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1.  RhoGDIα suppresses growth and survival of prostate cancer cells 
The Prostate  2011;72(4):392-398.
Background
Treatment for primary prostate cancer (CaP) is the withdrawal of androgens. However, CaP eventually progresses to grow in a castration-resistant state. The mechanisms involved in the development and progression of castration-resistant prostate cancer (CRPC) remain unknown. We have previously generated LNCaP-IL6+ cells by treating LNCaP cells chronically with interleukin-6 (IL-6), which have acquired the ability to grow in androgen-deprived conditions.
Methods
We compared the protein expression profile of LNCaP and LNCaP-IL6+ cells using two-dimensional gel electrophoresis. The gels were then silver stained in order to visualize proteins and the differentially expressed spots were identified and characterized by micro sequencing using MALDI-PMF mass spectrometry.
Results
In this study, we have identified RhoGDIα (GDIα) as a suppressor of prostate cancer growth. Expression of GDIα was reduced in LNCaP-IL6+ cells and was downregulated in more aggressive prostate cancer cells compared to LNCaP cells. Over expression of GDIα inhibited the growth of prostate cancer cells and caused LNCaP-IL6+ cells reversal to androgen-sensitive state, while down regulation of GDIα enhanced growth of androgen-sensitive LNCaP prostate cancer cells in androgen-deprived conditions. In addition, GDIα suppressed the tumorigenic ability of prostate tumor xenografts in vivo.
Conclusions
These results demonstrate that loss of GDIα expression promotes the development and progression of prostate cancer.
doi:10.1002/pros.21441
PMCID: PMC3961824  PMID: 21681778
Prostate cancer; RhoGDIα; IL-6
2.  NF-κB2/p52 Induces Resistance to Enzalutamide in Prostate Cancer: Role of Androgen Receptor and Its Variants 
Molecular cancer therapeutics  2013;12(8):1629-1637.
Resistance of prostate cancer cells to the next-generation antiandrogen enzalutamide may be mediated by a multitude of survival signaling pathways. In this study, we tested whether increased expression of NF-κB2/p52 induces prostate cancer cell resistance to enzalutamide and whether this response is mediated by aberrant androgen receptor (AR) activation and AR splice variant production. LNCaP cells stably expressing NF-κB2/p52 exhibited higher survival rates than controls when treated with enzalutamide. C4-2B and CWR22Rv1 cells chronically treated with enzalutamide were found to express higher levels of NF-κB2/p52. Downregulation of NF-κB2/p52 in CWR22Rv1 cells chronically treated with enzalutamide rendered them more sensitive to cell growth inhibition by enzalutamide. Analysis of the expression levels of AR splice variants by quantitative reverse transcription PCR and Western blotting revealed that LNCaP cells expressing p52 exhibit higher expression of AR splice variants. Downregulation of expression of NF-κB2/p52 in VCaP and CWR22Rv1 cells by short hairpin RNA abolished expression of splice variants. Downregulation of expression of either full-length AR or the splice variant AR-V7 led to an increase in sensitivity of prostate cancer cells to enzalutamide. These results collectively demonstrate that resistance to enzalutamide may be mediated by NF-κB2/p52 via activation of AR and its splice variants.
doi:10.1158/1535-7163.MCT-13-0027
PMCID: PMC3941973  PMID: 23699654
3.  RhoGDIα Downregulates Androgen Receptor Signaling in Prostate Cancer Cells 
The Prostate  2013;73(15):1614-1622.
INTRODUCTION
Treatment of primary prostate cancer (CaP) is the withdrawal of androgens. However, CaP eventually progresses to grow in a castration-resistant state due to aberrant activation of androgen receptor (AR). Understanding the mechanisms leading to the aberrant activation of AR is critical to develop effective therapy. We have previously identified Rho GDP Dissociation Inhibitor alpha (GDIα) as a novel suppressor in prostate cancer. In this study, we examine the effect of GDIα on AR signaling in prostate cancer cells.
METHODS
GDIα was transiently or stably transfected into several prostate cancer cell lines including LNCaP, C4-2, CWR22Rv1, and DU145. The regulation of AR expression by GDIα was analyzed by qRT-PCR and Western blot. AR activity was measured by luciferase reporter assays and electrophoretic mobility shift analysis (EMSA). Immunofluorescence assay was performed to study AR nuclear translocation. The interaction between GDIα and AR was examined by co-immunoprecipitation assays.
RESULTS
In this study, we have identified GDIα as a negative regulator of AR signaling pathway. Overexpression of GDIα downregulates AR expression at both mRNA and protein levels. Overexpression of GDIα is able to prevent AR nuclear translocation and inhibit trans-activation of AR target genes. Co-immunoprecipitation assays showed that GDIα physically interacts with the N-terminal domain of AR.
CONCLUSIONS
GDIα suppresses AR signaling through inhibition of AR expression, nuclear translocation, and recruitment to androgen-responsive genes. GDIα regulatory pathway may play a critical role in regulating AR signaling and prostate cancer growth and progression.
doi:10.1002/pros.22615
PMCID: PMC3941975  PMID: 23922223
prostate cancer; RhoGDIα; AR
4.  Functional p53 Determines Docetaxel Sensitivity in Prostate Cancer Cells 
The Prostate  2012;73(4):418-427.
BACKGROUND
Docetaxel is the first line treatment for castration resistant prostate cancer (CRPC). However, docetaxel resistance rapidly develops. Identifying the critical mechanisms giving rise to docetaxel resistance is the major challenge in advanced prostate cancer.
METHODS
The effects of docetaxel on human DU145, PC3, LNCaP, and C4-2 prostate cancer cells were examined in cell culture, and p53 expression were analyzed by Western blot analysis. The potential role of p53 in docetaxel sensitivity in prostate cancer cells was tested by either p53 silencing using shRNA or p53 overexpression by introducing wild-type p53.
RESULTS
We found that DU145 (mutant p53) and PC3 (p53 null) cells were less sensitive than LNCaP and C4-2 cells expressing functional p53 in response to docetaxel. Docetaxel treatment induces considerably higher apoptosis in LNCaP and C4-2 cells than in DU145 and PC3 cells in a dose dependent manner. Docetaxel increases the levels of ser15 phosphorylation of p53 in a dose dependent manner in both LNCaP and C4-2 cells, while has no effect on the levels of ser15 phosphorylation of p53 in DU145 cells. These results suggest that p53 phosphorylation is associated with docetaxel sensitivity in prostate cancer cells. To further confirm whether p53 activation can induce cell sensitivity to docetaxel treatment, we used p53 shRNA to knock down p53 expression in C4-2 cells and determined the cells response to docetaxel treatment. Knockdown of p53 significantly down regulated p53 phosphorylation and blocked docetaxel induced apoptotic cell death compared to the vector control. To further confirm this observation, we established a stable knock out p53 in C4-2 cells. Down regulation of p53 in the stable p53 knock out C4-2 cells significantly inhibited docetaxel induced apoptotic cell death. We also used wild-type (WT) p53 to over express p53 in DU145 cells, and found that expression of WT-p53 in DU145 cells increased their sensitivity to docetaxel.
CONCLUSIONS
These results demonstrate that docetaxel induces p53 phosphorylation and that p53 status is a crucial determinant of docetaxel sensitivity in prostate cancer cells.
doi:10.1002/pros.22583
PMCID: PMC3938015  PMID: 22996738
prostate cancer; docetaxel; p53
5.  Inhibition of Stat3 Activation by Sanguinarine Suppresses Prostate Cancer Cell Growth and Invasion 
The Prostate  2011;72(1):82-89.
BACKGROUND
Signal transducer and activator of transcription 3 (Stat3) is an oncogenic transcriptional factor that plays a critical role in carcinogenesis and cancer progression and is a potential therapeutic target. Sanguinarine, a benzophenanthridine alkaloid derived primarily from the bloodroot plant, was identified previously as a novel inhibitor of survivin that selectively kills prostate cancer cells over “normal” prostate epithelial cells.
METHODS
DU145, C4-2B, and LNCaP cells were treated with sanguinarine. The phosphorylation status of Stat3 and related proteins were measured with Western blots. Activation of transcription by Stat3 was measured with luciferase reporter assay. The effect of sanguinarine on anchorage-independent growth was examined with soft agar assay, and on cell migration and invasion of DU145 cells were measured with scratch assay and invasion assay, respectively.
RESULTS
In this study, we identified sanguinarine as a potent inhibitor of Stat3 activation which was able to suppress prostate cancer growth, migration, and invasion. Sanguinarine inhibits constitutive as well as IL6-induced phosphorylation of Stat3 at both Tyr705 and Ser727 in prostate cancer cells. The inhibition of Stat3 phosphorylation by sanguinarine correlates with reduction of Janus-activated Kinase 2 (Jak2) and Src phosphorylation. Sanguinarine downregulates the expression of Stat3-mediated genes such as c-myc and survivin and inhibits the Stat3 responsive element luciferase reporter activity. Sanguinarine inhibits the anchorage-independent growth of DU145 and LN-S17 cells expressing constitutively activated Stat3. Migration and invasion abilities of DU145 cells were also inhibited by sanguinarine in a manner similar to the dominant negative form of Stat3.
CONCLUSIONS
These data demonstrate that sanguinarine is a potent Stat3 inhibitor and it could be developed as a therapeutic agent for prostate cancer with constitutive activation of Stat3.
doi:10.1002/pros.21409
PMCID: PMC3938016  PMID: 21538419
prostate cancer; Stat3; sanguinarine; invasion
6.  LNCaP Prostate Cancer Cells with Autocrine Interleukin-6 Expression Are Resistant to IL-6-induced Neuroendocrine Differentiation due to Increased Expression of Suppressors of Cytokine Signaling 
The Prostate  2011;72(12):1306-1316.
BACKGROUND
Neuroendocrine differentiation (NED) is one of the mechanisms underlying development of castration-resistant prostate cancer. In this study, we investigated IL-6-induced NED in two LNCaP sublines.
METHODS
LNCaP-S17, an LNCaP subline that secretes IL-6, and LNCaP-C3, a control subline that does not express IL-6, were analyzed for IL-6-induced NED, activation of JAK2 and STAT3 pathways, and expression of IL-6/IL-6R signaling proteins and downstream target genes.
RESULTS
IL-6 did not induce NED in LNCaP-S17 cells, even though IL-6 induced NED in LNCaP-C3 cells. IL-6 activated JAK2 and STAT3 pathways in LNCaP-C3 cells but not in LNCaP-S17 cells. IL-6 did not activate ERK1/2, AKT, or NF-κB pathways in either cell line. Both LNCaP-C3 and LNCaP-S17 cell lines expressed IL-6R, gp130, and TYK2 at almost the same levels and did not express JAK1 or JAK3. The basal level of JAK2 expression was slightly higher in LNCaP-C3 cells than in LNCaP-S17 cells. Two suppressors of cytokine signaling, SOCS7 and CIS, were expressed constitutively at higher levels in LNCaP-S17 cells than in LNCaP-C3 cells, while SOCS1 to SOCS6 were expressed at approximately the same levels. Using siRNA to knockdown SOCS7 and CIS expression in LNCaP-S17 cells led to increased phosphorylation of STAT3 upon IL-6 stimulation.
CONCLUSIONS
LNCaP-S17 cells are resistant to exogenous IL-6-induced NED due to increased levels of CIS/SOCS7 that block activation of JAK2-STAT3 pathways.
doi:10.1002/pros.22479
PMCID: PMC3665156  PMID: 22213096
IL-6; neuroendocrine; prostate cancer
7.  Aberrant Activation of the Androgen Receptor by NF-κB2/p52 in Prostate Cancer Cells 
Cancer research  2010;70(8):3309-3319.
Prostate cancer initiation and progression are uniquely dependent on the androgen receptor (AR). Even when the cancer progresses to a castration-resistant stage, AR signaling remains active via a variety of mechanisms. In the present study, we showed that NF-κB/p52 can activate the AR, resulting in increased transactivation of AR-responsive genes, such as PSA and NKX3.1, in a ligand-independent manner. NF-κB2/p52 enhances nuclear translocation and activation of AR by interacting with its NH2-terminal domain and enhances the recruitment of coactivators such as p300 to the promoters of AR-dependent genes. These results were confirmed in three different prostate cancer cell lines: LAPC-4 (wild-type AR), LNCaP (mutant AR), and C4-2 (castration resistant). Transfection of p52 into LAPC-4 and LNCaP cells (which express low levels of p52) showed increased activation of the endogenous AR. Downregulation of endogenous p52 in C4-2 cells resulted in abrogation of AR constitutive activation. Comparison of the relative effects of p52 and p65 (RelA) showed that p52, but not p65, could activate the AR. Collectively, these findings, together with previous reports that the levels of NF-κB2/p52 are elevated in prostate cancer cells and that active NF-κB2/p52 promotes prostate cancer cell growth in vitro and in vivo, suggest that NF-κB2/p52 may play a critical role in the progression of castration-resistant prostate cancer.
doi:10.1158/0008-5472.CAN-09-3703
PMCID: PMC3041633  PMID: 20388792
8.  Interleukin-6 Regulates Androgen Synthesis in Prostate Cancer Cells 
Purpose
The standard systemic treatment for prostate cancer patients is androgen deprivation therapy. Although serum testosterone concentrations were significantly reduced after androgen deprivation therapy, levels of intraprostatic androgens are reproducibly measured at concentrations sufficient to activate androgen receptor and stimulate tumor growth, suggesting that prostate cancer cells may survive androgen deprivation therapies by increasing intracrine androgen synthesis within the prostate. However, factors that regulate de novo intracrine androgen synthesis have not been identified. Interleukin-6 (IL-6) has been implicated in the modulation of androgen receptor activation and growth and differentiation in prostate cancer. In this study, we investigate whether IL-6 regulates intraprostatic androgen synthesis in prostate cancer cells.
Experimental Design
Quantitative reverse transcription-PCR and Western blotting were done to detect expression levels of steroidogenic enzymes. AKR1C3 promoter reporter was constructed and analyzed for IL-6–mediated AKR1C3 transcriptional activity. IL-6–mediated signaling was knocked down using small interfering RNA specific to IL-6 receptor and gp130, and the effect on AKR1C3 expression was examined. Intraprostatic androgen levels in prostate cancer cells in culture and in tumors were measured by an enzyme immunoassay (Testosterone EIA kit).
Results
We found that IL-6 increases the expression of genes encoding many steroidogenic enzymes, including HSD3B2 and AKR1C3, involved in androgen biosynthesis. Down-regulation of IL-6 receptor and gp130 expression using specific small interfering RNA abolished IL-6–mediated AKR1C3 expression, suggesting that IL-6 signaling is responsible for AKR1C3 expression. IL-6 increases AKR1C3 promoter activity, indicating that the increase in IL-6–mediated AKR1C3 expression is in part at the transcriptional level. Treatment of IL-6 increased testosterone level in LNCaP cells. The tumor testosterone levels were detected at 378 pg/g in tumors generated from IL-6–overexpressing LNCaP-IL6+ cells inoculated orthotopically into the prostates of castrated male nude mice.
Conclusions
These results suggest that IL-6 increases levels of intracrine androgens through enhanced expression of genes mediating androgen metabolism in prostate cancer cells.
doi:10.1158/1078-0432.CCR-09-0640
PMCID: PMC3041150  PMID: 19638459
9.  Interleukin-6 increases prostate cancer cells resistance to bicalutamide via TIF2 
Molecular cancer therapeutics  2009;8(3):665-671.
The standard treatment for advanced, androgen-responsive prostate cancer is androgen deprivation therapy with or without a nonsteroidal antiandrogen, such as bicalutamide. Although maximal androgen blockade exhibits favorable responses in the majority of patients, prostate cancer eventually progresses to an androgen-refractory stage. The mechanism underlying bicalutamide resistance in the course of prostate cancer progression is incompletely understood. However, interleukin-6 (IL-6) plays a critical role in the development and progression of CRPC. Herein, we explored an association between IL-6 and bicalutamide resistance. To study this, series of lower and higher passages of LNCaP cell sublines generated by long-term exposure to IL-6 were used. The cells from higher passages of LNCaP treated with IL-6 developed resistance to bicalutamide treatment compared with parental LNCaP cells. The levels of transcriptional intermediary factor 2 (TIF2) in IL-6-treated LNCaP cells were found to be significantly higher than parental LNCaP cells. Down-regulation of TIF2 expression via short hairpin RNA in IL-6-treated LNCaP cells sensitized these cells to bicalutamide treatment, whereas overexpression of TIF2 in the parental LNCaP cells increased resistance to bicalutamide. Furthermore, overexpression of IL-6 attenuated bicalutamide-mediated blockage of androgen-induced androgen receptor nuclear translocation and recruitment. These results show that overexpression of IL-6 increases the resistance of prostate cancer cells to bicalutamide via TIF2. Overexpression of IL-6 not only plays an important role in prostate cancer progression but also contributes to bicalutamide resistance. Our studies suggest that bicalutamide-IL-6-targeted adjunctive therapy may lead to a more effective intervention than bicalutamide alone.
doi:10.1158/1535-7163.MCT-08-0823
PMCID: PMC3041173  PMID: 19240160
10.  Sanguinarine Suppresses Prostate Tumor Growth and Inhibits Survivin Expression 
Genes & cancer  2010;1(3):283-292.
Prostate cancer is a frequently occurring disease and is the second leading cause of cancer-related deaths of men in the United States. Current treatments have proved inadequate in curing or controlling prostate cancer, and a search for agents for the management of this disease is urgently needed. Survivin plays an important role in both progression of castration-resistant prostate cancer and resistance to chemotherapy. Altered expression of survivin in prostate cancer cells is associated with cancer progression, drug/radiation resistance, poor prognosis, and short patient survival. In the present study, the authors performed a cell-based rapid screen of the Prestwick Chemical Library consisting of 1120 Food and Drug Administration–approved compounds with known safety and bioavailability in humans to identify potential inhibitors of survivin and anticancer agents for prostate cancer. Sanguinarine, a benzophenanthridine alkaloid derived primarily from the bloodroot plant, was identified as a novel inhibitor of survivin that selectively kills prostate cancer cells over “normal” prostate epithelial cells. The authors found that sanguinarine inhibits survivin protein expression through protein degradation via the ubiquitin-proteasome system. Sanguinarine induces apoptosis and inhibits growth of human prostate cancer cells and in vivo tumor formation. Administration of sanguinarine, beginning 3 days after ectopic implantation of DU145 human prostate cancer cells, reduces both tumor weight and volume. In addition, sanguinarine sensitized paclitaxel-mediated growth inhibition and apoptosis, offering a potential therapeutic strategy for overcoming taxol resistance. These results suggest that sanguinarine may be developed as an agent either alone or in combination with taxol for treatment of prostate cancer overexpressing survivin.
doi:10.1177/1947601910368849
PMCID: PMC3036540  PMID: 21318089
prostate cancer; survivin; sanguinarine
11.  Andrographolide Targets Androgen Receptor Pathway in Castration-Resistant Prostate Cancer 
Genes & Cancer  2011;2(2):151-159.
Androgen receptor (AR) signaling not only plays a pivotal role in the development of androgen-dependent prostate cancer but is also important in the growth and survival of castration-resistant prostate cancer (CRPC). The first line of treatment of androgen-dependent prostate cancer is the use of androgen deprivation therapy. However, most patients will eventually relapse due to development of CRPC. Thus, development of a strategy to target AR for treatment of CRPC is urgently needed. The authors have previously identified andrographolide as an inhibitor of interleukin-6, which can suppress tumor growth of prostate cancer cells by screening compounds from the Prestwick Natural compound library. In this study, they identified that andrographolide can inhibit AR expression and prostate cancer cell growth and induce apoptosis. Andrographolide is able to down-regulate AR expression at both mRNA and protein levels, prevents its nuclear translocation, and inhibits transactivation of its target genes. Andrographolide prevents the binding of Hsp90 to AR, resulting in proteasome-mediated AR degradation. Furthermore, andrographolide inhibits castration-resistant C4-2 cell growth by reducing AR expression and activity. Thus, andrographolide can be developed as a potential therapeutic agent for prostate cancer by inhibition of androgen receptor signaling.
doi:10.1177/1947601911409744
PMCID: PMC3111248  PMID: 21779488
prostate cancer; androgen receptor; andrographolide
12.  Histone Methyltransferase NSD2/MMSET Mediates Constitutive NF-κB Signaling for Cancer Cell Proliferation, Survival, and Tumor Growth via a Feed-Forward Loop 
Molecular and Cellular Biology  2012;32(15):3121-3131.
Constitutive NF-κB activation by proinflammatory cytokines plays a major role in cancer progression. However, the underlying mechanism is still unclear. We report here that histone methyltransferase NSD2 (also known as MMSET or WHSC1), a target of bromodomain protein ANCCA/ATAD2, acts as a strong coactivator of NF-κB by directly interacting with NF-κB for activation of target genes, including those for interleukin-6 (IL-6), IL-8, vascular endothelial growth factor A (VEGFA), cyclin D, Bcl-2, and survivin, in castration-resistant prostate cancer (CRPC) cells. NSD2 is recruited to the target gene promoters upon induction and mediates NF-κB activation-associated elevation of histone H3K36me2 and H3K36me3 marks at the promoter, which involves its methylase activity. Interestingly, we found that NSD2 is also critical for cytokine-induced recruitment of NF-κB and acetyltransferase p300 and histone hyperacetylation. Importantly, NSD2 is overexpressed in prostate cancer tumors, and its overexpression correlates with NF-κB activation. Furthermore, NSD2 expression is strongly induced by tumor necrosis factor alpha (TNF-α) and IL-6 via NF-κB and plays a crucial role in tumor growth. These results identify NSD2 to be a key chromatin regulator of NF-κB and mediator of the cytokine autocrine loop for constitutive NF-κB activation and emphasize the important roles played by NSD2 in cancer cell proliferation and survival and tumor growth.
doi:10.1128/MCB.00204-12
PMCID: PMC3434505  PMID: 22645312
13.  Andrographolide, an Herbal Medicine, Inhibits Interleukin-6 Expression and Suppresses Prostate Cancer Cell Growth 
Genes & Cancer  2010;1(8):868-876.
Elevated interleukin-6 (IL-6), a major mediator of the inflammatory response, has been implicated in androgen receptor (AR) activation, cellular growth and differentiation, plays important roles in the development and progression of prostate cancer, and is a potential target in cancer therapy. Through drug screening using human prostate cancer cells expressing IL-6 autocrine loop, we found that andrographolide, a diterpenoid lactone isolated from a traditional Chinese and Indian medicinal plant Andrographis paniculata, could inhibit IL-6 expression and suppress IL-6–mediated signals. Andrographolide inhibits IL-6 expression at both mRNA and protein levels in a dose-dependent manner. Andrographolide suppresses both IL-6 autocrine loop– and paracrine loop–induced cell signaling including Stat3 and Erk phosphorylation. Furthermore, andrographolide inhibits cell viability and induces apoptotic cell death in both androgen-stimulated and castration-resistant human prostate cancer cells without causing significant toxicity to normal immortalized prostate epithelial cells. Moreover, treatment of andrographolide to mice bearing castration-resistant DU145 human prostate tumors that express constitutive IL-6 autocrine loop significantly suppresses tumor growth. Taken together, these results demonstrate that andrographolide could be developed as a therapeutic agent to treat both androgen-stimulated and castration-resistant prostate cancer possibly by suppressing IL-6 expression and IL-6–induced signaling.
doi:10.1177/1947601910383416
PMCID: PMC3063649  PMID: 21442031
prostate cancer; interleukin-6; andrographolide
14.  Sanguinarine Suppresses Prostate Tumor Growth and Inhibits Survivin Expression 
Genes & Cancer  2010;1(3):283-292.
Prostate cancer is a frequently occurring disease and is the second leading cause of cancer-related deaths of men in the United States. Current treatments have proved inadequate in curing or controlling prostate cancer, and a search for agents for the management of this disease is urgently needed. Survivin plays an important role in both progression of castration-resistant prostate cancer and resistance to chemotherapy. Altered expression of survivin in prostate cancer cells is associated with cancer progression, drug/radiation resistance, poor prognosis, and short patient survival. In the present study, the authors performed a cell-based rapid screen of the Prestwick Chemical Library consisting of 1120 Food and Drug Administration–approved compounds with known safety and bioavailability in humans to identify potential inhibitors of survivin and anticancer agents for prostate cancer. Sanguinarine, a benzophenanthridine alkaloid derived primarily from the bloodroot plant, was identified as a novel inhibitor of survivin that selectively kills prostate cancer cells over “normal” prostate epithelial cells. The authors found that sanguinarine inhibits survivin protein expression through protein degradation via the ubiquitin-proteasome system. Sanguinarine induces apoptosis and inhibits growth of human prostate cancer cells and in vivo tumor formation. Administration of sanguinarine, beginning 3 days after ectopic implantation of DU145 human prostate cancer cells, reduces both tumor weight and volume. In addition, sanguinarine sensitized paclitaxel-mediated growth inhibition and apoptosis, offering a potential therapeutic strategy for overcoming taxol resistance. These results suggest that sanguinarine may be developed as an agent either alone or in combination with taxol for treatment of prostate cancer overexpressing survivin.
doi:10.1177/1947601910368849
PMCID: PMC3036540  PMID: 21318089
prostate cancer; survivin; sanguinarine
15.  Selenium inhibition of survivin expression by preventing Sp1 binding to its promoter 
Survivin, an antiapoptotic protein highly expressed in cancer, regulates multiple cellular network associated with cancer cell viability and drug resistance. Inhibition of survivin expression has been pursued as a valid cancer therapeutic target. In this study, we showed that selenium, an effective chemopreventive agent for many types of cancers, down-regulated survivin expression. Selenium inhibited survivin expression in both mRNA and protein levels in a dose- and time-dependent manner. Using a series of survivin promoter–luciferase constructs, a 37-bp DNA element in the survivin core promoter region that mediates the ability of selenium to inhibit survivin transcription was identified. Gel mobility shift assays and chromatin immunoprecipitation analyses revealed that selenium prevents the binding of Sp1 or Sp1-like proteins to the 37-bp cis-acting DNA element in the survivin promoter. Furthermore, inhibition of survivin expression by small interfering RNA enhanced selenium’s inhibitory effects on cell growth, whereas overexpression of survivin in LNCaP human prostate cancer cells desensitized cancer cells to selenium effect, suggesting that the expression of survivin plays an important role in determining the response of cancer cells to selenium. Taken together, these results suggest that selenium down-regulated survivin expression by preventing the binding of Sp1 or Sp1-like proteins to the promoter of survivin, which contributes at least in part to the inhibitory effect of selenium on survivin gene transcription. In addition, down-regulation of survivin expression may account for one of the molecular mechanisms of the anticancer effects of selenium.
doi:10.1158/1535-7163.MCT-07-0172
PMCID: PMC2821810  PMID: 17876054
16.  MicroRNA let-7c Is Downregulated in Prostate Cancer and Suppresses Prostate Cancer Growth 
PLoS ONE  2012;7(3):e32832.
Purpose
Prostate cancer (PCa) is characterized by deregulated expression of several tumor suppressor or oncogenic miRNAs. The objective of this study was the identification and characterization of miR-let-7c as a potential tumor suppressor in PCa.
Experimental Design
Levels of expression of miR-let-7c were examined in human PCa cell lines and tissues using qRT-PCR and in situ hybridization. Let-7c was overexpressed or suppressed to assess the effects on the growth of human PCa cell lines. Lentiviral-mediated re-expression of let-7c was utilized to assess the effects on human PCa xenografts.
Results
We identified miR-let-7c as a potential tumor suppressor in PCa. Expression of let-7c is downregulated in castration-resistant prostate cancer (CRPC) cells. Overexpression of let-7c decreased while downregulation of let-7c increased cell proliferation, clonogenicity and anchorage-independent growth of PCa cells in vitro. Suppression of let-7c expression enhanced the ability of androgen-sensitive PCa cells to grow in androgen-deprived conditions in vitro. Reconstitution of Let-7c by lentiviral-mediated intratumoral delivery significantly reduced tumor burden in xenografts of human PCa cells. Furthermore, let-7c expression is downregulated in clinical PCa specimens compared to their matched benign tissues, while the expression of Lin28, a master regulator of let-7 miRNA processing, is upregulated in clinical PCa specimens.
Conclusions
These results demonstrate that microRNA let-7c is downregulated in PCa and functions as a tumor suppressor, and is a potential therapeutic target for PCa.
doi:10.1371/journal.pone.0032832
PMCID: PMC3316551  PMID: 22479342
17.  Effects of Triclocarban on Intact Immature Male Rat 
Reproductive Sciences  2011;18(2):119-127.
Triclocarban (TCC; 3,4,4′-trichlorocarbanilide) is an antimicrobial agent used widely in various personal hygiene products including soaps. Recently, TCC has been shown to enhance testosterone-induced effects in vitro and to enlarge accessory sex organs in castrated male rats. This study was designed to evaluate the effects of TCC on intact age-matched male rats and on human prostate LNCaP and C4–2B cells. Seven-week-old male Sprague-Dawley rats received either a normal diet or a diet supplemented with TCC (0.25% in diet) for 10 days. Triclocarban induced hyperplasia of accessory sex organs in the absence of significant qualitative histological changes. Serum luteinizing hormone (LH) and testosterone were not significantly altered by TCC treatment. In prostate cancer-derived LNCaP and C4–2B cells, TCC potentiated androgen actions via androgen receptor-dependent actions. In conclusion, TCC significantly affects intact male reproductive organs and potentiates androgen effects in prostate cancer cells.
doi:10.1177/1933719110382581
PMCID: PMC3031453  PMID: 20889956
triclocarban; androgen receptor; luteinizing hormone; testosterone; endocrine-disrupting substance
18.  Effects of triclocarban on intact immature male rat: augmentation of androgen action 
Triclocarban (TCC; 3,4,4′-trichlorocarbanilide) is an antimicrobial agent used widely in various personal hygiene products including soaps. Recently, TCC has been shown to enhance testosterone-induced effects in vitro and to enlarge accessory sex organs in castrated male rats. This study was designed to evaluate the effects of TCC on intact age matched male rats and on human prostate LNCaP and C4–2B cells. Seven-week old male Sprague-Dawley rats received either a normal diet or a diet supplemented with TCC (0.25% in diet) for ten days. TCC induced hyperplasia of accessory sex organs in the absence of significant qualitative histological changes. Serum LH and testosterone were not significantly altered by TCC treatment. In prostate cancer derived LNCaP and C4–2B cells, TCC potentiated androgen actions via androgen receptor-dependent actions. In conclusion, TCC significantly affects intact male reproductive organs and potentiates androgen effects in prostate cancer cells.
doi:10.1177/1933719110382581
PMCID: PMC3031453  PMID: 20889956
Triclocarban (TCC); androgen receptor (AR); Luteinizing hormone (LH); testosterone (T); endocrine-disrupting substance (EDS)
19.  Andrographolide, an herbal medicine, inhibits interleukin-6 expression and suppresses prostate cancer cell growth 
Genes & cancer  2010;1(8):868-876.
Elevated interleukin-6 (IL-6), a major mediator of the inflammatory response, has been implicated in androgen receptor (AR) activation, cellular growth and differentiation, plays important roles in the development and progression of prostate cancer, and is a potential target in cancer therapy. Through drug screening using human prostate cancer cells expressing IL-6 autocrine loop, we found that andrographolide, a diterpenoid lactone isolated from a traditional Chinese and Indian medicinal plant Andrographis paniculata, could inhibit IL-6 expression and suppress IL-6-mediated signals. Andrographolide inhibits IL-6 expression at both mRNA and protein levels in a dose-dependent manner. Andrographolide suppresses both IL-6 autocrine- and IL-6 paracrine-loop induced cell signaling including Stat3 and Erk phosphorylation. Furthermore, andrographolide inhibits cell viability and induces apoptotic cell death in both androgen-stimulated and castration-resistant human prostate cancer cells without causing significant toxicity to normal immortalized prostate epithelial cells. Moreover, treatment of andrographolide to mice bearing castration-resistant DU145 human prostate tumors that express constitutive IL-6 autocrine loop significantly suppresses tumor growth. Taken together, these results demonstrate that andrographolide could be developed as a therapeutic agent to treat both androgen-stimulated and castration-resistant prostate cancer possibly by suppressing IL-6 expression and IL-6-induced signaling.
doi:10.1177/1947601910383416
PMCID: PMC3063649  PMID: 21442031
Prostate cancer; Interleukin-6; Andrographolide
20.  Molecular mechanisms of castration-resistant prostate cancer progression 
Future oncology (London, England)  2009;5(9):1403-1413.
Hormone-refractory prostate cancer is the result of regrowth of prostate cancer cells that have adapted to the hormone-deprived environment of the prostate. The process by which castration-resistant prostate cancer (CRPC) cells are generated appears to be varied. The complex mechanism of hormone resistance has been the topic of research in most laboratories that have analyzed the process from different angles. This review compiles research findings that explain the methods of development of hormone resistance in prostate cancer. Research data show many different processes to be involved in the acquisition of hormone resistance. Interestingly, one observes interdependence between these processes, indicating a complex network at play in the development of hormone resistance. Cytokines such as IL-6 have been shown to initiate an alternative signaling pathway, compared with the androgen receptor signaling pathway, in CRPC. IL-6 has been proposed to be the effector of the intracrine signaling pathway by influencing the levels of metabolic enzymes. Neuroendocrine cells are present at low levels in normal prostate, and signify the transitory phase of normal hormone-sensitive cells to hormone-refractory cells. IL-6 induces growth of neuroendocrine cells or neuroendocrine-like features in cells in CRPC. The increased presence of neuroendocrine cells in CRPC signifies a change in the prostate cell microenvironment. The stromal microenvironment also influences the development of CRPC in the hormone-refractory stage. In addition, intracrine androgen metabolic enzymes play a significant role in the development of the hormone refractory process. Despite hormone ablation, there is a residual level of hormones in cells due to active intracrine metabolic pathways. It is acknowledged that the androgen receptor plays the most influential role in development of prostate cancer. In addition to mutation and amplification, the androgen receptor has been characterized and shown to differ in sequence in CRPC compared with the androgen-sensitive prostate cancer cells. These variants of the androgen receptor through sequence changes may preserve the basic function of the molecule, but have far-reaching consequences on the cell as a whole. A multicombinatorial drug treatment approach has been suggested to target these multiple pathways in an effort to reduce the possibility of recurrence of CRPC.
doi:10.2217/fon.09.117
PMCID: PMC3041149  PMID: 19903068
androgen receptor; AR; AR variant; castration-resistant prostate cancer; CRPC; IL-6; interleukin-6; intracrine signaling
21.  Interleukin-4 activates androgen receptor through CBP/p300 
The Prostate  2009;69(2):126-132.
Background
Aberrant activation of androgen receptor (AR) plays an important role in the progression of castration resistant prostate cancer. Interleukin-4 (IL-4) enhances AR activation in the absence of androgen and stimulates castration resistant growth of androgen-sensitive prostate cancer cells. However, the mechanism of IL-4 mediated AR activation has not yet been revealed.
Methods
The effect of IL-4 on CBP/p300 expression was examined by Western blot analysis. The effect of IL-4 on the interactions of AR and CBP/p300 was examined by co-immunoprecipitation and ChIP assays. CBP/p300 siRNA was used to knockdown CBP/p300 expression to examine the role of CBP/p300 expression on IL-4 mediated AR activation.
Results
We found that IL-4 increases CBP/p300 protein expression and enhances interaction of AR with CBP/p300 proteins through an increase in the recruitment of CBP/p300 protein to the androgen responsive elements in the promoters of androgen responsive genes. Down regulation of CBP/p300 expression using CBP/p300 specific siRNA abolished IL-4 mediated AR activation, suggesting that CBP/p300 is responsible for AR activation induced by IL-4. Furthermore, AR activation can be enhanced by AR acetylation induced by IL-4 in prostate cancer cells. The IL-4 mediated AR acetylation can be blocked by knocking down CBP/p300 expression using CBP/p300 specific siRNA.
Conclusion
These results suggest that IL-4 activates AR through enhanced expression of CBP/p300 and its histone acetyltransferase activity.
doi:10.1002/pros.20865
PMCID: PMC3035998  PMID: 18819102
Interleukin-4; CBP/p300; AR; prostate cancer
22.  Transcriptional regulation of human RANK ligand gene expression by E2F1 
Receptor activator of nuclear factor kappa B ligand (RANKL) is a critical osteoclastogenic factor involved in the regulation of bone resorption, immune function, the development of mammary gland and cardiovascular system. To understand the transcriptional regulation of RANKL, we amplified and characterized a 1890 bp 5′-flanking sequence of human RANKL gene (-1782 bp to +108 bp relative to the transcription start site). Using a series of deletion mutations of the 1890 bp RANKL promoter, we identified a 72 bp region (-172 to -100 bp) mediating RANKL basal transcriptional activity. Sequence analysis revealed a putative E2F binding site within this 72 bp region in the human RANKL promoter. Over expression of E2F1 increased RANKL promoter activity, while down regulation of E2F1 expression by small interfering RNA decreased RANKL promoter activity. RT-PCR and Enzyme linked immunosorbent assays (ELISA) further demonstrated that E2F1 induced the expression of RANKL. Electrophoretic gel mobility shift assays (EMSA) and antibody competition assays confirmed that E2F1 proteins bind to the consensus E2F binding site in the RANKL promoter. Mutation of the E2F consensus binding site in the RANKL promoter profoundly reduced the basal promoter activity and abolished the transcriptional modulation of RANKL by E2F1. These results suggest that E2F1 plays an important role in regulating RANKL transcription through binding to the E2F consensus binding site.
doi:10.1016/j.bbrc.2008.03.106
PMCID: PMC2950805  PMID: 18381203
RANK; RANKL; E2F1; Transcriptional regulation
23.  LIGHT, a member of the TNF superfamily, activates Stat3 mediated by NIK pathway 
Stat3, a member of the signal transducers and activators of transcription (STAT) family, is a key signal transduction protein activated by numerous cytokines, growth factors and oncoproteins that controls cell proliferation, differentiation, development, survival and inflammation. Constitutive activation of Stat3 has been found frequently in a wide variety of human tumors and induces cellular transformation and tumor formation. In this study, we demonstrated that LIGHT, a member of tumor necrosis factor superfamily, activates Stat3 in cancer cells. LIGHT induces dose dependent activation of Stat3 by phosphorylation at both the tyrosine 705 and serine 727 residues. The activation of Stat3 by LIGHT appears to be mediated by NIK phosphorylation. Expression of a kinase-inactive NIK mutant abolished LIGHT induced Stat3 activation. Overexpression of an active NIK induces Stat3 activation by phosphorylation at the both tyrosine 705 and serine 727 residues. Activation of Stat3 by NIK requires NIK kinase activity as showed by kinase assays. In addition, LIGHT increases the expression of Stat3 target genes including cyclin D1, survivin, and Bcl-xL, and stimulates human LNCaP prostate cancer cell growth in vitro which can by blocked by expression of a dominant-negative Stat3 mutant. Taken together, these results indicate that in addition to activating NF-κB/p52, LIGHT also activates Stat3. Activation of Stat3 together with activating non-canonical NF-κB/p52 signaling by LIGHT may maximize its effects on cellular proliferation, survival, and inflammation.
doi:10.1016/j.bbrc.2007.05.119
PMCID: PMC2062522  PMID: 17543278
Stat3; LIGHT; NIK; Prostate
24.  Microarray Data Mining for Potential Selenium Targets in Chemoprevention of Prostate Cancer 
Cancer genomics & proteomics  2005;2(2):97-114.
Background
A previous clinical trial showed that selenium supplementation significantly reduced the incidence of prostate cancer. We report here a bioinformatics approach to gain new insights into selenium molecular targets that might be relevant to prostate cancer chemoprevention.
Materials and Methods
We first performed data mining analysis to identify genes which are consistently dysregulated in prostate cancer using published datasets from gene expression profiling of clinical prostate specimens. We then devised a method to systematically analyze three selenium microarray datasets from the LNCaP human prostate cancer cells, and to match the analysis to the cohort of genes implicated in prostate carcinogenesis. Moreover, we compared the selenium datasets with two datasets obtained from expression profiling of androgen-stimulated LNCaP cells.
Results
We found that selenium reverses the expression of genes implicated in prostate carcinogenesis. In addition, we found that selenium could counteract the effect of androgen on the expression of a subset obtained from androgen-regulated genes.
Conclusions
The above information provides us with a treasure of new clues to investigate the mechanism of selenium chemoprevention of prostate cancer. Furthermore, these selenium target genes could also serve as biomarkers in future clinical trials to gauge the efficacy of selenium intervention.
PMCID: PMC2424238  PMID: 18548127
Selenium; chemoprevention; prostate carcinogenesis; androgen receptor
25.  Stat3 enhances transactivation of steroid hormone receptors 
Nuclear Receptor  2003;1:3.
Background
Steroid hormone receptors (SHRs) are members of the superfamily of ligand-activated transcription factors that regulate many biological processes. Co-regulators act as bridging molecules between the SHR and general transcription factors to enhance transactivation of target genes. Previous studies demonstrated that Stat3 is constitutively activated in prostate cancer and can enhance prostate specific antigen (PSA) expression and promote androgen independent growth. In this study, we investigate whether Stat3 can enhance steroid hormone receptors activation.
Methods
CV-1 cells in which plasmids expressing androgen receptor (AR), glucocorticoid receptor (GR), progesterone receptor (PR) or estrogen receptor (ER) were cotransfected with a constitutively active STAT3 mutant.
Results
Stat3 stimulates the transcriptional activity of all four SHR tested, AR, GR, PR and ER, in a hormone-dependent manner. Stat3 acts in a synergistic fashion with other coactivators such as SRC-1, pCAF, CBP, and TIF-2 on the transcriptional activity of these SHR. In addition, Stat3 significantly enhanced the sensitivity of androgen receptor in response to androgen. STAT3 did not affect the specificity of AR for other steroid hormones other than androgen or binding of AR to other hormone responsive elements.
Conclusions
These findings suggest that Stat3 can enhance the transactivation of AR, GR, PR and ER, and activated Stat3 could have a role in the development or progression of a hypersensitive AR.
doi:10.1186/1478-1336-1-3
PMCID: PMC179876  PMID: 12904256

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