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Azadirachta indica, commonly known as neem, has gained worldwide prominence because of its medical properties, namely antitumor, antiviral, anti-inflammatory, antihyperglycemic, antifungal, and antibacterial activities. Despite these promising results, gaps remain in our understanding of the molecular mechanism of action of neem compounds and their potential for use in clinical trials. We investigated supercritical extract of neem leaves (SENL) for the following: molecular targets in vitro, in vivo efficacy to inhibit tumor growth, and bioactive compounds that exert antitumor activity. Treatment of LNCaP-luc2 prostate cancer cells with SENL suppressed dihydrotestosterone-induced androgen receptor and prostate-specific antigen levels. SENL inhibited integrin β1, calreticulin, and focal adhesion kinase activation in LNCaP-luc2 and PC3 prostate cancer cells. Oral administration of SENL significantly reduced LNCaP-luc2 xenograft tumor growth in mice with the formation of hyalinized fibrous tumor tissue, reduction in the prostate-specific antigen, and increase in AKR1C2 levels. To identify the active anticancer compounds, we fractionated SENL by high-pressure liquid chromatography and evaluated 16 peaks for cytotoxic activity. Four of the 16 peaks exhibited significant cytotoxic activity against prostate cancer cells. Mass spectrometry of the isolated peaks suggested the compounds with cytotoxic activity were nimbandiol, nimbolide, 2′,3′-dihydronimbolide, and 28-deoxonimbolide. Analysis of tumor tissue and plasma samples from mice treated with SENL indicated 28-deoxonimbolide and nimbolide as the bioactive compounds. Overall, our data revealed the bioactive compounds in SENL and suggested that the anticancer activity could be mediated through alteration in androgen receptor and calreticulin levels in prostate cancer. Mol Cancer Ther; 13(5); 1067–77. ©2014 AACR.

PDF - 69K, Mass spectrometric analysis of SENL fractions 5, 6, and 7 obtained from HPLC. The total ion chromatogram retention time and the mass spectrum of individual peaks for the fractions 5, 6, and 7 are depicted in the panel A to C, respectively.

PDF - 64K, Mass spectrometric analysis of SENL fractions 9, 11, and 12 obtained from HPLC. The total ion chromatogram retention time and the mass spectrum of individual peaks for the fractions 9, 11, and 12 are depicted in the panel A to C, respectively.

PDF - 185K, The peaks labeled in the HPLC chromatogram were evaluated for cytotoxic activity against PC3 cells. The fractions collected from HPLC were concentrated using SpeedVac at 4 degrees C, and equal weights of the fractions were reconstituted in ethanol. PC3 cells were treated with serial dilutions of the fractions for 24 hours, and the viability was determined using the MTS assay.

PDF - 71K, Legends to Supplementary Figures 1 through 8.

PDF - 89K, HPLC and Mass spectrometric analysis of mice plasma and tumor tissues for the bioavailability of SENL compounds after oral administration of SENL, 200 mg/kg body weight. A, HPLC chromatogram of plasma of mice treated with SENL. B, HPLC chromatogram of LNCaP xenograft tumor tissue of mice treated with SENL. C, The mass spectrometric analysis of mice tumor tissue fraction 1 obtained from HPLC depicts the M ion at 488.1824 m/z, suggestive by mass alone as the sodium salt of nimbolide. D, The mass spectrometric analysis of mice tumor tissue fraction 2 obtained from HPLC depicts the M ion at 452.2195 m/z, suggestive by mass alone as the 28-deoxonimbolide. HPLC and mass spectrometric analysis was performed in duplicate, representative chromatograms were shown. The difference between the measured value for the sodium salt of nimbolide standard from the measured value in plasma and tumor tissue is less than 2.8 ppm and for 28-deoxonimbolide in the tumor tissues from the calculated value is less than 1 ppm.

PDF - 113K, Fractionation of SENL was performed using a Kinetex C18 column on a Shimadzu HPLC system. The experiments were performed in triplicate; a representative HPLC chromatogram of SENL is shown.

PDF - 103K, Molecular structures for the compounds shown in the mass spectrum are depicted.

PDF - 79K, Mass spectrometric analysis of SENL fractions 2, 3, and 4 obtained from HPLC. The total ion chromatogram retention time and the mass spectrum of individual peaks for the fractions 2, 3, and 4 are depicted in the panel A to C, respectively.

PDF file - 70K, Significance of PTGS2 methylation in different groups of prostate cancer tissues

PDF file - 172K, Prostate cancer tissues used for methylation analysis by pyrosequencing

PDF file - 419K, Significantly methylated genes in prostate cancer tissues from the binary group comparisons

PDF file - 273K, Significance of methylation in prostate cancer tissue samples from 4 microarrays with the batch effect

XLS file - 34K, Most significantly methylated genes associated with prostate cancer recurrence from the Cox univariate analysis

XLS file - 43K, Most significantly methylated genes associated with prostate cancer recurrence from the Cox multivariate model

Anterior gradient 2 (AGR2), a protein disulfide isomerase (PDI), is a well-established oncogene. Here, we found that Agr2-/- mice had a decreased fat mass and hepatic and serum lipid levels compared with their wild-type littermates after fasting, and exhibited reduced high-fat diet (HFD)-induced fat accumulation. Transgenic mice overexpressing AGR2 (Agr2/Tg) readily gained fat weight on a HFD but not a normal diet. Proteomic analysis of hepatic samples from Agr2-/- mice revealed that depletion of AGR2 impaired long-chain fatty acid uptake and activation but did not affect de novo hepatic lipogenesis. Further investigations led to the identification of several effector substrates, particularly fatty acid binding protein-1 (FABP1) as essential for the AGR2-mediated effects. AGR2 was coexpressed with FABP1, and knockdown of AGR2 resulted in a reduction in FABP1 stability. Physical interactions of AGR2 and FABP1 depended on the PDI motif in AGR2 and the formation of a disulfide bond between these two proteins. Overexpression of AGR2 but not a mutant AGR2 protein lacking PDI activity suppressed lipid accumulation in cells lacking FABP1. Moreover, AGR2 deficiency significantly reduced fatty acid absorption in the intestine, which might be resulted from decreased fatty acid transporter CD36 in mice. These findings demonstrated a novel role of AGR2 in fatty-acid uptake and activation in both the liver and intestine, which contributed to the AGR2-mediated lipid accumulation, suggesting that AGR2 is an important regulator of whole-body lipid metabolism and down-regulation of AGR2 may antagonize the development of obesity.

Jungermannenone A and B (JA, JB) are new ent-kaurane diterpenoids isolated from Chinese liverwort Jungermannia fauriana, which show anti-proliferation activities in cancer cells. In this study we investigated the mechanisms underlying the anticancer action of JA and JB in PC3 human prostate cancer cells in vitro. A panel of 9 human cancer cell lines was tested. Cell proliferation was assessed with a real-time cell analyzer and MTT assay. Cell apoptosis, cell cycle distribution and ROS levels were measured using cytometry. Mitochondrial damage was examined by transmission electron microscopy. DNA damage was detected with comet assay. Apoptotic, DNA damage- and cell cycle-related proteins were analyzed using Western blotting. The expression of DNA repair genes was measured with qRT-PCR. Both JA and JB exerted potent anti-proliferative action against the 9 cancer cell lines, and PC3 cells were more sensitive with IC50 values of 1.34±0.09 and 4.93±0.20 μmol/L, respectively. JA (1.5 μmol/L) and JB (5 μmol/L) induced PC3 cell apoptosis, which was attenuated by the caspase inhibitor Z-VAD. Furthermore, both JA and JB caused mitochondrial damage and ROS accumulation in PC3 cells, whereas vitamin C blocked the ROS accumulation and attenuated the cytotoxicity of JA and JB. Moreover, both JA and JB induced DNA damage, accompanied by downregulated DNA repair proteins Ku70/Ku80 and RDA51. JA induced marked cell cycle arrest at the G0/G1 phase, which was related to c-Myc suppression, whereas JB enforced the cell cycle blockade in the G2/M phase, which associated with activation of the JNK signaling. Both JA and JB induce prostate cancer apoptosis via ROS accumulation and induction of cell cycle arrest.

Although several mechanisms behind resistance to docetaxel in castration-refractory prostate cancer (CRPC) have been investigated, molecular determinants of evolved resistance are still not entirely understood. Proteomics-based analysis in this study revealed that SOD2, associated with downregulation of reactive oxygen species (ROS), was significantly up-regulated in docetaxel-resistant (PC3/Doc) cells if compared to sensitive cells, and the expression of redox-regulated genes such as IGF-1R, CXCR4, and BCL2 was increased as well. Forced expression of SOD2 in sensitive cells led to the increase of IGF-1R and association with drug resistance, whereas silencing of SOD2 resulted in the decrease of IGF-1R at the protein level in resistant cells. Further study revealed that SOD2 acted as a negative regulator of β-arrestin1 that is an important adaptor responsible for degradation of IGF-1R via the changes in ROS, as evidenced by observations that an antioxidant agent substantially attenuated β-arrestin1 expression in vitro and in vivo. Finally, we found that blocking of IL6 that was up-regulated in resistant cells resulted in attenuation of SOD2 and STAT3, and simultaneously in increased expression of β-arrestin1. The modulation consequently led to the decreased IGF-1R at both protein and transcription levels. Together, our data provide a novel explanation that high level of IL6 stimulated SOD2 expression that, at least partially, contributed to the low level of ROS that would likely result in a sustained increase in the expression of IGF-1R through abolishment of β-arrestin1 in docetaxel resistant cells.

Retigeric acid B (RB) has been reported to exhibit its anti-tumor activity in vitro and in vivo. Here, we found that RB significantly inhibited activity of topoisomerase IIα (Topo IIα), leading to remarkable DNA damage in prostate cancer (PCa) cells as evidenced by a strong induction of γH2AX and DNA fragmentation. Activation of ATM and ATR sequentially led to induction of phospho-Chk1/2 and phospho-Cdc25 in response to RB. Blockade of ATM/ATR signaling resulted in the attenuation of RB-induced γH2AX, and partially rescued RB-mediated cell death. RB treatment also resulted in inactivation of DNA repair proteins such as phospho-BRCA1, impairment of HR, and NHEJ repair as indicated by DNA end-joining assays. Meanwhile, a stress-responsive gene activating transcription factor 3 (ATF3) was noted for its predominant expression in response to RB-induced DNA damage. Knockdown of ATF3 inhibited the RB-induced expression changes of cell cycle- and apoptosis-related genes such as DR5, DDIT4, CDC25A, GADD45A, and partially blocked RB-mediated inhibition on cell proliferation and induction of apoptosis, suggesting the crucial involvement of ATF3 in this event. Microarray data displayed that RB caused changes of genes required for damaged-DNA binding and repair, as well as ATF3 and its target genes. Our data firstly demonstrated that RB was a novel DNA Topo II inhibitor and triggered cell death by inducing DNA damage and stress-response, suggesting a promising anticancer agent.

Advanced prostate cancer has significant long-term morbidity, and there is a growing interest in alternative and complimentary forms of therapy that will improve the outcomes of patients. Azadirachta indica (common name: neem) contains multiple active compounds that have potent anti-inflammatory and anticancer properties. The present study investigates the novel targets of the anticancer activity of ethanol extract of neem leaves (EENL) in vitro and evaluates the in vivo efficacy in the prostate cancer models. Analysis of the components in the EENL by mass spectrometry suggests the presence of 2′,3′-dehydrosalannol, 6-desacetyl nimbinene, and nimolinone. Treatment of C4-2B and PC-3M-luc2 prostate cancer cells with EENL inhibited the cell proliferation. Genome-wide expression profiling, using oligonucleotide microarrays, revealed genes differentially expressed with EENL treatment in prostate cancer cells. Functional analysis unveiled that most of the up-regulated genes were associated with cell death, and drug metabolism, and the down-regulated genes were associated with cell cycle, DNA replication, recombination, and repair functions. Quantitative PCR confirmed significant up-regulation of 40 genes and immunoblotting revealed increase in the protein expression levels of HMOX1, AKR1C2, AKR1C3, and AKR1B10. EENL treatment inhibited the growth of C4-2B and PC-3M-luc2 prostate cancer xenografts in nude mice. The suppression of tumor growth is associated with the formation of hyalinized fibrous tumor tissue and the induction of cell death by apoptosis. These results suggest that EENL-containing natural bioactive compounds could have potent anticancer property and the regulation of multiple cellular pathways could exert pleiotrophic effects in prevention and treatment of prostate cancer. Electronic supplementary material The online version of this article (doi:10.1208/s12248-011-9279-4) contains supplementary material, which is available to authorized users.

To examine whether two naturally occurring sesquiterpenoids (ST1 and ST2) with anti-proliferative activity in prostate cancer cells inhibit androgen receptor (AR) signaling.Human prostate cancer cell lines LNCaP and PC3 were used. The expression of AR, AR translocation into the nucleus, and expression levels of AR coactivators ARA70 and steroid receptor coactivator-1 (SRC-1) in LNCaP cells were examined using real-time PCR and Western blot. Changes in prostate-specific antigen (PSA) protein levels, PSA promoter activity, and androgen response element (ARE)-mediated reporter gene activity were examined using enzyme-linked immunoabsorbent assay (ELISA) and transient transfection assays. Co-immunoprecipitation was performed to analyze the interaction between AR and the AR coactivators in ST1- and ST2-treated cells.In LNCaP cells, ST1 and ST2 (40 μmol/L) led to a significant decrease in the expression of AR as well as a reduction of AR translocation into the nucleus, but had no effect on AR protein translation. ST1 and ST2 treatment also resulted in a significant decrease in the level of PSA protein secreted into the medium and was able to suppress PSA promoter-dependent and ARE-dependent luciferase activity. Furthermore, decreased expression of ARA70 and SRC-1 was observed when LNCaP cells were exposed to ST1 and ST2, which interfered with their ability to interact with AR.The observations suggest that suppression of AR transactivation by ST1 and ST2 may be mediated, in part, by inhibiting AR nuclear translocation and/or interfering with the interaction between AR and its coactivators ARA70 and SRC-1. Therefore, sesquiterpenoids could be developed as novel therapeutic agents for treating prostate cancer.

We previously demonstrated that ectopic expression of neurotrophic peptide (NP) derived from saposin C promotes androgen receptor (AR) expression and transactivation in human prostate cancer cells. This prompted us to investigate how NP or saposin C can function in cells. We constructed plasmids expressing saposin C or a chimeric peptide of a viral TAT transduction domain and saposin C (TAT-saposin C) with His-tag. Intracellular localization of saposin C and NP was predominantly shown in transfected cells, while TAT-saposin C was detected around membrane and in cytosol by immunofluorescence staining. Furthermore, induction of the AR expression and activation of the AR transcriptional function were observed in cells transfected with saposin C or TAT-saposin C, compared to control cells transfected with an empty plasmid. The effects of saposin C and TAT-saposin C on AR activity were examined in the presence of inhibitors of GPCR, MAPK1/2, and PI3K/Akt. Interestingly, we found that these inhibitors only affect AR activities in cells with TAT-saposin C expression but not with saposin C expression. Immunostaining images showed that co-localization of saposin C, Src, and the AR occurred in transfected cells. Physical interactions of saposin C/NP, Src, and the AR were then demonstrated by co-immunoprecipitation assays. Blockage of Src activity by specific inhibitor led to a decrease in the saposin C-mediated enhancement of AR transactivity, suggesting that intracellular expression of saposin C caused stimulation of AR expression and activity by associations with Src in LNCaP cells. This effect may not be mediated by GPCR. J. Cell. Biochem. 112: 818–828, 2011. © 2010 Wiley-Liss, Inc.

Retigeric acid B (RB), a naturally occurring pentacyclic triterpenic acid, has been noted for its antifungal properties in vitro. Here, we observed that RB inhibited prostate cancer cell proliferation and induced cell death in a dose-dependent manner, but exerted very little inhibitory effect on noncancerous prostate epithelial cell viability. Treatment of androgen-independent PC-3 cells with RB caused a moderate increase in p21(Cip1), and enforced the cell cycle arrest in the S phase. A block of S phase was accompanied with decreases in cyclin B, and increases in cyclin E and cyclin A proteins and phosphorylated retinoblastoma protein (pRb), whereas the expression of cdk2 remained almost unchanged in PC-3 cells exposed to RB. Moreover, RB significantly inhibited DNA synthesis with a dose-dependent reduction in the incorporation of BrdU into DNA, and enhanced apoptosis of PC-3 cells with induction of a higher ratio of Bax/Bcl-2 proteins, and activation of caspase-3 which, in turn, promoted the cleavage of poly (ADP-ribose) polymerase (PARP). However, pretreatment with the pan-caspase inhibitor z-VAD-fmk only partially alleviated RB-triggered apoptosis in PC-3 cells, suggesting the involvement of both caspase-dependent and caspase-independent pathways. Additionally, treatment of androgen-sensitive LNCaP cells with RB led to a reduction in the expression of androgen receptor (AR), and subsequently decreased the transactivity of AR. These observations help to support the search for promising candidates to treat prostate cancer.

Androgen deprivation therapy has been the major treatment for advanced prostate cancer (PCa) and has shown to prolong life. However, remissions are temporary and patients almost inevitably progress to become castration-resistant prostate cancer (CRPC). CRPC is almost incurable even when treated with docetaxel that may have a slight life prolonging effect on CRPC patients. Interestingly, most of CRPC still express androgen receptor (AR) and depend on the AR for growth. Recently it has been suggested that AR may act as a tumor suppressor in normal prostatic epithelial cells, while in PCa cells AR becomes oncogenic, even under androgen deprivation states. The mechanisms for the latter are still under intensive investigations. A number of studies showed that, in fact, AR signaling is increased under an androgen-depleted environment. The mechanisms suggested in these studies including AR mutations, AR overexpression by gene amplification and other mechanisms that allow activation by low androgen levels or by other endogenous steroids, increased local de novo synthesis of androgens will be discussed. Moreover, developments and tests in clinical trials in CRPC of a number of novel agents interrupting AR signaling mediated PCa growth will also be discussed.

We have previously reported that the increase in c-Jun expression induced by quercetin inhibited androgen receptor (AR) transactivation, and Sp1 was involved in quercetin-mediated downregulation of AR activity. Transient transfection assays in this work revealed that co-expression of c-Jun quenched Sp1-induced production of luciferase activity driven by AR promoter or three copies of Sp1 binding elements in the AR promoter. Moreover, c-Jun repressed AR-mediated luciferase activity via androgen-response elements (AREs) of the hK2 gene, while this suppression could be restored partially by cotransfection of Sp1 expression plasmid. The physical associations of c-Jun, Sp1, and AR induced by quercetin were further demonstrated by co-immunoprecipitation experiments. In addition, quercetin-mediated repression of AR expression and activity was partially reversed by blocking of JNK signaling pathway. These results suggested that c-Jun might play an important role in the suppression of AR expression and activity in the presence of quercetin, and association of a c-Jun/Sp1/AR protein complex induced by quercetin represented a novel mechanism that was involved in down-regulation of the AR function in prostate cancer cells.

BACKGROUND Hypermethylation of CpG islands is a common epigenetic alteration associated with cancer. Tumor suppressor genes retinoic acid receptor beta (RARβ) and PDLIM4 are hypermethylated and silenced in prostate cancer (PCa) tissues and PCa cell lines compared to normal prostate cells. METHODS In this study, a benign prostate epithelial cell line RWPE1 was used as a model to study the epigenetic regulation of Myc on the RARβ and PDLIM4 promoters. Forced Myc overexpression inhibited the RARβ and PDLIM4 expression. RESULTS Pyrosequencing study showed that Myc overexpression increased methylation in several CpG sites of both promoters. A DNA methylation inhibitor 5-aza-2′-deoxycytidine reversed the epigenetic alteration effect of Myc on both RARβ and PDLIM4. CONCLUSION The epigenetic regulation of Myc may be related to its up-regulation of the DNA methyltransferase DNMT3a and DNMT3b. Prostate 69: 1643–1650, 2009. © 2009 Wiley-Liss, Inc.

This study was undertaken to investigate the mechanism by which phenethyl isothiocyanate (PEITC), a natural compound from cruciferous vegetables, exhibits antitumor effect on prostate cancer cells. Cell proliferation, cell cycle, western blot, gene transfer and reporter assays were used to test the effects of PEITC on the growth and IL6/JAK/STAT3 pathway in prostate cancer. The result showed that PEITC significantly inhibited DU145 cell proliferation in a dose-dependent manner and induced the cell arrest at G2-M phase. PEITC inhibited both constitutive and interleukin 6 (IL-6)-induced STAT3 activity in DU145 cells. IL-6-stimulated phosphorylation of JAK2, an STAT3 upstream kinase, was also attenuated by PEITC. Moreover, an antioxidant reagent, N-acetyl-l-cysteine (NAC) which suppresses reactive oxygen species (ROS) generation, reversed the early inhibitory effects of PEITC on cell proliferation, constitutive or IL-6-mediated JAK-STAT3 phosphorylation in PCa cells. Taken together, our data demonstrated that PEITC can inhibit the activation of the JAK-STAT3 signal-cascade in prostate cancer cells and the underlying mechanism may be partially involved with blocking cellular ROS production during the early stage of the signaling activation by IL-6.

Aberrant activation of the androgen receptor (AR) by the ErbB2/ErbB3 heterodimer contributes to the development of hormone resistance in prostate cancer. EBP1, an ErbB3-binding protein, acts as an AR corepressor. As EBP1 is decreased in preclinical models of hormone-refractory prostate cancer, we studied the expression of EBP1 in human prostate cancer. We found that the expression of the EBP1 gene was significantly decreased in prostate cancer tissues compared with benign prostate at both mRNA and protein levels. Restoration of EBP1 expression in the hormone-refractory LNCaP C81 cell line led to an amelioration of the androgen-independent phenotype based on established biological criteria and a reduction in the expression of a cohort of AR target genes. The ability of the ErbB3 ligand heregulin (HRG) to stimulate growth and AKT phosphorylation of hormone-refractory prostate cancer cells was abolished. Abrogation of EBP1 expression by short hairpin RNA in hormone-dependent LNCaP cells, which undergo apoptosis in response to HRG, resulted in HRG-stimulated cell growth. Restoration of EBP1 expression decreased the tumorigenicity of C81 xenografts in female mice, whereas elimination of EBP1 expression enhanced the ability of LNCaP cells to grow in female mice. Our data support a role for EBP1 in the development of hormone-refractory prostate cancer via inhibition of both AR- and HRG-stimulated growth and present a novel strategy for treating androgen-refractory prostate cancer. [Mol Cancer Ther 2008;7(10):3176–86]

Androgen receptor (AR)-mediated signaling is crucial for the development and progression of prostate cancer (PCa). Naturally occurring phytochemicals that target the AR signaling offer significant protection against this disease. Acetyl-11-keto-beta-boswellic acid (AKBA), a compound isolated from the gum-resin of Boswellia carterii, caused G1-phase cell cycle arrest with an induction of p21(WAF1/CIP1), and a reduction of cyclin D1 as well in prostate cancer cells. AKBA-mediated cellular proliferation inhibition was associated with a decrease of AR expression at mRNA and protein levels. Furthermore, the functional biomarkers used in evaluation of AR transactivity showed suppressions of prostate-specific antigen promoter-dependent and androgen responsive element-dependent luciferase activities. Additionally, down-regulation of an AR short promoter mainly containing a Sp1 binding site suggested the essential role of Sp1 for the reduction of AR expression in cells exposed to AKBA. Interruption effect of AKBA on Sp1 binding activity but not Sp1 protein levels was further confirmed by EMSA and transient transfection with a luciferase reporter driven by three copies of the Sp1 binding site of the AR promoter. Therefore, anti-AR properties ascribed to AKBA suggested that AKBA-containing drugs could be used for the development of novel therapeutic chemicals.

Chemoprevention is presumably one of most effective means to combat prostate cancer (PCa). Patients usually require more than a decade to develop a clinically significant Pca, therefore, an ideal target for chemoprevention. This review will focus on recent findings of a group of naturally occurring chemicals, carotenoids, for potential use in reducing PCa risk.

By means of protein expression profile, mass spectral and/or RT-PCR analyses we found for the first time IgG Fc binding protein (Fc gammaBP), distinct from Fc gamma receptors is expressed in both human and mouse prostates. There is a strong correlation between downregulation of Fc gammaBP and the progression of prostate cancer in Transgenic Adenocarcinoma Mouse Prostate (TRAMP) mice. Fc gammaBP gene expression is significantly reduced or lost in human prostate tumor tissues and prostate cell lines. The differentially expressed Fc gammaBP could reflect its potential role in prostate malignancy as well as an indicator for progression of the cancer.

Aim: To determine the effects of the functional domain of saposin C (neurotrophic peptide [NP]) on androgen receptor (AR) expression and transcriptional activity. Methods: We constructed DNA vectors expressing NP or a chimeric peptide of the viral TAT transduction domain and NP (TAT-NP) using gene cloning technology. The effects of ectopic expression of NP or TAT-NP on cell growth were examined by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) assay. Reverse transcription-polymerase chain reaction (RT-PCR), Western blot, transient transfection and reporter gene assays were used to determine the effects of NP on AR expression and activation. Results: NP stimulated proliferation of androgen responsive LNCaP cells in the absence of androgens. RT-PCR and Western blot analyses showed that ectopic expression of NP resulted in induction of AR gene expression, and that the NP-stimulated expression of AR could be synergistically enhanced in the presence of androgens. Furthermore, reporter gene assay results showed that NP could enhance AR transactivation by increasing androgen-inducible gene reporter activity. Conclusion: We provided evidence that ectopic expression of saposin C-originated NP could upregulate AR gene expression and activate the AR transcriptional function in an androgen-independent manner in prostate cancer cells.

It has been suggested that interleukin-6 (IL-6) may modulate androgen receptor (AR) action to accelerate prostate cancer (PCa) progression. Selenium compounds are highly recommended as a promising chemopreventive agent for PCa. This study was to determine if selenium can repress IL-6 mediated AR action in PCa progression.Cell proliferation, prostate-specific antigen, gene transfer, and Western blot assays were used to study the effects of sodium selenite and methylseleninic acid on IL-6 mediated AR action on an AR expressing human prostate cancer cell line, LNCaP.We found that sodium selenite, but not methylseleninic acid, significantly (p0.05) inhibited IL-6-induced trans-activating activity of AR and cell proliferation in LNCaP cells. Interestingly, although sodium selenite did not show effect on activation of both STAT3 and ERK1/2 in the presence of IL-6, an increased expression of c-Jun was detected in cells after treatment with sodium selenite. Indeed, we showed overexpression of c-Jun blocked IL-6-induced AR activation.Taken together, our results suggest that sodium selenite not methylseleninic acid can inhibit IL-6-mediated AR activation by increased c-Jun in LNCaP cells. Sodium selenite may be a proper selenium form for further testing its potency on intervening IL-6-mediated PCa progression.

Aim: To elucidate the effect and the mechanisms of curcumin on the expression of the human homeobox gene NKX3.1 in the prostate cancer cell LNCaP. Methods: The expression change of NKX3.1 in cells incubated with varying concentrations of curcumin was observed by Western blotting and RT-PCR. A dual luciferase reporter assay was used to test the effect of curcumin on the activity of the NKX3.1 1040 bp promoter. Curcumin-treated cells disposed to a designated amount of androgen analog R1881 and the androgen receptor (AR) antagonist flutamide, then the expression of NKX3.1 or the activity of the NKX3.1 promoter were investigated by Western blotting or reporter gene assay, respectively. Finally, Western blotting and electrophoretic mobility shift assay were performed to demonstrate the effect of curcumin on the expression of AR and its binding activity to the androgen response element (ARE). Results: Curcumin downregulated the expression of NKX3.1 and the activity of the NKX3.1 1040 bp promoter in LNCaP cells. R1881 increased the expression of NKX3.1 , and the AR antagonist flutamide decreased the expression of NKX3.1 in LNCaP cells, while curcumin could inhibit androgen-AR mediated induction of NKX3.1 expression. Curcumin decreased the expression of AR and the binding activity to ARE directly. Conclusion: Curcumin could downregulate NKX3.1 expression in LNCaP cells. It could also inhibit the androgen-AR mediated induction of NKX3.1 expression by downregulating AR expression and blocking its DNA binding activity.

Bisbibenzyl compounds have gained our interests for their potential antitumor activity in malignant cell-types.The objective of this study is to investigate the effect of bisbibenzyl compounds riccardin C (RC), marchantin M (MM), and riccardin D (RD) on androgen receptor (AR) in prostate cancer (PCa) cells.After exposure to 10 μM of the compounds for 24 h, cell cycle and cell survival analyses were performed using FACS and MTT assay to confirm the effect of these bisbibenzyls on PCa LNCaP cells. Changes in the AR expression and function, as the result of exposure to the compounds, were investigated using real-time PCR, ELISA, transient transfection, western blotting (WB), immunoprecipitation, and immunofluorescence staining (IF). Chemical-induced autophagy was examined by WB, IF, and RNAi.RC, MM, and RD reduced the viability of LNCaP cells accompanied with arrested cell cycle in the G0/G1 phase and induction of apoptosis. Further investigation revealed that these compounds significantly inhibited AR expression at mRNA and protein levels, leading to the suppression of AR transcriptional activity. Moreover, inhibition of proteasome activity by bisbibenzyls, which in turn caused the induction of autophagy, as noted by induction of LC3B expression, conversion, and accumulation of punctate dots in treated cells. Co-localization of AR/LC3B and AR/Ub suggested that autophagy contributed to the degradation of polyubiquitinated-AR when proteasome activity was suppressed by the bisbibenzyls.Suppression of proteasome activity and induction of autophagy were involved in bisbibenzyl-mediated modulation of AR activities and apoptosis, suggesting their potential in treating PCa.

We have recently identified ZNF185 as a gene that is downregulated in prostate cancer (PCa), in part via epigenetic alteration, and maybe associated with disease progression. In this study, we cloned the ZNF185 cDNA from normal human prostate tissues and investigated its biological function. We show that ZNF185 is a novel actin-cytoskeleton-associated Lin-l 1, Isl-1 and Mec-3 (LIM) domain-containing protein that localizes to F-actin structures, and is enriched at focal adhesions. We find that the NH(2)-terminal region, which we designate the actin-targeting domain, facilitates ZNF185 binding to actin in vitro and is both necessary and sufficient to mediate actin-cytoskeleton targeting of ZNF185, whereas the LIM domain, which is localized in the COOH-terminus is dispensable for this phenomenon. Interestingly, ectopic expression of full-length ZNF185, but not a mutant lacking the actin-targeting domain, could suppress proliferation and anchorage-independent growth of PCa cells. Together, our data suggest that ZNF185 may function as a tumor-suppressor protein by associating with the actin-cytoskeleton.

Perillyl alcohol is a hydroxylated monocyclic monoterpene. In animal study, monoterpene has shown to have an anti-tumor effect. The aim of this study is to evaluate whether POH plays an important role in the development and progression of prostate cancer (pCa). We treated LNCaP cells with different concentrations of perillyl alcohol (POH). First of all, we performed cell proliferation assay and prostate-specific antigen (PSA) and human glandular kallikrein (hK2) quantification assays. LNCaP cells were treated with or without POH for Western blot analysis of androgen receptor (AR) and c-Jun. Finally, we performed transient transfection assay by transfecting LNCaP cells-which were treated with or without POH-with pGL-3 luciferase vector containing PSA promoter and AR promoter. We observed inhibition of the expression and function of the AR by POH, through inhibition of androgen-induced cell growth and androgen-stimulated secretion of prostate-specific antigen and hK2, in human pCa cell line LNCaP. In addition, we demonstrated, for the first time, that POH inhibits the transcription activities of the AR gene promoter by over-expression of c-Jun protein. These novel properties of POH strongly suggest that POH could be highly useful for intervention of pCa.

PURPOSE: We analyzed the expression of genes to identify reliable molecular markers in the diagnosis and progression of prostate cancer. EXPERIMENTAL DESIGN: Gene expression profiling was done using HG-U133 set microarrays in 32 prostate cancer and 8 benign tissues of patients with cancer. Expression levels of 11 genes were selected for quantitative real-time PCR evaluation in 52 prostate cancer and 20 benign tissues. Further, to assess transcriptional inactivation, we analyzed the promoter methylation of genes by quantitative methylation-specific PCR in 62 tumor and 36 benign tissues. RESULTS: Our results showed a significant down-regulation in the mRNA expression levels of PRIMA1, TU3A, PDLIM4, FLJ14084, SVIL, SORBS1, C21orf63, and KIAA1210 and up-regulation of FABP5, SOX4, and MLP in prostate cancer tissues by TaqMan real-time PCR. Quantitative methylation-specific PCR of PDLIM4, SVIL, PRIMA1, GSTP1, and PTGS2 detected prostate carcinoma with a sensitivity of 94.7%, 75.4%, 47.4%, 89.5%, and 87.7%, and a specificity of 90.5%, 75%, 54.2%, 95.8%, and 90.2%, respectively. Using this panel of methylation markers in combination, we were able to distinguish between prostate cancer and adjacent benign tissues with sensitivities and specificities of about 90% to 100%. Our data provide evidence of transcriptional repression of the putative tumor suppressor gene PDLIM4 by hypermethylation. CONCLUSIONS: Our analysis revealed differential expression of eight down-regulated and three up-regulated genes, implicating their role in prostate cancer development and progression. We further showed that the hypermethylation of PDLIM4 gene could be used as a sensitive molecular tool in detection of prostate tumorigenesis.

Accumulating evidence suggests that the androgen receptor (AR) may play an important role in the development and progression of prostate cancer. To find new, useful compounds that effectively may attenuate the function of AR in prostate cancer cells, the authors investigated the effect of gum mastic, a natural resin, on AR activity. An androgen-responsive prostate cancer cell line LNCaP was used as a model for this study. Gene transfer, reverse transcriptase-polymerase chain reaction analysis, electrophoretic mobility shift assay, and Western blot analysis were used to test the effect of gum mastic on the expression and function of the AR. To demonstrate the inhibitory effect of gum mastic on the function of the AR, the expression of androgen-regulated genes, including prostate-specific antigen (PSA), human kallikrein 2 (hK2), and NKX3.1 were measured. In addition, transient transfection assays with the PSA promoter and the AR promoter also were used to test the effects of mastic. The results showed that gum mastic inhibited the expression of the AR at the transcriptional level, resulting in the down-regulation of both AR messenger RNA and protein levels. Therefore, the function of the AR was inhibited, as reflected by the reduced expression of NKX3.1 and PSA and by androgen-stimulated growth. Because gum mastic exhibited a strong in vitro potency to attenuate the expression and function of the AR, further investigation will be required to determine whether this naturally occurring substance has in vivo potency to inhibit prostate cancer development.

AGR2, the human homologue of Xenopus anterior gradient 2 (XAG2), was identified by a suppression subtractive hybridization-based technique as an androgen-inducible gene. There are two AGR2 transcripts, which encode the same secretory protein of 175 amino acids. The androgen induction was time- and dose-dependent, with more than a 10-fold increase in the level of AGR2 mRNA after 48 hr of treatment with 10(-9) M R1881. Expression of AGR2 mRNA was specifically detected in limited human tissue rich in epithelial cells, including the prostate gland. Analysis of 46 microdissected primary prostate adenocarcinoma samples showed that AGR2 mRNA expression was markedly elevated in the majority of tumors as compared to matched adjacent benign tissues. Androgen-induced AGR2 protein expression was demonstrated in LNCaP cells by Western blot analysis with an anti-AGR2 antibody. Immunohistochemistry analysis indicated that AGR2 protein expression was highly restricted to the secretory epithelial cells in the prostate gland. In tissue sections from radical prostatectomy specimens, immunohistochemical staining of AGR2 showed markedly increased expression in high-grade prostatic intraepithelial neoplasia and Gleason pattern 3-4 prostatic adenocarcinoma. Therefore, the androgen-induced secretory protein AGR2 may serve as a potential therapeutic target and/or molecular marker for prostate cancer.

It has been strongly suggested that androgens and the cognate receptor (AR) may play important roles in the development and progression of prostate cancer. The AR is a transcription factor consisting of three major domains, i.e., N-terminal transactivation, middle DNA binding, and C-terminal steroid binding domains. Molecular events of androgen induced activation of the AR include conformation change, phosphorylation, acetylation, genomic DNA binding, and co-regulator recruiting. Many of these events can be manipulated in certain prostate cancer cells in favor of their progression. Dietary compounds and certain herbs have recently drawn a great deal of attention because of their relevance to development of several cancers including prostate cancer. We discuss in depth the findings from our and other laboratories of effects of dietary factors or herbs on the function of the AR and potential mechanisms on expression of the AR and AR regulated genes. We further discuss the potential implication of these dietary chemicals on prevention of development and progression of prostate cancer.

Nkx3.1 is a prostate-specific homeobox gene related strongly to prostate development and prostate cancer. To study its regulation of transcription, 1.06 kb 5′ flanking region of Nkx3.1 gene and its 5′ deletion mutants (861, 617, 417 and 238 bp) were obtained by PCR and cloned into pGL 3 -basic, a promoter-less luciferase reporter vector, to examine their promoter activities driving the reporter gene transcription. pRL-TK, a Renilla luciferase reporter vector was used as internal control, and pGL 3 -control and pGL 3 -basic were used as positive and negative control respectively. The promoter activities were determined by dual-luciferase reporter assay 48 h after pGL 3 constructs were cotransfected with pRL-TK into prostate cancer cell LNCaP. The results showed that dual-luciferase reporter assay (M 1 /M 2 ) of pGL 3 -1.06 kb cotransfection with pRL-TK was 2.7, which was about 1.5-fold higher than that of pGL 3 -control cotransfection with pRL-TK and 50-fold higher than that of pGL 3 -basic cotransfection with pRL-TK. The results also showed that the relative activities (M 1 /M 2 ) were 0.71, 0.84, 0.44 and 2.07 respectively for pGL 3 -861 bp, pGL 3 -617 bp, pGL 3 -417 bp, pGL 3 -238 bp, the last one still had 80% promoter activity compared with pGL 3 -1.06 kb, which showed that deletion from 1.06 kb to 238 bp had small effects on promoter activity. The conclusion was that the 238 bp fragment containing a TATA box and two CAAT boxes had strong promoter activity. However, the deletion from 1.06 kb to 861 bp reduced activity 3.8-fold while the deletion from 417 bp to 238 bp enhanced activity 4.7-fold, which indicated that these deleted sequences might contain some important positive or negative regulatory elements. It will be important to identify the elements within the Nkx3.1 promoter that contribute to regulation of the gene transcription in the future studies.

Azadirachta indica, commonly known as neem, has gained worldwide prominence because of its medical properties, namely antitumor, antiviral, anti-inflammatory, antihyperglycemic, antifungal, and antibacterial activities. Despite these promising results, gaps remain in our understanding of the molecular mechanism of action of neem compounds and their potential for use in clinical trials. We investigated supercritical extract of neem leaves (SENL) for the following: molecular targets in vitro, in vivo efficacy to inhibit tumor growth, and bioactive compounds that exert antitumor activity. Treatment of LNCaP-luc2 prostate cancer cells with SENL suppressed dihydrotestosterone-induced androgen receptor and prostate-specific antigen levels. SENL inhibited integrin β1, calreticulin, and focal adhesion kinase activation in LNCaP-luc2 and PC3 prostate cancer cells. Oral administration of SENL significantly reduced LNCaP-luc2 xenograft tumor growth in mice with the formation of hyalinized fibrous tumor tissue, reduction in the prostate-specific antigen, and increase in AKR1C2 levels. To identify the active anticancer compounds, we fractionated SENL by high-pressure liquid chromatography and evaluated 16 peaks for cytotoxic activity. Four of the 16 peaks exhibited significant cytotoxic activity against prostate cancer cells. Mass spectrometry of the isolated peaks suggested the compounds with cytotoxic activity were nimbandiol, nimbolide, 2′,3′-dihydronimbolide, and 28-deoxonimbolide. Analysis of tumor tissue and plasma samples from mice treated with SENL indicated 28-deoxonimbolide and nimbolide as the bioactive compounds. Overall, our data revealed the bioactive compounds in SENL and suggested that the anticancer activity could be mediated through alteration in androgen receptor and calreticulin levels in prostate cancer. Mol Cancer Ther; 13(5); 1067–77. ©2014 AACR.

Chemoprevention is presumably one of most effective means to combat prostate cancer (PCa). Patients usually require more than a decade to develop a clinically significant PCa, therefore, an ideal target for chemoprevention. This review will focus on recent findings of a group of naturally occurring chemicals, carotenoids, for potential use in reducing PCa risk.

There is some epidemiological support for a protective influence of omega-3 fatty acids against prostate cancer. We wanted to explore whether omega-3 polyunsaturated fatty acids such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) can affect androgen receptor function in prostate cancer cells. Our study showed that both DHA and EPA inhibit androgen-stimulated cell growth. Androgenic induction of prostate-specific antigen (PSA) protein was repressed by DHA and EPA in a dose-dependent manner. The mRNA levels of five androgen up-regulated genes, PSA, ornithine decarboxylase, NKX 3.1, immunophilin fkbp 51 and Drg-1, were decreased with DHA treatment in the presence of androgens. Transfection experiments using a DNA vector containing androgen-responsive elements demonstrated that both DHA and EPA could interfere with transactivation activities of the androgen receptor (AR). However, western blot analysis of AR protein showed that DHA and EPA treatments did not change AR expression levels. Interestingly, the proto-oncoprotein c-jun was increased by DHA treatment. A transient transfection found that forced expression of c-jun inhibited AR transactivation activity. Thus, this study found that the inhibitory effects of omega-3 polyunsaturated fatty acids on AR-mediated actions are due, at least in part, to an increase in c-jun protein.

Tissue-specific transcriptional regulatory elements can increase the safety of gene therapy vectors. Unlike prostate-specific antigen (PSA/hK3), whose expression displays an inverse correlation with prostate cancer grade and stage, human glandular kallikrein 2 (hK2) is upregulated in higher grade and stage disease. Therefore, our goal was to develop a strong and prostate-specific hK2-based promoter for targeted gene therapy. We identified the minimum "full-strength" hK2 enhancer and built transcriptional regulatory elements composed of multiple tandem copies of this 1.2-kb enhancer, fused to the hK2 minimal promoter. Relative to the weak induction of the minimal hK2 promoter by androgen analog (R1881) in androgen receptor (AR)-positive LNCaP cells, transcriptional activity was increased by 25-, 44-, 81-, and 114-fold when one to four enhancers were spliced to the hK2 promoter, respectively. In contrast, the enhancer/promoter elements were inactive in the AR(-) prostate cancer line PC-3 and in a panel of nonprostate lines, including 293, U87, MCF-7, HuH-7, and HeLa cells. Furthermore, we generated a recombinant adenovirus, ADV.hK2-E3/P-EGFP, expressing enhanced green fluorescent protein (EGFP) under the control of the hK2 triplicate enhancer/promoter, and compared its properties with ADV.CMV-EGFP expressing EGFP under the control of the cytomegalovirus (CMV) enhancer/promoter. Unlike the CMV promoter, the hK2-E3/P promoter was at least 100-fold inducible by R1881 in the adenoviral backbone. Compared with in situ injection of subcutaneous LNCaP tumors with ADV.CMV-EGFP, which led to detectable EGFP expression in tumor, liver, and brain tissue, ADV.hK2-E3/P-EGFP injection led to robust but tumor-restricted EGFP expression. These results suggest that the hk2 multienhancer/promoter should be a powerful novel reagent for safer targeted gene therapy of prostate cancer.

Objectives. Human glandular kallikrein 2 (hK2) and prostate-specific antigen (PSA) are members of a multigene family of serine proteases that share approximately 80% sequence homology. Both are expressed in the prostate epithelium, are under androgen regulation, are present in serum and seminal fluid, and can form complexes with endogenous protease inhibitors (eg, alpha2-macroglobulin and alpha1-antichymotrypsin). Differences in immunohistochemistry and substrate specificity suggest hK2 may provide unique information for early detection and characterization of prostate cancer. Methods. Nine hundred thirty-seven archived serum samples from men treated at two academic institutions were studied. All men underwent biopsy, had a histologically confirmed diagnosis of cancer or noncancer, and a total PSA level greater than 2 ng/mL. Samples were tested in Hybritech’s Tandem-R PSA and Tandem-R free PSA (fPSA) assays and a research prototype assay for total hK2 (thK2). Results. The thK2/fPSA ratio provided additional specificity for cancer detection over PSA and the percentage of fPSA (%fPSA). A model for cancer detection using %fPSA and the thK2/fPSA ratio when PSA is 2 to 4 ng/mL is proposed that would identify as many as 40% of the cancers and would require biopsy in only 16.5% of the men in this PSA range. Conclusions. In this study, %fPSA and thK2/fPSA provided unique information for prostate cancer detection and increased the specificity of cancer detection.