Proliferation Of LNCaP Prostate Cancer Cells Research Articles

Core Structure-Activity Relationship Studies of 5,7,20-O-Trimethylsilybins in Prostate Cancer Cell Models.

Silibinin, also known as silybin, is isolated from milk thistle (Silybum marianum). Silibinin has been demonstrated to be a good lead compound due to its potential to prevent and treat prostate cancer. Its moderate potency and poor pharmacokinetic profile hindered it from moving forward to therapeutic use. Our research group has been working on optimizing silibinin for the potential treatment of castration-resistant prostate cancer. Our previous studies established 5,7,20-O-trimethylsilybins as promising lead compounds as they can selectively suppress androgen receptor (AR)-positive LNCaP cell proliferation. Encouraged by the promising data, the present study aims to investigate the relationships between the core structure of 5,7,20-O-trimethylsilybin and their antiproliferative activities towards AR-positive (LNCaP) and AR-negative prostate cancer cell lines (PC-3 and DU145). The structure-activity relationships among the four different core structures (including flavanonol-type flavonolignan (silibinin), flavone-type flavonolignan (hydnocarpin D), chalcone-type flavonolignan, and taxifolin (a flavonolignan precursor) indicate that 5,7,20-O-trimethylsilybins are the most promising scaffold to selectively suppress AR-positive LNCaP prostate cancer cell proliferation. Further investigation on the antiproliferative potency of their optically enriched versions of the most promising 5,7,20-O-trimethylsilybins led to the conclusion that (10R,11R) derivatives (silybin A series) are more potent than (10S,11S) derivatives (silybin B series) in suppressing AR positive LNCaP cell proliferation.

23-O-Substituted-2,3-Dehydrosilybins Selectively Suppress Androgen Receptor-Positive LNCaP Prostate Cancer Cell Proliferation

As part of our ongoing project to search for natural product-based antiandrogens, nine derivatives of 2,3-dehydrosilybin have been synthesized for the evaluation of its antiproliferative activity in an androgen receptor-positive prostate cancer cell model. Specifically, 3,5,7,20- O-tetramethyl-2,3-dehydrosilybin was synthesized through two approaches, and eight 23- O-substituted-3,5,7,20- O-tetramethyl-2,3-dehydrosilybins were achieved from 3,5,7,20- O-tetramethyl-2,3-dehydrosilybin. The antiproliferative potency of 3,5,7,20- O-tetramethyl-2,3-dehydrosilybin and its eight derivatives were assessed in an androgen receptor (AR)-positive LNCaP prostate cancer cell line, as well as in two AR-negative (PC-3 and DU145) prostate cancer cell models as a comparison. Our WST cell proliferation assay data indicate 3,5,7,20- O-tetramethyl-2,3-dehydrosilybin and most of its 23- O-substituents can selectively inhibit AR-positive LNCaP prostate cancer cell proliferation. Our data suggest that 3,5,7,20- O-tetramethyl-2,3-dehydrosilibins could serve as a natural product-based scaffold for new antiandrogens for lethal castration-resistant prostate cancer.

SGK3 is an androgen-inducible kinase promoting prostate cancer cell proliferation through activation of p70 S6 kinase and up-regulation of cyclin D1.

Both androgen and phosphatidylinositol 3-kinase (PI3K) signaling are critical for cell proliferation of androgen receptor (AR)-positive prostate cancer cells, but the underlying mechanisms are still not fully understood. Here we report that serum- and glucocorticoid-inducible kinase 3 (SGK3), a Ser/Thr kinase functioning downstream of PI3K, is an AR transcriptional target and promotes prostate cancer cell proliferation. SGK3 expression is up-regulated by androgen DHT via AR. We identified an AR-binding region at the sgk3 locus, which confers androgen responsiveness of sgk3 promoters. Interestingly, we found that androgen/AR-dependent SGK3 expression requires estrogen receptor (ER) (including both isoforms, ERα and ERβ). Depletion of ER blocked DHT-induced SGK3 expression. Functionally, knockdown of SGK3 expression significantly decreased LNCaP prostate cancer cell proliferation by inhibiting G1 to S phase cell cycle progression. We further provided evidence that SGK3 promotes p70 S6 kinase (p70S6K) activation and increases cyclin D1 levels. In summary, our study identifies SGK3 as an AR target and provides a novel androgen-induced cell proliferation mechanism mediated by the AR-SGK3-p70S6K-cyclin D1 pathway in prostate cancer cells.

Dutasteride and Metformin Reduce the Growth of LNCaP Cells and Alter the SREBP-1 Pathway

Prostate cancer (PCa) is the second most prevalent cancer in men after lung cancer. Prostate cancer develop- ment and progression is associated with the dysregulation of a number of molecular pathways; hence, therapeutic strate- gies targeting such pathways bring great promise. Recently we have shown that metformin, the anti-diabetic drug, can in- hibit tumor progression when combined with dutasteride, a 5-alpha-reductase inhibitor (5ARI). Interestingly, both met- formin and dutasteride have been reported to alter the Sterol Regulatory Element Binding Proteins (SREBP) Fatty Acid Synthase (FASN) pathway. The SREBP pathway is involved with lipid and energy homeostasis. In our present study, we investigated if dutasteride in combination with metformin can reduce the proliferation of LNCaP PCa cells, and whether this is mediated through the SREBP-1/FASN pathway. Human PCa cells were treated with either dutasteride (0-100 µM) or metformin (0-50 mM) alone or in combination. The treated cells were then incubated for up to 24 hours, and prolifera- tion assessed using the MTS 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazo- lium) assay. Western blot analysis was performed on cell lysates to assess alterations in key signaling molecules including cleaved SREBP-1, FASN, AR, PSA, pAMPK and apoptotic markers. Results revealed that there was a significant (p<0.05) decrease in cellular proliferation of LNCaP cells treated with a combination of metformin and dutasteride, this effect being greater than either treatment alone. Treating LNCaP cells with both metformin and dutasteride reduced the expression of FASN, cleaved SREBP-1 and pro-caspase-3 with expression of cleaved PARP; suggesting a possible inter- action between FASN and apoptosis. All treatments resulted in reductions in AR, PSA and an upregulation of pAMPK, with the highest expression seen in combination treatment. We report for the first time that metformin and dutasteride in combination can reduce the proliferation of androgen-sensitive cell line through the activation of p-AMPK, and SREBP- 1/FASN pathway and highlight the importance of targeting the SREBP-1 pathway, for improving future therapeutic strate- gies for prostate cancer.

Differential effects of exogenous and autocrine growth hormone on LNCaP prostate cancer cell proliferation and survival

The prostate gland is regulated by multiple hormones and growth factors that may also affect prostate tumorigenesis. Growth hormone (GH) contributes to prostate development and function, but the direct effects of GH on prostate cancer cells are not well understood. The expression of endogenous GH in prostate cancer cell lines has also been observed, suggesting the potential for an effect of autocrine GH. In the present study, we measure the levels of GH and GH receptor (GHR) mRNA in multiple prostate cancer and normal prostate-derived cell lines, and compare the effects of exogenous and autocrine GH on LNCaP prostate cancer cell proliferation and apoptosis, and the associated signal transduction pathways. We found that GHR and GH expression were higher in the prostate cancer cell lines, and that exogenous GH increased LNCaP cell proliferation, but had no effect on apoptosis. In contrast, autocrine GH overexpression reduced LNCaP cell proliferation and increased apoptosis. The distinct actions of exogenous and autocrine GH were accompanied by differences in the involvement of GHR-associated signal transduction pathways, and were paralleled by an alteration in the subcellular localization of GHR, in which autocrine GH appeared to sequester GHR in the Golgi and endoplasmic reticulum. This alteration of GHR trafficking may underlie a distinct mode of GH-mediated signaling associated with the effect of autocrine GH. These findings clarify the potential effects of GH on prostate cancer cell function, and indicate that the activity of autocrine GH may be distinct from that of endocrine GH in prostate cancer cells.

Abstract 3577: The role of Checkpoint Kinase 2 in the regulation of androgen receptor signaling and prostate cancer cell growth during progression to castration resistance.

Abstract As the second leading cause of cancer deaths in men, prostate cancer is a persistent, significant challenge for clinicians and researchers. Almost all patients with disseminated prostate cancer initially respond to androgen deprivation therapy. However, virtually every patient will relapse due to the growth of castration-resistant cancer cells and develop metastatic and lethal disease. Even with the recent development of new androgen ablation treatments such as Abiraterone and Enzalutamide, castration-resistant prostate cancer is still incurable. We hypothesize that compensatory signaling mechanisms that limit the effectiveness of androgen ablation can be overcome with therapeutic strategies targeting kinase cascades. In order to identify signaling pathways that regulate AR activity and prostate cancer cell growth we screened a panel of shRNAs targeting the human kinome in LNCaP prostate cancer cells grown in the presence and absence of androgen. We discovered that knockdown of Checkpoint Kinase 2 (CHK2) dramatically increased LNCaP prostate cancer cell proliferation. This observation is clinically relevant since CHK2 inactivating mutations arise in over 10% of prostate cancer patients and CHK2 expression decreases as prostate cancer progresses to a castration-resistant disease. Consistent with these clinical observations, CHK2 knockdown did not affect cell growth in the castration resistant C4-2 cell line, suggesting that the selection of the castration resistant C4-2 line from LNCaP involved the same effector as observed in clinical specimens. Consistent with this, CHK2 expression is lower in C4-2 cells compared to LNCaP. We determined that CHK2 knockdown increases androgen receptor (AR) transcriptional activity on both androgen activated and androgen repressed genes, providing evidence that CHK2 affects prostate cancer cell proliferation, at least in part, through the AR. These data suggest that CHK2 is a negative regulator of androgen sensitivity and prostate cancer cell growth and that CHK2 is lost during the progression to castration resistance. We are examining the role of CHK2 in regulating androgen-AR signaling, growth, and survival of prostate cancer cells; specifically we are identifying the crucial intracellular proteins that mediate CHK2 signaling and growth regulation of prostate cancer cells. Since CHK2 signaling is activated by DNA damage, such as that triggered by radiation therapy and brachytherapy, understanding how CHK2 signaling regulates the AR will provide critical insights into how we can combine current therapies with androgen blockade for greater clinical effectiveness. Citation Format: Huy Q. Ta, Melissa L. Ivey, Daniel Gioeli. The role of Checkpoint Kinase 2 in the regulation of androgen receptor signaling and prostate cancer cell growth during progression to castration resistance. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3577. doi:10.1158/1538-7445.AM2013-3577

EFFECTS OF GENISTEIN ON REGULATION OF DIHYDROTESTOSTERONE-INDUCED CELL PROLIFERATION IN ENDOTHELIAL AND PROSTATE CANCER CELLS

ObjectivesThere is currently no cure therapy available once prostate cancer is metastasised, and androgen deprivation is one of the standard therapies. However, the long-term use of oestrogens in treatment of...

110 EFFECTS OF GENISTEIN ON REGULATION OF DIHYDROTESTOSTERONE-INDUCED CELL PROLIFERATION IN ENDOTHELIAL AND PROSTATE CANCER CELLS

Grant Supports: This study was supported in part by a grant from National Institutes of Health (NIH) (UL1 RR024996) and a grant from the National Natural Science Foundation of China (No. 30873126) and a grant from the National Basic Research Program of China (2011CB5120010. Background: Androgen deprivation therapy of prostate cancer with estrogens shows significant cardiovascular side effects. This study was to examine the effect of genistein comparing to 17β − estradiol on modulation of androgen actions in human aortic endothelial cells (HAECs) and prostate cancer LAPC-4 and LNCaP cells. Methods: MTS, RT-PCR, and Western blot were used to detect cell proliferation, mRNA and protein expression of estrogen receptor (ER) gene, and cyclin A genes, respectively. Specific ERβ siRNA was synthesized and transfected to knockdown ERβ expression in prostate cancer cells. Results: Dihydrotestosterone (DHT) produced a time and dose-dependent induction of cell proliferation in HAEC, LAPC-4 and LNCaP cells. These DHT actions were inhibited by genistein in LAPC-4 and LNCaP cells but not in HAEC cells. While βE2 only attenuated the DHT-induced cell proliferation in HAEC and LAPC-4 cells without any inhibition of DHT-induced LNCaP cell proliferation. In LAPC-4 cells, knockdown of ERβ expression partially eliminated the blockade of DHT-induced cell proliferation. Conclusion: This study demonstrates that genistein may be a potential agent for prostate cancer therapy since genistein inhibits DHT-induced LAPC-4 and LNCaP prostate cancer cell proliferation but not HAEC cell growth. 17β − estradiol completely blocked DHT-induced cell growth in HAECs while inhibiting LAPC-4 cell proliferation, accounting for the side-effect of cardiovascular in Androgen deprivation therapy of prostate cancer. DHT-induced LNCaP prostate cancer cell proliferation can’t be attenuated by 17β − estradiol, suggesting 17β − estradiol is inactive in treating metastasized prostate cancer. ERβ played an important role in the modulation of androgen receptor actions.

Structure–activity studies of lGnRH‐III through rational amino acid substitution and NMR conformational studies

Lamprey gonadotropin-releasing hormone type III (lGnRH-III) is an isoform of GnRH isolated from the sea lamprey (Petromyzon marinus) with negligible endocrine activity in mammalian systems. Data concerning the superior direct anticancer activity of lGnRH-III have been published, raising questions on the structure-activity relationship. We synthesized 21 lGnRH-III analogs with rational amino acid substitutions and studied their effect on PC3 and LNCaP prostate cancer cell proliferation. Our results question the importance of the acidic charge of Asp⁶ for the antiproliferative activity and indicate the significance of the stereochemistry of Trp in positions 3 and 7. Furthermore, conjugation of an acetyl-group to the side chain of Lys⁸ or side chain cyclization of amino acids 1-8 increased the antiproliferative activity of lGnRH-III demonstrating that the proposed salt bridge between Asp⁶ and Lys⁸ is not crucial. Conformational studies of lGnRH-III were performed through NMR spectroscopy, and the solution structure of GnRH-I was solved. In solution, lGnRH-III adopts an extended backbone conformation in contrast to the well-defined β-turn conformation of GnRH-I.

Tea Polyphenols and Theaflavins Are Present in Prostate Tissue of Humans and Mice after Green and Black Tea Consumption

Green and black tea have shown promise in the chemoprevention of prostate cancer. The objective of this study was to determine the bioavailability and bioactivity of tea polyphenols (PP) and theaflavins in human serum and human and mouse tissues. A decaffeinated black tea diet was administered to C57BL/6 mice. PPs and theaflavins were found in the small and large intestine, liver, and prostate in conjugated and free forms. The relative prostate bioavailability of theaflavin was 70% higher than that of epigallocatechin gallate (EGCG). In the second mouse study, a green tea (GT) diet was administered followed by the control diet for 1-5 d. Epicatechin (EC), EGCG, and epicatechin gallate (ECG) concentrations in prostate tissue were significantly decreased after 1 d of consuming the control diet. Epigallocatechin gallate (EGC), however, did not decrease significantly. For the human study, 20 men scheduled for surgical prostatectomy were randomly assigned to consume 1.42 L daily of GT, BT, or a caffeine-matched soda control (SC) for 5 d before radical prostatectomy. Tea PPs were greater in prostate samples from men consuming BT and GT than in men consuming SC (P = 0.0025). Although tea PP were not detectable in serum, ex vivo LNCaP prostate cancer cell proliferation was less when cells were grown in media containing patient serum collected after BT (P < 0.001) and GT (P = 0.025) consumption relative to baseline serum This is the first human study to show that tea polyphenols and theaflavins are bioavailable in the prostate where they may be active in the prevention of prostate cancer.

Effect of Calcitriol on Prostate-Specific Antigen In vitro and in Humans

Calcitriol, the natural ligand for the vitamin D receptor, has significant potential in prostate cancer treatment. Measurement of its antineoplastic activity in prostate cancer clinical trials may be complicated by effects of calcitriol on prostate-specific antigen (PSA) production. We examined the effects of calcitriol at similar concentration on cell proliferation, androgen receptor (AR) expression, and PSA production in vitro and on PSA concentrations in prostate cancer patients. LNCaP prostate cancer cell proliferation was examined by cell counts 6 days after exposure to a range of concentrations of calcitriol. AR and PSA protein was quantified in LNCaP cells over 96 hours after exposure to 1 nmol/L calcitriol. Serum PSA and free PSA was serially measured by immunoassay over a period of 8 days in patients with hormone-naïve prostate cancer after a single dose of 0.5 microg/kg calcitriol. Calcitriol treatment resulted in dose-dependent growth inhibition of LNCaP with approximately 50% growth inhibition at the clinically achievable concentration of 1 nmol/L. Time-dependent up-regulation of AR expression and of PSA production in LNCaP cells was shown at the same concentration. No significant change in serum PSA or free PSA over 8 days was seen in eight subjects treated with a single dose of 0.5 microg/kg calcitriol. The analysis was powered to detect a 1.23-fold change between the baseline and day 8 serum PSA. At clinically achievable concentrations, calcitriol inhibits growth and induces AR and PSA expression in LNCaP cells. We did not detect similar changes in serum PSA or free PSA in patients exposed to similar concentrations of calcitriol. Thus, a PSA flare, predicted by preclinical systems, is unlikely to occur in patients and therefore unlikely to complicate interpretation of clinical trial outcomes.

Equiguard™ suppresses androgen-dependent LNCaP prostate cancer cell proliferation by targeting cell cycle control via down regulation of the retinoblastoma protein Rb and induction of apoptosis via the release of cytochrome c

Lack of life-prolonging therapies has provided much of the impetus for seeking complementary and alternative management/treatment options by prostate cancer (CaP) patients. Among these, the use of dietary supplements and botanical products has been showing a sustained increase in recent years, owing in part to some encouraging pre-clinical and clinical data shown in a limited number of herbal products. Notably, however, the majority of herbal and dietary supplement products have not been rigorously studied with regard to their efficacy. In vitro mechanistic experiments are considered essential preludes and requisites to more lengthy and costly animal and human studies, in that they may provide relevant insights and scientific basis for effects some of these products purportedly might demonstrate. In vitro studies in our laboratory have shown that a polyherbal supplement, Equiguard, exhibits anti-tumor activity against hormone dependent LNCaP cells cultured in both androgen-proficient (FBS) and -deficient (CS-FBS) conditions. Clinically relevant anti-prostate cancer effects of Equiguard are vividly illustrated by growth suppression and down regulated expression of prostate specific genes, respectively, androgen receptor (AR) and prostate specific antigen (PSA). However, the mechanistic bases contributing to these effects have not been well characterized. This communication describes experiments aimed at further understanding growth arrest elicited by Equiguard in LNCaP cells cultured in FBS and CS-FBS conditions. We have focused on aspects of cell cycle control and induction of apoptosis. Regulation of cell cycle progression by Equiguard was analyzed by examining changes in the expression of Rb and cyclins D/E. Using Western blot analysis, we showed that treatment caused inhibition of Rb phosphorylation, which was accompanied by the reduction of cyclins D/E expression, in both culture conditions. Moreover, cells treated with Equiguard and cultured with FBS-supplemented media showed up-regulation of cyclin-dependent kinase inhibitor Kip1/p27. These results support the interpretation that suppression of Rb phosphorylation mediated the observed growth arrest induced by Equiguard under androgen-proficient condition. In contrast, Equiguard-treated cells cultured in CS-FBS had lowered expression of the Kip1/p27, suggesting that different control mechanisms, possibly evoked by changes in cellular microenvironments, contributed to growth suppression by Equiguard. The growth suppressive effects of Equiguard in both culture conditions were also evaluated with respect to induction of apoptosis. While Equiguard elicited apoptosis was accompanied by an increase in the level of cytosolic cytochrome c, the relative accumulation of cytochrome c in the cytosol was unaffected by culture conditions. These results suggest that the ability to trigger apoptosis as one aspect of the control of cell growth by Equiguard is integrally linked to the release of cytochrome c, by a mechanism largely independent of the presence of androgens.