Benzyldihydroxyoctenone,a novel anticancer agent,induces apoptosis via mitochondrial-mediated pathway in androgen-sensitive LNCaP prostate cancer cells

This study was aimed to evaluate detailed mechanisms on the apoptotic induction of benzyldihydroxyoctenone, a novel compound isolated from Streptomyces sp. KACC91015, in androgen-sensitive LNCaP prostate cancer cells. Benzyldihydroxyoctenone, designated as F3-2-5 in the current study, caused accumulation of apoptotic sub-G₁ phase in the flow cytometric analysis using propidium iodide staining. Moreover, the typical apoptotic DNA fragmentation of the LNCaP cells treated with 30 μM of F3-2-5 was confirmed using the TUNEL assay. This apoptotic induction of F3-2-5 in the LNCaP cells was associated with the cytochrome c release from mitochondria to cytosol, and the activation of procaspase-8, -9, and -3, as well as the specific proteolytic cleavage of poly(ADP-ribose) polymerase (PARP). In addition, F3-2-5 treatment caused the down-regulation of the antiapoptotic protein, such as Bcl-2 and Bcl-X_L, but the proapoptotic protein, such as Bax, was not influenced. To investigate whether apoptotic induction by F3-2-5 is also due to the down-regulation of androgen receptor (AR), Western blot analysis and quantitative RT-PCR were conducted in F3-2-5-treated LNCaP prostate cancer cells. We found that F3-2-5 significantly inhibited the expression levels of AR and prostate-specific antigen (PSA) proteins in a time-dependent manner, as well as F3-2-5 abrogated the up-regulation of AR and PSA genes with and without DHT. Therefore, F3-2-5 has been shown to be an androgen antagonist, suggesting that F3-2-5 could be a potent agent for the treatment of both androgen-dependent and hormone-refractory prostate cancer.     

Conversion of Androgen Receptor Signaling From a Growth Suppressor in Normal Prostate Epithelial Cells to an Oncogene in Prostate Cancer Cells Involves a Gain of Function in c-Myc Regulation

In normal prostate, androgen-dependent androgen receptor (AR) signaling within prostate stromal cells induces their secretion of paracrine factors, termed “andromedins” which stimulate growth of the epithelial cells. The present studies demonstrate that androgen-dependent andromedin-driven growth stimulation is counter-balanced by androgen-induced AR signaling within normal adult prostate epithelial cells resulting in terminal G₀ growth arrest coupled with terminal differentiation into ΔNp63-negative, PSA-expressing secretory luminal cells. This cell autonomous AR-driven terminal differentiation requires DNA-binding of the AR protein, is associated with decreases in c-Myc m-RNA and protein, are coupled with increases in p21, p27, and SKP-2 protein expression, and does not require functional p53. These changes result in down-regulation of Cyclin D₁ protein and RB phosphoryation. shRNA knockdown documents that neither RB, p21, p27 alone or in combination are required for such AR-induced G₀ growth arrest. Transgenic expression of a constitutive vector to prevent c-Myc down-regulation overrides AR-mediated growth arrest in normal prostate epithelial cells, which documents that AR-induced c-Myc down-regulation is critical in terminal growth arrest of normal prostate epithelial cells. In contrast, in prostate cancer cells, androgen-induced AR signaling paradoxically up-regulates c-Myc expression and stimulates growth as documented by inhibition of both of these responses following exposure to the AR antagonist, bicalutamide. These data document that AR signaling is converted from a growth suppressor in normal prostate epithelial cells to an oncogene in prostate cancer cells during prostatic carcinogenesis and that this conversion involves a gain of function for regulation of c-Myc expression.     

Cytosolic phospholipase A2α sustains pAKT,pERK and AR levels in PTEN-null/mutated prostate cancer cells

Constitutive phosphorylation of protein kinase B (AKT) is a common feature of cancer caused by genetic alteration in the phosphatase and tensin homolog (PTEN) gene and is associated with poor prognosis. This study determined the role of cytosolic phospholipase A₂α (cPLA₂α) in AKT, extracellular signal-regulated kinase (ERK) and androgen receptor (AR) signaling in PTEN-null/mutated prostate cancer cells. Doxycycline (Dox)-induced expression of cPLA₂α led to an increase in pAKT, pGSK3β and cyclin D1 levels in LNCaP cells that possess a PTEN frame-shift mutation. In contrast, silencing cPLA₂α expression with siRNA decreased pAKT, pGSK3β and cyclin D1 levels in both PC-3 (PTEN deletion) and LNCaP cells. Silencing of cPLA₂α decreased pERK and AR protein levels. The inhibitory effect of cPLA₂α siRNA on pAKT and AR protein levels was reduced by the addition of arachidonic acid (AA), whereas the stimulatory effect of AA on pAKT, pERK and AR levels was decreased by an inhibitor of 5-hydroxyeicosatetraenoic acid production. Pharmacological blockade of cPLA₂α with Efipladib reduced pAKT and AR levels with a concomitant inhibition of PC-3 and LNCaP cell proliferation. These results demonstrate an important role for cPLA₂α in sustaining AKT, ERK and AR signaling in PTEN-null/mutated prostate cancer cells and provide a potential molecular target for treating prostate cancer.     

Similarities and Distinctions in Actions of Surface-Directed and Classic Androgen Receptor Antagonists

The androgen receptor (AR) surface-directed antagonist MJC13 inhibits AR function and proliferation of prostate cancer (PC) cells. These effects are related to arrest of an AR/chaperone complex in the cytoplasm. Here, we compared MJC13 and classic AR antagonists such as flutamide and bicalutamide. Microarray analysis and confirmatory qRT-PCR reveals that MJC13 and flutamide inhibit dihydrotestosterone (DHT)-dependent genes in LNCaP PC cells. Both compounds are equally effective on a genome wide basis and as effective as second generation AR antagonists (MDV3100, ARN-509) at selected genes. MJC13 inhibits AR binding to the prostate specific antigen (PSA) promoter more strongly than flutamide, consistent with different mechanisms of action. Examination of efficacy of MJC13 in conditions that reflect aspects castrate resistant prostate cancer (CRPC) reveals that it inhibits flutamide activation of an AR mutant (ART877A) that emerges during flutamide withdrawal syndrome, but displays greatly restricted gene-specific activity in 22Rv1 cells that express a constitutively active truncated AR and is inactive against glucocorticoid receptor (GR), which can co-opt androgen-dependent signaling networks in CRPC. Importantly, MJC13 inhibits AR interactions with SRC2 and β-catenin in the nucleus and, unlike flutamide, strongly inhibits amplification of AR activity obtained with transfected SRC2 and β-catenin. MJC13 also inhibits DHT and β-catenin-enhanced cell division in LNCaP cells. Thus, a surface-directed antagonist can block AR activity in some conditions in which a classic antagonist fails and may display utility in particular forms of CRPC.     

The PPARγ ligand ciglitazone regulates androgen receptor activation differently in androgen-dependent versus androgen-independent human prostate cancer cells

The androgen receptor (AR) regulates growth and progression of androgen-dependent as well as androgen-independent prostate cancer cells. Peroxisome proliferator-activated receptor gamma (PPARγ) agonists have been reported to reduce AR activation in androgen-dependent LNCaP prostate cancer cells. To determine whether PPARγ ligands are equally effective at inhibiting AR activity in androgen-independent prostate cancer, we examined the effect of the PPARγ ligands ciglitazone and rosiglitazone on C4-2 cells, an androgen- independent derivative of the LNCaP cell line. Luciferase-based reporter assays and Western blot analysis demonstrated that PPARγ ligand reduced dihydrotestosterone (DHT)-induced increases in AR activity in LNCaP cells. However, in C4-2 cells, these compounds increased DHT-induced AR driven luciferase activity. In addition, ciglitazone did not significantly alter DHT-mediated increases in prostate specific antigen (PSA) protein or mRNA levels within C4-2 cells. siRNA-based experiments demonstrated that the ciglitazone-induced regulation of AR activity observed in C4-2 cells was dependent on the presence of PPARγ. Furthermore, overexpression of the AR corepressor cyclin D1 inhibited the ability of ciglitazone to induce AR luciferase activity in C4-2 cells. Thus, our data suggest that both PPARγ and cyclin D1 levels influence the ability of ciglitazone to differentially regulate AR signaling in androgen-independent C4-2 prostate cancer cells.     

Structure-based virtual screening and identification of a novel androgen receptor antagonist

Hormonal therapies, mainly combinations of anti-androgens and androgen deprivation, have been the mainstay treatment for advanced prostate cancer because the androgen-androgen receptor (AR) system plays a pivotal role in the development and progression of prostate cancers. However, the emergence of androgen resistance, largely due to inefficient anti-hormone action, limits the therapeutic usefulness of these therapies. Here, we report that 6-(3,4-dihydro-1H-isoquinolin-2-yl)-N-(6-methylpyridin-2-yl)nicotinamide (DIMN) acts as a novel anti-androgenic compound that may be effective in the treatment of both androgen-dependent and androgen-independent prostate cancers. Through AR structure-based virtual screening using the FlexX docking model, fifty-four compounds were selected and further screened for AR antagonism via cell-based tests. One compound, DIMN, showed an antagonistic effect specific to AR with comparable potency to that of the classical AR antagonists, hydroxyflutamide and bicalutamide. Consistent with their anti-androgenic activity, DIMN inhibited the growth of androgen-dependent LNCaP prostate cancer cells. Interestingly, the compound also suppressed the growth of androgen-independent C4-2 and CWR22rv prostate cancer cells, which express a functional AR, but did not suppress the growth of the AR-negative prostate cancer cells PPC-1, DU145, and R3327-AT3.1. Taken together, the results suggest that the synthetic compound DIMN is a novel anti-androgen and strong candidate for useful therapeutic agent against early stage to advanced prostate cancer.     

Androgen receptor represses the neuroendocrine transdifferentiation process in prostate cancer cells

Androgen-ablation therapy is an effective method for treating prostate cancer. However, prostate tumors that survive long-term androgen-ablation therapy are classified as androgen-independent as they proliferate in the absence of androgens, and they tend to be enriched for neuroendocrine (NE) cells. Androgen withdrawal causes androgen-dependent prostate cancer cells to adopt a pronounced NE phenotype, suggesting that androgen receptor (AR) represses an intrinsic NE transdifferentiation process in prostate cancer cells. In this report we show that short interfering RNA-induced AR silencing induced a NE phenotype that manifested itself in the growth of dendritic-like processes in both the androgen-dependent LNCaP and androgen-independent LNCaP-AI human prostate cancer cells. Western blot analysis revealed that neuronal-specific enolase, a marker of the neuronal lineage, was increased by AR knockdown in LNCaP cells. The expression levels of the neuronal-specific cytoskeletal proteins beta-tubulin III, nestin, and glial acidic fibrillary protein were also characterized in AR knockdown cells. Most interestingly, AR silencing induced beta-tubulin III expression in LNCaP cells, while AR knockdown increased glial acidic fibrillary protein levels in both LNCaP and LNCaP-AI cells. Lastly, AR silencing reduced the proliferative capacity of LNCaP and LNCaP-AI cells. Our data demonstrate that AR actively represses an intrinsic NE transdifferentiation process in androgen-responsive prostate cancer cells and suggest a potential link between AR inactivation and the increased frequency of NE cells in androgen-independent tumors.     

A competitive inhibitor that reduces recruitment of androgen receptor to androgen-responsive genes

The androgen receptor (AR) has a critical role in the growth and progression of androgen-dependent and castration-resistant prostate cancers. To identify novel inhibitors of AR transactivation that block growth of prostate cancer cells, a luciferase-based high-throughput screen of ~160,000 small molecules was performed in cells stably expressing AR and a prostate-specific antigen (PSA)-luciferase reporter. CPIC (1-(3-(2-chlorophenoxy) propyl)-1H-indole-3-carbonitrile) was identified as a small molecule that blocks AR transactivation to a greater extent than other steroid receptors. CPIC inhibited AR-mediated proliferation of androgen-sensitive prostate cancer cell lines, with minimal toxicity in AR-negative cell lines. CPIC treatment also reduced the anchorage-independent growth of LAPC-4 prostate cancer cells. CPIC functioned as a pure antagonist by inhibiting the expression of AR-regulated genes in LAPC-4 cells that express wild-type AR and exhibited weak agonist activity in LNCaP cells that express the mutant AR-T877A. CPIC treatment did not reduce AR levels or alter its nuclear localization. We used chromatin immunoprecipitation to identify the site of action of CPIC. CPIC inhibited recruitment of androgen-bound AR to the PSA promoter and enhancer sites to a greater extent than bicalutamide. CPIC is a new therapeutic inhibitor that targets AR-mediated gene activation with potential to arrest the growth of prostate cancer.     

Vav3 oncogene is overexpressed and regulates cell growth and androgen receptor activity in human prostate cancer

The purpose of this research was to investigate the role of Vav3 oncogene in human prostate cancer. We found that expression of Vav3 was significantly elevated in androgen-independent LNCaP-AI cells in comparison with that in their androgen-dependent counterparts, LNCaP cells. Vav3 expression was also detected in other human prostate cancer cell lines (PC-3, DU145, and 22Rv1) and, by immunohistochemistry analysis, was detected in 32% (26 of 82) of surgical specimens of human prostate cancer. Knockdown expression of Vav3 by small interfering RNA inhibited growth of both androgen-dependent LNCaP and androgen-independent LNCaP-AI cells. In contrast, overexpression of Vav3 promoted androgen-independent growth of LNCaP cells induced by epidermal growth factor. Overexpression of Vav3 enhanced androgen receptor (AR) activity regardless of the presence or absence of androgen and stimulated the promoters of AR target genes. These effects of Vav3 could be attenuated by either phosphatidylinositol 3-kinase (PI3K) inhibitors or dominant-negative Akt and were enhanced by cotransfection of PI3K. Moreover, phosphorylation of Akt was elevated in LNCaP cells overexpressing Vav3, which could be blocked by PI3K inhibitors. Finally, we ascertained that the DH domain of Vav3 was responsible for activation of AR. Taken together, our data show that overexpression of Vav3, through the PI3K-Akt pathway, inappropriately activates AR signaling axis and stimulates cell growth in prostate cancer cells. These findings suggest that Vav3 overexpression may be involved in prostate cancer development and progression.     

Isolation of a New Quinoline, 2,8-Dihydroxycinchoninic Acid from Corn Steep Liquor

Androgen receptor (AR) signaling is the master regulator of prostate cell growth. Here, to better understand AR signaling, we searched for AR-interacting proteins by yeast two-hybrid screening and identified protein arginine methyltransferase 10 (PRMT10) as one of the interacting proteins. PRMT10 was highly expressed in reproductive tissues, such as prostate. Immunostaining showed that PRMT10 was expressed in the nucleus of both epithelia and stroma of rat prostate. In human prostate cancer LNCaP cells, PRMT10 co-immunoprecipitated with AR in both the presence and absence of dihydrotestosterone (DHT). Knockdown of PRMT10 by siRNA decreased DHT-dependent LNCaP cell growth and induction of prostate-specific antigen, an AR-target gene, without apparent loss of AR. DHT decreased PRMT10 at both the mRNA and protein levels. The decrease in PRMT10 was canceled by knockdown of AR or an AR antagonist. These results indicate that PRMT10 plays an important role in androgen-dependent proliferation of prostate cancer cells.     

Differential Redox Regulation of Ca2+ Signaling and Viability in Normal and Malignant Prostate Cells

In prostate cancer, reactive oxygen species (ROS) are elevated and Ca²⁺ signaling is impaired. Thus, several novel therapeutic strategies have been developed to target altered ROS and Ca²⁺ signaling pathways in prostate cancer. Here, we investigate alterations of intracellular Ca²⁺ and inhibition of cell viability caused by ROS in primary human prostate epithelial cells (hPECs) from healthy tissue and prostate cancer cell lines (LNCaP, DU145, and PC3). In hPECs, LNCaP and DU145 H₂O₂ induces an initial Ca²⁺ increase, which in prostate cancer cells is blocked at high concentrations of H₂O₂. Upon depletion of intracellular Ca²⁺ stores, store-operated Ca²⁺ entry (SOCE) is activated. SOCE channels can be formed by hexameric Orai1 channels; however, Orai1 can form heteromultimers with its homolog, Orai3. Since the redox sensor of Orai1 (Cys-195) is absent in Orai3, the Orai1/Orai3 ratio in T cells determines the redox sensitivity of SOCE and cell viability. In prostate cancer cells, SOCE is blocked at lower concentrations of H₂O₂ compared with hPECs. An analysis of data from hPECs, LNCaP, DU145, and PC3, as well as previously published data from naive and effector T_H cells, demonstrates a strong correlation between the Orai1/Orai3 ratio and the SOCE redox sensitivity and cell viability. Therefore, our data support the concept that store-operated Ca²⁺ channels in hPECs and prostate cancer cells are heteromeric Orai1/Orai3 channels with an increased Orai1/Orai3 ratio in cells derived from prostate cancer tumors. In addition, ROS-induced alterations in Ca²⁺ signaling in prostate cancer cells may contribute to the higher sensitivity of these cells to ROS.     

Restoration of cyclin D2 has an inhibitory potential on the proliferation of LNCaP cells

Despite well known oncogenic function of G1-S cell-cycle progression, cyclin D2 (CCND2) is often silenced epigenetically in prostate cancers. Here we show that CCND2 has an inhibitory potential on the proliferation of androgen receptor (AR)-dependent prostate cancer LNCaP cells. Forced expression of CCND2 suppressed the proliferative ability and induced cell death in LNCaP cells in a cdk-independent manner. Knocking down CCND2 restored the proliferation of LNCaP subclones with relatively high CCND2 expression and low proliferative profiles. Immunoprecipitation using deletion mutants of CCND2 indicated that a central domain of CCND2 is required for binding to AR. A deletion mutant lacking the central domain failed to hinder LNCaP cells. Collectively, our results indicated that CCND2 inhibits cell proliferation of AR-dependent prostate cancer through the interaction with AR. Our study suggests that restoration of CCND2 expression potentially prevents the carcinogenesis of prostate cancer, which is mostly AR-dependent in the initial settings.     

Capsaicin, a component of red peppers, induces expression of androgen receptor via PI3K and MAPK pathways in prostate LNCaP cells

In this study, capsaicin (trans-8-methyl-N-vanillyl-6-nonenamide) induced an increase in the cell viability of the androgen-responsive prostate cancer LNCaP cells, which was reversed by the use of the TRPV1 antagonists capsazepine, I-RTX and SB 366791. In further studies we observed that capsaicin induced a decrease in ceramide levels as well as Akt and Erk activation. To investigate the mechanism of capsaicin action we measured androgen (AR) receptor levels. Capsaicin induced an increase in the AR expression that was reverted by the three TRPV1 antagonists. AR silencing by the use of siRNA, as well as blocking the AR receptor with bicalutamide, inhibited the proliferative effect of capsaicin.