Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2015 Feb 1.
Published in final edited form as: Mol Cancer Ther. 2013 Nov 20;13(2):341–352. doi: 10.1158/1535-7163.MCT-13-0478

A Naturally-Derived Small Molecule Disrupts Ligand-Dependent and Ligand-Independent Androgen Receptor Signaling in Human Prostate Cancer Cells

Karishma S Amin 1, Shankar Jagadeesh 1,#, Gakul Baishya 2, Paruchuri G Rao 2, Nabin C Barua 2, Samir Bhattacharya 2,3, Partha P Banerjee 1,*
PMCID: PMC3945679  NIHMSID: NIHMS543107  PMID: 24258347

Abstract

Continued reliance on androgen receptor (AR) signaling is a hallmark of prostate cancer, including the development of castration-resistant prostate cancer (CRPC), making it an attractive therapeutic target for prostate cancer treatment. Mahanine is a novel carbazole alkaloid derived from the leaves of Murraya koenigii, commonly known as the curry leaf plant, which grows widely across East Asia. We show here that mahanine possesses the ability to inhibit ligand-dependent and ligand-independent AR transactivation, leading to a prominent decline in AR target gene expression. Mahanine treatment causes a time- and dose- dependent decline in AR protein levels, including truncated AR splice variants, in a panel of androgen-responsive and androgen-independent prostate cancer cells. The decrease in AR levels induced by mahanine occurs post-translationally by proteasomal degradation, without any change in AR gene expression. Mahanine treatment induces an outward movement of the AR from the nucleus to the cytoplasm, leading to an initial rise in cytoplasmic AR levels, followed by a gradual decline in the AR levels in both cellular compartments. Ligand-induced AR phosphorylation at Ser-81, a phospho-site associated with prostate cancer cell growth and AR transactivity, is greatly diminished in the presence of mahanine. The decline in AR phosphorylation at Ser-81 by mahanine occurs via the inactivation of mitotic kinase, CDK1. Collectively, our data demonstrate that mahanine strongly disrupts androgen receptor signaling and inhibits the growth of androgen-dependent and –independent prostate cancer cells, thereby implicating a therapeutic role for mahanine in prostate cancer treatment.

Keywords: Androgen Receptor, Anti-Androgen, CDK1, Prostate cancer, Mahanine

Introduction

Prostate cancer is estimated to represent 28% of new cancer cases diagnosed in men in the year 2013, making it the most widely diagnosed cancer in men 1. Radiation or hormonal therapy is the first line of treatment employed for early stages of prostate cancer, when the tumor is still confined within the prostatic capsule. While this approach is successful in causing initial tumor regression, the cancer is often found to reoccur, adopting an androgen-independent phenotype known as castration resistant prostate cancer, or CRPC.

The androgen receptor (AR) signaling axis remains intact in CRPC despite the low levels of circulating androgen in these patients. The illicit activity of the AR can be attributed to several different molecular mechanisms; including intracrine steroidogenesis within the tumor microenvironment, AR gene amplification, leading to increased sensitivity to low levels of circulating androgens, AR mutations resulting in a gain-of-function or an ability to promiscuously respond to other steroids and growth factors, and androgen-independent AR activation by various cellular kinases such as MAPK, Akt, Src (2–5). While there may not be a single definitive mechanism to explain the transition of a hormone sensitive state to a hormone refractory one, it is evident that uninterrupted AR signaling plays a central role in the development of CRPC.

The AR is a steroidal receptor which exists in the cytosol in an inactive conformation bound to chaperone proteins such as Hsp90 6. Ligand binding causes the AR to adopt an altered conformation, allowing it to disengage from its interaction with chaperones, and undergo various post-translational modifications which facilitate its export to the nucleus, where it executes its transcriptional effects in conjunction with co-activator proteins. In addition to androgen, various growth factors such as EGF and IL-6 are known to modulate the activity and stability of the AR via diverse signaling cascades (7–9).

Phosphorylation is the most common post-translational modification associated with AR in response to stimulation by androgen and other growth factors 10. Majority of the known phospho-sites are concentrated in the N-terminal domain (NTD) of the AR, which also contains the activation of function-1 (AF-1) domain, although residues within the DNA binding domain (DBD) and C-terminal ligand binding domain (LBD) have also been reported to undergo phosphorylation (3, 11, 12). The effects of AR phosphorylation are varied, and can result in AR transactivation, nuclear translocation, DNA binding and degradation, depending on the kinase cascade and site of phosphorylation involved (11, 13–15). Phosphorylation of the AR on its Ser-81 site occurs in a time-dependent manner in response to androgen, with maximal phosphorylation after 6–8 hours, and has been reported to be the mostly abundantly phosphorylated site following ligand stimulation (16, 17). Phosphorylation of Ser-81 is associated with nuclear localization and chromatin binding of the AR, thereby influencing the transcriptional output in androgen stimulated cells 14. Furthermore, Ser-81 phosphorylation governs the stability of the AR and prevents its proteasomal degradation 18. Independent studies have demonstrated the importance of AR Ser-81 phosphorylation in regulating prostate cancer cell growth in response to ligand and soluble stromal factors (19, 20). Taken together, these findings suggest that Ser-81 phosphorylation is an important determinant of AR stability and activity, and its inhibition alters AR transcriptional output and affects prostate cancer cell growth.

CDK1, in conjunction with its binding partner, Cyclin B1 is essential to drive the G2/M transition of the cell cycle and was the first kinase identified to phosphorylate AR Ser-81. CDK1 over-expression has been reported in prostate cancer, and is associated with disease recurrence and resistance to therapy (21, 22). Recent evidence indicates that CDK1 also plays a role in promoting prostate cancer cell migration, suggesting its involvement in disease progression 23. Several naturally derived, synthetic and molecular agents targeting CDK1 have been assessed for use in prostate cancer therapy and are found to significantly decrease growth and proliferation (24–27). The elevated activity of CDK1 in CRPC and its ability to phosphorylate multiple sites on the AR, such as Ser-81 and Ser-515 28 implicate the possibility of crosstalk between CDK1 and AR signaling pathways, resulting in increased AR activity and cell proliferation.

In the present study, we investigated the effects of a novel anti-cancer compound, mahanine on the AR signaling pathway in human prostate cancer cells. Mahanine is a carbazole alkaloid drug derived from the leaves of Murraya koenigii, also known as the curry leaf plant, which grows widely across East Asia 29. Mahanine has been shown to possess cytotoxic, anti-oxidative and anti-proliferative properties 30. Studies using human leukemic cell lines have shown that treatment with mahanine results in the apoptotic death of these cells via mitochondrial pathways (31, 32). Our prior work has shown that mahanine treatment restores the expression of the epigenetically silenced tumor suppressor gene, RASSF1A in a panel of cancer cell lines, including prostate cancer 33. A recent report demonstrates the ability of mahanine to induce cell death in pancreatic cancer cells via the induction of ROS production 34.

We show here that mahanine inhibits ligand-dependent and -independent transactivation of the AR leading to a significant decrease in the expression of androgen regulated genes. Furthermore, mahanine compromises the stability of the AR, resulting in its proteasomal degradation. Mahanine prevents DHT-induced phosphorylation of the AR on its Ser-81 residue, via the inactivation of CDK1. Taken together, our results suggest a potential therapeutic role for mahanine in the treatment of prostate cancer based on its ability to disrupt AR signaling, induce AR degradation and inactivate CDK1 in prostate cancer cells.

Materials and Methods

Cell culture and reagents

Prostate cancer cell lines (LNCaP, VCaP, LNCaP C4-2B, 22Rv1) were obtained from American Type Culture Collection (Manassas, VA) and were cultured in phenol-red free IMEM (Invitrogen, Carlsbad, CA) containing 10% fetal bovine serum (Atlanta Biologicals, Lawrenceville, GA), 2 mM glutamine, 100 U/ml penicillin G sodium, and 100 µg/ml streptomycin sulfate (Sigma, St Louis, MO) unless otherwise specified. All cell lines used were tested and authenticated at the Tissue Culture Shared Resource in Lombardi Comprehensive Cancer Center by DNA fingerprinting short-tandem repeat (STR) analysis. The chemical structure of mahanine and its purification protocol have been described previously (30, 35) (Supplementary Fig. 1). DHT (Steraloids, Newport, RI), forskolin (Sigma) and IL6 (Invitrogen) were used in the concentrations indicated below. Cyclohexamide and MG132 were obtained from Tocris Biosciences (Bristol, UK). CDK1-AF and Cyclin B1 expression vectors were obtained from Dr. Steven Balk from Harvard University 17.

Quantitative Real Time PCR (qRT-PCR)

Total cellular RNA was extracted using RNeasy Mini Kit (Qiagen) according to manufacturer’s instructions. 1 µg of RNA was converted to cDNA using RT2 First Strand kit (Qiagen), following which qRT-PCR analysis was carried out on the ABI 7900HT (Applied Biosystems) using RT2 SYBR Green qPCR Master mix from Qiagen. Primer sets were obtained from Integrated DNA Technologies (Coralville, IA) and primer sequences are provided in the supplementary information (Supplementary Table 1 and Supplementary Table 2). PSA, GAPDH and ARv7 qPCR primer sequences have been previously reported (36, 37).

Luciferase assay

Cells were transiently transfected with an androgen response element –luciferase reporter (ARR3-TK-Luc) construct (from Dr. Robert Matusik, Vanderbilt University, Nashville, TN) or a PSA promoter-luciferase reporter construct (from Dr. Donald Tindall, Mayo Clinic, Rochester, MN) (200ng) using Genejet vII transfection reagent (Signagen, Gaithersburg, MD), following which they were treated with DHT (1nM) and the indicated concentrations of mahanine. The Dual Luciferase Assay kit (Promega, Madison, WI) was used to measure luciferase activity, which was read using a microplate luminometer (Harta Instruments, Gaithersburg, MD). Readings were normalized to Renilla luciferase activity by co-transfection of pRL-TK-luciferase plasmid (50ng). AR-NTD-Gal4DBD and Gal4UAS-TATA-luciferase vectors were obtained from Dr. Marianne Sadar, University of British Columbia, Vancouver, Canada 38 and VP16-Gal4DBD was obtained from Dr. Winship Herr, UniversitÈ de Lausanne, Switzerland 39 and transfected into LNCaP cells as described above.

Western blot analysis

Cells were harvested to extract total cellular protein which was resolved using 8–12% SDS-PAGE and electro-transferred to nitrocellulose membranes (Amersham Biosciences, Pittsburg, PA). The membranes were blocked with a 5% solution of nonfat milk powder in Tris buffered saline (TBS) containing 0.1% Tween-20, and were incubated with respective primary antibodies overnight at 4°C. Following an hour long incubation with horseradish peroxide-conjugated secondary antibodies (1:5000 dilution) (Santa Cruz Biotechnology, Santa Cruz, CA), membranes were visualized using chemiluminescent reagent (Santa Cruz Biotechnology). Images of the membranes were captured using a Fuji LAS-1000 Imager (Tokyo, Japan). Androgen receptor (Epitomics, Burlingame, CA and Santa Cruz Biotechnology), PSA (Dako, Carpinteria, CA), pARS81 (Millipore, Billerica, MA), β-actin, nucleolin, Gal4DBD (Santa Cruz Biotechnology), tubulin (Sigma), pCDK1T161, pCDK1Y15, CDK1 (Cell Signaling Technologies, Danvers, MA) antibodies were used at concentrations recommended by the respective manufacturer.

Immunoprecipitation

Cells were washed with ice-cold PBS following which total protein was extracted and quantified and diluted to obtain a concentration of 1µg/µl. 500µl of cell lysate was subjected to pre-clearance using 50µl Protein A/G Plus Agarose beads (Santa Cruz Biotechnology) for 15 mins. The lysate was incubated at 4°C with androgen receptor antibody (441) (Santa Cruz Biotechnology) for a period of 16 hours with constant rotation. The antibody-protein complexes were pulled down using 50µl Protein A/G Plus Agarose beads for 5 hours, following which the beads were separated by centrifugation at 14,000g for 1min at 4°C. The beads were washed three times with cell lysis buffer after which they were boiled with Laemmli buffer to detach the adsorbed protein. The protein was resolved using SDS-PAGE as described above.

Immunofluorescence staining

LNCaP cells were grown on ECL (Upstate Biotechnology Inc, Lake Placid, NY) coated chamber slides in phenol-red free IMEM. After the indicated treatments, cells were fixed in methanol, air-dried and re-hydrated with phosphate buffered saline (PBS). 0.2% BSA was used to block the cells following which they were incubated overnight at 4 °C with primary antibody. Cells were washed three times with PBS and incubated with 4µg/ml Alexa†Fluor†488 or 594 conjugated secondary antibody (Invitrogen) for 1hr. Cells were washed three times with PBS and incubated with DAPI or propidium iodide for 5 min. Cells were washed once again PBS and mounted using 50% glycerol and were viewed using a fluorescent microscope (Olympus BX, Olympus Corp, Tokyo, Japan). Images were imported into Adobe Photoshop.

Nuclear-Cytoplasmic Separation

LNCaP cells were treated with mahanine or DMSO as described below and cytoplasmic and nuclear fractions were obtained using Nuclear Extract Kit (Active Motif, Carlsbad, CA) according to manufacturer’s protocol.

Statistical Analysis

All data were derived from at least three independent experiments and statistical analysis were conducted using the Prism 3 GraphPad software. Values were presented as means ± SEM. Significance level was calculated using the one-way analysis of variance (ANOVA) or Student’s t test as applicable. p value<0.05 was considered significant.

Results

Mahanine inhibits ligand-dependent AR transactivation

Prostate cancer cells often exhibit increased androgen receptor activity, leading to elevated expression of androgen regulated genes 40. To assess the ability of mahanine to attenuate ligand induced transcriptional activity of the AR, LNCaP cells were cultured in the absence of androgen (charcoal-stripped or CS media) for 4 days and transfected with an androgen response element-luciferase reporter construct (ARR3-TK-luc). Expectedly, the addition of DHT (1nM) significantly induced luciferase activity; however, this induction was inhibited in the presence of mahanine in a dose-dependent manner, suggesting that mahanine inhibits DHT- induced AR transactivation (Fig. 1A). A similar effect was observed when a PSA promoter-luciferase reporter construct was used (Fig. 1B). Additionally, we transfected LNCaP cells with hTERT-promoter luciferase construct and c-myc-promoter luciferase construct and assessed whether mahanine treatment altered promoter activity of these genes. We did not observe any significant change in luciferase activity upon mahanine treatment, indicating that mahanine does not ubiquitously decrease promoter-luciferase activity of all genes (Supplementary Fig. 2A and 2B).

Figure 1. Mahanine inhibits DHT induced AR transactivation.

Figure 1

Open in a new tab

LNCaP cells grown in CS media for 4 days were transfected with (A) ARR3-TK-luciferase reporter construct or (B) PSA-promoter luciferase reporter construct and Renilla luciferase plasmids (pRL-TK-Luc) and treated with DHT (1nM) for 24 hours with or without indicated doses of mahanine. Promoter activity was determined after normalization with Renilla luciferase activity. Columns, mean of three independent experiments with quadruplicate samples; bars, SEM. (C) LNCaP cells were grown in CS medium for 4 days and treated as indicated with DHT and mahanine (Mah). AR and PSA levels were assessed using Western blot. β-actin was used as a loading control. (D–H) Expression levels of androgen regulated genes in LNCaP cells treated with DHT in the presence of mahanine were measured by qRT-PCR and normalized to GAPDH levels. Columns, mean of three independent experiments with quadruplicate samples; bars, SD. *p <0.05 ***p value<0.0001.

To confirm that the observed decrease in ARR3-TK-luciferase and PSA promoter-luciferase activity could be attributed to the ability of mahanine to inhibit DHT induced AR transactivation, and was not a result of AR degradation in the presence of mahanine, we measured the cellular levels of AR and PSA in LNCaP cells which had been cultured in CS media and subsequently treated with DHT (1nM), with or without mahanine (10µM). Our results show that while PSA levels decline in a manner that mimics the effect of mahanine on PSA promoter-luciferase activity, the AR levels remain mostly unchanged during this time (Fig. 1C). This suggests that the decline in AR transcriptional activity can be attributed to the inactivation of the AR in the presence of mahanine, and is not because of a decline in cellular AR levels.

To further confirm the inhibitory effects of mahanine on AR signaling, the expression levels of a number of well-established androgen-regulated genes (GREB1, PSA, NDRG1, PMEPA1, SGK1) was measured in LNCaP using quantitative real time PCR and in LNCaP and VCaP cells using semi-quantitative RT-PCR. Cells were cultured in CS media to ensure low basal AR transcriptional activity and target gene expression. The increase in AR target gene expression upon addition of DHT was effectively repressed in the presence of mahanine, further confirming the ability of mahanine to disrupt ligand-induced AR signaling (Fig. 1D–H and Supplementary Fig. 3).

Mahanine inhibits ligand-independent AR transactivation

In addition to androgen, the AR can be activated by various growth factors and steroids 41. While androgen binds to the ligand binding domain located in the C-terminus of the AR, growth factors activate the N-terminal domain of the AR, which contains the activation of function-1 domain, via various signaling cascades. To assess the ability of mahanine to disrupt ligand-independent activation of the AR, we co-transfected LNCaP and VCaP cells with an expression vector encoding a fusion protein of the Gal4-DNA binding domain (Gal4DBD) and the N-terminal domain of the AR (AR-NTD) and a luciferase reporter vector containing the Gal4 binding site (Gal4UAS-TATA-luciferase). The addition of forskolin (50µM) or IL6 (50ng/ml) caused a robust increase in luciferase activity, indicating that these growth factors activate the AR-NTD. However, the presence of mahanine significantly inhibited the induction of luciferase activity by these growth factors, without affecting the levels of AR-NTD-Gal4DBD fusion protein, indicating that mahanine possesses the ability to inhibit ligand-independent transactivation of the AR (Fig. 2A and 2B and Supplementary Fig. 4A). Mahanine did not suppress the luciferase activity of a constitutively active VP16-Gal4DBD fusion protein, confirming that the inhibitory effect of mahanine on the AR-NTD does not involve the Gal4DBD (Fig. 2C). To further confirm this effect of mahanine, LNCaP and VCaP cells were transfected with an ARR3-TK-luciferase reporter construct and treated as described above with forskolin or IL6 in the absence and presence of mahanine. While both forskolin and IL6 significantly induced endogenous AR activation, this effect was significantly diminished in the presence of mahanine, thereby confirming the ability of mahanine to disrupt ligand-independent AR transactivation, without altering endogenous AR levels (Fig. 2D and 2E and Supplementary Fig. 4B).

Figure 2. Mahanine inhibits ligand-independent AR transactivation.

Figure 2

Open in a new tab

LNCaP cells were co-transfected with AR-NTD-Gal4DBD expression vector and Gal4UAS-TATA-luciferase reporter construct and treated with (A) Forskolin (FSK) (50µM) or (B) IL6 (50ng/ml) in the absence and presence of mahanine (10µM) for 24 hours. Luciferase activity was measured after normalization with Renilla luciferase. Western blot analysis was performed to show the levels of the AR-NTD-Gal4DBD fusion protein. (C) LNCaP cells were co-transfected with VP16 activation domain-Gal4DBD fusion protein and Gal4UAS-TATA-luciferase reporter and treated with mahanine (10µM) for 24 hours. Luciferase activity was measured after normalization with Renilla luciferase. LNCaP cells were transfected with ARR3-TK-luciferase reporter construct and treated with (D) Forskolin (FSK) (50µM) or (E) IL6 (50ng/ml) in the absence and presence of mahanine (10µM) for 24 hours. Luciferase activity was measured after normalization with Renilla luciferase. Western blot analysis was performed to show the levels of the endogenous AR. Columns, mean of three independent experiments with quadruplicate samples; bars, SEM. ***p value<0.0001.

Mahanine causes a dose- and time-dependent decline in cellular AR levels

Our experiments thus far have involved mahanine treatment for a period of 24 hours, during which we established that mahanine treatment at a dose of 10µM does not alter AR cellular levels (Fig. 1C and Fig. 2), but significantly disrupts ligand-dependent and -independent AR signaling. Next, we sought to determine whether longer periods of mahanine treatment affected cellular AR levels. To this end, we treated a panel of prostate cancer cells (LNCaP, VCaP, LNCaP C4-2B and 22Rv1) with various doses of mahanine (2.5, 5, 10µM) for 3 days and measured AR levels by Western blot. We found that the AR protein levels decreased in all cell lines in a dose-dependent manner. A similar decrease in AR levels was observed in LNCaP and LNCaP C4-2B cells treated for 1–3 days with mahanine (10µM), indicating that mahanine also works to decrease AR levels in a time-dependent fashion (Fig. 3A–C and Supplementary Fig. 5A–C). It is worth noting that mahanine treatment also decreased the levels of the 80 kDa splice variant, ARv7, of the androgen receptor expressed in 22Rv1 cells (Fig. 3C), indicating that mahanine induces degradation of not only full-length androgen receptor, but also decreases the levels of androgen receptor splice variants which are associated with aggressive forms of prostate cancer.

Figure 3. Mahanine induces proteasomal degradation of AR.

Figure 3

Open in a new tab

LNCaP cells were treated with (A) indicated doses of mahanine for 3 days or (B) mahanine (10µM) for 1–3 days and the cellular levels of AR were measured by Western blotting. β-actin was used as a loading control. (C) 22Rv1 cells were used to assess AR and Arv7 levels upon mahanine treatment as described in (A). (D) AR message levels were assessed using qRT-PCR in LNCaP cells treated with mahanine (10µM) for 3 days. (E) AR and ARv7 message levels were measured in 22Rv1 cells treated with mahanine (10µM) for 3 days using qRT-PCR. (F) LNCaP cells were treated with cyclohexamide (CHX) (10µg/ml) in the absence and presence of mahanine (10µM). The cellular levels of AR were measured by Western blotting. β-actin was used as a loading control. (G) LNCaP cells were treated as shown with mahanine (20µM) and MG132 (5µM) for a period of 12 hours. AR cellular levels were measured using Western blot. (H) AR was immunoprecipitated from LNCaP cells treated with mahanine (20µM) and MG132 (5µM) as indicated, following which Western blot analysis was carried out using anti-ubiquitin and anti-AR antibody.

To determine whether the observed decrease in AR protein levels was due to decreased AR gene expression, we assessed AR message levels in LNCaP and VCaP cells treated with mahanine (10µM) for 3 days. Our data showed that AR message levels remain mostly unchanged upon mahanine treatment (Fig. 3D and Supplementary Fig. 5D). In addition, we assessed AR and ARv7 message levels in 22Rv1 cells treated in the same manner as described above and once again we did not observe any significant changes in the message levels of these genes (Fig. 3E), which indicates that mahanine decreases AR and ARv7 protein levels post-translationally, without affecting AR gene transcription.

To assess the susceptibility of AR to degradation in the presence of mahanine, we treated LNCaP cells with cyclohexamide (CHX) (10µg/ml) over a period of 48 hrs, in the absence and presence of mahanine. The presence of mahanine accelerated AR degradation, confirming that mahanine compromises the stability of the AR (Fig. 3F).

Mahanine induces proteasomal degradation of the AR

The ubiquitin-proteasome pathway is the predominant mechanism for AR degradation. To explore the mechanism by which mahanine induces AR degradation, LNCaP cells were treated mahanine (20µM), with or without a proteasome inhibitor, MG132 (5µM), for 12 hrs. The decrease in AR levels upon mahanine treatment was significantly rescued when the proteasome was inhibited by MG132 (Fig. 3G), indicating that AR degradation in the presence of mahanine is mediated by the proteasome. In addition, a two-fold increase in ubiquitinated AR was observed in LNCaP cells treated with MG132 (5µM) and mahanine (20µM) for 12 hours, indicating that mahanine causes an increase in AR ubiquitination, leading to its proteasomal degradation (Fig. 3H). The interaction of AR with chaperone protein Hsp90 was unaffected by mahanine, suggesting that mahanine could be degrading a pool of AR which already exists in an unbound state (Supplementary Fig. 5E).

Mahanine prevents AR nuclear translocation and causes it to accumulate in the cytoplasm

Nuclear localization is an important prerequisite for the AR to exert its transcriptional effects. To evaluate the effects of mahanine on the cellular distribution of the AR, LNCaP cells were treated with DHT (1nM), with or without mahanine (10µM) and immunostained for AR. In untreated control cells, although the AR was found to exist predominantly in the nucleus, it was also detectable in the cytoplasm. The nuclear localization of the AR in control cells can be attributed to steroids and other growth factors present in the serum-supplemented media. Upon stimulation with DHT, all of the AR was found to localize in the cell nucleus; however, when the cells were treated with both, DHT and mahanine, the nuclear localization of the AR was greatly diminished, with a large proportion of the AR present in the cytoplasm (Fig. 4A). In order to determine whether mahanine had a similar effect on the cellular distribution of androgen receptor splice variant, ARv7, 22Rv1 cells were treated with mahanine for 24 hours and then immunostained using an ARv7 specific antibody. We found that mahanine treatment induced the relocalization of ARv7 from the nucleus to the cytoplasm, suggesting that mahanine affects the cellular distribution of not only full-length AR, but also its splice variant ARv7 (Fig. 4B).

Figure 4. Mahanine decreases AR nuclear localization, leading to its cytoplasmic accumulation and subsequent degradation.

Figure 4

Open in a new tab

(A) LNCaP cells were treated with DHT (1nM) with or without mahanine (10µM) for 24 hours. Immunofluorescence analysis was carried out to assess AR cellular localization. (B) 22Rv1 cells were treated with mahanine (10µM) for 24 hours. Immunofluorescence analysis was carried out to assess ARv7 localization. (C) Immunofluorescence analysis for AR localization was performed at intervals in LNCaP cells treated with mahanine (20µM) over a 12 hour time frame. (D) Nuclear and cytoplasmic fractions were separated at the indicated intervals of time from LNCaP cells treated with mahanine (20µM) over a 12 hour time frame. AR levels were assessed in each fraction by Western Blot. β-actin and nucleolin were used as cytoplasmic and nuclear loading controls, respectively. (E) LNCaP cells were treated as indicated with CHX, MG132 and mahanine. Nuclear and cytoplasmic fractions were separated after 3 hours treatment and analyzed by Western blotting for AR levels. β-actin and nucleolin were used as cytoplasmic and nuclear loading controls, respectively. (F) LNCaP cells which were cultured in CS media for 4 days were treated with DHT (1nM) for 6 hours in the absence and presence of mahanine (10µM). Nuclear and cytoplasmic fractions were separated and assessed for AR levels by Western Blot. β-actin and nucleolin were used as cytoplasmic and nuclear loading controls, respectively. *p <0.05

To further understand how mahanine affects the intracellular distribution of the AR, LNCaP cells were treated with mahanine (20µM) for a 12 hour window, during which the AR localization was monitored at specific intervals of time by immunofluorescence staining. Over a 12 hour treatment time, the AR content of the nucleus was progressively depleted, accompanied by an initial increase in the cytoplasmic levels of the AR after 3 hours of mahanine treatment, followed by a gradual decline at 6 and 12 hours (Fig. 4C). To confirm the above pattern of sub-cellular movement of the AR, the nuclear and cytoplasmic fractions of LNCaP cells treated in a similar manner with mahanine (20µM) over a 12 hour time frame were collected. The AR sub-cellular distribution patterns mimicked those observed by immunofluorescence staining, with a noticeable dip in the nuclear AR levels over a period of 12 hours, and a concomitant rise in the cytoplasmic AR levels after 3 hours of mahanine treatment, followed by a gradual decline (Fig. 4D).

To clarify that the observed increase in cytoplasmic AR levels following mahanine treatment is the result of AR shuttling out of the nucleus, and not due to increased AR synthesis in the cytoplasm, LNCaP cells were pre-treated with cyclohexamide (10µg/ml) and MG132 (5µM) for 1 hour, after which mahanine (20µM) was administered for 3 hours. The presence of cyclohexamide and MG132 ensured that no further AR was synthesized in the cell and the existing AR was not degraded by endogenous mechanisms or the presence of mahanine. Under these conditions, a distinct elevation in the cytoplasmic AR was still observed upon mahanine treatment, along with a decline in nuclear AR levels. These data confirm the ability of mahanine to cause a sub-cellular shift in the localization of AR, inducing its movement out of the nucleus and into the cytoplasm, and subsequently resulting in a decrease in the AR levels in both cellular compartments (Fig. 4E). Interestingly, in LNCaP cells cultured in CS media, which caused the AR to localize predominantly in the cytoplasm, mahanine did not prevent the shuttling of the AR from the cytoplasm to the nucleus upon stimulation with DHT. However, although the AR did localize to the nucleus in cells treated with DHT and mahanine, PSA expression was not induced in the mahanine treated cells and remained at the control levels, suggesting that the AR which translocated to the nucleus in the presence of mahanine was not transcriptionally active (Fig. 4F).

Mahanine inhibits DHT-induced phosphorylation of AR Ser-81

Phosphorylation is the most common post-translational modification associated with AR in response to stimulation by androgen and other growth factors. Prior studies have found that the cell cycle kinases such as CDK1 and CDK9 stimulate phosphorylation at AR Ser-81, thereby mediating cell growth, chromatin binding and transcriptional activity of the AR (17, 19). Our findings that mahanine regulates prostate cancer cell growth and AR transcriptional activity in response to DHT, led us to explore whether it modulated AR Ser-81 phosphorylation. Time-course studies were carried out in LNCaP cells cultured in CS medium for 4 days, following which DHT was added for regular time intervals up to 24 hrs. In accordance with others’ findings, AR Ser-81 phosphorylation was maximally induced between 6 and 12 hours of DHT treatment. Interestingly, in the presence of mahanine, the induction of AR Ser-81 phosphorylation was completely inhibited, with only a very slight induction noticeable at 24 hours (Fig. 5A). This indicates that mahanine inhibits DHT induced phosphorylation of AR Ser-81. The prevention of AR Ser-81 phosphorylation by mahanine was accompanied by an inhibition in the induction of PSA expression by DHT, which is consistent with the prior reports demonstrating the importance of AR Ser-81 phosphorylation for AR transcriptional activity 14.

Figure 5. Mahanine inhibits DHT induced AR Ser-81 phosphorylation via the inactivation of CDK1.

Figure 5

Open in a new tab

(A) LNCaP cells were cultured in CS media and subsequently treated with DHT with or without mahanine (10µM) for the indicated time periods. pARS81, AR and PSA levels were assessed by Western blotting. Tubulin was used as a loading control. (B) LNCaP cells were treated with nocodazole (100ng/ml) in the absence and presence of mahanine (10µM). pARS81, pCDK1T161, pCDK1Y15 and total CDK1 levels were assessed by Western blotting. β-actin was used as a loading control. (C) LNCaP cells were co-transfected with a constitutively active CDK1-AF expression vector and a cyclin B1 expression vector and treated with DHT and mahanine as indicated. pARS81, AR and PSA levels were assessed by Western blotting. Tubulin was used as a loading control.

Mahanine inhibits AR Ser-81 phosphorylation via the inactivation of CDK1

CDK1 has maximal activity at G2/M phase of the cell cycle, where it mediates entry into mitotic prophase in conjunction with its binding partner, Cyclin B1. In addition to mitotic protein targets, CDK1 is known to phosphorylate the AR at its Ser-81 site in an androgen-dependent manner. The ability of mahanine to modulate prostate cancer cell growth (Supplementary Fig. 6A and 6B) and cell cycle distribution (data not shown) led us to explore whether mahanine inhibits AR Ser-81 phosphorylation via CDK1 inactivation. We did not observe a direct inhibition of CDK1 activity by mahanine using an in-vitro kinase assay (Supplementary Fig. 7). Next, LNCaP cells were synchronized to the G2/M phase by 24 hour nocodazole treatment (100ng/ml) to attain maximal CDK1 activity, as indicated by an increase in activating T161 phosphorylation and a decrease in inhibitory Y15 phosphorylation in the synchronized cells compared to the asynchronized control cells. However, the activation of CDK1 by nocodazole was inhibited in the presence of mahanine. Furthermore, while nocodazole caused an induction in AR Ser-81 phosphorylation, this effect was significantly diminished in the presence of mahanine (Fig. 5B).

To confirm that mahanine inhibits AR Ser-81 phosphorylation via CDK1 inactivation, we co-transfected LNCaP cells with an expression vector encoding constitutively active CDK1 (CDK1-AF) and an expression vector for its binding partner, Cyclin B1. The cells were treated with DHT in the absence and presence of mahanine. While mahanine completely inhibited DHT induced AR Ser-81 phosphorylation, the presence of constitutively active CDK1 rescued the decline in AR Ser-81 phosphorylation induced by mahanine, demonstrating that mahanine inactivates CDK1 and thereby prevents DHT induced AR Ser-81 phosphorylation (Fig. 5C).

Discussion

The central role of the AR in the progression of CRPC has become increasingly evident, making it a highly attractive therapeutic target for CRPC therapy. Recent approaches in CRPC therapy include the use of new generation anti-androgen drugs such as MDV3100 and ARN509, which specifically target the ligand-binding domain of the AR, or drugs which block intratumoral androgen synthesis, such as abiraterone (42, 43). While these newly approved drugs improved median overall survival by 3 to 5 months in Phase III clinical trials, their mechanism of action is limited to targeting the canonical, ligand-dependent activation of the AR 44. However, AR signaling is not solely mediated by androgen; crosstalk of the AR axis with various signaling cascades triggered by growth factors activates the AR even in the absence of circulating androgens. In addition, the generation of AR splice variants, majority of which lack the ligand binding domain and are thereby androgen-independent, is associated with disease progression and resistance to the newly approved drugs, MDV3100 and abiraterone, and adds another layer of complexity in the development of therapeutic agents which are effective in CRPC. Drugs which disrupt AR signaling, irrespective of its manner of activation, and thereby inhibit its downstream effects such as prostate cancer growth and progression, would more effectively prevent growth of the androgen-dependent and -independent cell types of which the prostatic tumor is comprised. To this end, recent reports have described the use of agents which down-regulate the levels of AR splice variants and effectively prevent the growth of drug-resistant prostate cancer cells for CRPC therapy (45, 46).

Naturally derived, phytochemical compounds from diverse sources have demonstrated anti-tumorigenic properties in prostate cancer (47–50). We show here that mahanine, a carbazole alkaloid derived from the curry leaf plant, possesses the ability to inhibit the growth of androgen-dependent and androgen-independent prostate cancer cells (Supplementary Fig. 6A and 6B). Interestingly, mahanine did not demonstrate growth suppressive effects on RWPE1 cells, a non-tumorigenic prostatic epithelial cell line, and while it did decrease slightly the AR levels in these cells, it did not have a pronounced effect on AR nuclear localization as seen in prostate cancer cell lines (LNCaP and 22Rv1), suggesting a differential effect of mahanine on normal versus cancerous cells of the prostate (Supplementary Fig. 6A and Supplementary Fig. 8).

Mahanine blocked ligand-dependent activation of AR signaling, effectively inhibiting the expression of downstream target genes of the AR, including PSA. Importantly, the activation of the N-terminal domain of the AR was prevented in the presence of mahanine, suggesting that its inhibitory effects on AR signaling include the segment involved in androgen-independent activation of the AR. The ability to prevent ligand-independent activation of the AR, specifically by inhibiting the AR NTD, is of importance in CRPC patients who have previously undergone androgen deprivation therapy but continue to retain high AR activity due to promiscuous activation of the AR by paracrine or autocrine growth factors, or due to splice variants which lack the LBD and are constitutively active.

Our findings indicate that mahanine inactivates the AR at first, within 24 hours of treatment with 10µM of mahanine, following which the AR undergoes proteasomal degradation after about 48 hours and continues to decline in the cells up to 72 hours after treatment. The ability of mahanine to inactivate ligand-dependent and –independent activity of the AR, prevent its nuclear localization and subsequently induce its degradation highlight the various levels at which mahanine intercepts the AR axis. Most importantly, mahanine also induces degradation of truncated AR splice variants, demonstrating its effectiveness against a key mediator of castration resistance and aggressive prostate cancer.

Another interesting effect of mahanine on the AR signaling pathway is its ability to completely inhibit AR Ser-81 phosphorylation. The Ser-81 site is the most abundantly phosphorylated site in response to androgen and has been shown to be imperative for AR nuclear localization, chromatin binding, transcriptional activation and growth. Our data demonstrates a striking inhibition of DHT-induced phosphorylation at AR Ser-81 by mahanine, accompanied by a decline in transcriptional activation of the AR, exemplified by decreased PSA expression. In addition, we found that although mahanine does not prevent DHT induced nuclear translocation of the AR (Fig. 4E), it inhibits AR Ser-81 phosphorylation to a large extent and thereby prevents phosphorylated AR Ser-81 from accumulating in the nucleus (Supplementary Fig. 9). In spite of the AR localizing in the nucleus, PSA expression was not induced in the presence of mahanine, which correlates well with others’ findings that phosphorylation at the AR Ser-81 site is important for AR transactivation in response to ligand 14. Although phosphorylation at this site can be induced by androgen at any phase of the cell cycle, mahanine mediated inhibition of AR Ser-81 phosphorylation depends on the inactivation of mitotic kinase CDK1, which is maximally active at the G2/M transition and was the first kinase reported to phosphorylate this site. This suggests a potential link between the growth inhibitory effects of mahanine and the disruption of AR signaling. However, since several different signaling cascades are known to mediate the growth inhibitory effects of mahanine, it is likely that the inhibition of AR signaling is also a result of the combined effect of mahanine on various pathways involved in AR activation, and does not solely depend on CDK1 inactivation. Nevertheless, since AR Ser-81 is known to be important for AR nuclear localization and transcriptional activity, and CDK1 activity is found to be elevated in CRPC, this finding is of importance in establishing the potential of mahanine for prostate cancer therapy.

The current lack of epidemiological data on mahanine makes it difficult to estimate physiologically achievable concentrations of mahanine. Further studies in animal models would be essential to evaluate the in vivo effectiveness of mahanine in disrupting AR signaling. Taken together, our findings demonstrate that mahanine intercepts the AR signaling pathway at multiple levels, prevents phosphorylation at the most abundantly phosphorylated site on the AR, and ultimately induces the degradation of both full-length AR and its splice variants thereby highlighting the therapeutic potential of mahanine in patients with prostate cancer and CRPC, where the AR plays a central role in tumor growth and progression.

Supplementary Material

1
8
9
10
11
2
3
4
5
6
7

Acknowledgements

The authors thank Dr. Steven Balk of Harvard University for his generous gift of the CDK1-AF and Cyclin B1 expression vectors. We also thank Dr. Robert Matusik of Vanderbilt University for androgen response element–luciferase reporter (ARR3-TK-Luc) construct, Dr. Donald Tindall of Mayo Clinic for PSA promoter-luciferase reporter construct, Dr. Satoru Kyo of Kanazawa University for the hTERT promoter luciferase reporter construct, Dr. Linda Penn of Ontario Cancer Institute for the c-myc promoter luciferase reporter construct, Dr. Marianne Sadar of University of British Columbia for AR-NTD-Gal4DBD and Gal4UAS-TATA-luciferase vectors, and Dr. Winship Herr of Université de Lausanne for VP16-Gal4DBD vector. The authors acknowledge the use of the Genomics and Epigenomics Shared Resource and the Tissue Culture Shared Resource in Lombardi Comprehensive Cancer Center for various studies.

Grant Support

This work was supported by a NIH grant R01CA131123 awarded to Dr. Partha P. Banerjee.

Footnotes

Conflict of interest: All authors declare no conflict of interest.

Authors’ Contributions

Conception and design: KS. Amin and PP. Banerjee

Development of methodology: KS. Amin, S. Jagadeesh, G. Baishya, PG Rao, NC Barua, S. Bhattacharya and PP. Banerjee

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): KS. Amin, and PP. Banerjee

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): KS. Amin and PP. Banerjee

Writing, review, and/or revision of the manuscript: KS. Amin and PP. Banerjee

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): KS. Amin and PP. Banerjee

Study supervision: PP. Banerjee

References

  • 1.Siegel R, Naishadham D, Jemal A. Cancer statistics, 2013. CA Cancer J Clin. 2013;63:11–30. doi: 10.3322/caac.21166. [DOI] [PubMed] [Google Scholar]
  • 2.Ueda T, Bruchovsky N, Sadar MD. Activation of the androgen receptor N-terminal domain by interleukin-6 via MAPK and STAT3 signal transduction pathways. J Biol Chem. 2002;277:7076–7085. doi: 10.1074/jbc.M108255200. [DOI] [PubMed] [Google Scholar]
  • 3.Wen Y, Hu MC, Makino K, Spohn B, Bartholomeusz G, Yan DH, et al. HER-2/neu promotes androgen-independent survival and growth of prostate cancer cells through the Akt pathway. Cancer Res. 2000;60:6841–6845. [PubMed] [Google Scholar]
  • 4.Guo Z, Dai B, Jiang T, Xu K, Xie Y, Kim O, et al. Regulation of androgen receptor activity by tyrosine phosphorylation. Cancer Cell. 2006;10:309–319. doi: 10.1016/j.ccr.2006.08.021. [DOI] [PubMed] [Google Scholar]
  • 5.Montgomery RB, Mostaghel EA, Vessella R, Hess DL, Kalhorn TF, Higano CS, et al. Maintenance of intratumoral androgens in metastatic prostate cancer: a mechanism for castration-resistant tumor growth. Cancer Res. 2008;68:4447–4454. doi: 10.1158/0008-5472.CAN-08-0249. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Marivoet S, Van Dijck P, Verhoeven G, Heyns W. Interaction of the 90-kDa heat shock protein with native and in vitro translated androgen receptor and receptor fragments. Mol Cell Endocrinol. 1992;88:165–174. doi: 10.1016/0303-7207(92)90021-w. [DOI] [PubMed] [Google Scholar]
  • 7.Migliaccio A, Castoria G, Di Domenico M, Ciociola A, Lombardi M, De Falco A, et al. Crosstalk between EGFR and extranuclear steroid receptors. Ann N Y Acad Sci. 2006;1089:194–200. doi: 10.1196/annals.1386.006. [DOI] [PubMed] [Google Scholar]
  • 8.Nazareth LV, Weigel NL. Activation of the human androgen receptor through a protein kinase A signaling pathway. J Biol Chem. 1996;271:19900–19907. doi: 10.1074/jbc.271.33.19900. [DOI] [PubMed] [Google Scholar]
  • 9.Chen T, Wang LH, Farrar WL. Interleukin 6 activates androgen receptor-mediated gene expression through a signal transducer and activator of transcription 3-dependent pathway in LNCaP prostate cancer cells. Cancer Res. 2000;60:2132–2135. [PubMed] [Google Scholar]
  • 10.Gioeli D, Paschal BM. Post-translational modification of the androgen receptor. Mol Cell Endocrinol. 2011;352:70–78. doi: 10.1016/j.mce.2011.07.004. [DOI] [PubMed] [Google Scholar]
  • 11.Gioeli D, Black BE, Gordon V, Spencer A, Kesler CT, Eblen ST, et al. Stress kinase signaling regulates androgen receptor phosphorylation, transcription, and localization. Mol Endocrinol. 2006;20:503–515. doi: 10.1210/me.2005-0351. [DOI] [PubMed] [Google Scholar]
  • 12.Ponguta LA, Gregory CW, French FS, Wilson EM. Site-specific androgen receptor serine phosphorylation linked to epidermal growth factor-dependent growth of castration-recurrent prostate cancer. J Biol Chem. 2008;283:20989–21001. doi: 10.1074/jbc.M802392200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Lin HK, Wang L, Hu YC, Altuwaijri S, Chang C. Phosphorylation-dependent ubiquitylation and degradation of androgen receptor by Akt require Mdm2 E3 ligase. Embo J. 2002;21:4037–4048. doi: 10.1093/emboj/cdf406. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Chen S, Gulla S, Cai C, Balk SP. Androgen receptor serine 81 phosphorylation mediates chromatin binding and transcriptional activation. J Biol Chem. 2012;287:8571–8583. doi: 10.1074/jbc.M111.325290. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Shu SK, Liu Q, Coppola D, Cheng JQ. Phosphorylation and activation of androgen receptor by Aurora-A. J Biol Chem. 2010;285:33045–33053. doi: 10.1074/jbc.M110.121129. [DOI] [PMC free article] [PubMed] [Google Scholar] [Retracted]
  • 16.Gioeli D, Ficarro SB, Kwiek JJ, Aaronson D, Hancock M, Catling AD, et al. Androgen receptor phosphorylation. Regulation and identification of the phosphorylation sites. J Biol Chem. 2002;277:29304–29314. doi: 10.1074/jbc.M204131200. [DOI] [PubMed] [Google Scholar]
  • 17.Chen S, Xu Y, Yuan X, Bubley GJ, Balk SP. Androgen receptor phosphorylation and stabilization in prostate cancer by cyclin-dependent kinase 1. Proc Natl Acad Sci U S A. 2006;103:15969–15974. doi: 10.1073/pnas.0604193103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Hsu FN, Chen MC, Chiang MC, Lin E, Lee YT, Huang PH, et al. Regulation of androgen receptor and prostate cancer growth by cyclin-dependent kinase 5. J Biol Chem. 2011;286:33141–33149. doi: 10.1074/jbc.M111.252080. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Gordon V, Bhadel S, Wunderlich W, Zhang J, Ficarro SB, Mollah SA, et al. CDK9 regulates AR promoter selectivity and cell growth through serine 81 phosphorylation. Mol Endocrinol. 2010;24:2267–2280. doi: 10.1210/me.2010-0238. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Shigemura K, Isotani S, Wang R, Fujisawa M, Gotoh A, Marshall FF, et al. Soluble factors derived from stroma activated androgen receptor phosphorylation in human prostate LNCaP cells: roles of ERK/MAP kinase. Prostate. 2009;69:949–955. doi: 10.1002/pros.20944. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Tsaur I, Makarevic J, Hudak L, Juengel E, Kurosch M, Wiesner C, et al. The cdk1-cyclin B complex is involved in everolimus triggered resistance in the PC3 prostate cancer cell line. Cancer Lett. 2011;313:84–90. doi: 10.1016/j.canlet.2011.08.026. [DOI] [PubMed] [Google Scholar]
  • 22.Kallakury BV, Sheehan CE, Ambros RA, Fisher HA, Kaufman RP, Jr, Ross JS. The prognostic significance of p34cdc2 and cyclin D1 protein expression in prostate adenocarcinoma. Cancer. 1997;80:753–763. doi: 10.1002/(sici)1097-0142(19970815)80:4<753::aid-cncr15>3.0.co;2-s. [DOI] [PubMed] [Google Scholar]
  • 23.Manes T, Zheng DQ, Tognin S, Woodard AS, Marchisio PC, Languino LR. Alpha(v)beta3 integrin expression up-regulates cdc2, which modulates cell migration. J Cell Biol. 2003;161:817–826. doi: 10.1083/jcb.200212172. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Takeshita F, Patrawala L, Osaki M, Takahashi RU, Yamamoto Y, Kosaka N, et al. Systemic delivery of synthetic microRNA-16 inhibits the growth of metastatic prostate tumors via downregulation of multiple cell-cycle genes. Mol Ther. 2010;18:181–187. doi: 10.1038/mt.2009.207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Rigas AC, Robson CN, Curtin NJ. Therapeutic potential of CDK inhibitor NU2058 in androgen-independent prostate cancer. Oncogene. 2007;26:7611–7619. doi: 10.1038/sj.onc.1210586. [DOI] [PubMed] [Google Scholar]
  • 26.Xiao D, Johnson CS, Trump DL, Singh SV. Proteasome-mediated degradation of cell division cycle 25C and cyclin-dependent kinase 1 in phenethyl isothiocyanate-induced G2-M-phase cell cycle arrest in PC-3 human prostate cancer cells. Mol Cancer Ther. 2004;3:567–575. [PubMed] [Google Scholar]
  • 27.Xiao D, Srivastava SK, Lew KL, Zeng Y, Hershberger P, Johnson CS, et al. Allyl isothiocyanate, a constituent of cruciferous vegetables, inhibits proliferation of human prostate cancer cells by causing G2/M arrest and inducing apoptosis. Carcinogenesis. 2003;24:891–897. doi: 10.1093/carcin/bgg023. [DOI] [PubMed] [Google Scholar]
  • 28.Willder JM, Heng SJ, McCall P, Adams CE, Tannahill C, Fyffe G, et al. Androgen receptor phosphorylation at serine 515 by Cdk1 predicts biochemical relapse in prostate cancer patients. Br J Cancer. 2013;108:139–148. doi: 10.1038/bjc.2012.480. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Ramsewak RS, Nair MG, Strasburg GM, DeWitt DL, Nitiss JL. Biologically active carbazole alkaloids from Murraya koenigii. J Agric Food Chem. 1999;47:444–447. doi: 10.1021/jf9805808. [DOI] [PubMed] [Google Scholar]
  • 30.Tachibana Y, Kikuzaki H, Lajis NH, Nakatani N. Antioxidative activity of carbazoles from Murraya koenigii leaves. J Agric Food Chem. 2001;49:5589–5594. doi: 10.1021/jf010621r. [DOI] [PubMed] [Google Scholar]
  • 31.Bhattacharya K, Samanta SK, Tripathi R, Mallick A, Chandra S, Pal BC, et al. Apoptotic effects of mahanine on human leukemic cells are mediated through crosstalk between Apo-1/Fas signaling and the Bid protein and via mitochondrial pathways. Biochem Pharmacol. 2010;79:361–372. doi: 10.1016/j.bcp.2009.09.007. [DOI] [PubMed] [Google Scholar]
  • 32.Roy MK, Thalang VN, Trakoontivakorn G, Nakahara K. Mahanine, a carbazole alkaloid from Micromelum minutum, inhibits cell growth and induces apoptosis in U937 cells through a mitochondrial dependent pathway. Br J Pharmacol. 2005;145:145–155. doi: 10.1038/sj.bjp.0706137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Jagadeesh S, Sinha S, Pal BC, Bhattacharya S, Banerjee PP. Mahanine reverses an epigenetically silenced tumor suppressor gene RASSF1A in human prostate cancer cells. Biochem Biophys Res Commun. 2007;362:212–217. doi: 10.1016/j.bbrc.2007.08.005. [DOI] [PubMed] [Google Scholar]
  • 34.Sarkar S, Dutta D, Samanta SK, Bhattacharya K, Pal BC, Li J, et al. Oxidative inhibition of Hsp90 disrupts the super-chaperone complex and attenuates pancreatic adenocarcinoma in vitro and in vivo. Int J Cancer. 2013;132:695–706. doi: 10.1002/ijc.27687. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Sinha S, Pal BC, Jagadeesh S, Banerjee PP, Bandyopadhaya A, Bhattacharya S. Mahanine inhibits growth and induces apoptosis in prostate cancer cells through the deactivation of Akt and activation of caspases. Prostate. 2006;66:1257–1265. doi: 10.1002/pros.20415. [DOI] [PubMed] [Google Scholar]
  • 36.Chan SC, Li Y, Dehm SM. Androgen receptor splice variants activate androgen receptor target genes and support aberrant prostate cancer cell growth independent of canonical androgen receptor nuclear localization signal. J Biol Chem. 2012;287:19736–19749. doi: 10.1074/jbc.M112.352930. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Hu R, Dunn TA, Wei S, Isharwal S, Veltri RW, Humphreys E, et al. Ligand-independent androgen receptor variants derived from splicing of cryptic exons signify hormone-refractory prostate cancer. Cancer Res. 2009;69:16–22. doi: 10.1158/0008-5472.CAN-08-2764. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Andersen RJ, Mawji NR, Wang J, Wang G, Haile S, Myung JK, et al. Regression of castrate-recurrent prostate cancer by a small-molecule inhibitor of the amino-terminus domain of the androgen receptor. Cancer Cell. 2010;17:535–546. doi: 10.1016/j.ccr.2010.04.027. [DOI] [PubMed] [Google Scholar]
  • 39.Das G, Hinkley CS, Herr W. Basal promoter elements as a selective determinant of transcriptional activator function. Nature. 1995;374:657–660. doi: 10.1038/374657a0. [DOI] [PubMed] [Google Scholar]
  • 40.Xu LL, Shanmugam N, Segawa T, Sesterhenn IA, McLeod DG, Moul JW, et al. A novel androgen-regulated gene, PMEPA1, located on chromosome 20q13 exhibits high level expression in prostate. Genomics. 2000;66:257–263. doi: 10.1006/geno.2000.6214. [DOI] [PubMed] [Google Scholar]
  • 41.Culig Z, Hobisch A, Cronauer MV, Radmayr C, Trapman J, Hittmair A, et al. Androgen receptor activation in prostatic tumor cell lines by insulin-like growth factor-I, keratinocyte growth factor, and epidermal growth factor. Cancer Res. 1994;54:5474–5478. [PubMed] [Google Scholar]
  • 42.Tran C, Ouk S, Clegg NJ, Chen Y, Watson PA, Arora V, et al. Development of a second-generation antiandrogen for treatment of advanced prostate cancer. Science. 2009;324:787–790. doi: 10.1126/science.1168175. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Clegg NJ, Wongvipat J, Joseph JD, Tran C, Ouk S, Dilhas A, et al. ARN-509: a novel antiandrogen for prostate cancer treatment. Cancer Res. 2012;72:1494–1503. doi: 10.1158/0008-5472.CAN-11-3948. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Hoffman-Censits J, Kelly WK. Enzalutamide: a novel antiandrogen for patients with castrate-resistant prostate cancer. Clin Cancer Res. 2013;19:1335–1339. doi: 10.1158/1078-0432.CCR-12-2910. [DOI] [PubMed] [Google Scholar]
  • 45.Kuruma H, Matsumoto H, Shiota M, Bishop J, Lamoureux F, Thomas C, et al. A Novel Antiandrogen, Compound 30, Suppresses Castration-Resistant and MDV3100-Resistant Prostate Cancer Growth In Vitro and In Vivo. Mol Cancer Ther. 2013 doi: 10.1158/1535-7163.MCT-12-0798. [DOI] [PubMed] [Google Scholar]
  • 46.Li X, Liu Z, Xu X, Blair CA, Sun Z, Xie J, et al. Kava components down-regulate expression of AR and AR splice variants and reduce growth in patient-derived prostate cancer xenografts in mice. PLoS One. 2012;7:e31213. doi: 10.1371/journal.pone.0031213. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Cha TL, Qiu L, Chen CT, Wen Y, Hung MC. Emodin down-regulates androgen receptor and inhibits prostate cancer cell growth. Cancer Res. 2005;65:2287–2295. doi: 10.1158/0008-5472.CAN-04-3250. [DOI] [PubMed] [Google Scholar]
  • 48.Harada N, Murata Y, Yamaji R, Miura T, Inui H, Nakano Y. Resveratrol down-regulates the androgen receptor at the post-translational level in prostate cancer cells. J Nutr Sci Vitaminol (Tokyo) 2007;53:556–560. doi: 10.3177/jnsv.53.556. [DOI] [PubMed] [Google Scholar]
  • 49.Liu S, Yuan Y, Okumura Y, Shinkai N, Yamauchi H. Camptothecin disrupts androgen receptor signaling and suppresses prostate cancer cell growth. Biochem Biophys Res Commun. 2010;394:297–302. doi: 10.1016/j.bbrc.2010.02.164. [DOI] [PubMed] [Google Scholar]
  • 50.Li J, Cao B, Liu X, Fu X, Xiong Z, Chen L, et al. Berberine suppresses androgen receptor signaling in prostate cancer. Mol Cancer Ther. 2011;10:1346–1356. doi: 10.1158/1535-7163.MCT-10-0985. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1
NIHMS543107-supplement-1.pdf (71.6KB, pdf)
8
NIHMS543107-supplement-8.pdf (273.8KB, pdf)
9
NIHMS543107-supplement-9.pdf (95.4KB, pdf)
10
NIHMS543107-supplement-10.pdf (19.9KB, pdf)
11
NIHMS543107-supplement-11.pdf (19.8KB, pdf)
2
NIHMS543107-supplement-2.pdf (29KB, pdf)
3
NIHMS543107-supplement-3.pdf (189.4KB, pdf)
4
NIHMS543107-supplement-4.pdf (77.3KB, pdf)
5
NIHMS543107-supplement-5.pdf (223.2KB, pdf)
6
NIHMS543107-supplement-6.pdf (87.3KB, pdf)
7
NIHMS543107-supplement-7.pdf (101.4KB, pdf)

RESOURCES