice with PBS and fixed with 10% formalin. After permeabilized with 0.1% Triton-X 100 in PBS, cells were blocked with rabbit serum in 0.1% BSA/PBS and incubated with AR specific antibody (SantaCruz, CA) for overnight. After cells were washed with PBS, labeled with Alexa Fluor 488 anti-rabbit IgG (Invitrogen, NY) and incubated with 10 lg/ml Hoechst33342 (Invitrogen, NY) for 5 min, AR translocation was evaluated by scanning cells and measuring the fluorescent intensity of channels with Cellomics ArrayScan (ThermoScientific, PA). Significance was determined by Student’s t-test and differences were considered significant when P <0.05. 25. AR reporter luciferase assay—LNCap cells were seeded in a 12-well plate at 8,104 cells/well. After 24 h, cells were tranduced by SureENTRY transduction reagent with Cignal Lenti AR reporter (5 105 TU; SABioscience, MD) in RPMI1640 with 10% FBS according to the manufacturer’s protocol. After 2 days, medium was changed to RPMI1640 with 10% FBS, 1% antibiotics and 1% NEAA, and then tranduced cells were selected in culture medium with 30 lg/ml of puromycin (Sigma, MO). After puromycin selection, selected cells (4,104 cells/ well) were incubated in a 96-well plate for 1 day, cultured in androgendepleted medium (RPMI1640 with 10% CD-FBS) for 1 day, and treated with CX4945 in androgen depleted medium with 0.1% CD-FBS in the absence of presence of 0.1 lM DHT for 1 day. Then, AR activation and cell viability were evaluated by using luciferase reporter assay (Promega, WI) and CCK-8 kit, respectively. Significance was determined by Student’s t-test and differences were considered significant when P <0.05. 26. Real-time PCR analysis—LNCap cells were cultured in RPMI1640 supplemented with 5% CD-FBS and incubated with CX4945 in the absence or presence of 1 lM of DHT for 8 hr. Then, total RNA was isolated by using TRIzol reagent (Life Technologies, MD) and the first strand cDNA was synthesized using 2 lg of total RNA and 1 lM of oligo-dT18 primer and Omniscript Reverse Transcriptase (Qiagen, CA, USA) as described previously; Hwang, M. K.; Min, Y. K.; Kim, S. H. Biochem. Cell Biol. 2009, 87, 919. SYBR green-based quantitative PCR was performed using the Stratagene Mx3000P Real-Time PCR system and Brilliant SYBR Green Master Mix (Stratagene, CA, USA). Primers used in this study were designed using an online primer design program; Rozen, S.; Skaletsky, H. Methods Mol. Biol. 2000, 132, 365. The primers used for PCR were as follows: PSA, 50 -GAT GAA ACA CCC TGT GCC G-30 (forward) and 50 -CCT CAC AGC TAC CCA CTG CA-30 (reverse); TMPRSS2, 50 -CGA GGA GAA AGG GAA GAC CT-30 (forward) and 50 -TAT CCC CTA TCA GCC ACC AG-30 (reverse); GAPDH, 50 -AAC TTT GGC ATT GTG GAA GG-30 (forward) and 50 -ACA CAT TGG GGG TAG GAA CA30 (reverse). GAPDH was used as the control gene. All reactions were run in triplicate, and data were analyzed by the 2CT method; Livak, K. J.; Schmittgen, T. D. Method. Methods. 2001, 25, 402. Significance was determined with GAPDH-normalized 2CT values. Significance was determined by Student’s ttest and differences were considered significant when P <0.05. 27. (a) Zhu, Y. S.; Cai, L. Q.; You, X.; Cordero, J. J.; Huang, Y.; Imperato-McGinley, J. J. Androl. 2003, 24, 681; (b) Zhuo, M.; Zhu, C.; Sun, J.; Weis, W. I.; Sun, Z. Mol. Endocrinol. 2011, 25, 1677; (c) Liu, Y.; Majumder, S.; McCall, W.; Sartor, C. I.; Mohler, J. L.; Gregory, C. W.; Earp, H. S.; Whang, Y. E. Cancer Res. 2005, 65, 3404; (d) Rigas, A. C.; Ozanne, D. M.; Neal, D. E.; Robson, C. N. J. Biol. Chem. 2003, 278, 46087.
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Anti-androgen receptor activity of apoptotic CK2 inhibitor CX4945 in human prostate cancer LNCap cells Byung Jun Ryu a, , Seung-hwa Baek a, , Jiyeon Kim a, Su Jung Bae a, Sung-Youn Chang b, Jung-Nyoung Heo b, Hyuk Lee b, Sang Yeol Lee c, Seong Hwan Kim a,d,⇑ a
Laboratory of Translational Therapeutics, Pharmacology Research Center, Yuseong-gu, Daejeon 305-600, Republic of Korea Medicinal Chemistry Center, Bio-Organic Science Division, Korea Research Institute of Chemical Technology, PO Box 107, Yuseong-gu, Daejeon 305-600, Republic of Korea c Department of Life Science, Gachon University, Sungnam 461-701, Republic of Korea d Department of Pharmaceutical and Medical Chemistry, Korea University of Science and Technology, Daejeon 305-350, Republic of Korea b
a r t i c l e
i n f o
Article history: Received 2 March 2012 Revised 26 June 2012 Accepted 9 July 2012 Available online 14 July 2012 Keywords: CK2 CX4945 Androgen receptor Apoptosis Prostate cancer
a b s t r a c t Androgen receptor (AR) is crucial for transcriptional signaling in prostate cancers. The anti-cancer activity of protein kinase CK2 (formerly called casein kinase 2)-specific small molecule inhibitors have been reported in several cancers including prostate cancers. The orally available CX4945, a potent and selective small molecule inhibitor of CK2, has advanced into human clinical trials and has exhibited strong antitumor activity. The inhibition of CK2 leads to a down-regulation of the AR-dependent transcription, but the functional relevance of CX4945 to AR-dependent transcription in AR-positive LNCap cells has not been studied yet. Our observation of inhibitory effects of CX4945 on the expression or phosphorylation levels of CK2a, Akt and anti-apoptotic molecules including IAP family members agreed with a previous study showing the effect of CK2 inhibition in cancer cells. This study also provides novel information on the impact of CX4945 in the inhibition of AR-dependent transcriptional activation in LNCap cells via its down-regulation. Pharmacologic inhibition experiment revealed that CX4945 could exhibit its anti-cancer activity in LNCap cells via the independent inhibitions of AR and Akt-survivin signalings. Ó 2012 Elsevier Ltd. All rights reserved.
Androgen receptor (AR) is crucial for transcriptional signaling in prostate cancer, one of the most commonly diagnosed cancers in elderly men.1 Testosterone and its reduced metabolite dihydrotestosterone (DHT) are the two major growth factors of prostate cells. DHT, as the principal androgen, mainly regulates intraprostatic androgen-mediated cellular events. In the absence of the ligand binding, the cytoplasmic AR is held inactive, but upon androgen binding, the androgen–AR complex enters the nucleus and then triggers the transcription of androgen-regulated genes.2 Typical androgen-regulated genes such as prostate-specific antigen (PSA) and transmembrane serine protease 2 (TMPRSS2) are induced by DHT or synthetic androgen in AR-positive human prostate cancer LNCap cells.3 Since androgenic stimulation in prostate cancer is the major cause of its progression, the suppression of plasma testosterone by medical or surgical castration or the interruption of androgenic stimulation by AR antagonists or inhibitors of AR signaling leads to symptom reduction in men with advanced or metastatic prostate cancers.
⇑ Corresponding author. Tel.: +82 42 860 7687.
E-mail address: [email protected] (S.H. Kim). These authors contributed equally to this study.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmcl.2012.07.031
Protein kinase CK2 (formerly called casein kinase 2) is a ubiquitous serine/threonine kinase that controls a broad spectrum of cellular processes through phosphorylation of thousands of protein substrates involved in the regulation of the cell cycle, proliferation, and apoptosis.4 Constitutively active CK2 is elevated in several types of cancers where it protects cells from apoptosis, and its over-expression results in tumorigenesis in cellular and animal models.5,6 Since the inhibition of CK2 has been shown to induce apoptotic cell death of tumor cells, it has been considered as a promising druggable target for cancer therapy. The inhibition of CK2 activity or its expression decreases cellular proliferation as well as induces apoptosis in cancer cells including prostate cancers.4d,7 Decreased CK2 activity either by a specific antisense oligonucleotide or by a selective small molecule inhibitor of CK2 has been shown to reduce prostate cancer cell growth and inhibit prostate tumor growth in xenograft models.7,8 Recently, orally available CX4945 (Fig. 1A), a potent and selective small molecule inhibitor of CK2, was advanced into human clinical trials. This compound exhibited a dose-dependent anti-tumor activity in AR-independent PC-3 cells-inoculated xenograft model with the reduction in the phosphorylation of the biomarker p21 at T145.8c The apoptotic susceptibility of AR-sensitive prostate cancer LNCap cells to the knock-down of CK2 was similar to that of
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Figure 1. CX4945 induces the death of LNCap cells with caspase-3 activation. The effects of CX4945 (its structure in A) on the viability of LNCap cells (B) and RWPE-1 cells (C), the induction of caspase-3 activity (D), and the induction of annexin V-positive apoptotic cells (E) were evaluated. ⁄⁄, P <0.01; ⁄⁄⁄, P <0.001.
AR-refractory prostate cancer PC-3 cells, suggesting the AR-independent anti-prostate cancer activity of CK2.8a However, several studies have shown that the inhibition of CK2 by its specific inhibitors provoked different responses in LNCap cells and in PC-3 cells.8b,9 In contrast to PC-3 cells, LNCap cells responded to CK2 inhibition with apoptosis, suggesting that the mechanism of action on the apoptotic activity of CK2 inhibition might differ in the two types of cells. This suggestion in conjunction with the result showing that the inhibition of CK2 led to a down-regulation of the ARdependent transcription led us to investigate the functional relevance of CX4945 to AR-dependent transcription in LNCap cells.10 Herein, we have evaluated the anti-AR activity of CX4945 in LNCap cells. CX4945 was synthesized as described in a previous study and according to the protocol used in Cylene Pharmaceuticals where CX4945 was developed, cells were treated with CX4945 for 4 days and then cell viability was measured.8d,11,12 The IC50 value of CX4945 in LNCap cells was 4.59 lM (Fig. 1B), comparable to the IC50 values in several cancer cells (around 1.71–20.01 lM) reported by Cylene Pharmaceuticals. In normal prostate RWPE-1 cells, CX4945 did not affect the cell viability when compared to its activity in LNCap cells (Fig. 1C); at 10 lM, CX4945 time-dependently inhibited the viability of LNCap cells, but RWPE-1 cells survived 94% even incubated with CX4945 for 3 days.12 Since LNCap cells have been shown to respond to CK2 inhibition with apoptosis, we extended our study to examine the effect of CX4945 on the induction of caspase-3 activity.8b,9,12 Consistent with previous reports, CX4945 at levels greater than 10 lM significantly increased caspase-3 activity (Fig. 1D), suggesting that the cytotoxicity of CX4945 could be due to its potential to trigger apoptotic cell death.11 Additionally, the previous study showed that all normal cell lines failed to show a detectable change of caspase 3/7 activity as high as 100 lM of CX4945.11 The effect of CX4945 on the induction of apoptotic death in LNCap cells was also confirmed by staining annexin V that is used as a probe to detect
cells expressing phosphatidylserine (PS).13 When cells undergo apoptosis, PS becomes exposed on the surface of cells. As shown in Fig. 1E, the annexin V-positive LNCap cells were found when treated with CX4945. The apoptotic activity of CX4945 was further evaluated by measuring the CX4945-induced aberrant expressions of CK2a, CX4945related biomarkers and apoptosis-related molecules.14 These measurements could be helpful to explain the anti-apoptotic activity of CX4945 in LNCap cells. Since 72% of LNCap cells survived when treated with 10 lM CX4946 for 1 day (data not shown), CX4945 was used at concentration 10 lM or below in the following experiments. An evaluation of the effect of CX4945 on the expression of its target CK2 revealed that CX4945 reduced the protein level of CK2a in both the cytosolic and nuclear fractions (Fig. 2A). The immunohistochemical expression of nuclear CK2a was greater in tumor tissues than in adenomas and normal colorectal tissues and furthermore, a strong association between the nuclear localization of CK2a and poor prognostic factors in human prostate adenocarcinomas has been also reported.15,16 Considering these results suggesting the importance of nuclear CK2a levels in cancers, CX4945, with the potential to reduce the nuclear level of CK2a, might be an optimum drug candidate for treating cancers. The down-regulation of CK2 expression has been shown to induce apoptosis in several cancer cells including prostate cancers.4d,17 We then evaluated the effect of CX4945 on the phosphorylated levels of p21 and Akt. The phosphorylated forms of both are considered as biomarkers indicating the anti-cancer activity of CX4945 since it has been reported to inhibit the phosphorylations of p21 (at threonine 145) and Akt (at the canonical serine 473) in xenograft models.8c,11 Here, in LNCap cells, CX4945 strongly inhibited the phosphorylations of p21 and Akt (Fig. 2A). Akt signaling is required for the survival of cancers and has been shown to regulate the expression of survivin. Additionally, CK2 has been shown to functionally interact with Akt to promote the
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of CK2 in relation to the pathobiology of androgen-dependent and -independent prostate cancer has been identified with the inhibition of CK2 leading to a down-regulation of the AR-dependent transcription in LNCap cells.10,23 Thus, we investigated the functional involvement of CX4945 in AR signaling to better understand the underlying mechanism responsible for CX4945-mediated apoptosis in AR-positive prostate cancers. We conducted Western blot and high-content screening (HCS) analyses to elucidate the functional involvement of CX4945 in AR signaling.13,24 Western blot analysis revealed that CX4945 completely inhibited the nuclear translocation of AR via its down-regulation (Fig. 3A). The inhibitory effect of CX4945 on the nuclear translocation of AR was also confirmed by HCS analysis (Fig. 3B and C); AR translocation was significantly induced by DHT treatment, while its induction was inhibited by CX4945 in a dose dependent fashion. Even in the absence of DHT, CX4945 significantly inhibited the nuclear translocation of AR in LNCap cells. Additionally, consistent with the outcome of the studies in Fig. 3A, the reduced level of AR translocation by CX4945 was less than its basal level, suggesting that CX4945 could inhibit the nuclear translocation of AR via its down-regulation.
Figure 2. Aberrant levels of CX4945-responsive biomarkers in LNCap cells. (A) The effects of CX4945 on the phosphorylation of p21 and Akt, and the expression of CHOP and survivin were evaluated by Western blot analysis. (B) The effect of Akt inhibitor LY294002 on the expressions of survivin and AR was evaluated by Western blot analysis.
expression of survivin and enhance cell survival.18 We observed the down-regulation of survivin by CX4945 (Fig. 2A). Also, the levels of the anti-apoptotic X-linked inhibitor of apoptosis protein (XIAP) were reduced by CX4945. Survivin and XIAP are members of the class of inhibitors of apoptosis proteins (IAPs) that are elevated in cancers and have been proposed to block caspase activity.19 The inhibition or down-regulation of CK2 has been shown to reduce the expression of IAP proteins, suggesting that CK2 could sensitize cells toward induction of apoptosis by attenuating IAPs expression.20 Another IAP, cIAP-1, but not cIAP-2, was shown to be reduced by TBB in ALVA-41 prostate cancer cells, but neither of these IAPs was affected by CX4945 in LNCap cells (data not shown).20 The Akt-dependent expression of survivin was further supported by a pharmacologic study; Akt inhibitor LY294002 blocked the phosphorylation of Akt and the expression of survivin in LNCap cells (Fig. 2B). CK2 inhibition in LNCap cells has been shown to induce apoptosis via the endoplasmic reticulum (ER) stress response.8b To determine whether CX4945 could trigger the ER stress response, we measured the level of transcription factor C/EBP-homologous protein (CHOP), a protein known to be involved in the ER stress response and to induce apoptosis.21 CX4945 strongly induced CHOP protein expression (Fig. 2A). Since CHOP has been involved in ER stress-induced apoptosis by enhancing DR5 expression in human carcinoma cells, we also investigated the effect of CX4945 on DR5 expression.22 CX4945 had no effect on DR5 expression (Fig. 2A), suggesting that CX4945-induced CHOP might regulate the transcriptional levels of genes other than DR5. The development and progression of prostate cancer are dependent on AR signaling. Since androgens are the primary regulators of prostate cancer cell growth and proliferation, androgen ablation by orchiectomy or reduction of androgen levels by administration of anti-androgens that act as AR antagonists or inhibitors of AR signaling have been common therapeutic approaches for treating patients with prostate cancers. In fact, significant functional activity
Figure 3. Anti-AR activity of CX4945 in LNCap cells. The effect of CX4945 on the nuclear translocation of AR was evaluated by (A) Western blot analysis and (B) HCS analysis. (C) Fluorescence intensity of AR-GFP was measured with Cellomics ArrayScan. Control vs. treated group: ⁄, P <0.05; ⁄⁄, P <0.01; ⁄⁄⁄, P <0.001. DHTtreated group versus DHT plus CX4945-treatd group:#, P <0.05; ##, P <0.01; ###, P <0.001.
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Figure 4. Effect of CX4945 on AR activity. The effects of CX4945 on AR activity were evaluated by (A) AR reporter luciferase activity assay and (B) by measuring the ARresponsive gene expression based on real-time PCR. Control versus treated group: ⁄⁄, P <0.01; ⁄⁄⁄, P <0.001. DHT-treated group versus DHT plus CX4945-treatd group: #, P <0.05; ##, P <0.01; ###, P <0.001.
Since the inhibitory effect of CX4945 on the nuclear translocation of AR could strongly affect its role in transcriptional regulation, the effect of CX4945 on AR activity was further evaluated by AR reporter luciferase activity assay and also by quantifying the mRNA expression levels of the most typical androgen-regulated genes such as PSA and TMPRSS2 by the real-time PCR.25,26 In the absence of DHT, CX4945 significantly inhibited AR reporter luciferase activity in a dose-dependent manner and it also significantly inhibited DHT-mediated activation of AR (Fig. 4A). Considering that CX4945 did not significantly affect cell viability, the CX4945-mediated reduction of AR activity was not due to its cytotoxicity. Additionally, real-time PCR analysis revealed that the mRNA levels of PSA and TMPRSS2 were significantly reduced by 10 lM CX4945 in the absence of DHT. Consistent with DHT-mediated activation of AR, the expressions of both genes were strongly induced by DHT, but those inductions were inhibited by CX4945; importantly, DHT-induced mRNA expression of TMPRSS2 was significantly and dose-dependently inhibited by CX4945 (Fig. 4B). DHT-induced mRNA of PSA was significantly inhibited by CX4945 only at 3 lM. DHT is a major mediator of androgen induction of PSA gene expression in LNCap cells, but several signaling pathways including CK2 signaling and their cross-talk with AR signaling have been shown to be involved in the regulation of PSA gene expression.27 This might be the reason that CX4945 could not strongly inhibit the DHT-induced PSA mRNA expression. Apparently, the activation of protein kinase C or the inhibition of HER-2 signaling led to impairment of PSA expression in LNCap cells, and furthermore, the cross talk between androgen and Wnt signaling pathways has been reported by showing that inhibitor of b-catenin and T-cell factor (ICAT) enhanced expression of endogenous PSA. ICAT is a b-catenin binding protein that inhibits the canonical Wnt/b-catenin signaling pathway. In summary, our observations on the inhibitory effects of CX4945 on the expression or phosphorylation levels of CK2a, Akt, and anti-apoptotic molecules including IAP family members agree with a previous study showing the effect of CK2 inhibition in cancer cells. Our results provide novel information on the impact of CX4945 in the inhibition of AR-dependent transcriptional activation in LNCap cells, suggesting that the apoptotic activity of CX4945 could be attributed to its potential to inhibit transcriptional activity of AR via down-regulation of its expression in LNCap cells. Also, considering that LY294002 decreased the expression of survivin, but not of AR (Fig. 2B), the anti-cancer properties of CX4945 in LNCap cells may function by the independent inhibition of AR and Akt-survivin signalings. The further development of CK2 inhibition by specific inhibitors like CX4945 might lead to a
frontline strategy for the clinical treatment of patients with prostate cancers. Acknowledgments This work was supported by KRICT’s project, SI-1205 funded by the Ministry of Knowledge Economy, Republic of Korea. AR translocation was the result of a study on the ‘Joint Research Support Project’, supported by University of Science and Technology (UST). References and notes 1. Jemal, A.; Siegel, R.; Ward, E.; Hao, Y.; Xu, J.; Murray, T.; Thun, M. J. CA Cancer J. Clin. 2008, 58, 71. 2. (a) Evans, R. M. Science 1988, 240, 889; (b) Chatterjee, B. Mol. Cell Biochem. 2003, 253, 89; (c) Vis, A. N.; Schröder, F. H. BJU Int. 2009, 104, 438. 3. (a) Zhu, Y. S.; Cai, L. Q.; You, X.; Cordero, J. J.; Huang, Y.; Imperato-McGinley, J. J. Androl. 2003, 24, 681; (b) Barbier, O.; Bélanger, A. Best Pract. Res. Clin. Endocrinol. Metab. 2008, 22, 259; (c) Tran, C.; Ouk, S.; Clegg, N. J.; Chen, Y.; Watson, P. A.; Arora, V.; Wongvipat, J.; Smith-Jones, P. M.; Yoo, D.; Kwon, A.; Wasielewska, T.; Welsbie, D.; Chen, C. D.; Higano, C. S.; Beer, T. M.; Hung, D. T.; Scher, H. I.; Jung, M. E.; Sawyers, C. L. Science 2009, 324, 787. 4. (a) Allende, J. E.; Allende, C. C. FASEB J. 1995, 9, 313; (b) Litchfield, D. W. Biochem. J. 2003, 369, 1; (c) Meggio, F.; Pinna, L. A. FASEB J. 2003, 17, 349; (d) Wang, G.; Unger, G.; Ahmad, K. A.; Slaton, J. W.; Ahmed, K. Mol. Cell Biochem. 2005, 274, 77; (e) Homma, M. K.; Homma, Y. Mol Cell Biochem. 2008, 316, 49. 5. (a) Prowald, K.; Fischer, H.; Issinger, O. G. FEBS Lett. 1984, 176, 479; (b) Guerra, B.; Issinger, O. G. Electrophoresis 1999, 20, 391; (c) Tawfic, S.; Yu, S.; Wang, H.; Faust, R.; Davis, A.; Ahmed, K. Histol. Histopathol. 2001, 16, 573. 6. (a) Channavajhala, P.; Seldin, D. C. Oncogene 2002, 21, 5280; (b) LandesmanBollag, E.; Song, D. H.; Romieu-Mourez, R.; Sussman, D. J.; Cardiff, R. D.; Sonenshein, G. E.; Seldin, D. C. Mol. Cell Biochem. 2001, 227, 153. 7. Slaton, J. W.; Unger, G. M.; Sloper, D. T.; Davis, A. T.; Ahmed, K. Mol. Cancer Res. 2004, 2, 712. 8. (a) Wang, H.; Davis, A.; Yu, S.; Ahmed, K. Mol. Cell Biochem. 2001, 227, 167; (b) Hessenauer, A.; Schneider, C. C.; Götz, C.; Montenarh, M. Cell Signal. 2011, 23, 145; (c) Pierre, F.; Chua, P. C.; O’Brien, S. E.; Siddiqui-Jain, A.; Bourbon, P.; Haddach, M.; Michaux, J.; Nagasawa, J.; Schwaebe, M. K.; Stefan, E.; Vialettes, A.; Whitten, J. P.; Chen, T. K.; Darjania, L.; Stansfield, R.; Bliesath, J.; Drygin, D.; Ho, C.; Omori, M.; Proffitt, C.; Streiner, N.; Rice, W. G.; Ryckman, D. M.; Anderes, K. Mol. Cell Biochem. 2011, 356, 37; (d) Pierre, F.; Chua, P. C.; O’Brien, S. E.; Siddiqui-Jain, A.; Bourbon, P.; Haddach, M.; Michaux, J.; Nagasawa, J.; Schwaebe, M. K.; Stefan, E.; Vialettes, A.; Whitten, J. P.; Chen, T. K.; Darjania, L.; Stansfield, R.; Anderes, K.; Bliesath, J.; Drygin, D.; Ho, C.; Omori, M.; Proffitt, C.; Streiner, N.; Trent, K.; Rice, W. G.; Ryckman, D. M. J. Med. Chem. 2011, 54, 635. 9. Hessenauer, A.; Montenarh, M.; Götz, C. Int. J. Oncol. 2003, 22, 1263. 10. Götz, C.; Bachmann, C.; Montenarh, M. Prostate 2007, 67, 125. 11. Siddiqui-Jain, A.; Drygin, D.; Streiner, N.; Chua, P.; Pierre, F.; O’Brien, S. E.; Bliesath, J.; Omori, M.; Huser, N.; Ho, C.; Proffitt, C.; Schwaebe, M. K.; Ryckman, D. M.; Rice, W. G.; Anderes, K. Cancer Res. 2010, 70, 10288. 12. (a) Cell culture - Human prostate cancer LNCap cells were cultured in RPMI1640 (Hyclone, UT) supplemented with 10% fetal bovine serum (FBS, Hyclone), 100 U/ml of penicillin and 100 lg/ml of streptomycin in humidified atmosphere of 5% CO2 at 37 °C. The culture medium was changed every 3 days. Human normal prostate RWPE-1 cells were cultured in Keratinocyte-Serum Free Medium (Invitrogen) with 0.05 mg/ml of bovine pituitary extract and
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5 ng/ml of human recombinant epidermal growth factor; (b) Cell viability assay - LNCap cells were seeded in a 96-well plate at 3 103 cells/well, cultured for 24 h and then incubated with CX4945 for 4 days. Then, cell viability was measured in triplicates by the Cell Counting Kit-8 (Dojindo Molecular Technologies, ML) according to the manufacturer’s protocol. Absorbance was measured by using Wallac EnVision microplate reader (PerkinElmer, Finland). RWPE-1 cells were seeded in a 96-well plate at 1 104 cells/well, cultured for 24 h and then incubated with CX4945 for 1-3 days. Then, cell viability was measured in triplicates by CCK-8. (c) Caspase-3 activity assay – LNCap cells were seeded in a 96-well plate at 1 104 cells/well, cultured for 24 h and then incubated with CX4945 for 1 or 2 days. Then, caspase-3 activity was measured in triplicates by Caspase-3 activity assay kit (Promega, WI) according to the manufacturer’s protocol. Significance was determined by Student’s t-test and differences were considered significant when P < 0.05. Immunocytochemistry - LNCaP cells (5 104 cells) were plated on the 1% gelatin coated coverslips and treated with CX-4945 (30 lM) for 72 hrs. After incubation, cells were rinsed and stained with Annexin V staining kit (Clonetech, CA, USA) according to the manufacturer’s protocol. All cells were then fixed with 2% formaldehyde and the annexin V-positive cells were visualized under the fluorescent microscope. Nuclei were stained with 10 lg/ ml DAPI. Scale bars represent 200 lm. Western blot analysis - Western blot was performed as described in a previous study; Hwang, M. K.; Min, Y. K.; Kim, S. H. Biochem. Cell Biol. 2009, 87, 919. Briefly, cells were homogenized and centrifuged at 10,000 g for 15 min. The supernatant was used as the cytoplasmic protein fraction and nuclear proteins were extracted using NucBuster Protein Extraction kit (Novagen, Germanay). Denatured proteins were separated on the gels and transferred onto PVDF (Millipore, CA). After incubation with antibody, membranes were developed with Pierce SuperSignal West Femto Maximum Sensitivity Substrate (Fisher Scientific, PA) using the LAS-3000 luminescent image analyzer (Fuji Photo Film Co., Ltd., Japan). Antibodies against p21, Akt, p-Akt, CHOP, DR5, XIAP and survivin were purchased from Cell Signaling Technology Inc. (MA). Antibodies against AR, actin, histone H3, p-p21, cIAP-1, cIAP-2 and secondary antibodies were purchased from Santa Cruz Biotechnology, Inc. (CA). Antibody against CK2a was purchased from Millipore (CA). Zou, J.; Luo, H.; Zeng, Q.; Dong, Z.; Wu, D.; Liu, L. J. Transl. Med. 2011, 9, 97. Laramas, M.; Pasquier, D.; Filhol, O.; Ringeisen, F.; Descotes, J. L.; Cochet, C. Eur. J. Cancer. 2007, 43, 928. Ahmad, K. A.; Wang, G.; Slaton, J.; Unger, G.; Ahmed, K. Anticancer Drugs. 2005, 1037, 16. Ponce, D. P.; Yefi, R.; Cabello, P.; Maturana, J. L.; Niechi, I.; Silva, E.; Galindo, M.; Antonelli, M.; Marcelain, K.; Armisen, R.; Tapia, J. C. Mol. Cell Biochem. 2011, 356, 127. (a) Bortner, C. D.; Cidlowski, J. A. Annu. Rev. Pharmacol. Toxicol. 2002, 42, 259; (b) McEleny, K. R.; Watson, R. W.; Coffey, R. N.; O’Neill, A. J.; Fitzpatrick, J. M. Prostate 2002, 51, 133; (c) Krajewska, M.; Krajewski, S.; Banares, S.; Huang, X.; Turner, B.; Bubendorf, L.; Kallioniemi, O. P.; Shabaik, A.; Vitiello, A.; Peehl, D.; Gao, G. J.; Reed, J. C. Clin. Cancer Res. 2003, 9, 4914. Wang, G.; Ahmad, K. A.; Ahmed, K. Cancer Res. 2006, 66, 2242. Maytin, E. V.; Ubeda, M.; Lin, J. C.; Habener, J. F. Exp. Cell Res. 2001, 267, 193. Yamaguchi, H.; Wang, H. G. J. Biol. Chem. 2004, 279, 45495. Wang, G.; Ahmad, K. A.; Unger, G.; Slaton, J. W.; Ahmed, K. J. Cell Biochem. 2006, 99, 382. HCS analysis—LNCap cells were seeded in a black clear 96-well plate (Greiner Bio One, NC) at 2.5104 cells/well in RPMI1640 with 10% charcoal-stripped,
dextran-treated FBS (CD-FBS, Sigma, MO). After 1 day incubation, media were changed to RPMI1640 with 0.1% CD-FBS and cells were cultured for 16 hr. Cells were pre-treated with CX4945 for 9 hr before incubating with 10 nM DHT (Tokyo Chemical Industry, Japan) for 3 h and then, cells were washed twice with PBS and fixed with 10% formalin. After permeabilized with 0.1% Triton-X 100 in PBS, cells were blocked with rabbit serum in 0.1% BSA/PBS and incubated with AR specific antibody (SantaCruz, CA) for overnight. After cells were washed with PBS, labeled with Alexa Fluor 488 anti-rabbit IgG (Invitrogen, NY) and incubated with 10 lg/ml Hoechst33342 (Invitrogen, NY) for 5 min, AR translocation was evaluated by scanning cells and measuring the fluorescent intensity of channels with Cellomics ArrayScan (ThermoScientific, PA). Significance was determined by Student’s t-test and differences were considered significant when P <0.05. 25. AR reporter luciferase assay—LNCap cells were seeded in a 12-well plate at 8,104 cells/well. After 24 h, cells were tranduced by SureENTRY transduction reagent with Cignal Lenti AR reporter (5 105 TU; SABioscience, MD) in RPMI1640 with 10% FBS according to the manufacturer’s protocol. After 2 days, medium was changed to RPMI1640 with 10% FBS, 1% antibiotics and 1% NEAA, and then tranduced cells were selected in culture medium with 30 lg/ml of puromycin (Sigma, MO). After puromycin selection, selected cells (4,104 cells/ well) were incubated in a 96-well plate for 1 day, cultured in androgendepleted medium (RPMI1640 with 10% CD-FBS) for 1 day, and treated with CX4945 in androgen depleted medium with 0.1% CD-FBS in the absence of presence of 0.1 lM DHT for 1 day. Then, AR activation and cell viability were evaluated by using luciferase reporter assay (Promega, WI) and CCK-8 kit, respectively. Significance was determined by Student’s t-test and differences were considered significant when P <0.05. 26. Real-time PCR analysis—LNCap cells were cultured in RPMI1640 supplemented with 5% CD-FBS and incubated with CX4945 in the absence or presence of 1 lM of DHT for 8 hr. Then, total RNA was isolated by using TRIzol reagent (Life Technologies, MD) and the first strand cDNA was synthesized using 2 lg of total RNA and 1 lM of oligo-dT18 primer and Omniscript Reverse Transcriptase (Qiagen, CA, USA) as described previously; Hwang, M. K.; Min, Y. K.; Kim, S. H. Biochem. Cell Biol. 2009, 87, 919. SYBR green-based quantitative PCR was performed using the Stratagene Mx3000P Real-Time PCR system and Brilliant SYBR Green Master Mix (Stratagene, CA, USA). Primers used in this study were designed using an online primer design program; Rozen, S.; Skaletsky, H. Methods Mol. Biol. 2000, 132, 365. The primers used for PCR were as follows: PSA, 50 -GAT GAA ACA CCC TGT GCC G-30 (forward) and 50 -CCT CAC AGC TAC CCA CTG CA-30 (reverse); TMPRSS2, 50 -CGA GGA GAA AGG GAA GAC CT-30 (forward) and 50 -TAT CCC CTA TCA GCC ACC AG-30 (reverse); GAPDH, 50 -AAC TTT GGC ATT GTG GAA GG-30 (forward) and 50 -ACA CAT TGG GGG TAG GAA CA30 (reverse). GAPDH was used as the control gene. All reactions were run in triplicate, and data were analyzed by the 2CT method; Livak, K. J.; Schmittgen, T. D. Method. Methods. 2001, 25, 402. Significance was determined with GAPDH-normalized 2CT values. Significance was determined by Student’s ttest and differences were considered significant when P <0.05. 27. (a) Zhu, Y. S.; Cai, L. Q.; You, X.; Cordero, J. J.; Huang, Y.; Imperato-McGinley, J. J. Androl. 2003, 24, 681; (b) Zhuo, M.; Zhu, C.; Sun, J.; Weis, W. I.; Sun, Z. Mol. Endocrinol. 2011, 25, 1677; (c) Liu, Y.; Majumder, S.; McCall, W.; Sartor, C. I.; Mohler, J. L.; Gregory, C. W.; Earp, H. S.; Whang, Y. E. Cancer Res. 2005, 65, 3404; (d) Rigas, A. C.; Ozanne, D. M.; Neal, D. E.; Robson, C. N. J. Biol. Chem. 2003, 278, 46087.