Androgen-insensitive Prostate Cancer Cells Research Articles

Magnolol Causes Alterations in the Cell Cycle in Androgen Insensitive Human Prostate Cancer Cells In Vitro by Affecting Expression of Key Cell Cycle Regulatory Proteins

Prostate cancer, one of the most common cancers in the Western world, affects many men worldwide. This study investigated the effects of magnolol, a compound found in the roots and bark of the magnolia tree Magnolia officinalis, on the behavior of 2 androgen insensitive human prostate cancer cell lines, DU145 and PC3, in vitro. Magnolol, in a 24-h exposure at 40 and 80 μM, was found to be cytotoxic to cells. Magnolol also affected cell cycle progression of DU145 and PC3 cells, resulting in alterations to the cell cycle and subsequently decreasing the proportion of cells entering the G2/M-phase of the cell cycle. Magnolol inhibited the expression of cell cycle regulatory proteins including cyclins A, B1, D1, and E, as well as CDK2 and CDK4. Protein expression levels of pRBp107 decreased and pRBp130 protein expression levels increased in response to magnolol exposure, whereas p16INK4a, p21, and p27 protein expression levels were apparently unchanged post 24-h exposure. Magnolol exposure at 6 h did increase p27 protein expression levels. This study has demonstrated that magnolol can alter the behavior of androgen insensitive human prostate cancer cells in vitro and suggests that magnolol may have potential as a novel anti-prostate cancer agent.

Epibrassinolide-induced apoptosis regardless of p53 expression via activating polyamine catabolic machinery, a common target for androgen sensitive and insensitive prostate cancer cells.

Epibrassinolide (EBR), is a member of the brassinosteroids (BR), has been shown as an apoptotic inducer in different cancer cell lines. We previously showed that EBR induced apoptosis by activating polyamine catabolic pathway, which lead to the accumulation of cytotoxic compounds such as hydrogen peroxide and aldehydes in LNCaP and DU 145 prostate cancer cells. However, we found that LNCaP prostate cancer cells expressing functional androgen receptor (AR) was found more sensitive to EBR than those with non-functional AR (DU 145 cells). To better understand the apoptotic effect of EBR, we aimed to investigate the cellular responses in p53 null, PC3 prostate cancer cells. We showed that EBR induced mitochondria-mediated and caspase-dependent apoptosis in wt and p53 stable transfected PC3 cells, which suggesting that EBR-induced apoptosis regardless of p53 expression. In addition, inhibition of p53 by pifithrin-α orthe activation of Mdm2 by Nutlin-3 co-treatment did not alter EBR induced PARP cleavage. Furthermore, EBR treatment was also induced apoptosis in both LNCaP(wt p53) and DU 145 (mt p53)cells, respectively. These all findings verified that EBR-induced apoptosis regardless of p53 expression. The PA catabolic pathway was also altered in PC3 cells causing the generation of reactive oxygen species (ROS) and intracellular PA pool decrease. However, the silencing of spermidine-spermineacetyltransferase (SSAT), a key enzyme at polyamine catabolic machinery prevented the EBR-induced apoptosis. Therefore, we concluded that EBR-induced apoptosis was mainly related with PA catabolic pathway and independent from p53 expression.

ERBB2 increases metastatic potentials specifically in androgen-insensitive prostate cancer cells.

Despite all the blood-based biomarkers used to monitor prostate cancer patients, prostate cancer remains as the second common cause of cancer mortality in men in the United States. This is largely due to a lack of understanding of the molecular pathways that are responsible for the aggressive forms of prostate cancers, the castrate-resistant prostate cancer and the metastatic prostate cancer. Cell signaling pathways activated by the ERBB2 oncogene or the RAS oncogene are frequently found to be altered in metastatic prostate cancers. To evaluate and define the role of the ERBB2/RAS pathway in prostate cancer metastasis, we have evaluated the impact of ERBB2- or RAS-overexpression on the metastatic potentials for four prostate cancer cell lines derived from tumors with different androgen sensitivities. To do so, we transfected the human DU145, LnCaP, and PC3 prostate cancer cells and the murine Myc-CaP prostate cancer cells with the activated form of ERBB2 or H-RAS and assessed their metastatic potentials by three complementary assays, a wound healing assay, a transwell motility assay, and a transwell invasion assay. We showed that while overexpression of ERBB2 increased the metastatic potential of the androgen-insensitive prostate cancer cells (i.e. PC3 and DU145), it did not affect metastatic potentials of the androgen-sensitive prostate cancer cells (i.e. LnCaP and Myc-CaP). In contrast, overexpression of H-RAS only increased the cell motility of Myc-CaP cells, which overexpress the human c-MYC oncogene. Our data suggest that ERBB2 collaborates with androgen signaling to promote prostate cancer metastasis, and that although RAS is one of the critical downstream effectors of ERBB2, it does not phenocopy ERBB2 for its impact on the metastatic potentials of prostate cancer cell lines.

Sodium meta-arsenite induces reactive oxygen species-dependent apoptosis, necrosis, and autophagy in both androgen-sensitive and androgen-insensitive prostate cancer cells

Sodium meta-arsenite (NaAsO2), a novel compound synthesized by Komipham International Co. Ltd, is an orally bioavailable, water-soluble trivalent arsenical that has shown potent cytotoxic activity in human solid cancer cells in vitro and in vivo, and is currently undergoing phase I/II clinical trials for the treatment of prostate cancer. In this study, mechanisms of cell death induced by sodium meta-arsenite were investigated. Sodium meta-arsenite reduced cell viability and increased the sub-G1 population in cell cycle analysis in both androgen-sensitive LNCaP and androgen-insensitive CWR22RV1 cells. The apoptosis induced by sodium meta-arsenite was associated with cleavage of caspases 3, 8, and 9, and poly (ADP-ribose) polymerase (PARP) and increased annexin V-positive cells, and was inhibited by the pan-caspase inhibitor Z-VAD-fmk. Sodium meta-arsenite also increased the level of the autophagy marker microtubule-associated protein 1 light chain 3 (LC3)-II and the number of autophagic vacuoles as shown by electron microscopy. Both the autophagy inhibitor 3-methyladenine and the necrosis inhibitor necrostatin-1 blocked cell death induced by sodium meta-arsenite. Moreover, sodium meta-arsenite led to the accumulation of intracellular reactive oxygen species (ROS) and N-acetyl-L-cysteine (NAC), a ROS scavenger, decreased sodium meta-arsenite-induced levels of cleaved PARP and LC3-II. Propidium iodide (PI) staining also showed that NAC restored membrane integrity, damaged by sodium meta-arsenite. Therefore, these results suggest that sodium meta-arsenite induces apoptotic, necrotic, and autophagic cell death through intracellular ROS accumulation in both androgen-sensitive and androgen-insensitive prostate cancer cells and may be used as a new anticancer drug for the treatment of prostate cancer.

Regulation of TRPM8 Channel Activity in Prostate Cancer by Androgens

The TRPM8 protein is a Ca2+-permeable ion channel and may therefore play significant role in Ca2+-homeostasis. The role of TRPM8 in peripheral nervous system is well established as a cold and menthol receptor. Unlike the sensory neurons, expression and physiological designation of TRPM8 channels in the normal prostate function and in the development of prostate cancer are entirely unclear. Previous studies showed that androgens (testosterone and 5-alpha-dihydrotestosterone) induced the mRNA expression of TRPM8 in prostate cancer cells. However, the mechanism behind their synergistic regulation and the molecular staging of prostate cancer is not fully understood. In the present study, we first investigated the intracellular location of TRPM8 in a panel of 60 cases of prostate adenocarcinoma (grade 1-4) and 9 cases of normal prostate tissues. We identified that unlike prostate cancer cell lines, prostate tumor tissues show high levels of TRPM8 which was predominantly localized as the plasma membrane protein at the tumor periphery. We also showed the expression of TRPM8 protein and its association with an androgen-sensitive (LNCaP, RWPE-2) and an androgen-insensitive (PC-3) prostate cancer and epithelial cells. Further, using chromatin-immunoprecipitation (ChIP) and calcium-imaging experiments, we identified that TRPM8 Ca2+ channel induction and functional activation is regulated by androgens in prostate. We propose for the first time a physiological role of androgen and its receptor in the induction and stabilization of TRPM8 protein in carcinogenesis. Finally, our studies suggest that TRPM8 may have a new therapeutic application for prostate cancer patients in the future.

Angiogenin Mediates Androgen-Stimulated Prostate Cancer Growth and Enables Castration Resistance

The androgen receptor (AR) is a critical effector of prostate cancer development and progression. Androgen-dependent prostate cancer is reliant on the function of AR for growth and progression. Most castration-resistant prostate cancer (CRPC) remains dependent on AR signaling for survival and growth. Ribosomal RNA (rRNA) is essential for both androgen-dependent and castration-resistant growth of prostate cancer cells. During androgen-dependent growth of prostate cells, androgen-AR signaling leads to the accumulation of rRNA. However, the mechanism by which AR regulates rRNA transcription is unknown. Here, investigation revealed that angiogenin (ANG), a member of the secreted ribonuclease superfamily, is upregulated in prostate cancer and mediates androgen-stimulated rRNA transcription in prostate cancer cells. Upon androgen stimulation, ANG undergoes nuclear translocation in androgen-dependent prostate cancer cells, where it binds to the rDNA promoter and stimulates rRNA transcription. ANG antagonists inhibit androgen-induced rRNA transcription and cell proliferation in androgen-dependent prostate cancer cells. Interestingly, ANG also mediates androgen-independent rRNA transcription through a mechanism that involves its constitutive nuclear translocation in androgen-insensitive prostate cancer cells, resulting in a constant rRNA overproduction and thereby stimulating cell proliferation. Critically, ANG overexpression in androgen-dependent prostate cancer cells enables castration-resistant growth of otherwise androgen-dependent cells. Thus, ANG-stimulated rRNA transcription is not only an essential component for androgen-dependent growth of prostate cancer but also contributes to the transition of prostate cancer from androgen-dependent to castration-resistant growth status. The ability of angiogenin to regulate rRNA transcription and prostate cancer growth makes it a viable target for therapy.

43. Targeted modulation of integrin expression increases freeze sensitivity of androgen-insensitive prostate cancer

Cryoablation has emerged as a primary therapy to treat prostate cancer. While effective, the assumption that freezing serves as a ubiquitous lethal stress is challenged by clinical experience and experimental evidence demonstrating time temperature related cell death dependence. The age-related transformation from an androgen-sensitive (AS) to an androgen-insensitive (AI) phenotype is a major challenge in the management of prostate cancer. AI cells exhibit morphological changes and resistance to many treatment therapies. This resistance has been linked with α 6 β 4 integrin overexpression as a result of androgen receptor (AR) loss. As such, we investigated the influence of increased α 6 β 4 integrin expression as a result of AR loss, on the reported increased freeze tolerance of AI prostate cancer. Further, we evaluated the targeted modulation of integrin expression in combination with cryoablation on human prostate cancer cell death. A series of studies using established AS (LNCaP LP and PC-3 AR) and AI (LNCaP HP and PC-3) cell lines were designed to investigate the cellular mechanisms contributing to variations in freezing response. Samples were frozen, thawed, and temporally assessed using fluorescence microscopy, flow cytometry and immunoblotting. Investigation into α 6 β 4 integrin expression confirmed that AI cell lines overexpressed this protein, thereby altering cellular morphology and increasing adhesion characteristics. For instance, following freezing to −15 °C, AI cells were found to exhibit increased resistance to freezing injury compared to AS cells (55% vs. 18%, respectively). Molecular investigations revealed a significant decrease in caspase 8, 9, and 3 levels in AI cells following freezing. Inhibition of α 6 β 4 integrin in AI samples resulted in increased caspase activity and enhanced cell death. These studies demonstrate that integrin expression significantly influences cell tolerance to cryoablation. The data show that the inhibition of α 6 β 4 integrin function results in a significant increase in freeze sensitivity of AI prostate cancer cells. Given these results, understanding the role of androgen-receptor related integrin expression in cell response to freezing may lead to novel options for neo-adjunctive approaches to treat prostate cancer.

Abstract 5455: Differential regulation of E2A (TCF3) by androgens in prostate cancer cells.

Abstract Introduction: E2A (TCF3) is a multifunctional basic helix loop helix (bHLH) transcription factor. E2A promotes cell differentiation, acts as a negative regulator of cell proliferation in normal cells and cancer cell lines and is required for normal B-cell development. Previous studies from our laboratory has shown that E2A expression is highly increased in prostate cancer as compared to normal prostate and that it acts as a tumor promoter in prostate cancer. Given the diverse biological pathways regulated/ influenced by E2A little is known about its regulation in prostate cancer. Experimental design: E2A expression in androgen sensitive LNCaP and insensitive C81 prostate cancer cell lines was determined by western blot after treatments with androgen receptor (AR) agonist R1881 and antagonist casodex. Putative Androgen Response Elements (ARE) were identified in the first intronic region of the E2A gene using some of the online bioinformatics tools and confirmed by Chromatin Immunoprecipitation (ChIP) with AR antibody and Luciferase reporter assays on the above mentioned cell lines after treatments with R1881 and casodex. Results: E2A expression was found to increase with the increasing aggrasiveness of prostate cancer cell lines as compared to normal prostate epithelial cell line RWPE1. When androgen responsive cell line LNCaP was treated with R1881 there was an increased expression of E2A which decreased upon treatment with antiandrogen casodex whereas the E2A expression remained unaltered upon similar treatments in androgen insensitive cell line C81. The first intronic region of the E2A gene was predicted to contain two putative ARE sites. ChIP after treatment of LNCaP and C81 cells with R1881 and casodex showed that the intronic region was bound by AR in LNCaP cells only in the presence of R1881, whereas C81 cells showed a pulldown with AR in presence as well as absence of R1881. Similar results were observed in luciferase reporter assays indicating that E2A is transactivated by AR in LNCaP cell lines whereas it is independent of androgens in C81 cell line. Furthermore, Luciferase reporter assays also confirmed that only one of the two predicted putative AREs was functionally active and responsible for the androgen mediated regulation of E2A. Conclusion: Our results indicate that E2A is differentially regulated in Prostate cancer cell lines. The increased expression of E2A and its role as a tumor promoter in prostate cancer cell lines may be contributed to its loss of androgen dependence as is evident in its progression from androgen dependent LNCaP to independent C81 cells. Acknowledgements: This study was supported by NIH/NCI RO1 CA128914 and NIH/NCRR/RCMI G12RR03062. Citation Format: Divya Patel, Jaideep Chaudhary. Differential regulation of E2A (TCF3) by androgens in prostate cancer cells. [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 5455. doi:10.1158/1538-7445.AM2013-5455

Abstract 4771: Regulatory role of an orphan nuclear receptor LRH-1 in castration-resistant growth of prostate cancer cells.

Abstract The advanced development of castration-resistant prostate cancer (CRPC) in patients upon androgen-deprivation therapy is generally believed to be mostly mediated by reactivation of androgen receptor (AR) signaling or its bypass with mechanisms involved, including clonal selection of androgen-independent or stem cell-like cell populations, AR hypersensitivity due to AR overexpression, and ligand promiscuity or independence by AR mutations, intratumoral conversion of adrenal androgens to high affinity AR ligands. Besides, the de novo steroid synthesis from cholesterol in CRPC has also been proposed. In this study, we found that the nuclear receptor liver receptor homolog-1 (LRH-1) might contribute to the CRPC growth or de novo steroid synthesis in prostate cancer cells through its positive regulation on expression of several critical enzymes in steroidogenesis, including CYP17A1, HSD3B1, HSD3B2 and StAR. Functional analyses showed that overexpression of LRH-1 could induce higher resistance to antiandrogen bicalutamide and steroid-depleted culture condition in AR-positive androgen-sensitive (LNCaP) but not in AR-negative androgen-insensitive (DU145) prostate cancer cells. In vivo tumorigenicity study showed that LNCaP-LRH-1 cells grew more aggressively in castrated or intact SCID mice, contrary to LNCaP-vector control cells that did not grow in castrated mice. Importantly, liquid chromatography-tandem mass spectrometry showed that intratumoral androgen concentrations (testosterone and DHT) were significantly higher in tumors formed by LNCaP-LRH-1 cells than those by LNCaP-vector control cells. Treatment of LNCaP-LRH-1 cells with a CYP17A1 inhibitor abiraterone could restore their sensitivity to bicalutamide and steroid-depleted culture condition. Gene knockdown of endogenous LRH-1 in AR-positive VCaP prostate cancer cells could not only suppress the mRNA expression of steroidogenic enzymes but also enhance their sensitivity to antiandrogen. Together, our results indicated that LRH-1 might play a regulatory role in advanced castration-resistant growth of prostate cancer via positive control on de novo steroid synthesis in prostate cancer cells. (This work was supported by a Direct Grant for Research 2011/2012 and a RGC-GRF grant 2009/2010) Citation Format: Lijia Xiao, Shan Yu, Wendy W.L. Hsiao, Franky Leung Chan. Regulatory role of an orphan nuclear receptor LRH-1 in castration-resistant growth of prostate cancer cells. [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 4771. doi:10.1158/1538-7445.AM2013-4771

Abstract 3883: Robo1, a new target, by Wnt and androgen receptor signaling in castration resistant prostate cancer.

Abstract Androgen receptor (AR) and β-catenin/TCF-4 signaling both play a critical role during prostate cancer progression to castration-resistance and cross-talk with each other. Therefore, identifying novel down-stream targets that are co-regulated by AR and β-catenin/TCF-4 signaling is important for understanding the development of castration-resistant prostate cancer (CRPC). Robo1, a transmembrane receptor of Slit, is known for its roles in neurogenesis and angiogenesis. Previous studies have shown that Robo/Slits are aberrantly expressing in different cancers. We have shown that Robo1 is over-expressed in androgen-insensitive prostate cancer cell lines (C4-2B, PC3, 22RV1) compared to androgen-sensitive lines (LNCaP and LAPC-4), and absent in normal prostate epithelial cells. Androgen deprivation resulted in enhanced expression of Robo1 and addition of synthetic androgen R1881decreased Robo1 expression in LNCaP cells. In addition, Wnt 3A and 5A conditioned medium stimulation increased Robo1 mRNA expression in LNCaP cells. Inhibition of Wnt signaling by overexpression of secreted Wnt antagonists Wnt-inhibitory factor-1 (WIF-1) and Frzb/sFRP3 in two CRPC cell lines, decreased the expression of Robo1. Using GenBank and MatInspector software, we have identified potential binding sites for TCF-4 in the 5’ flanking promoter region of the ROBO1 gene. Chromatin immunoprecipitation followed by real-time PCR analysis revealed that TCF-4 binds to the ROBO1 promoter at two predicted sites, and that WIF-1 and Frzb/sFRP3 expression inhibited its promoter binding. Overexpression of WIF-1 and Frzb/sFRP3 also inhibited the promoter activity of Robo1. Knock-down of Robo1 by short-hairpin RNA in PC3 cells resulted in dramatic morphological changes with increased cell aggregation and adhesion. Taken together, our results indicate that Robo1 is a co-target of Wnt and AR signaling. Further studies are in progress to define the functional role of Robo1 in CRPC. Citation Format: Noriko N. Yokoyama, Zheng Sun, Toshinori Sakai, Bang H. Hoang, Xiaolin Zi. Robo1, a new target, by Wnt and androgen receptor signaling in castration resistant prostate cancer. [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 3883. doi:10.1158/1538-7445.AM2013-3883

Heterocycles 23: Synthesis, characterization and anticancer activity of new hydrazinoselenazole derivatives

A series of novel functionalized 1,3-selenazole was synthesized by Hantzsch-type condensation reaction and evaluated for their in vitro cytotoxic activity against two human cancer cell lines. The structures of the synthesized selenosemicarbazones and functionalized 2-hydrazinyl-1,3-selenazole derivatives were assigned based on IR and NMR spectroscopic investigations, mass spectrometry, and elemental analysis data. Some of the synthesized compounds showed significant cytotoxic activities, the IC50 values below or around 10 μM were recorded with compounds 1d and 4b on both androgen-insensitive prostate cancer cells (DU-145) and hepatocarcinoma (Hep-G2) cell lines.

Distribution and Possible Function of Cannabinoid Receptor Subtype 1 in the Human Prostate

Background: Cannabinoid receptor subtype 1 (CB1) has a relationship to the proliferation of various cells including malignant tumoral cells. We investigated and compared the expression of CB1 in benign and malignant human prostate tissues and in benign and malignant human prostate cell lines, as well as its function for the proliferation of human prostate cancer cells. Methods: Real-time quantitative PCR was performed to compare its expressions in human prostate tissues (normal, benign hyperplasia, and cancer) and prostate cell lines (3 normal and 3 malignant). For localization of CB1, immunofluorescent staining with rabbit anti-CB1 polyclonal antibodies and tetramethyl isothiocyanate (TRITC)-labeled swine anti-rabbit immunoglobulin (DAKO) were used under fluorescence microscope. To further analyze whether cell death was induced by anandamide (non-selective agonist for CB1/CB2) via a receptor dependent mechanism, the viability of DU145 cells, which is known as androgen-insensitive prostate cancer cell, was measured using MTT assay. Results: CB1mRNA was found to be expressed in the all 3 human prostate tissues, however, CB1 protein was expressed in BPH and low grade malignant PC tissues, but not in high grade malignant PC tissues. CB1 as for cell lines, the expression of CB1 was low in malignant cell lines except for DU145. Anandamide elicited cell death, which was significantly inhibited by AM251 (selective antagonist for CB1), indicating that cell death induced by anandamide in DU145 cells was mediated by CB1. Anandamide time-dependently elicits up-regulation of CB1 in DU145 cells. Conclusions: CB1 may be an inhibitory regulator of androgen-insensitive human prostate cancer epithelial cell growth.

Increased Akt Signaling Resulting from the Loss of Androgen Responsiveness in Prostate Cancer

The mechanisms responsible for the switch of prostate cancer from androgen-sensitive (AS) to androgen-insensitive (AI) form are not well understood. Regulation of androgen receptor (AR), through which androgens control the expression of genes involved in prostate cells proliferation, migration and death also involves its cross-talk with the other signaling pathways, transcription factors and coregulatory proteins, such as β-catenin. With the aim to determine their possible contribution in triggering the switch from AS to AI form, which occurs upon androgen deprivation therapy - AR, Akt and β-catenin expression were knocked-down with respective siRNAs. Treatment of LNCaP prostate cells with siRNA for AR significantly reduced their proliferation (45-70%), expression of nuclear β- catenin, cyclin-D1, cyclin-G1, c-Myc as well as activity of metalloproteinases (MMPs) -2,-7,-9 and cell migration. Surprisingly, after longer (over 72 hrs) silencing of AR in LNCaP cells, elevated levels of p-Akt were detected and enhanced proliferation as well as expression of nuclear β-catenin, cyclin-D1, c-Myc and activity of MMPs were observed. Such effects were not observed in either PC-3 or DU145 AI cells. However, silencing of Akt and /or β-catenin in those as well as in LNCaP cells led to their decreased proliferation and migration. Our findings suggest that in prostate cancer cells, either AR or Akt signaling prevails, depending on their initial androgen sensitivity and its availability. In AI prostate cancer cells, Akt takes over the role of AR and more effectively contributes through the same signaling molecule, β-catenin, to AI cancer progression.

Plk1 is upregulated in androgen-insensitive prostate cancer cells and its inhibition leads to necroptosis.

Castration-resistant prostate cancer (PCa) is refractory to hormone therapy and new strategies for treatment are urgently needed. We found that androgen-insensitive (AI) PCa cells, LNCaP-AI, are reprogrammed to upregulate the mitotic kinase Plk1 and other M phase cell cycle proteins, which may underlie AI PCa growth. In androgen-depleted media, LNCaP-AI cells showed exquisite sensitivity to growth inhibition by subnanomolar concentrations of a small molecule inhibitor of Plk1, BI2536, suggesting that these cells are dependent on Plk1 for growth. In contrast, the androgen-responsive parental LNCaP cells showed negligible responses to BI2536 treatment under the same condition. BI2536 treatment of LNCaP-AI cells resulted in an increase in cell death marker PARP-1 but did not activate caspase-3, an apoptosis marker, suggesting that the observed cell death was caspase-independent. BI2536-treated LNCaP-AI cells formed multinucleated giant cells that contain clusters of nuclear vesicles indicative of mitotic catastrophe. Live-cell time-lapse imaging revealed that BI2536-treated giant LNCaP-AI cells underwent necroptosis, as evidenced by “explosive” cell death and partial reversal of cell death by a necroptosis inhibitor. Our studies suggest that LNCaP-AI cells underwent reprogramming in both their cell growth and cell death pathways, rendering them highly sensitive to Plk1 inhibition that induces necroptosis. Harnessing necroptosis through Plk1 inhibition may be explored for therapeutic intervention of castration-resistant PCa.

Telomere and Microtubule Targeting in Treatment-Sensitive and Treatment-Resistant Human Prostate Cancer Cells

Modulating telomere dynamics may be a useful strategy for targeting prostate cancer cells, because they generally have short telomeres. Because a plateau has been reached in the development of taxane-based treatments for prostate cancer, this study was undertaken to evaluate the relative efficacy of targeting telomeres and microtubules in taxane-sensitive, taxane-resistant, androgen-sensitive, and androgen-insensitive prostate cancer cells. Paclitaxel- and docetaxel-resistant DU145 cells were developed and their underlying adaptive responses were evaluated. Telomere dynamics and the effects of targeting telomeres with sodium meta-arsenite (KML001) (an agent undergoing early clinical trials), including combinations with paclitaxel and docetaxel, were evaluated in parental and drug-resistant cells. The studies were extended to androgen-sensitive LNCaP cells and androgen-insensitive LNCaP/C81 cells. Both P-glycoprotein (Pgp)-dependent and non-Pgp-dependent mechanisms of resistance were recruited within the same population of DU145 cells with selection for drug resistance. Wild-type DU145 cells have a small side population (SP) (0.4-1.2%). The SP fraction increased with increasing drug resistance, which was correlated with enhanced expression of Pgp but not breast cancer resistance protein. Telomere dynamics remained unchanged in taxane-resistant cells, which retained sensitivity to KML001. Furthermore, KML001 targeted SP and non-SP fractions, inducing DNA damage signaling in both fractions. KML001 induced telomere erosion, decreased telomerase gene expression, and was highly synergistic with the taxanes in wild-type and drug-resistant DU145 cells. This synergism extended to androgen-sensitive and androgen-insensitive LNCaP cells under basal and androgen-deprived conditions. These studies demonstrate that KML001 plus docetaxel and KML001 plus paclitaxel represent highly synergistic drug combinations that should be explored further in the different disease states of prostate cancer.

The Role of Hypoxia-Inducible Factor-1α and -2α in Androgen Insensitive Prostate Cancer Cells

ObjectivesThe aim of this study was to investigate the effects of induction and knocking down of hypoxia-inducible factor (HIF)-1α and/or -2α on tumor biology in androgen insensitive prostate cancer cell lines. Materials and methodsThe induction patterns of HIF-1α and -2α after treatment with ZnSO4 were evaluated in PC3 and DU145 cells. Both cell lines were transfected with siRNA targeted against HIF-1α and/or -2α, and the expression patterns of these 2 HIF isoforms were examined. We next performed cell counting Kit-8 (CCK-8) assays and matrigel invasion assays. Potential additive effects of HIF blockade to chemotherapy (docetaxel) or target agents (sunitinib and sorafenib) were examined. In addition, gene expression changes were determined in ZnSO4-treated DU145 cells using Western blotting. ResultsZnSO4 affected the expression of HIF in a dose-dependent manner. HIF expression was increased within the first 3 hours but then decreased. Cells in which HIF-1α and/or -2α had been knocked down using siRNA showed decreased cell viability. Invasion abilities were increased by ZnSO4 treatment in both cell lines overexpressing HIF. However, invasion potencies were decreased in response to treatment with HIF siRNAs. Blocking HIF prominently augmented the antitumor effects of target agents. The underlying mechanism could be associated with p21, cMET, IGF-1, and GLUT-1. ConclusionsOur results demonstrate that HIF-1α and -2α are important for cell proliferation and invasion ability in prostate cancer. Together, our results indicate that combinations of target agents with HIF knockdown may represent a promising strategy for the treatment of prostate cancer.

Abstract LB-161: Metformin increases lipolytic metabolism and PEDF expression in prostate cancer cells

Abstract Obesity increases the risk of prostate cancer (PCa) progression; however, the mechanism remains unclear. Obesity results in metabolic dysfunction, including increases in circulating and tissue lipid levels. A common sequela of obesity is Type II diabetes, and some studies suggest that PCa patients on the antidiabetic drug, metformin, have a reduced PCa risk. The antitumor mechanism of metformin is largely unknown. Metformin exerts its antidiabetic function via AMPK activation-dependent inhibition of hepatic gluconeogenesis, with secondary decrease in serum insulin/IGF-1 levels that may represent an indirect mechanism for its proposed antitumor effects. Previous studies have shown that metformin directly inhibits PCa cell proliferation and cyclin D1 expression in vitro. Expression of pigment epithelium-derived factor (PEDF), a potent angiogenesis inhibitor, is decreased in PCa tissues, and PEDF inhibits PCa cell proliferation. Interestingly, PEDF knockout mice have defects in lipid metabolism and develop prostate hyperplasia, so we hypothesized that metformin's antitumor mechanism could be mediated, in part, through regulation of PEDF expression and lipolytic metabolism. To simulate obesity-induced cellular triglyceride (TG) accumulation, we treated cells with oleic acid (OA, 1.0 mM) ± PEDF (10 nM) or metformin (5 mM). Proliferation (cell count or MTT assay) and lipid content (TG isolation) were quantified, and lipid and protein were assessed by lipid staining and immunocytochemistry or Western blot, respectively. In androgen-insensitive PCa cells (PC-3 and DU145), OA treatment promoted TG accumulation, cyclin D1 expression and proliferation, with increased expression of lipid droplet-associated proteins, adipose triglyceride lipase (ATGL) and CGI-58, and conversely suppressed PEDF expression. Furthermore, PEDF treatment decreased basal TG levels (32.89 ± 1.0 control vs. 17.3 ± 0.6 in PEDF; P<0.0002) and suppressed OA-induced TG accumulation (130.33 ± 1.2 μg/1x106 cells in OA vs. 72.92 ± 3.5 OA + PEDF; P<0.0002) and proliferation (P<0.05), concomitantly decreasing ATGL and CGI-58 expression. Interestingly, metformin blocked OA-induced proliferation (P<0.02), decreased ATGL and CGI-58 expression, and increased PEDF expression. When free glycerol release was quantified as a measure of TG lipolysis, both metformin and PEDF increased PCa cell TG turnover compared to control cells (P<0.05). These data suggest that metformin exerts its anti-proliferative effect on PCa cells, in part, through upregulation of lipolytic metabolism via increased PEDF expression. These data provide a novel pharmacodynamic biomarker for mechanistic studies in patients, and platform for rational design of “personalized” cancer therapies targeting altered lipid metabolism in obesity-induced cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-161. doi:1538-7445.AM2012-LB-161

796 CORTISOL SERUM LEVELS ARE INCREASED AND INDUCE GENOMIC INSTABILITY IN ADVANCED PROSTATE CANCER

INTRODUCTION AND OBJECTIVES: Cortisol, also known as stress hormone, is involved with chronic stress; it appears to be associated with higher levels of prostate-specific antigen (PSA) and benign prostate enlargement (BPE). To assess the relationship between cortisol and prostate cancer (PCa), serum cortisol levels were measured in normal men and those with primary or metastatic PCa. In addition, the effect of cortisol on genomic instability was investigated by evaluating DNA mismatch repair (MMR) pathway in androgen-deprived benign or malignant prostate cells. METHODS: Serum cortisol and creatinine levels were determined using ELISA assay. Immunohistochemical staining for MMR proteins was performed on tissue microarray slides. Genomic instability was evaluated by analyzing the microsatellite instability (MSI) using PCR and the National Cancer Institute (NCI)-recommended panel of five MS markers including two mononucleotide repeat (BAT25 and BAT26) and three dinucleotide repeats (D2S123, D5S346, and D17S250). Genomic DNAs for PCR were extracted from AR-positive (LNCaP, E006AA, CWR-22RV1, C4-2B, MDA-PCa2b) and androgeninsensitive (PC-3 and DU145) PCa cell lines and normal prostate epithelial cells that were treated with cortisol and androgen-deprived for 2 to 20 weeks. Purified PCR fragments were separated on ABI 3730XL DNA Analyzer and genotyped using the ABI Gene Mapper ID, V3.2 software. MMR genes and protein expression levels in the cells were examined with qRT-PCR and western blotting, respectively. RESULTS: Serum cortisol levels were in rank order metastatic castrate-resistant prostate cancer (mCRPCa) primary PCa BPE. Serum cortisol levels were higher in African Americans (AAs) than Caucasian Americans (CAs) in BPE, primary PCa, and mCRPCa. Chronic treatment of PCa cells with cortisol increased growth rate and PSA expression. Cortisol induced high-frequency MSI as evidenced by the insertion or deletion of 1-5 bps in at least 2 of 5 MS markers in both androgen-responsive and androgen-insensitive PCa cell lines. Analysis of the DNA mismatch repair pathway in PCa cells and tissues originated from AAs and CAs showed alterations in the expression levels of PMS1, PMS2, MLH1, MSH2, MSH3, MSH6, that belong to the MMR system. CONCLUSIONS: These data support a non-immunological role for cortisol in promoting genomic instability in androgen-deprived PCa cells that might lead to hyper-mutator phenotype or accumulation of adoptive-genetic changes in response to the stress that results from androgen-deprivation therapy for advanced PCa.

Chemopreventative phytochemical 3,3′‐diindolylmethane inhibits histone deacetylases in prostate cancer cells

Increased consumption of cruciferous vegetables is associated with a reduced risk of prostate cancer. Indole‐3‐carbinol (I3C) and 3, 3′‐diindolylmethane (DIM), phytochemicals derived from cruciferous vegetables, have shown promise in inhibiting prostate cancer in experimental models. Histone deacetylase (HDAC) inhibition is an emerging target for cancer prevention and therapy. We sought to examine the effects of I3C and DIM on HDACs in prostate cancer cells. I3C had a minimal effect on cell proliferation and had no effect on HDAC activity or expression in androgen insensitive (PC‐3) prostate cancer cells. In contrast, DIM significantly decreased cell proliferation, HDAC activity, as well as HDAC2 and HDAC8 expression in PC‐3 cells. A trend of decreasing expression was observed for HDAC4 and HDAC6. Surprisingly, DIM treatment caused a significant reduction in acetylated histone H4 but an increase in acetylation of the HDAC6 target gene, tubulin. DIM also significantly inhibited HDAC activity in androgen sensitive LnCap cells. These results show that DIM inhibits HDAC activity and may contribute to the anti‐proliferative effects of DIM in the prostate. The ability of DIM to target aberrant epigenetic patterns, in addition to its effects on detoxification, may make it an effective chemopreventive agent by targeting multiple stages of prostate carcinogenesis.Grant Funding Source : P01CA090890

Abstract C151: Maspin is regulated by the alternative NF-κB subunit in androgen-insensitive prostate cancer cell lines.

Abstract Dysregulation of Maspin expression and constitutive activation of NF-κB subunits are important events in tumorigenesis of prostate cancer. Recent finding points that RelB, which contributes to the alternative NF-κB activity, interferes with carcinogenesis in the prostate. We report here, that both the classical (predominantly p50/RelA heterodimers) and the alternative NF-κB activities (p52/RelB heterodimers) are constitutively present in androgen-insensitive human prostate cancer cells. An inverse relationship of Maspin and RelB expression is observed in prostate cancer tissues at the later stage. TNF-α signaling triggers the nuclear accumulation of RelB and the concomitant reduction of Maspin expression in a time-dependent manner. In addition, the proteasome inhibitor-induced Maspin expression is accompanied by the reduction of RelB expression. A successful depletion of RelB expression, but not RelA expression, induces Maspin expression. RelB-deficiency abrogates the proteasome inhibitor-induced Maspin expression. Moreover, we demonstrate that the enforced expression of RelB protein in prostate cancer cells inhibits Maspin expression. We propose that RelB is an essential molecule controlling the endogenous and the proteasome inhibitor-induced Maspin expression. Developing a RelB-targeted therapeutic intervention, which might be coupled with the induction of a tumor suppressor Maspin, is valuable in treating advanced, metastatic prostate cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C151.