- Email: [email protected]
Antioxidant Therapy Alleviates Oxidative Stress by Androgen Deprivation and Prevents Conversion From Androgen Dependent to Castration Resistant Prostate Cancer
Antioxidant Therapy Alleviates Oxidative Stress by Androgen Deprivation and Prevents Conversion From Androgen Dependent to Castration Resistant Prostate Cancer Masaki Shiota,* YooHyun Song,* Ario Takeuchi,* Akira Yokomizo,† Eiji Kashiwagi, Kentaro Kuroiwa, Katsunori Tatsugami, Takeshi Uchiumi, Yoshinao Oda and Seiji Naito From the Departments of Urology (MS, YS, AT, AY, EK, KK, KT, SN), Anatomic Pathology (YO) and Clinical Chemistry and Laboratory Medicine (TU), Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan
Purpose: Prostate cancer progression from androgen dependence to castration resistance results at least in part from oxidative stress induced by androgen deprivation therapy. We elucidated the state and the role of oxidative stress induced by androgen deprivation therapy and the possibility of antioxidant therapy in human prostate cancer. Materials and Methods: We investigated 4-HNE (4-hydroxy-2-nonenal histidine adduct) staining, and Twist1, YB-1 and androgen receptor expression by immunohistochemistry in prostate cancer samples treated with or without neoadjuvant androgen deprivation therapy. Intracellular reactive oxygen species and protein expression were examined by CM-H2DCFDA and Western blot analysis, respectively. A cell proliferation assay and a mouse xenograft model were used to assess tumor growth. Results: Androgen deprivation therapy increased oxidative stress, as shown by 4-HNE staining in human prostate cancer tissue. Twist1 and YB-1 expression was up-regulated by androgen deprivation, resulting in androgen receptor over expression. In LNCaP and 22Rv1 cells androgen deprivation increased intracellular reactive oxygen species and evoked Twist1, YB-1 and androgen receptor over expression, resulting in cell growth in a castration resistant manner. Growth was alleviated by N-acetyl-cysteine, an electrophile that supports glutathione production. N-acetyl-cysteine also decreased LNCaP and 22Rv1 tumor growth in castrated and noncastrated mice. Conclusions: Androgen deprivation therapy induced oxidative stress in in vitro and human prostate cancer. Antioxidant therapy using N-acetyl-cysteine appears to be a promising therapeutic modality for prostate cancer.
Abbreviations and Acronyms 4-HNE ⫽ 4-hydroxy-2-nonenal histidine adduct ADT ⫽ androgen deprivation therapy AR ⫽ androgen receptor CRPC ⫽ castration resistant prostate cancer NAC ⫽ N-acetyl-cysteine PCa ⫽ prostate cancer PSA ⫽ prostate specific antigen ROS ⫽ reactive oxygen species YB-1 ⫽ Y-box binding protein-1 Submitted for publication May 13, 2011. Study received Kyushu University institutional review board approval. * Equal study contribution. † Correspondence: Department of Urology, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan (telephone: ⫹81-92-642-5603; FAX: ⫹81-92-642-5618; e-mail: [email protected]. kyushu-u.ac.jp).
Key Words: prostate, prostatic neoplasms, acetylcysteine, androgen antagonists, disease progression PROSTATE cancer is the most common noncutaneous cancer and the second leading cause of cancer related death in men in developed countries.1,2 Most PCa cases are androgen dependent at diagnosis and most respond well to ADT. However, most PCa eventually
relapses to CRPC after ADT.3 Currently there are few successful therapies for CRPC other than docetaxel. Thus, modalities to prevent or overcome CRPC are needed. The androgen/AR signaling pathway is recognized as key in PCa de-
0022-5347/12/1872-0707/0 THE JOURNAL OF UROLOGY® © 2012 by AMERICAN UROLOGICAL ASSOCIATION EDUCATION
Vol. 187, 707-714, February 2012 Printed in U.S.A. DOI:10.1016/j.juro.2011.09.147
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velopment and progression. The progression of androgen dependent PCa to CRPC could be associated with increased AR expression.4 –9 Recently we reported that androgen deprivation induced an increase in intracellular ROS in human androgen dependent LNCaP PCa cells. Also, Twist1 transcription factor induced by oxidative stress regulates AR transcription, resulting in AR over expression in CRPC.10 More recently we found that YB-1, a major target gene of Twist1,11–13 is also implicated in progression to CRPC via AR transcription regulation.14 However, to our knowledge the status of oxidative stress, and Twist1 and YB-1 expression remains unknown in human PCa tissue treated with ADT. ADT decreases circulating testosterone to about 10% and androgen in prostate tissue to 15% to 40%. Although complete androgen deprivation evoked oxidative stress in androgen dependent LNCaP cells in vitro, to our knowledge whether low level, long-term androgen deprivation induces oxidative stress in vitro and whether ADT induces oxidative stress in human PCa remain unknown. We investigated whether ADT induces oxidative stress, as represented by 4-HNE, and expression of the oxidative stress related molecules Twist1 and YB-1 in human PCa tissue. We also examined whether low level, long-term androgen deprivation induces oxidative stress, and Twist1, YB-1 and AR up-regulation in vitro. In addition, we investigated whether NAC, an electrophile supporting glutathione production that functions as an antioxidant, could alleviate the unfavorable effects of androgen deprivation. We further pursued the possibility of antioxidant therapy as a prophylactic modality against the progression of androgen dependent PCa to CRPC.
MATERIALS AND METHODS Tissues and Clinical Data Included in this study were 31 patients with PCa who underwent radical prostatectomy with or without neoadjuvant ADT at Kyushu University Hospital, Japan, between 1997 and 2001, for whom there were enough carcinoma areas for immunohistochemical evaluation. All patients underwent surgery for clinically localized prostate cancer, as determined by preoperative PSA, digital rectal examination and prostate needle biopsy. Neoadjuvant ADT was done during 1 to 11 months. The indication for neoadjuvant ADT did not depend on patient risk criteria but rather on the policy of each home physician. The background of patients with vs without neoadjuvant ADT was similar. Preoperative PSA in patients with and without neoadjuvant ADT was 2.7 to 57.4 (median 12.7) and 0.61 to 22.5 ng/ml (median 11.7), respectively.
Slides for this study were prepared from prostate blocks containing the largest representative area of tumor and adjacent normal epithelium. The study was approved by the Kyushu University institutional review board.
Immunohistochemistry Immunohistochemistry was done as described previously.14 –16 The primary antibodies used were anti-4-HNE (Nikken Seil, Fukuroi, Japan), anti-Twist1 (Sigma®), antiYB-1 (Epitomics®) and anti-AR (Dako, Glostrup, Denmark). Antigen retrieval was done by microwave heating in citrate buffer (pH 6.0) for 20 minutes for anti-YB-1 antibody.
Immunohistochemical Analysis Twist1, YB-1 and AR expression was evaluated by the proportion and intensity of positively staining carcinoma cells. A proportion score was assigned to represent the estimated proportion of positively stained carcinoma cells, including 0 —none, 1—fewer than 1/100, 2—1/100 to 1/10, 3—1/10 to 1/3, 4 —1/3 to 2/3 and 5— greater than 2/3. The average estimated intensity of staining in positive carcinoma cells was assigned an intensity score of 0 —none, 1—weak, 2—intermediate or 3—strong. Proportion and intensity scores were added to obtain a total score of 0 to 8.17 Immunohistochemical results were classified based on total scores, including 0 to 4 — low and 5 to 8 — high expression. Immunohistochemical results were judged by 2 well trained pathologists (YS and YO).
Cell Culture Human PCa LNCaP and 22Rv1 cells were cultured under RPMI 1640 medium (Invitrogen™) containing 10% fetal bovine serum. The androgen reduced medium was supplemented with 8.75% charcoal stripped serum and 1.25% fetal bovine serum. LNCaP and 22Rv1 cells were cultured under this androgen reduced medium with or without 5 mM NAC for 2, 4, 8 and 12 weeks, respectively. LNCaP and 22Rv1 cells cultured for 0 weeks were used for experiments under medium containing 10% fetal bovine serum while those for 2, 4, 8 and 12 weeks were used for experiments under androgen reduced medium with or without 5 mM NAC. Cell lines were maintained in a 5% CO2 atmosphere at 37C.
Intracellular ROS Measurement Intracellular ROS was measured as described previously.10 The indicated LNCaP or 22Rv1 cells (1.0 ⫻ 103) were seeded in 96-well plates and incubated for 48 hours. Intracellular ROS was measured using CM-H2DCFDA (Invitrogen) according to the manufacturer protocol. Results represent at least 3 independent experiments.
Western Blot Western blot analysis was done as described previously.10,14 –16 We used antibodies against AR (sc-815) and Twist1 (sc-81417) (Santa Cruz Biotechnology, Santa Cruz, California), and antibodies against YB-1 (Epitomics) and lamin B1 (Abcam®).
Cell Proliferation Assay Cell proliferation assay was done as described previously.10,14,15 Briefly, the indicated LNCaP or 22Rv1 cells (2.5 ⫻ 104) were seeded in 12-well plates and incubated
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with 0 hour considered 12 hours after transfection. Cells were harvested with trypsin and counted daily using a cell counter (Beckman Coulter, Fullerton, California). Results were normalized to cell counts at 0 hours and represent at least 3 independent experiments.
Mouse Xenograft Model We used 6 to 8-week-old male NCr-nu/nu mice (Charles River Laboratories Japan, Yokohama, Japan). LNCaP (5.0 ⫻ 107) or 22Rv1 (5.0 ⫻ 107) cells were inoculated subcutaneously into the lateral flank of 20 mice each. At 7 days the mice were randomly divided into 4 groups, including castration or no castration with or without NAC. For castration or sham operation mice were anesthetized with intraperitoneal injection of ketamine/xylazine mixture. For castration each testis was removed surgically 7 days after tumor inoculation. NAC (100 mg/kg) was administered intraperitoneally daily. The size of inoculated tumors was determined every 2 to 3 days using calipers. Tumor volume was calculated using the formula, V ⫽ (A ⫻ B2)/2, where V represents volume in mm3, and A and B represent the long and the short diameter in mm, respectively.
Statistical Analysis The Wilcoxon test was used to examine pairwise correlations of immunohistochemical staining scores. Fisher’s exact test was used for statistical analysis of correlations between neoadjuvant ADT status and immunohistochemical staining scores. In vitro and in vivo experiments were analyzed by the t test with 2-sided p ⬍0.05 considered statistically significant.
RESULTS ADT induced oxidative stress, and Twist1 and YB-1 expression, resulting in AR over expression in human PCa tissue. Staining with the oxidative stress marker 4-HNE in PCa tissue receiving neoadjuvant ADT was increased compared with that in PCa tissue without neoadjuvant ADT (table 1 and fig. 1, A). Similarly Twist1 and YB-1 expression was up-regulated in PCa tissue with vs without neoadjuvant
Table 1. Neoadjuvant ADT and 4-HNE staining, Twist1, YB-1 and AR
4-HNE: Low High Twist1: Low High YB-1: Low High AR: Low High
No.
No Neoadjuvant ADT
Neoadjuvant ADT
14 17
11 5
3 12
16 15
11 5
5 10
15 16
11 5
4 11
13 18
8 8
5 10
p Value 0.0113*
0.0756
0.0320*
0.473
* Statistically significant.
Figure 1. Treating human PCa tissue with vs without ADT revealed that ADT induced oxidative stress, and Twist1 and YB-1 expression, resulting in AR over expression. A, 4-HNE. B, Twist1, C, YB-1. D, AR. Reduced from ⫻400.
ADT, although statistical significance was not attained for Twist1 expression (table 1 and fig. 1, B and C). Neoadjuvant ADT duration did not affect 4-HNE staining or Twist1 and YB-1 expression (data not shown). Also, 4-HNE staining correlated with Twist1 and YB-1 expression (r ⫽ 0.6240 and 0.4651, respectively, table 2), indicating that these molecules are associated with oxidative stress, as shown previously.10 AR expression in PCa tissue with neoadjuvant ADT was increased compared with tissue without neoadjuvant ADT, although statistical significance was not attained (table 1 and fig. 1, D). AR expression also correlated with Twist1 and YB-1 expression (r ⫽ 0.4227 and 0.4131, respectively, table 2), supporting our previous reports that Twist1 and YB-1 regulate AR expression.10,15 Also, 4-HNE staining among patients not treated with
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mor growth (fig. 4, A). Similar results were noted in the 22Rv1 xenograft model (fig. 4, B).
Table 2. Twist1, YB-1 and AR, and 4-HNE staining correlation coefficients
4-HNE Twist1 YB-1 AR
4-HNE
Twist1
YB-1
— 0.624 0.465 0.214
— — 0.425 0.422
— — — 0.413
neoadjuvant ADT did not correlate with Gleason score (data not shown). Low level and long-term androgen deprivation induced intracellular ROS, resulting in castration resistance, which was alleviated by adding NAC to LNCaP cells. Intracellular ROS gradually increased up to 4 weeks and gradually decreased thereafter to about baseline at 12 weeks (fig. 2, A). The increase in intracellular ROS was almost completely alleviated by adding NAC (fig. 2, A). AR expression was increased 8 and 12 weeks after androgen deprivation. Simultaneously Twist1 and YB-1 were also induced while ROS decreased as Twist1 and YB-1 expression was up-regulated. This may have resulted from the antioxidative properties of Twist1 and YB-1. In contrast, increases in AR, Twist1 and YB-1 after androgen deprivation decreased when NAC was added to androgen deprivation (fig. 2, B). LNCaP cells gained castration resistance as the duration of androgen deprivation increased, while LNCaP cells remained androgen dependent when NAC was added to androgen deprivation (fig. 2, C). Low level and long-term androgen deprivation induced intracellular ROS, resulting in castration resistance, which was alleviated by adding NAC to 22Rv1 cells, another PCa cell line that expresses functional AR and is sensitive to androgen in cell proliferation (data not shown).18,19 Similar to LNCaP cells, intracellular ROS in 22Rv1 cells increased up to 4 and 8 weeks and decreased 12 weeks after androgen deprivation (fig. 3, A). AR expression in 22Rv1 cells was prominently induced 12 weeks after androgen deprivation with the increases in Twist1 and YB-1, although adding NAC abolished these reactions, similar to LNCaP cells (fig. 3, B). Adding NAC alleviated the circumvention of androgen sensitivity, similar to LNCaP cells (fig. 3, C). NAC administration suppressed androgen dependent LNCaP and androgen sensitive 22Rv1 tumor growth in vivo. As expected, in the mouse xenograft model castration prominently suppressed LNCaP tumor growth. NAC administration significantly suppressed LNCaP tumor growth in castrated mice. NAC also suppressed LNCaP tumor growth in noncastrated mice. As a result, castration combined with NAC most effectively suppressed LNCaP tu-
DISCUSSION ADT induced oxidative stress, as represented by 4-HNE staining in human PCa tissue. Under physiological conditions the cellular concentration of 4-HNE is 0.1 to 0.3 M. However, under conditions of oxidative stress it accumulates at concentrations of 10 to 5,000 M.20,21 Thus, 4-HNE is a known, established marker of oxidative stress. Furthermore, in human PCa tissue Twist1 and YB-1, which are transcription factors regulating AR transcription, were up-regulated by ADT. A functional link between these molecules and oxidative stress was strengthened by the finding that 4-HNE staining correlated with Twist1 and YB-1 expression. It is known that 4-HNE facilitates activation of the NF-E2 related factor 2 signaling pathway, induces a cluster of phase II enzymes and readily activates various protein kinase pathways.22 However, to our knowledge regulation of the Twist1/YB1/AR signaling pathway by 4-HNE has not been reported. Thus, these interactions should be clarified in future studies. On the other hand, a functional link between Twist1 and YB-1 molecules with AR was indicated, supporting our previous findings.10,15 Immunohistochemistry using human PCa tissue treated vs not treated with ADT revealed that oxidative stress by ADT induces Twist1/YB-1 over expression, resulting in AR over expression. This is our hypothesis of the mechanism of progression of androgen dependent PCa to CRPC, although our study was limited by its small sample size. After obtaining these findings we confirmed this notion in vitro and investigated whether antioxidant NAC could alleviate oxidative stress by androgen deprivation, of Twist1, YB-1 and AR induction, and the gain of castration resistance. We noted successful alleviation of oxidative stress by androgen deprivation. Thus, NAC could suppress the increased Twist1, YB-1 and AR expression caused by androgen deprivation and, thus, prevent progression to castration resistance in vitro. Subsequently we confirmed the successful results of antioxidant therapy against progression to CRPC in a mouse xenograft model, although the additive tumor suppressive effect by NAC with castration may have derived from the direct tumor suppressive effect of NAC alone, as described. NAC administration alone suppressed PCa tumor growth. NAC inhibits the mitogenic activity of v-HRas in NIH3T3, as judged by short-term assay of [3H] thymidine incorporation during 4 hours.23 NAC has also had a chemopreventive effect on Kaposi
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Figure 2. Low level, long-term androgen deprivation induced intracellular ROS, resulting in castration resistance, which was alleviated by adding NAC in LNCaP cells. A, cells were seeded in 96-well plates and incubated. At 48 hours fluorescence intensity was measured with intensity at 0 week considered 1. Boxes represent mean. Bars represent ⫾ SD. Asterisk indicates p ⬍0.05 vs 0 week. B, whole cell extracts were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Western blot was done using indicated antibodies. C, cells were seeded in 12-well plates and incubated. After indicated hours cell number was counted and results were normalized to cell number at hour 0. Bars represent ⫾ SD. Asterisk indicates p ⬍0.05 vs 0 week.
sarcoma progression by decreasing the expression of vascular endothelial growth factor from tumor cells, thus, suppressing the growth of tumors transplanted into mice.24 Havre et al reported that NAC induced apoptosis in human tumor cells.25 On the other hand, Zhang et al reported that NAC enhanced the growth of BCR-ABL transformed cells.26 These controversies may have resulted from the cell type or the NAC dose used in experiments. High NAC doses may induce apoptosis while lower doses induce cell growth.25,26 This suggests that
the biological effects of NAC depend on the dose and should be carefully assessed in different cellular conditions. We used a NAC concentration of 5 mM in vitro. At this concentration LNCaP and 22Rv1 cell proliferation was not affected by adding NAC (data not shown). However, adding NAC prominently suppressed the gain of castration resistance, suggesting that 5 mM NAC did not simply suppress cell growth but rather specifically prevented progression to CRPC. On the other hand, in vivo NAC suppressed tumor growth in intact and castrated mice, suggesting that NAC
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Figure 3. Low level, long-term androgen deprivation induced intracellular ROS, resulting in castration resistance, which was alleviated by adding NAC in 22Rv1 cells. A, cells were seeded in 96-well plates and incubated. At 48 hours fluorescence intensity was measured with intensity at 0 week considered 1. Boxes represent mean. Bars represent ⫾ SD. Asterisk indicates p ⬍0.05 vs 0 week. B, whole cell extracts were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Western blot was done with indicated antibodies. C, cells were seeded in 12-well plates and incubated. After indicated hours cell number was counted and results were normalized to cell number at hour 0. Bars represent ⫾ SD. Asterisk indicates p ⬍0.05 vs 0 week.
might suppress neoangiogenesis by inhibiting vascular endothelial growth factor production in vivo. Although we used NAC, which is an agent that functions as an antioxidant, other antioxidants such as vitamin C, vitamin E, polyphenols and carotenoids are available. Previously lycopene reportedly decreased serum PSA and improved survival combined with castration,27 although evidence is insufficient to draw firm conclusions.28 The antioxidant ␣-tocopherol was suggested to increase survival in patients with PCa, and higher serum ␣-tocopherol and ␣-tocopherol intervention contributed to im-
proved PCa survival, although -carotene and retinol did not.29 Thus, other antioxidants might be more effective than NAC to treat PCa and prevent PCa progression. We should confirm the efficacy of antioxidant therapy for human PCa and develop more valid antioxidant therapy for PCa and its progression to CRPC.
CONCLUSIONS ADT increased oxidative stress in human PCa tissue, which may lead to Twist1 and YB-1 over expression, resulting in AR over expression in
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Figure 4. NAC suppressed androgen dependent and androgen sensitive tumor growth in vivo in male ncr-nu/nu mice injected subcutaneously. Five mice per group were assigned to no castration or NAC (circles), NAC without castration (squares), castration without NAC (triangles) and castration with NAC (inverted triangles). NAC was administered 7 days after inoculation. Tumor volume was inspected every 2 to 3 days. A, LNCaP cells. B, 22Rv1 cells. Bars represent ⫾ SEM. Asterisk indicates p ⬍0.05.
PCa. In androgen dependent and androgen sensitive PCa cells low level and long-term androgen deprivation caused the over expression of Twist1, YB-1 and AR, and provoked castrate resistant growth, which was abolished by the antioxidant NAC. These findings suggest that oxidative stress may be involved in the pathogenesis of CRPC in human PCa and indicate that new therapies tar-
geting oxidative stress using an antioxidant may prevent the conversion of androgen dependent or androgen sensitive PCa to CRPC.
ACKNOWLEDGMENTS Noriko Hakoda and Seiko Kamori provided technical assistance.
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3. Debes JD and Tindall DJ: The role of androgens and the androgen receptor in prostate cancer. Cancer Lett 2002; 187: 1. 4. Gregory CW, Hamil KG, Kim D et al: Androgen receptor expression in androgen-independent prostate cancer is associated with increased expression of androgen-regulated genes. Cancer Res 1998; 58: 5718. 5. Zegarra-Moro OL, Schmidt LJ, Huang H et al: Disruption of androgen function inhibits proliferation of androgen-refractory prostate cancer cells. Cancer Res 2002; 62: 1008. 6. Chen CD, Welsbie DS, Tran C et al: Molecular determinants of resistance to antiandrogen therapy. Nat Med 2004; 10: 33. 7. Scher HI and Sawyers CL: Biology of progressive, castration-resistant prostate cancer: directed therapies targeting the androgen-receptor signaling axis. J Clin Oncol 2005; 23: 8253. 8. Shiota M, Yokomizo A and Naito S: Increased androgen receptor transcription: a cause of castration-resistant prostate cancer and a possible
10. Shiota M, Yokomizo A, Tada Y et al: Castration resistance of prostate cancer cells caused by castration-induced oxidative stress through Twist1 and androgen receptor overexpression. Oncogene 2010; 29: 237. 11. Shiota M, Izumi H, Onitsuka T et al: Twist promotes tumor cell growth through YB-1 expression. Cancer Res 2008; 68: 98. 12. Shiota M, Izumi H, Onitsuka T et al: Twist and p53 reciprocally regulate target genes via direct interaction. Oncogene 2008; 27: 5543. 13. Shiota M, Izumi H, Tanimoto A et al: Programmed cell death protein 4 down-regulates Y-box binding protein-1 expression via a direct interaction with Twist1 to suppress cancer cell growth. Cancer Res 2009; 69: 53148. 14. Shiota M, Song Y, Yokomizo A et al: Human heterochromatin protein 1 isoform HP1 enhances androgen receptor activity and is implicated in prostate cancer growth. Endocr Relat Cancer 2010; 17: 455.
15. Shiota M, Takeuchi A, Song Y et al: Y-box binding protein-1 promotes castration-resistant prostate cancer growth via androgen receptor expression. Endocr Relat Cancer 2011; 18: 505. 16. Shiota M, Song Y, Yokomizo A et al: Foxo3a suppression promotes urothelial cancer invasion through Twist1, Y-box-binding protein 1 and Ecadherin regulation. Clin Cancer Res 2010; 16: 5654. 17. Allred DC, Harvey JM, Berardo M et al: Prognostic and predictive factors in breast cancer by immunohistochemical analysis. Mod Pathol 1998; 11: 155. 18. Tepper CG, Boucher DL, Ryan PE et al: Characterization of a novel androgen receptor mutation in a relapsed CWR22 prostate cancer xenograft and cell line. Cancer Res 2002; 62: 6606. 19. Dehm SM, Schmidt LJ, Heemers HV et al: Splicing of a novel androgen receptor exon generates a constitutively active androgen receptor that mediates prostate cancer therapy resistance. Cancer Res 2008; 68: 5469. 20. Uchida K: 4-Hydroxy-2-nonenal: a product and mediator of oxidative stress. Prog Lipid Res 2003; 42: 318. 21. Esterbauer H, Schaur RJ and Zollner H: Chemistry and biochemistry of 4-hydroxynonenal, malonal-
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dehyde and related aldehydes. Free Radic Biol Med 1991; 11: 81. 22. Ishii T, Itoh K, Ruiz E et al: Role of Nrf2 in the regulation of CD36 and stress protein expression in murine macrophages: activation by oxidatively modified LDL and 4-hydroxynonenal. Circ Res 2004; 94: 609. 23. Irani K, Xia Y, Zweier JL et al: Mitogenic signaling mediated by oxidants in Ras-transformed fibroblasts. Science 1997; 275: 1649.
24. Albini A, Morini M, D’Agostini F et al: Inhibition of angiogenesis-driven Kaposi’s sarcoma tumor growth in nude mice by oral N-acetylcysteine. Cancer Res 2001; 61: 8171.
27. Ansari MS and Gupta NP: A comparison of lycopene and orchidectomy vs orchidectomy alone in the management of advanced prostate cancer. BJU Int 2003; 92: 375.
25. Havre PA, O’Reilly S, McCormick JJ et al: Transformed and tumor-derived human cells exhibit preferential sensitivity to the thiol antioxidants, N-acetyl cysteine and penicillamine. Cancer Res 2002; 62: 1443.
28. Haseen F, Cantwell MM, O’Sullivan JM et al: Is there a benefit from lycopene supplementation in men with prostate cancer? A systematic review. Prostate Cancer Prostatic Dis 2009; 12: 325.
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Purpose: Prostate cancer progression from androgen dependence to castration resistance results at least in part from oxidative stress induced by androgen deprivation therapy. We elucidated the state and the role of oxidative stress induced by androgen deprivation therapy and the possibility of antioxidant therapy in human prostate cancer. Materials and Methods: We investigated 4-HNE (4-hydroxy-2-nonenal histidine adduct) staining, and Twist1, YB-1 and androgen receptor expression by immunohistochemistry in prostate cancer samples treated with or without neoadjuvant androgen deprivation therapy. Intracellular reactive oxygen species and protein expression were examined by CM-H2DCFDA and Western blot analysis, respectively. A cell proliferation assay and a mouse xenograft model were used to assess tumor growth. Results: Androgen deprivation therapy increased oxidative stress, as shown by 4-HNE staining in human prostate cancer tissue. Twist1 and YB-1 expression was up-regulated by androgen deprivation, resulting in androgen receptor over expression. In LNCaP and 22Rv1 cells androgen deprivation increased intracellular reactive oxygen species and evoked Twist1, YB-1 and androgen receptor over expression, resulting in cell growth in a castration resistant manner. Growth was alleviated by N-acetyl-cysteine, an electrophile that supports glutathione production. N-acetyl-cysteine also decreased LNCaP and 22Rv1 tumor growth in castrated and noncastrated mice. Conclusions: Androgen deprivation therapy induced oxidative stress in in vitro and human prostate cancer. Antioxidant therapy using N-acetyl-cysteine appears to be a promising therapeutic modality for prostate cancer.
Abbreviations and Acronyms 4-HNE ⫽ 4-hydroxy-2-nonenal histidine adduct ADT ⫽ androgen deprivation therapy AR ⫽ androgen receptor CRPC ⫽ castration resistant prostate cancer NAC ⫽ N-acetyl-cysteine PCa ⫽ prostate cancer PSA ⫽ prostate specific antigen ROS ⫽ reactive oxygen species YB-1 ⫽ Y-box binding protein-1 Submitted for publication May 13, 2011. Study received Kyushu University institutional review board approval. * Equal study contribution. † Correspondence: Department of Urology, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan (telephone: ⫹81-92-642-5603; FAX: ⫹81-92-642-5618; e-mail: [email protected]. kyushu-u.ac.jp).
Key Words: prostate, prostatic neoplasms, acetylcysteine, androgen antagonists, disease progression PROSTATE cancer is the most common noncutaneous cancer and the second leading cause of cancer related death in men in developed countries.1,2 Most PCa cases are androgen dependent at diagnosis and most respond well to ADT. However, most PCa eventually
relapses to CRPC after ADT.3 Currently there are few successful therapies for CRPC other than docetaxel. Thus, modalities to prevent or overcome CRPC are needed. The androgen/AR signaling pathway is recognized as key in PCa de-
0022-5347/12/1872-0707/0 THE JOURNAL OF UROLOGY® © 2012 by AMERICAN UROLOGICAL ASSOCIATION EDUCATION
Vol. 187, 707-714, February 2012 Printed in U.S.A. DOI:10.1016/j.juro.2011.09.147
AND
RESEARCH, INC.
www.jurology.com
707
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ANTIOXIDANT THERAPY PREVENTS CASTRATION RESISTANT PROSTATE CANCER
velopment and progression. The progression of androgen dependent PCa to CRPC could be associated with increased AR expression.4 –9 Recently we reported that androgen deprivation induced an increase in intracellular ROS in human androgen dependent LNCaP PCa cells. Also, Twist1 transcription factor induced by oxidative stress regulates AR transcription, resulting in AR over expression in CRPC.10 More recently we found that YB-1, a major target gene of Twist1,11–13 is also implicated in progression to CRPC via AR transcription regulation.14 However, to our knowledge the status of oxidative stress, and Twist1 and YB-1 expression remains unknown in human PCa tissue treated with ADT. ADT decreases circulating testosterone to about 10% and androgen in prostate tissue to 15% to 40%. Although complete androgen deprivation evoked oxidative stress in androgen dependent LNCaP cells in vitro, to our knowledge whether low level, long-term androgen deprivation induces oxidative stress in vitro and whether ADT induces oxidative stress in human PCa remain unknown. We investigated whether ADT induces oxidative stress, as represented by 4-HNE, and expression of the oxidative stress related molecules Twist1 and YB-1 in human PCa tissue. We also examined whether low level, long-term androgen deprivation induces oxidative stress, and Twist1, YB-1 and AR up-regulation in vitro. In addition, we investigated whether NAC, an electrophile supporting glutathione production that functions as an antioxidant, could alleviate the unfavorable effects of androgen deprivation. We further pursued the possibility of antioxidant therapy as a prophylactic modality against the progression of androgen dependent PCa to CRPC.
MATERIALS AND METHODS Tissues and Clinical Data Included in this study were 31 patients with PCa who underwent radical prostatectomy with or without neoadjuvant ADT at Kyushu University Hospital, Japan, between 1997 and 2001, for whom there were enough carcinoma areas for immunohistochemical evaluation. All patients underwent surgery for clinically localized prostate cancer, as determined by preoperative PSA, digital rectal examination and prostate needle biopsy. Neoadjuvant ADT was done during 1 to 11 months. The indication for neoadjuvant ADT did not depend on patient risk criteria but rather on the policy of each home physician. The background of patients with vs without neoadjuvant ADT was similar. Preoperative PSA in patients with and without neoadjuvant ADT was 2.7 to 57.4 (median 12.7) and 0.61 to 22.5 ng/ml (median 11.7), respectively.
Slides for this study were prepared from prostate blocks containing the largest representative area of tumor and adjacent normal epithelium. The study was approved by the Kyushu University institutional review board.
Immunohistochemistry Immunohistochemistry was done as described previously.14 –16 The primary antibodies used were anti-4-HNE (Nikken Seil, Fukuroi, Japan), anti-Twist1 (Sigma®), antiYB-1 (Epitomics®) and anti-AR (Dako, Glostrup, Denmark). Antigen retrieval was done by microwave heating in citrate buffer (pH 6.0) for 20 minutes for anti-YB-1 antibody.
Immunohistochemical Analysis Twist1, YB-1 and AR expression was evaluated by the proportion and intensity of positively staining carcinoma cells. A proportion score was assigned to represent the estimated proportion of positively stained carcinoma cells, including 0 —none, 1—fewer than 1/100, 2—1/100 to 1/10, 3—1/10 to 1/3, 4 —1/3 to 2/3 and 5— greater than 2/3. The average estimated intensity of staining in positive carcinoma cells was assigned an intensity score of 0 —none, 1—weak, 2—intermediate or 3—strong. Proportion and intensity scores were added to obtain a total score of 0 to 8.17 Immunohistochemical results were classified based on total scores, including 0 to 4 — low and 5 to 8 — high expression. Immunohistochemical results were judged by 2 well trained pathologists (YS and YO).
Cell Culture Human PCa LNCaP and 22Rv1 cells were cultured under RPMI 1640 medium (Invitrogen™) containing 10% fetal bovine serum. The androgen reduced medium was supplemented with 8.75% charcoal stripped serum and 1.25% fetal bovine serum. LNCaP and 22Rv1 cells were cultured under this androgen reduced medium with or without 5 mM NAC for 2, 4, 8 and 12 weeks, respectively. LNCaP and 22Rv1 cells cultured for 0 weeks were used for experiments under medium containing 10% fetal bovine serum while those for 2, 4, 8 and 12 weeks were used for experiments under androgen reduced medium with or without 5 mM NAC. Cell lines were maintained in a 5% CO2 atmosphere at 37C.
Intracellular ROS Measurement Intracellular ROS was measured as described previously.10 The indicated LNCaP or 22Rv1 cells (1.0 ⫻ 103) were seeded in 96-well plates and incubated for 48 hours. Intracellular ROS was measured using CM-H2DCFDA (Invitrogen) according to the manufacturer protocol. Results represent at least 3 independent experiments.
Western Blot Western blot analysis was done as described previously.10,14 –16 We used antibodies against AR (sc-815) and Twist1 (sc-81417) (Santa Cruz Biotechnology, Santa Cruz, California), and antibodies against YB-1 (Epitomics) and lamin B1 (Abcam®).
Cell Proliferation Assay Cell proliferation assay was done as described previously.10,14,15 Briefly, the indicated LNCaP or 22Rv1 cells (2.5 ⫻ 104) were seeded in 12-well plates and incubated
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with 0 hour considered 12 hours after transfection. Cells were harvested with trypsin and counted daily using a cell counter (Beckman Coulter, Fullerton, California). Results were normalized to cell counts at 0 hours and represent at least 3 independent experiments.
Mouse Xenograft Model We used 6 to 8-week-old male NCr-nu/nu mice (Charles River Laboratories Japan, Yokohama, Japan). LNCaP (5.0 ⫻ 107) or 22Rv1 (5.0 ⫻ 107) cells were inoculated subcutaneously into the lateral flank of 20 mice each. At 7 days the mice were randomly divided into 4 groups, including castration or no castration with or without NAC. For castration or sham operation mice were anesthetized with intraperitoneal injection of ketamine/xylazine mixture. For castration each testis was removed surgically 7 days after tumor inoculation. NAC (100 mg/kg) was administered intraperitoneally daily. The size of inoculated tumors was determined every 2 to 3 days using calipers. Tumor volume was calculated using the formula, V ⫽ (A ⫻ B2)/2, where V represents volume in mm3, and A and B represent the long and the short diameter in mm, respectively.
Statistical Analysis The Wilcoxon test was used to examine pairwise correlations of immunohistochemical staining scores. Fisher’s exact test was used for statistical analysis of correlations between neoadjuvant ADT status and immunohistochemical staining scores. In vitro and in vivo experiments were analyzed by the t test with 2-sided p ⬍0.05 considered statistically significant.
RESULTS ADT induced oxidative stress, and Twist1 and YB-1 expression, resulting in AR over expression in human PCa tissue. Staining with the oxidative stress marker 4-HNE in PCa tissue receiving neoadjuvant ADT was increased compared with that in PCa tissue without neoadjuvant ADT (table 1 and fig. 1, A). Similarly Twist1 and YB-1 expression was up-regulated in PCa tissue with vs without neoadjuvant
Table 1. Neoadjuvant ADT and 4-HNE staining, Twist1, YB-1 and AR
4-HNE: Low High Twist1: Low High YB-1: Low High AR: Low High
No.
No Neoadjuvant ADT
Neoadjuvant ADT
14 17
11 5
3 12
16 15
11 5
5 10
15 16
11 5
4 11
13 18
8 8
5 10
p Value 0.0113*
0.0756
0.0320*
0.473
* Statistically significant.
Figure 1. Treating human PCa tissue with vs without ADT revealed that ADT induced oxidative stress, and Twist1 and YB-1 expression, resulting in AR over expression. A, 4-HNE. B, Twist1, C, YB-1. D, AR. Reduced from ⫻400.
ADT, although statistical significance was not attained for Twist1 expression (table 1 and fig. 1, B and C). Neoadjuvant ADT duration did not affect 4-HNE staining or Twist1 and YB-1 expression (data not shown). Also, 4-HNE staining correlated with Twist1 and YB-1 expression (r ⫽ 0.6240 and 0.4651, respectively, table 2), indicating that these molecules are associated with oxidative stress, as shown previously.10 AR expression in PCa tissue with neoadjuvant ADT was increased compared with tissue without neoadjuvant ADT, although statistical significance was not attained (table 1 and fig. 1, D). AR expression also correlated with Twist1 and YB-1 expression (r ⫽ 0.4227 and 0.4131, respectively, table 2), supporting our previous reports that Twist1 and YB-1 regulate AR expression.10,15 Also, 4-HNE staining among patients not treated with
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mor growth (fig. 4, A). Similar results were noted in the 22Rv1 xenograft model (fig. 4, B).
Table 2. Twist1, YB-1 and AR, and 4-HNE staining correlation coefficients
4-HNE Twist1 YB-1 AR
4-HNE
Twist1
YB-1
— 0.624 0.465 0.214
— — 0.425 0.422
— — — 0.413
neoadjuvant ADT did not correlate with Gleason score (data not shown). Low level and long-term androgen deprivation induced intracellular ROS, resulting in castration resistance, which was alleviated by adding NAC to LNCaP cells. Intracellular ROS gradually increased up to 4 weeks and gradually decreased thereafter to about baseline at 12 weeks (fig. 2, A). The increase in intracellular ROS was almost completely alleviated by adding NAC (fig. 2, A). AR expression was increased 8 and 12 weeks after androgen deprivation. Simultaneously Twist1 and YB-1 were also induced while ROS decreased as Twist1 and YB-1 expression was up-regulated. This may have resulted from the antioxidative properties of Twist1 and YB-1. In contrast, increases in AR, Twist1 and YB-1 after androgen deprivation decreased when NAC was added to androgen deprivation (fig. 2, B). LNCaP cells gained castration resistance as the duration of androgen deprivation increased, while LNCaP cells remained androgen dependent when NAC was added to androgen deprivation (fig. 2, C). Low level and long-term androgen deprivation induced intracellular ROS, resulting in castration resistance, which was alleviated by adding NAC to 22Rv1 cells, another PCa cell line that expresses functional AR and is sensitive to androgen in cell proliferation (data not shown).18,19 Similar to LNCaP cells, intracellular ROS in 22Rv1 cells increased up to 4 and 8 weeks and decreased 12 weeks after androgen deprivation (fig. 3, A). AR expression in 22Rv1 cells was prominently induced 12 weeks after androgen deprivation with the increases in Twist1 and YB-1, although adding NAC abolished these reactions, similar to LNCaP cells (fig. 3, B). Adding NAC alleviated the circumvention of androgen sensitivity, similar to LNCaP cells (fig. 3, C). NAC administration suppressed androgen dependent LNCaP and androgen sensitive 22Rv1 tumor growth in vivo. As expected, in the mouse xenograft model castration prominently suppressed LNCaP tumor growth. NAC administration significantly suppressed LNCaP tumor growth in castrated mice. NAC also suppressed LNCaP tumor growth in noncastrated mice. As a result, castration combined with NAC most effectively suppressed LNCaP tu-
DISCUSSION ADT induced oxidative stress, as represented by 4-HNE staining in human PCa tissue. Under physiological conditions the cellular concentration of 4-HNE is 0.1 to 0.3 M. However, under conditions of oxidative stress it accumulates at concentrations of 10 to 5,000 M.20,21 Thus, 4-HNE is a known, established marker of oxidative stress. Furthermore, in human PCa tissue Twist1 and YB-1, which are transcription factors regulating AR transcription, were up-regulated by ADT. A functional link between these molecules and oxidative stress was strengthened by the finding that 4-HNE staining correlated with Twist1 and YB-1 expression. It is known that 4-HNE facilitates activation of the NF-E2 related factor 2 signaling pathway, induces a cluster of phase II enzymes and readily activates various protein kinase pathways.22 However, to our knowledge regulation of the Twist1/YB1/AR signaling pathway by 4-HNE has not been reported. Thus, these interactions should be clarified in future studies. On the other hand, a functional link between Twist1 and YB-1 molecules with AR was indicated, supporting our previous findings.10,15 Immunohistochemistry using human PCa tissue treated vs not treated with ADT revealed that oxidative stress by ADT induces Twist1/YB-1 over expression, resulting in AR over expression. This is our hypothesis of the mechanism of progression of androgen dependent PCa to CRPC, although our study was limited by its small sample size. After obtaining these findings we confirmed this notion in vitro and investigated whether antioxidant NAC could alleviate oxidative stress by androgen deprivation, of Twist1, YB-1 and AR induction, and the gain of castration resistance. We noted successful alleviation of oxidative stress by androgen deprivation. Thus, NAC could suppress the increased Twist1, YB-1 and AR expression caused by androgen deprivation and, thus, prevent progression to castration resistance in vitro. Subsequently we confirmed the successful results of antioxidant therapy against progression to CRPC in a mouse xenograft model, although the additive tumor suppressive effect by NAC with castration may have derived from the direct tumor suppressive effect of NAC alone, as described. NAC administration alone suppressed PCa tumor growth. NAC inhibits the mitogenic activity of v-HRas in NIH3T3, as judged by short-term assay of [3H] thymidine incorporation during 4 hours.23 NAC has also had a chemopreventive effect on Kaposi
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Figure 2. Low level, long-term androgen deprivation induced intracellular ROS, resulting in castration resistance, which was alleviated by adding NAC in LNCaP cells. A, cells were seeded in 96-well plates and incubated. At 48 hours fluorescence intensity was measured with intensity at 0 week considered 1. Boxes represent mean. Bars represent ⫾ SD. Asterisk indicates p ⬍0.05 vs 0 week. B, whole cell extracts were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Western blot was done using indicated antibodies. C, cells were seeded in 12-well plates and incubated. After indicated hours cell number was counted and results were normalized to cell number at hour 0. Bars represent ⫾ SD. Asterisk indicates p ⬍0.05 vs 0 week.
sarcoma progression by decreasing the expression of vascular endothelial growth factor from tumor cells, thus, suppressing the growth of tumors transplanted into mice.24 Havre et al reported that NAC induced apoptosis in human tumor cells.25 On the other hand, Zhang et al reported that NAC enhanced the growth of BCR-ABL transformed cells.26 These controversies may have resulted from the cell type or the NAC dose used in experiments. High NAC doses may induce apoptosis while lower doses induce cell growth.25,26 This suggests that
the biological effects of NAC depend on the dose and should be carefully assessed in different cellular conditions. We used a NAC concentration of 5 mM in vitro. At this concentration LNCaP and 22Rv1 cell proliferation was not affected by adding NAC (data not shown). However, adding NAC prominently suppressed the gain of castration resistance, suggesting that 5 mM NAC did not simply suppress cell growth but rather specifically prevented progression to CRPC. On the other hand, in vivo NAC suppressed tumor growth in intact and castrated mice, suggesting that NAC
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Figure 3. Low level, long-term androgen deprivation induced intracellular ROS, resulting in castration resistance, which was alleviated by adding NAC in 22Rv1 cells. A, cells were seeded in 96-well plates and incubated. At 48 hours fluorescence intensity was measured with intensity at 0 week considered 1. Boxes represent mean. Bars represent ⫾ SD. Asterisk indicates p ⬍0.05 vs 0 week. B, whole cell extracts were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Western blot was done with indicated antibodies. C, cells were seeded in 12-well plates and incubated. After indicated hours cell number was counted and results were normalized to cell number at hour 0. Bars represent ⫾ SD. Asterisk indicates p ⬍0.05 vs 0 week.
might suppress neoangiogenesis by inhibiting vascular endothelial growth factor production in vivo. Although we used NAC, which is an agent that functions as an antioxidant, other antioxidants such as vitamin C, vitamin E, polyphenols and carotenoids are available. Previously lycopene reportedly decreased serum PSA and improved survival combined with castration,27 although evidence is insufficient to draw firm conclusions.28 The antioxidant ␣-tocopherol was suggested to increase survival in patients with PCa, and higher serum ␣-tocopherol and ␣-tocopherol intervention contributed to im-
proved PCa survival, although -carotene and retinol did not.29 Thus, other antioxidants might be more effective than NAC to treat PCa and prevent PCa progression. We should confirm the efficacy of antioxidant therapy for human PCa and develop more valid antioxidant therapy for PCa and its progression to CRPC.
CONCLUSIONS ADT increased oxidative stress in human PCa tissue, which may lead to Twist1 and YB-1 over expression, resulting in AR over expression in
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Figure 4. NAC suppressed androgen dependent and androgen sensitive tumor growth in vivo in male ncr-nu/nu mice injected subcutaneously. Five mice per group were assigned to no castration or NAC (circles), NAC without castration (squares), castration without NAC (triangles) and castration with NAC (inverted triangles). NAC was administered 7 days after inoculation. Tumor volume was inspected every 2 to 3 days. A, LNCaP cells. B, 22Rv1 cells. Bars represent ⫾ SEM. Asterisk indicates p ⬍0.05.
PCa. In androgen dependent and androgen sensitive PCa cells low level and long-term androgen deprivation caused the over expression of Twist1, YB-1 and AR, and provoked castrate resistant growth, which was abolished by the antioxidant NAC. These findings suggest that oxidative stress may be involved in the pathogenesis of CRPC in human PCa and indicate that new therapies tar-
geting oxidative stress using an antioxidant may prevent the conversion of androgen dependent or androgen sensitive PCa to CRPC.
ACKNOWLEDGMENTS Noriko Hakoda and Seiko Kamori provided technical assistance.
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