The JNK inhibitor AS602801 Synergizes with Enzalutamide to Kill Prostate Cancer Cells In Vitro and In Vivo and Inhibit Androgen Receptor Expression

In our previous study, we observed that androgen deprivation therapy (ADT) may induce a compensatory increase in MAPK or JNK signaling. Here, we tested the effects of the MEK inhibitors PD0325901 and GSK1120212, ERK1/2 inhibitor GDC-0994, and the JNK inhibitor AS602801 alone and in combination with the AR inhibitor enzalutamide (ENZ) in androgen-sensitive LNCaP cells and androgen-resistant C4-2 and 22Rv1 cells. Enzalutamide combined with AS602801 synergistically killed LNCaP, C4-2, and 22Rv1 cells, and decreased migration and invasion of LNCaP and C4-2 cells. We studied the combination of enzalutamide with AS602801 in vivo using luciferase labeled LNCaP xenografts, and observed that combination of ENZ with AS602801 significantly suppressed tumor growth compared with either drug alone. Importantly, combination therapy resulted in dramatic loss of AR mRNA and protein. Surprisingly, mechanistic studies and Nanostring data suggest that AS602801 likely activates JNK signaling to induce apoptosis. Since AS602801 had sufficient safety and toxicity profile to advance from Phase I to Phase II in clinical trials, repurposing of this compound may represent an opportunity for rapid translation for clinical therapy of CRPC patients.     

Radiopotentiation of enzalutamide over human prostate cancer cells as assessed by real-time cell monitoring

AimTo evaluate the radiopotentiation of enzalutamide in human prostate cancer cells.BackgroundWhile radiotherapy is the first line of treatment for prostate cancer, androgen blockade therapies are demonstrating significant survival benefit as monotherapies. As androgen blockade can cause cell death by apoptosis, it is likely that androgen blockade will potentiate the cytotoxic activities of radiotherapy.Materials and methodsHere, we tested the potential synergistic effects of these two treatments over two human metastatic prostate cancer cells by real-time cell analysis (RTCA), androgen-sensitive LNCaP cells (Lymph Node Carcinoma of the Prostate) and androgen-independent PC-3. Both cell lines were highly resistant to high doses of radiotherapy.ResultsA pre-treatment of LNCaP cells with IC50 concentrations of enzalutamide significantly sensitized them to radiotherapy through enhanced apoptosis. In contrast, enzalutamide resistant PC-3 cells were not sensitized to radiotherapy by androgen blockade.ConclusionsThese results provide evidence that the enzalutamide/radiotherapy combination could maximize therapeutic responses in patients with enzalutamide-sensitive prostate cancer.     

Androgen receptor agonist and antagonist reduce response of cytokine-induced killer cells on prostate cancer cells

Despite many advances, prostate cancer (PCa) is still the second most frequently diagnosed cancer and fifth leading cause of cancer death in men worldwide. So far, the promising field of onco-immunology has not yet provided a satisfactory treatment option for PCa. Here we show that the ex vivo expansion and activation of cytokine-induced killer (CIK) cells isolated from primary peripheral blood mononuclear cells induce immune-mediated apoptosis in both human PCa LNCaP and C4-2 cells. Interestingly, pretreating LNCaP and C4-2 cells with either androgen or the androgen receptor (AR) antagonist enzalutamide mediates resistance to this immunogenic attack. This is associated with a reduction of both total cell loss and apoptosis levels suggesting one possible mechanism blunting onco-immunological activity. The data also suggest that secreted factors from AR ligand-treated PCa cell suppress lymphocyte proliferation. Further, we analysed immune-mediated killing activity using conditioned media from LNCaP and C4-2 treated cells. The obtained data suggest that the conditioned media from PCa treated cells does not influence a measurable lymphocyte-mediated apoptosis. However, analysing clonal expansion of activated lymphocytes, the androgen-derived conditioned media suppresses lymphocyte proliferation/expansion suggesting inhibition of onco-immunological activity by pretreatment of PCa cells with AR ligands.     

The androgen receptor antagonist enzalutamide induces apoptosis,dysregulates the heat shock protein system,and diminishes the androgen receptor and estrogen receptor β1 expression in prostate cancer cells

Enzalutamide's accepted mode of action is by targeting the androgen receptor's (AR) activity. In clinical practice, enzalutamide demonstrates a good benefit-risk profile for the treatment of advanced prostate cancer (PC), even after poor response to standard antihormonal treatment. However, since both, well-established antiandrogens and enzalutamide, target AR functionality, we hypothesized that additional unknown mechanisms might be responsible for enzalutamide's superior anticancer activity. In the current study, PC cells were incubated with enzalutamide and enzalutamide-dependent modulation of apoptotic mechanisms were assessed via Western blot analysis, TDT-mediated dUTP-biotin nick end-labeling assay, and nuclear morphology assay. Alterations of heat shock protein (HSP), AR, and estrogen receptor (ER) expression were examined by Western blot analysis. Enzalutamide attenuated the proliferation of PC cells in a time- and dose-dependent manner. In the presence of enzalutamide, apoptosis occurred which was shown by increased BAX expression, decreased Bcl-2 expression, nuclear pyknosis, and genomic DNA fragmentation. Moreover, enzalutamide inhibited the expression of HSPs primarily involved in steroid receptor stabilization and suppressed AR and ERβ1 expression. This study demonstrates for the first time that enzalutamide treatment of PC cells triggers varying molecular mechanisms resulting in antiproliferative effects of the drug. In addition to the well-characterized antagonistic inhibition of AR functionality, we have shown that enzalutamide also affects the intracellular synthesis of steroid receptor-associated HSPs, thereby diminishing the expression of AR and ERβ1 proteins and inducing apoptotic pathways. According to an indirect attenuation of HSP-associated factors such as steroid receptors, endometrial carcinoma, uterine leiomyosarcoma, and mamma carcinoma cells also demonstrated inhibited cell growth in the presence of enzalutamide. Our data, therefore, suggest that enzalutamide's high efficacy is at least partially independent of AR and p53 protein expression, which are frequently lost in advanced PC.     

Pomegranate polyphenols down-regulate expression of androgen-synthesizing genes in human prostate cancer cells overexpressing the androgen receptor

Prostate cancer is dependent on circulating testosterone in its early stages and is treatable with radiation and surgery. However, recurrent prostate tumors advance to an androgen-independent state in which they progress in the absence of circulating testosterone, leading to metastasis and death. During the development of androgen independence, prostate cancer cells are known to increase intracellular testosterone synthesis, which maintains cancer cell growth in the absence of significant amounts of circulating testosterone. Overexpression of the androgen receptor (AR) occurs in androgen-independent prostate cancer and has been proposed as another mechanism promoting the development of androgen independence. The LNCaP-AR cell line is engineered to overexpress AR but is otherwise similar to the widely studied LNCaP cell line. We have previously shown that pomegranate extracts inhibit both androgen-dependent and androgen-independent prostate cancer cell growth. In this study, we examined the effects of pomegranate polyphenols, ellagitannin-rich extract and whole juice extract on the expression of genes for key androgen-synthesizing enzymes and the AR. We measured expression of the HSD3B2 (3beta-hydroxysteroid dehydrogenase type 2), AKR1C3 (aldo-keto reductase family 1 member C3) and SRD5A1 (steroid 5alpha reductase type 1) genes for the respective androgen-synthesizing enzymes in LNCaP, LNCaP-AR and DU-145 human prostate cancer cells. A twofold suppression of gene expression was considered statistically significant. Pomegranate polyphenols inhibited gene expression and AR most consistently in the LNCaP-AR cell line (P=.05). Therefore, inhibition by pomegranate polyphenols of gene expression involved in androgen-synthesizing enzymes and the AR may be of particular importance in androgen-independent prostate cancer cells and the subset of human prostate cancers where AR is up-regulated.     

Synthesis and preliminary investigations into novel 1,2,3-triazole-derived androgen receptor antagonists inspired by bicalutamide

A versatile and high yielding synthesis of novel androgen receptor (AR) antagonists is presented. Using this methodology, six 1,4-substituted-1,2,3-triazole derived bicalutamide mimics were synthesised in five steps and in isolated overall yields from 41% to 85%. Evaluation of these compounds for their anti-proliferative properties against androgen dependent (LNCaP) and independent (PC-3) cells showed promising IC₅₀ values of 34–45 μM and 29–151 μM, respectively. The data suggest that the latter compounds may be an excellent starting point for the development of prostate cancer therapeutics for both androgen dependent and independent forms of this disease. Docking of these compounds (each enantiomer) in silico into the T877A mutated androgen receptor, as possessed by LNCaP cells, was also undertaken.     

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.     

PLCε knockdown overcomes drug resistance to androgen receptor antagonist in castration-resistant prostate cancer by suppressing the wnt3a/β-catenin pathway

Most prostate cancers (Pcas) develop into castration-resistant prostate cancer (CRPC) after receiving androgen deprivation therapy (ADT). The expression levels of PLCε and wnt3a are increased in Pca and regulate androgen receptor (AR) activity. However, the biological function and mechanisms of PLCε and wnt3a in CRPC remain unknown. In this study, we found that the expression levels of PLCε, wnt3a, and AR were significantly increased in CRPC tissues as well as bicalutamide-resistant-LNCaP and enzalutamide-resistant-LNCaP cells. In addition, PLCε knockdown partly restored the sensitivity of drug-resistant cells to bicalutamide and enzalutamide by inhibiting the activity of the wnt3a/β-catenin/AR signaling axis. Interestingly, the resistance of LNCaP cells docetaxel is related to PLCε but not the wnt3a/β-catenin pathway. We also found that the combination of PLCε knockdown and enzalutamide treatment synergistically suppressed cell proliferation, tumor growth, and bone metastasis using in vitro and in vivo experiments. Our study revealed that PLCε is involved in the progression of drug-resistance in CRPC and could be a new target for the treatment of CRPC.     

The significance of Her2 on androgen receptor protein stability in the transition of androgen requirement in prostate cancer cells

Androgen ablation therapy is the most common strategy for suppressing prostate cancer progression; however, tumor cells eventually escape androgen dependence and progress to an androgen-independent phase. The androgen receptor (AR) plays a pivotal role in this transition. To address this transition mystery in prostate cancer, we established an androgen-independent prostate cancer cell line (LNCaPdcc), by long-term screening of LNCaP cells in androgen-deprived conditions, to investigate changes of molecular mechanisms before and after androgen withdrawal. We found that LNCaPdcc cells displayed a neuroendocrine morphology, less aggressive growth, and lower expression levels of cell cycle-related factors, although the cell cycle distribution was similar to parental LNCaP cells. Notably, higher protein expression of AR, phospho-Ser(81)-AR, and PSA in LNCaPdcc cells were observed. The nuclear distribution and protein stability of AR increased in LNCaPdcc cells. In addition, cell proliferation results exhibited the biphasic nature of the androgen (R1881) effect in two cell lines. On the other hand, LNCaPdcc cells expressed higher levels of Her2, phospho-Tyr(1221/1222)-Her2, ErbB3, and ErbB4 proteins than parental LNCaP cells. These two cell lines exhibited distinct responses to Her2 activation (by heregulin treatment) on Her2 phosphorylation and Her2 inhibition (by AG825 or Herceptin treatments) on proliferation. In addition, the Her2 inhibitor more effectively caused AR degradation and diminished AR Ser(81) phosphorylation in LNCaPdcc cells. Taken together, our data demonstrate that Her2 plays an important role in the support of AR protein stability in the transition of androgen requirement in prostate cancer cells. We hope these findings will provide novel insight into the treatment of hormone-refractory prostate cancer.     

Pentafluorophenyl Substitution of Natural Di(indol‐3‐yl)methane Strongly Enhances Growth Inhibition and Apoptosis Induction in Various Cancer Cell Lines

Di(indol‐3‐yl)methane (=3,3′‐methanediyldi(1H‐indole), DIM, 1 ) is a known weakly antitumoral compound formed by digestion of indole‐3‐carbinol (=1H‐indol‐3‐ylmethanol), an ingredient of various Brassica vegetables. Out of a series of nine fluoroaryl derivatives of 1 , three pentafluorophenyl derivatives 2c , 2h , and 2i were identified that exhibited a two to five times greater anti‐proliferative effect and an increased apoptosis induction when compared with 1 in the following carcinoma cell lines: BxPC‐3 pancreas, LNCaP prostate, C4‐2B prostate, PC3 prostate and the triple‐negative MDA‐MB‐231 breast carcinoma. Compound 2h was particularly efficacious against androgen‐refractory C4‐2B prostate cancer cells (IC₅₀=6.4 μm ) and 2i against androgen‐responsive LNCaP cells (IC₅₀=6.2 μm ). In addition, 2c and 2h exhibited distinct activity in three cancer cell lines resistant to 1 .     

Syntheses and potential anti-prostate cancer activities of ionone-based chalcones

We report the SAR studies of 43 ionone-based chalcones that demonstrate substantial in vitro anti-proliferative activities in LNCaP, MDA-PCa-2b, 22Rv1, C4-2B and PC-3 prostate cancer cell lines. Compound 25 with an IC₅₀ value of 0.74 μM in LNCaP cells potently antagonizes DHT-induced transactivation of the wild type and the clinically relevant T877A, W741C and H874Y mutated androgen receptors, representing a novel chalcone as pan-antagonist of androgen receptor.     

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.     

Overexpression of aldo-keto reductase 1C3 (AKR1C3) in LNCaP cells diverts androgen metabolism towards testosterone resulting in resistance to the 5α-reductase inhibitor finasteride

Type 5 17β-hydroxysteroid dehydrogenase (AKR1C3) is the major enzyme in the prostate that reduces 4-androstene-3,17-dione (Δ(4)-Adione) to the androgen receptor (AR) ligand testosterone. AKR1C3 is upregulated in prostate cancer (PCa) and castrate resistant prostate cancer (CRPC) that develops after androgen deprivation therapy. PCa and CRPC often depend on intratumoral androgen biosynthesis and upregulation of AKR1C3 could contribute to intracellular synthesis of AR ligands and stimulation of proliferation through AR signaling. To test this hypothesis, we developed an LNCaP prostate cancer cell line overexpressing AKR1C3 (LNCaP-AKR1C3) and compared its metabolic and proliferative responses to Δ(4)-Adione treatment with that of the parental, AKR1C3 negative LNCaP cells. In LNCaP and LNCaP-AKR1C3 cells, metabolism proceeded via 5α-reduction to form 5α-androstane-3,17-dione and then (epi)androsterone-3-glucuronide. LNCaP-AKR1C3 cells made significantly higher amounts of testosterone-17β-glucuronide. When 5α-reductase was inhibited by finasteride, the production of testosterone-17β-glucuronide was further elevated in LNCaP-AKR1C3 cells. When AKR1C3 activity was inhibited with indomethacin the production of testosterone-17β-glucuronide was significantly decreased. Δ(4)-Adione treatment stimulated cell proliferation in both cell lines. Finasteride inhibited LNCaP cell proliferation, consistent with 5α-androstane-3,17-dione acting as the major metabolite that stimulates growth by binding to the mutated AR. However, LNCaP-AKR1C3 cells were resistant to the growth inhibitory properties of finasteride, consistent with the diversion of Δ(4)-Adione metabolism from 5α-reduced androgens to increased formation of testosterone. Indomethacin did not result in differences in Δ(4)-Adione induced proliferation since this treatment led to the same metabolic profile in LNCaP and LNCaP-AKR1C3 cells. We conclude that AKR1C3 overexpression diverts androgen metabolism to testosterone that results in proliferation in androgen sensitive prostate cancer. This effect is seen despite high levels of uridine glucuronosyl transferases suggesting that AKR1C3 activity can surmount the effects of this elimination pathway. Treatment options in prostate cancer that target 5α-reductase where AKR1C3 co-exists may be less effective due to the diversion of Δ(4)-Adione to testosterone.