Androgen-independent Prostate Cancer Research Articles

Peri-Prostatic Adipocyte-Released TGFβ Enhances Prostate Cancer Cell Motility by Upregulation of Connective Tissue Growth Factor

Periprostatic adipose tissue (PPAT) has emerged as a key player in the prostate cancer (PCa) microenvironment. In this study, we evaluated the ability of PPAT to promote PCa cell migration, as well as the molecular mechanisms involved. Methods: We collected conditioned mediums from in vitro differentiated adipocytes isolated from PPAT taken from PCa patients during radical prostatectomy. Migration was studied by scratch assay. Results: Culture with CM of human PPAT (AdipoCM) promotes migration in two different human androgen-independent (AI) PCa cell lines (DU145 and PC3) and upregulated the expression of CTGF. SB431542, a well-known TGFβ receptor inhibitor, counteracts the increased migration observed in presence of AdipoCM and decreased CTGF expression, suggesting that a paracrine secretion of TGFβ by PPAT affects motility of PCa cells. Conclusions: Collectively, our study showed that factors secreted by PPAT enhanced migration through CTGF upregulation in AI PCa cell lines. These findings reveal the potential of novel therapeutic strategies targeting adipocyte-released factors and TGFβ/CTGF axis to fight advanced PCa dissemination.

Induction of Endoplasmic Reticulum Stress-Mediated Apoptosis by Aminosteroid RM-581 Efficiently Blocks the Growth of PC-3 Cancer Cells and Tumors Resistant or Not to Docetaxel.

Aminosteroid derivative RM-581 was previously identified as an endoplasmic-reticulum (ER) stress inducer with potent in vitro and in vivo anticancer activities. We report its evaluation in androgen-independent prostate cancer (PC-3) cells. RM-581 efficiently blocks PC-3 cell proliferation with stronger activity than that of a selection of known antineoplastic agents. This later also showed a synergistic effect with docetaxel, able to block the proliferation of docetaxel-resistant PC-3 cells and, contrary to docetaxel, did not induce cell resistance. RM-581 induced an increase in the expression level of ER stress-related markers of apoptosis, potentially triggered by the presence of RM-581 in the ER of PC-3 cells. These in vitro results were then successfully translated in vivo in a PC-3 xenograft tumor model in nude mice, showing superior blockade than that of docetaxel. RM-581 was also able to stop the progression of PC-3 cells when they had become resistant to docetaxel treatment. Concomitantly, we observed a decrease in gene markers of mevalonate and fatty acid pathways, and intratumoral levels of cholesterol by 19% and fatty acids by 22%. Overall, this work demonstrates the potential of an ER stress inducer as an anticancer agent for the treatment of prostate cancers that are refractory to commonly used chemotherapy treatments.

Effect of Combining Traditional Chinese Medicine with Hormonal Therapy on Quality of Life and Tumor Markers of Prostate Cancer Patients.

Objective To explore the effect of combining traditional Chinese medicine (TCM) with hormonal therapy on the quality of life and tumor markers of prostate cancer patients. Methods A total of 60 prostate cancer patients treated in Zibo Central Hospital from June 2017 to June 2021 were selected for the retrospective analysis study and divided into the control group and experimental group based on whether applying TCM treatment, with 30 cases each. The patients in the experimental group received the combined treatment of TCM and hormonal therapy, and those in the control group only accepted the hormonal therapy, so as to analyze their clinical efficacy and tumor marker levels after treatment. Results The patients' general information were not statistically different (P > 0.05); after treatment, the levels and ratios of total prostate-specific antigen (TPSA) and free prostate-specific antigen (FPSA) of patients in both groups were improved, and the experimental group obtained significantly lower TPSA and FPSA levels (P < 0.05) and higher TPSA/FPSA ratios (P < 0.05); the incidence of androgen-independent prostate cancer was significantly lower in the experimental group than in the control group (8 vs. 16, P < 0.05); in addition, the time to develop into the androgen-independent prostate cancer was longer in patients of the experimental group than those of the control group (P < 0.05); the treated patients in the experimental group obtained obviously higher Functional Assessment of Cancer Therapy-Prostate (FACT-P) quality of life scores and Karnofsky score (KPS) than those in the control group (P < 0.05), and the number of patients with recovered PSA levels was significantly higher in the experimental group than in the control group (P < 0.05). Conclusion Combining self-made TCM formula with hormonal therapy can effectively improve the levels of prostate tumor markers and postpone the progress of developing from prostate cancer to androgen-independent prostate cancer, which is conducive to promoting the patients' quality of life.

KLF9 suppresses cell growth and induces apoptosis via the AR pathway in androgen-dependent prostate cancer cells

Kruppel-like factors (KLFs) play an important role in many biological processes including cell proliferation, differentiation and development. Our study showed that the level of KLF9 is lower in PCa cell lines compared to a benign prostate cell line; the androgen-independent cell line PC3 expresses significantly lower KLF9 than the androgen-dependent cell line, LNCaP. Forced overexpression of KLF9 suppressed cell growth, colony formation, and induced cell apoptosis in LNCaP cells. We also found that KLF9 expression was induced in response to apoptosis caused by flutamide, and further addition of dihydrotestosterone antagonized the action of flutamide and significantly decreased KLF9 expression. Furthermore, activation of the androgen receptor (AR) was inhibited by the overexpression of KLF9. Our research shows that KLF9 is lower in androgen-independent cell lines than in androgen-dependent cell lines; Overexpression of KLF9 dramatically suppresses the proliferation, anchorage-independent growth, and induces apoptosis in androgen-dependent cells; KLF9 inhibition on prostate cancer cell growth may be acting through the AR pathway. Our results therefore suggest that KLF9 may play a significant role in the transition from androgen-dependent to androgen-independent prostate cancer and is a potential target of prevention and therapy.

Caryophyllene Oxide, the Active Compound Isolated from Leaves of Hymenaea courbaril L. (Fabaceae) with Antiproliferative and Apoptotic Effects on PC-3 Androgen-Independent Prostate Cancer Cell Line.

Cancer treatment frequently carries side effects, therefore, the search for new selective and effective molecules is indispensable. Hymenaea courbaril L. has been used in traditional medicine in South America to treat several diseases, including prostate cancer. Leaves’ extracts from different polarities were evaluated using the 3-(4,5-methyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) cell viability assay to determine the cytotoxicity in prostate p53-null cells, followed by bio-guided fractionations to obtain the most cytotoxic fraction considering the selectivity index. The most cytotoxic fraction was analyzed by GC/MS to identify the active compounds. The majority compound, caryophyllene oxide, induced early and late apoptosis, depolarized the mitochondrial membrane, leading to several morphological changes and shifts in apoptotic proteins, and caspases were evidenced. Depolarization of the mitochondrial membrane releases the pro-apoptotic protein Bax from Bcl-xL. The apoptosis process is caspase-7 activation-dependent. Caryophyllene oxide is a safe anti-proliferative agent against PC-3 cells, inducing apoptosis with low toxicity towards normal cells.

Safety and efficacy of high dose pulse calcitriol and docetaxel for androgen-independent prostate cancer

Abstract Background: Previous therapy usually included prostatectomy or radiation to remove or destroy the cancerous cells within the prostate capsule. But, many patients eventually fail therapy and die of recurrent androgen-independent prostate cancer (AIPC). To date, there is no effective cure for prostate cancer. This study aims to evaluate the clinical safety and efficacy of high-dose pulse calcitriol and docetaxel for androgen-independent prostate cancer to guide the clinician's drug use. Methods: Seven Eight databases included PubMed, PubMed Central, EMbase, Medline, China National Knowledge Infrastructure, WAN FANG Database, and Web of Science will be systematically retrieved from January 1st, 2010 to May 30th, 2021. We will screen eligible studies to meet the inclusion criteria. Two independent reviewers will extract data, evaluate the risk of bias and analyze data independently. The quality of the included studies will be evaluated by the RevMan 5.4 software. The Cochrane risk of bias tool will be used to assess the quality of the studies. Results: This systematic review will provide high-quality evidence for the safety and efficacy of high-dose pulse calcitriol and docetaxel for AIPC. Conclusions: The findings of the study will provide scientific evidence of the safety and efficacy of high dose pulse calcitriol, and docetaxel for AIPC to guide the clinician's drug use. Ethics and dissemination: Not applicable INPLASY registration number: INPLASY202170028.

MP33-11 OBESITY-ASSOCIATED PROINFLAMMATORY CYTOKINES REGULATE PROSTATE CANCER CELL PROLIFERATION AND MIGRATION

You have accessJournal of UrologyProstate Cancer: Basic Research & Pathophysiology I (MP33)1 Sep 2021MP33-11 OBESITY-ASSOCIATED PROINFLAMMATORY CYTOKINES REGULATE PROSTATE CANCER CELL PROLIFERATION AND MIGRATION Xiaobo Wu, Christina Sharkey, Zongwei Wang, and Aria Olumi Xiaobo WuXiaobo Wu More articles by this author , Christina SharkeyChristina Sharkey More articles by this author , Zongwei WangZongwei Wang More articles by this author , and Aria OlumiAria Olumi More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002042.11AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Previous studies revealed that obesity is significantly associated with a higher incidence of prostate cancer (PCa). Meanwhile, obesity-associated inflammation plays an important role in tumorigenesis. Here we wished to identify the downstream signaling pathways of obesity-associated proinflammatory cytokines IL-6 and TNF-α’s effects on prostate cancer cell proliferation and migration. METHODS: Adipocytes and THP-1 macrophages were differentiated and treated with myristic acid (MA) to induce inflammatory cytokines, and conditioned media (CM) were collected to treat prostate epithelial cancer, androgen-dependent LNCaP and androgen-independent PC-3 cell lines. Cells were also treated with interleukin-6 (IL-6) 10ng/ml, tumor necrosis factor-alpha (TNF-α) 10ng/ml, oncometabolite R-2-hydroxyglutarate 50µM (R-2-HG) and Dutasteride 10µM (SRD5A1 inhibitor). Prostate cancer cell migration and proliferation were assessed by scratch assay (0h, 24h, 48h) and MTT assay (0h, 24h, 48h, 72h) and analyzed by 2-way ANOVA. Enzymes regulating steroid hormone metabolism SRD5A1, AKR1C1, AKR1C3, HSD3B2, CYP17A1 and UGT2B15 were determined by immunocytochemistry and qPCR. The protein level of SRD5A1 was measured by western blot. RESULTS: The proliferation of LNCaP and PC-3 cells was significantly stimulated by adding of CM for 24h, 48h, and 72h. The migration of PC-3 cells was also promoted by CM. Addition of testosterone did not change the effect of CM. Administration of IL-6, TNF-α and R-2-HG significantly increased the proliferation and migration of LNCaP and PC-3 cells. IL-6 also increased the expression of SRD5A1.. The expression of AKR1C1 and HSD3B2 was downregulated, and UGT2B15 was upregulated by TNF-α. SRD5A1 was upregulated either by R-2-HG alone or combining with IL-6 and TNF-α, but downregulated by Dutasteride alone. CONCLUSIONS: Our study suggests that obesity-associated proinflammatory cytokines, IL-6 and TNF-α, affect prostate cancer cells’ proliferation and migration in an androgen-independent manner. Synergistic targeting oncometabolite R-2-HG and proinflammatory cytokines may improve the management of androgen-independent prostate cancer. Source of Funding: NIH/NIDDK © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e610-e611 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Xiaobo Wu More articles by this author Christina Sharkey More articles by this author Zongwei Wang More articles by this author Aria Olumi More articles by this author Expand All Advertisement Loading ...

632P Differential transcriptomic profiling of BCL2-related genes in primary tumor (PT) and metastatic sites (MS) of prostate cancer (PCa)

Bcl-2 anti-apoptotic and pro-apoptotic proteins regulate sensitivity to apoptosis stimuli. Overexpression of Bcl-2 and resistance to apoptosis have been implicated in the development of androgen-independent PCa and disease progression. This project aimed to characterize the transcriptomic profile of BCL2-related genes and investigate distinct sensitivity to apoptosis in PT and MS.

Innovative C2-symmetric testosterone and androstenedione dimers: Design, synthesis, biological evaluation on prostate cancer cell lines and binding study to recombinant CYP3A4.

The synthesis of two isomeric testosterone dimers and an androstenedione dimer is reported. The design takes advantage of an efficient transformation of testosterone leading to the synthesis of the key diene, 7α-(buta-1,3-dienyl)-4-androsten-17β-ol-3-one, through an elimination reaction. It was found that in some instances the same reaction led to partial epimerization of the 17β-hydroxyl group into the 17α-hydroxyl group. The specific orientation of the hydroxyl function was confirmed by NMR spectroscopy. Capitalizing on this unforeseen side reaction, several dimers were assembled using an olefin metathesis reaction with Hoveyda-Grubbs catalyst. This led to the formation of two isomeric testosterone dimers with 17α-OH or 17β-OH (14α and 14β) as well as an androstenedione dimer (14). The new dimers and their respective precursors were tested on androgen-dependent (LNCaP) and androgen independent (PC3 and DU145) prostate cancer cells. It was discovered that the most active dimer was made of the natural hormone testosterone (14β) with an average IC50 of 13.3μM. In LNCaP cells, 14β was ∼5 times more active than the antiandrogen drug cyproterone acetate (IC50 of 12.0μM vs. 59.6μM, respectively). At low concentrations (0.25-0.5μM), 14α and 14β were able to completely inhibit LNCaP cell growth induced by testosterone or dihydrotestosterone. Furthermore, cross-reactivity of androgen-based dimers with sterol-metabolizing cytochrome P450 3A4 was explored and the results are disclosed herein.

SHH-N non-canonically sustains androgen receptor activity in androgen-independent prostate cancer cells

Prostate cancer is the second most frequent cancer diagnosed in men worldwide. Localized disease can be successfully treated, but advanced cases are more problematic. After initial effectiveness of androgen deprivation therapy, resistance quickly occurs. Therefore, we aimed to investigate the role of Hedgehog-GLI (HH-GLI) signaling in sustaining androgen-independent growth of prostate cancer cells. We found various modes of HH-GLI signaling activation in prostate cancer cells depending on androgen availability. When androgen was not deprived, we found evidence of non-canonical SMO signaling through the SRC kinase. After short-term androgen deprivation canonical HH-GLI signaling was activated, but we found little evidence of canonical HH-GLI signaling activity in androgen-independent prostate cancer cells. We show that in androgen-independent cells the pathway ligand, SHH-N, non-canonically binds to the androgen receptor through its cholesterol modification. Inhibition of this interaction leads to androgen receptor signaling downregulation. This implies that SHH-N activates the androgen receptor and sustains androgen-independence. Targeting this interaction might prove to be a valuable strategy for advanced prostate cancer treatment. Also, other non-canonical aspects of this signaling pathway should be investigated in more detail and considered when developing potential therapies.

Diarylpentanoid (1,5-bis(4-hydroxy-3-methoxyphenyl)-1,4-pentadiene-3-one) (MS13) Exhibits Anti-proliferative, Apoptosis Induction and Anti-migration Properties on Androgen-independent Human Prostate Cancer by Targeting Cell Cycle-Apoptosis and PI3K Signalling Pathways.

Diarylpentanoids exhibit a high degree of anti-cancer activity and stability in vitro over curcumin in prostate cancer cells. Hence, this study aims to investigate the effects of a diarylpentanoid, 1,5-bis(4-hydroxy-3-methoxyphenyl)-1,4-pentadiene-3-one (MS13) on cytotoxicity, anti-proliferative, apoptosis-inducing, anti-migration properties, and the underlying molecular mechanisms on treated androgen-independent prostate cancer cells, DU 145 and PC-3. A cell viability assay has shown greater cytotoxicity effects of MS13-treated DU 145 cells (EC50 7.57 ± 0.2 µM) and PC-3 cells (EC50 7.80 ± 0.7 µM) compared to curcumin (EC50: DU 145; 34.25 ± 2.7 µM and PC-3; 27.77 ± 6.4 µM). In addition, MS13 exhibited significant anti-proliferative activity against AIPC cells compared to curcumin in a dose- and time-dependent manner. Morphological observation, increased caspase-3 activity, and reduced Bcl-2 protein levels in these cells indicated that MS13 induces apoptosis in a time- and dose-dependent. Moreover, MS13 effectively inhibited the migration of DU 145 and PC-3 cells. Our results suggest that cell cycle-apoptosis and PI3K pathways were the topmost significant pathways impacted by MS13 activity. Our findings suggest that MS13 may demonstrate the anti-cancer activity by modulating DEGs associated with the cell cycle-apoptosis and PI3K pathways, thus inhibiting cell proliferation and cell migration as well as inducing apoptosis in AIPC cells.

Abstract 2459: Altered bioenergetics profile in prostate cancer cells treated with andrographolide

Abstract Prostate cancer (PCa) is the second leading cause of cancer deaths in the United States alone. An emerging area of cancer therapeutics is the combination of the standard-of-care treatment with natural products. Andrographolide, a labdane diterpenoid and the main bioactive component of the medicinal plant Andrographis paniculata, has shown a wide range of therapeutic potential, including cancer. Previous studies from our laboratory have demonstrated that Andrographolide induces a DNA damage response, including ATM gene upregulation, involving cell growth suppression, cell cycle arrest, and apoptosis in PCa in vitro. The ATM gene is an important regulator of p53 and is consider a master switch for the p53-family member p63. The p63 is a natural marker of the basal lamina in the normal prostate, while its expression is lost in 99% of prostatic adenocarcinomas. Like p53, the p63 protein is implicated in tumor suppression and inhibition of metastasis, and also plays a role in energy metabolism. In this study, we aim to (1) determine the role of Andrographolide on the expression of p63, and (2) determine the bioenergetics profile of PCa cells. Androgen-dependent (22Rv1) and androgen-independent (PC3) PCa cell lines were treated with Andrographolide (25µM) for 24 h and 48 h. The gene expression of TP63 was assessed by real-time quantitative PCR (RT-qPCR). At the protein level, the expression of p63 was evaluated by immunofluorescence. The bioenergetics profile was performed using the Agilent Seahorse XF Real-Time ATP Rate Assay to determine the ATP production from glycolysis and mitochondria in living PC3 and 22Rv1 PCa cells. RT-PCR analysis showed a significant upregulation of the TP63 gene with a fold-change of 2.2 (p&amp;lt; 0.05) and immunofluorescence analysis showed an increase in expression of p63 protein with a fold-change of 1.8 (p&amp;lt; 0.05) in 22Rv1 cells treated with Andrographolide. The bioenergetics profile of PC3 cells, 48 h after Andrographolide treatment, revealed a shift in the cytoplasmic to mitochondrial ATP production rate from 54.4% to 72.6% and from 45.6% to 27.4%, respectively. Similar results were obtained in 22Rv1 cells. These results confirmed that Andrographolide induces the expression of the tumor protein p63 in androgen-dependent 22Rv1 PCa cells and alters the bioenergetics profile of both PC3 and 22Rv1 PCa cells. Our findings suggest a novel mechanism in which Andrographolide promotes the expression of p63 as in normal cells, and transforms the bioenergetics by increasing glycolytic-ATP. Citation Format: Jesus Sosa, María Sánchez Vázquez, Sylvette Ayala-Peña, Carlos Torres Ramos, Magaly Martínez Ferrer. Altered bioenergetics profile in prostate cancer cells treated with andrographolide [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2459.

Abstract 1035: MS13 (1, 5-bis (4-hydroxy-3-methanoxyphenyl)-1, 4-pentadiene-3-one) exhibits anti-cancer properties in androgen-independent prostate cancer cells

Abstract Prostate cancer (PCa) is the most frequent malignancy among men. Most PCa patients initially respond to androgen deprivation therapy (ADT) but eventually gain androgen-independent properties (AIPC), which are currently incurable and develop resistance to treatment. Thus, novel therapeutic agents with fewer side effects to treat AIPC is urgently needed. Diarylpentanoid [1, 5-bis (4-hydroxy-3-methanoxyphenyl)-1, 4-pentadiene-3-one] (MS13), a curcumin analogue, demonstrated dose- and time-dependent growth inhibitory effects on AIPC cells. However, the anti-cancer effects of MS13 on AIPC cells have not been extensively studied. This study aims to investigate the expression of genes associated with Notch, Hedgehog, and chromatin condensation pathways in MS13-treated DU 145 cells. In our previous study, MS13 exhibited greater cytotoxicity effects than curcumin in DU 145 cells with lower EC50 values (approximately 8 µM and 35 µM, respectively). The gene expression analysis of MS13-treated DU 145 cells was further explored using nCounter PanCancer Pathway Panel (Nanostring Technologies, USA). Briefly, DU 145 cells were treated with two times of EC50 value (16 µM) of MS13 for 24 hours. The RNA was extracted from treated and controls cells using the RNeasy Mini Kit (Qiagen, USA). Genes with a filter of two-fold-cut-off, p. adjusted ≤ 0.05, and FDR≤ 0.05 were defined as significant differentially expressed genes (DEGs). The finding demonstrated that several genes were significantly altered in MS13-treated DU 145 cells at 16 µM for 24 hours, in comparison with the controls. A total of 7 significantly DEGs associated with Notch, Hedgehog, and chromatin condensation pathways were selected for further analysis. The DEGs associated with Notch pathway including Jagged Canonical Notch Ligand 1 (JAG1), Delta Like Canonical Notch Ligand 4 (DLL4) and Notch Receptor 1 (NOTCH1) were upregulated, and shown to mediate anti-proliferative, apoptosis as well as inhibit metastasis and angiogenic properties in cancer cells. Meanwhile, the DEGs associated with Hedgehog pathway; Protein Kinase CAMP-Activated Catalytic Subunit Beta (PRKACB), Wnt Family Member 7B (WNT7B), Wnt Family Member 5B (WNT5B) and Chromatin condensation; Histone Deacetylase 6 (HDAC6) were downregulated and implicated in cancer progression, tumorigenesis, metastasis, and cell migration. Moreover, genes such as NOTCH1, PRKACB, WNT7B, and WNT5B were involved in multiple signaling pathways, displaying greater growth inhibition. In conclusion, the findings suggest that MS13 may demonstrate anti-cancer effects through modulating key canonical pathways that enhances tumor-suppressive and inhibits oncogenic genes, indicating a potential therapeutic agent for AIPC. Citation Format: Nurul Azwa Abd. Wahab, Iekhsan Othman, Faridah Abas, Rakesh Naidu. MS13 (1, 5-bis (4-hydroxy-3-methanoxyphenyl)-1, 4-pentadiene-3-one) exhibits anti-cancer properties in androgen-independent prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1035.

MiR-221 regulates proliferation, invasion, apoptosis and progression of prostate cancer cells by modulating E-cadherin/Wnt/β catenin axis

MicroRNA-221 (miR-221) is aberrantly expressed in various tumours including prostate carcinoma and can act as either tumour suppressor or oncogene depending on the tumour system and stage. Epithelial-mesenchymal transition (EMT) is a plastic transition in tumour progression characterized by decreased expression of epithelial markers and increased expression of mesenchymal markers and has been known to play a critical role in promoting metastases. In this study, we have demonstrated the functional role of miR-221 in regulating epithelial-mesenchymal transition in prostate cancer. Expression of miR-221 was found to be downregulated in androgen-independent prostate cancer cells and was found to be inversely correlated with EMT. Ectopic expression of miR-221 potentially inhibited invasion and migration of androgen-independent prostate cancers cells and inhibition of miR-221 expression rescued the anti-invasive effects of miR-221. Flow cytometric analysis further revealed that miR-221 could induce cell cycle arrest with simultaneous induction of apoptosis. Additionally, transfection with miR-221 mimic upregulated negative regulators of Wnt/β catenin pathway such as GSK-3β thereby inhibiting it and downregulated mesenchymal markers viz., N-cadherin and Vimentin with simultaneous upregulation of epithelial marker E-cadherin as revealed by real-time PCR and Western blot analysis. A study using xenograft model further confirmed the anti-tumorigenic and anti-metastatic role of miR-221 in prostate cancer. We believe that his study is the first report documenting the miR-221- Wnt/β catenin signaling-EMT regulatory circuit in prostate cancer.

Abstract 1935: Antitumor effect mechanism of statin in PCa cells focusing on autophagy

Abstract Antitumor effect mechanism of statin in prostate cancer cells focusing on autophagy Background:Statins have recently been studied for their proapoptotic and antimetastatic effects. However, the exact mechanisms of their anticancer actions remain unclear. We have studied the novel castration resistant prostate cancer (CRPC) treatment with statins. Using microarrays, we discovered up-regulation of ATG4B (Autophagy related 4 homolog B) in PC-3 cells after simvastatin treatment. In this study, we evaluated the effects of simvastatin focusing on androgen-independent prostate cancer cells. Methods:PC-3, LNCaP-LA, 22RV-1-LA (taht were derived from LNCaP and 22RV1 cells and cultured in 10% charcoal-stripped fetal bovine serum for 3 months), and DU145 human prostate cancer cell lines were used. Microarrays were performed using PC-3 untreated or administered with simvastatin (0 uM versus 5 uM). Proliferation, migration, and invasion were evaluated using MTS assay, Migration assay, and Invasion assay, respectively. The increases of autophagy were detected by western blot using microtubule associated protein 1 light chain 3 (LC-3) antibody and confirmed by a fluorescence microscope with staining autophagosomes by autophagy detection kit (abcam, Cambridge, UK). Results:Using microarrays, we discovered up-regulation of ATG4B in PC-3 cells after simvastatin treatment. Simvastatin inhibited the proliferation, migration, and invasion of PC-3, LNCaP-LA, 22RV1-LA and DU145 cells. The expression level of LC-3 was up-regulated by simvastatin in PC-3, LNCaP-LA, 22RV1-LA and DU145 cells. In PC-3 cells treated with statin, a significant increase in autophagosomes was confirmed by fluorescence microscopy. The expression of LC-3 and autophagosomes were increased in a concentration-dependent manner. Conclusions:It was found that statin administration enhances autophagy induction in a prostate cell line. We plan to study the combined effect with other drugs that induce autophagy. We would like to develop CRPC treatment by inducing autophagy cell death. Citation Format: Yoshiyuki Miyazawa, Daisuke Oka, Hiroshi Nakayama, Yoshitaka Sekine, Seiji Arai, Kazuhiro Suzuki. Antitumor effect mechanism of statin in PCa cells focusing on autophagy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1935.

Alnus sibirica Compounds Exhibiting Anti-Proliferative, Apoptosis-Inducing, and GSTP1 Demethylating Effects on Prostate Cancer Cells.

Alnus sibirica (AS) is distributed in Korea, Japan, China, and Russia and has reported anti-oxidant, anti-inflammatory, and reducing activities on atopic dermatitis-like skin lesions, along with other beneficial health properties. In the present study, we tried to prove the cancer-preventive activity against prostate cancer. The extracted and isolated compounds, oregonin (1), hirsutenone (2), and hirsutanonol (3), which were isolated from AS, were tested for anti-proliferative activity. To do this, we used the MTT assay; NF-κB inhibitory activity, using Western blotting; apoptosis-inducing activity using flow cytometry; DNA methylation activity, using methylation-specific polymerase chain reaction in androgen-dependent (LNCaP) and androgen-independent (PC-3) prostate cancer cell lines. The compounds (1–3) showed potent anti-proliferative activity against both prostate cancer cell lines. Hirsutenone (2) exhibited the strongest NF-κB inhibitory and apoptosis-inducing activities compared with oregonin (1) and hirsutanonol (3). DNA methylation activity, which was assessed for hirsutenone (2), revealed a concentration-dependent enhancement of the unmethylated DNA content and a reduction in the methylated DNA content in both PC-3 and LNCaP cells. Overall, these findings suggest that hirsutenone (2), when isolated from AS, may be a potential agent for preventing the development or progression of prostate cancer.

AMPK/SIRT1 signaling through p38MAPK mediates Interleukin-6 induced neuroendocrine differentiation of LNCaP prostate cancer cells

Neuroendocrine Prostate Cancer (NEPC) is an aggressive form of androgen independent prostate cancer (AIPC), correlated with therapeutic resistance. Interleukin (IL)-6 promotes proliferation and neuroendocrine differentiation (NED) of androgen dependent LNCaP cells. We treated LNCaP cells with IL-6 and observed for in vitro NED of cells and also expression of NE markers βIII tubulin, neuron-specific enolase (NSE) and chromogranin A (ChA). Here we investigated the proteins and/or pathways involved in NED of LNCaP cells induced by IL-6 and characterized their role in NED of PCa cells. We found that the altered proteins modulated AMPK signaling pathway in NE cells. Remarkably, IL-6 induces NED of LNCaP cells through activation of AMPK and SIRT1 and also both of these are co-regulated while playing a predominant role in NED of LNCaP cells. Of the few requirements of AMPK-SIRT1 activation, increased eNOS is essential for NED by elevating Nitric oxide (NO) levels. Pleiotropic effects of NO ultimately regulate p38MAPK in IL-6 induced NED. Hence, IL-6 induced AMPK-SIRT1 activation eventually transfers its activation signals through p38MAPK for advancing NED of LNCaP cells. Moreover, inactivation of p38MAPK with specific inhibitor (SB203580) attenuated IL-6 induced NED of LNCaP cells. Therefore, IL-6 promotes NED of PCa cells via AMPK/SIRT1/p38MAPK signaling. Finally, targeting AMPK-SIRT1 or p38MAPK in androgen independent PC3 cells with neuroendocrine features reversed their neuroendocrine characteristics. Taken together these novel findings reveal that targeting p38MAPK mitigated NED of PCa cells, and thus it can be a favorable target to overcome progression of NEPC.

Effect of 1-Carbaldehyde-3,4-dimethoxyxanthone on Prostate and HPV-18 Positive Cervical Cancer Cell Lines and on Human THP-1 Macrophages.

Xanthone derivatives have shown promising antitumor properties, and 1-carbaldehyde-3,4-dimethoxyxanthone (1) has recently emerged as a potent tumor cell growth inhibitor. In this study, its effect was evaluated (MTT viability assay) against a new panel of cancer cells, namely cervical cancer (HeLa), androgen-sensitive (LNCaP) and androgen-independent (PC-3) prostate cancer, and nonsolid tumor derived cancer (Jurkat) cell lines. The effect of xanthone 1 on macrophage functions was also evaluated. The effect of xanthone 1-conditioned THP-1 human macrophage supernatants on the metabolic viability of cervical and prostate cancer cell lines was determined along with its interference with cytokine expression characteristic of M1 profile (IL-1 ≤ β; TNF-α) or M2 profile (IL-10; TGF-β) (PCR and ELISA). Nitric oxide (NO) production by murine RAW264.7 macrophages was quantified by Griess reaction. Xanthone 1 (20 μM) strongly inhibited the metabolic activity of the cell lines and was significantly more active against prostate cell lines compared to HeLa (p < 0.05). Jurkat was the cell most sensitive to the effect of xanthone 1. Compound 1-conditioned IL-4-stimulated THP-1 macrophage supernatants significantly (p < 0.05) inhibited the metabolic activity of HeLa, LNCaP, and PC-3. Xanthone 1 did not significantly affect the expression of cytokines by THP-1 macrophages. The inhibiting effect of compound 1 observed on the production of NO by RAW 264.7 macrophages was moderate. In conclusion, 1-carbaldehyde-3,4-dimethoxyxanthone (1) decreases the metabolic activity of cancer cells and seems to be able to modulate macrophage functions.

Anti-Tumor Effects of Ginsenoside 20(S)-Protopanaxadiol and 1,25-Dihydroxyvitamin D3 Combination in Castration Resistant Prostate Cancer.

In spite of possessing desirable anticancer properties, currently, limited clinical success has been achieved with 20(S)-protopanaxadiol (aPPD) and 1,25-dihydroxyvitamin D3 (calcitriol). This study is designed to evaluate if the combination of aPPD with calcitriol can inhibit human prostate cancer xenograft growth by using nuclear receptor signaling. Athymic male nude mice were utilized to establish an androgen-independent human prostate cancer C4-2 cell castration-resistant prostate cancer (CRPC) xenograft model. Mice were treated orally for six weeks with 70 mg/kg aPPD administered once daily or three times per week with 4 µg/kg calcitriol or in combination or only with vehicle control. Contrary to our expectations, calcitriol treatment alone increased C4-2 tumor growth. However, the addition of calcitriol substantially increased aPPD-mediated tumor growth suppression (76% vs. 53%, combination vs. aPPD alone). The combination treatment significantly increased levels of cleaved caspase-3 apoptotic marker compared to vehicle-treated or aPPD-treated C4-2 tumors. The mechanistic elucidations indicate that tumor inhibition by the aPPD and calcitriol combination was accompanied by elevated vitamin D receptor (VDR) protein expression. In silico data suggest that aPPD weakly binds to the native LBD pocket of VDR. Interestingly, the combination of aPPD and calcitriol activated VDR at a significantly higher level than calcitriol alone and this indicates that aPPD may be an allosteric activator of VDR. Overall, aPPD and calcitriol combination significantly inhibited tumor growth in vivo with no acute or chronic toxic effects in the C4-2 xenograft CRPC nude mice. The involvement of VDR and downstream apoptotic pathways are potential mechanistic routes of antitumor effects of this combination.

Qi Ling Inhibits Progression of Androgen-Independent Prostate Cancer via Negative Regulation of TRIM66/HP1γ/AR Axis

Aim: This study aimed to understand the molecular mechanism underlying the therapeutic effect of Qi Ling (QL) against androgen-independent prostate cancer. Methods: The relative expression of TRIM66 in prostate tumor was interrogated by microarray. Real-time polymerase chain reaction and Western blotting were performed to determine the transcript abundances and protein expressions of TRIM66, HP1γ, AR, c-Myc, and GAPDH. Cell proliferation and apoptosis were analyzed by cell counting kit-8 method and flow cytometry. The regulatory action of c-Myc on TRIM66 was interrogated with luciferase reporter plasmid and the direct binding was demonstrated by chromatin immunoprecipitation. The secretory prostate-specific antigen was quantified by enzyme-linked immunosorbent assay. Results: TRIM66 was aberrantly overexpressed in prostate cancer and associated with unfavorable prognosis. TRIM66/HP1γ/AR was upregulated during the androgen-independent transition in hormone-deprived medium. The TRIM66 level positively linked to cell proliferation and negatively linked to cell apoptosis in androgen-independent prostate cancer cells. QL treatment specifically inhibited c-Myc and therefore directly downregulated TRIM66 via binding to its promoter. Ectopic introduction of TRIM66 significantly reversed the anti-tumor effects of QL against androgen-independent prostate cancer. Conclusion: Our study uncovered the importance of downregulated TRIM66/HP1γ/AR signaling in mediating the anti-tumor properties of QL.