Androgen-independent Prostate Cancer Cell Lines Research Articles

Abstract 5695: miR-5787 suppresses cancer cell growth as a Fe3O4-nanoparticles induced pivotal miRNA

Abstract Nanotechnology offers great potential for cancer diagnosis and treatments. Especially, nanoparticles (NPs) have been examined and utilized as drug delivery systems, hyperthermia and combination therapy. We have examined and reported the combined effects of Fe3O4-nanoparticles (Fe3O4-NPs) with chemotherapeutic agents on prostate cancer cell in vitro. miRNAs are an important factor in cancer progression, and elucidating the miRNA-mediated intracellular network of cancer cells could suggest new cancer treatment strategies. In this study, to identify microRNAs (miRNAs) associated with anticancer effects of Fe3O4-NPs, the comprehensive expression of miRNAs by Microarray (Agilent G4780A SuperPrint G3 Human v16 miRNA 8x60K Microarray kit) in A549 (lung cancer cell line), LNCaP (androgen-dependent prostate cancer cell line) and DU145 (androgen-independent prostate cancer cell line) under Fe3O4-NPs exposure was performed. miR-5787 was one of the increased miRNAs in three cancer cell lines under Fe3O4-NPs, and its increase in miR-5787 was due to the reactive oxygen species (ROS) production by Fe3O4-NPs. The additional analysis suggested that miR-5787 might suppressed the cancer cell growth by targeting the eIF5. In this study, we identified miR5787 as a Fe3O4-NPs induced pivotal miRNA in cancer cells, and can be used as a therapeutic target. Citation Format: Masatoshi Watanabe, Hitoshi Nakano, Chise Matsuda, Eri Usugi, Hiroshi Imai, Yoshifumi Hirokawa, Kazutoshi Iijima. miR-5787 suppresses cancer cell growth as a Fe3O4-nanoparticles induced pivotal miRNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5695.

Antiproliferative, apoptosis-inducing, and GSTP1 demethylation activities of Ellagitannins isolated from Cornus alba L.

This study aimed to prove the prostate cancer chemopreventive activity of compounds isolated from CA. We evaluated these compounds using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and evaluated their NF-κB inhibitory activity and apoptosis-inducing activity using western blot analysis and flow cytometry, respectively. Their DNA methylation activity was also evaluated via a methylation-specific polymerase chain reaction in androgen-dependent (LNCaP) and androgen-independent (PC-3) prostate cancer cell lines. Camptothin B (1), cornusiin B (2), and cornusiin A (3), which were isolated in our previous work, relatively reduced the protein expression levels in PCa cells. Among them, cornusiin B (2) exhibited excellent NF-κB inhibitory activity. Also, concentration-dependently increased the unmethylated DNA content and decreased the methylated DNA content in both PC-3 and LNCaP cells. Therefore, cornusiin B (2), which was isolated from CA, has the potential to act as a chemopreventive agent for prostate cancer.

Photoactivation of pheophorbide-a utilizing 670 nm LEDs elicits cancer suppressive effects in androgen-independent prostate cancer cellular models.

Prostate cancer (PCa) is the most commonly diagnosed cancer and the second leading cause of cancer death in men in the USA. Photodynamic therapy (PDT) is a state-of-the-art treatment that combines high selectivity with minor side effects. Pheophorbide-a (Pheo) is a natural pigment with a photosensitizer property. Our study delved into the impact of Pheo alone or Pheo-PDT combination on the androgen-independent metastatic prostate cancer (AIPC) cell lines DU-145 and C4-2. Furthermore, an in-depth examination has been conducted on the photocytotoxicity mechanism of Pheo-PDT in these specific cell lines. In vitro studies were conducted using the AIPC cell lines. DU-145 and C4-2 cells were treated with Pheo at different concentrations for 60 min alone, or Pheo treatment followed by exposure to 670 nm illumination (60 mW/cm2 in 88 s pulses), producing 5 J/cm2 via portable light-emitting diode. Our results show that Pheo-PDT substantially inhibits cell viability, anchorage-independent growth, and migration capacities and induces autophagy and apoptosis via the over-production of reactive oxygen species that mediates endoplasmic reticulum stress in AIPC cell lines. Our study highlights the potential benefits of Pheo-PDT in metastatic hormone-insensitive PCa cell lines. It paves the way for treating localized and locally advanced PCa as a possible candidate for castration-resistant prostate cancer.

MicroRNA-147b induces neuroendocrine differentiation of prostate cancer cells by targeting ribosomal protein RPS15A.

Prostate cancer (PCa) is the leading cause of cancer related deaths in men, often androgen deprivation therapy (ADT) leads to the progression of androgen independent PCa (AIPC) which further leads to Neuroendocrine PCa (NEPC). Identifying the molecular mechanisms which navigate the neuroendocrine differentiation(NED) of PCa cells is clinically relevant. It has been suggested that the micro RNAs (miRNAs) play an important role in the regulation of intrinsic mechanisms relevant to tumor progression, resistance as a result leads to poor prognosis. miR-147b has been transpiring as one of the deregulated miRNAs associated with the occurrence of multiple cancers. The present study has studied the role of miRNA-147b in inducing NEPC. To investigate the functional role of miR-147b in NEPC, we have expressed miRNA mimics or inhibitors in PCa cells and monitored the progression of NEPC along with PCa cell proliferation and survival. The molecular mechanism miRNA-147b follows was studied using western blot and reverse transcriptionpolymerase chain analysis. miRNA target prediction using bioinformatics tools followed by target validation using luciferase reporter assays was performed. In the present study, we found that miR-147b is highly expressed in AIPC cell lines in particular neuroendocrine cells NCI-H660 and NE-LNCaP derived from LNCaP. Mechanistic studies revealed that overexpression of miR-147b or miRNA mimics induced NED in LNCaP cells in in-vitro while its inhibitor reversed the NE features (increased NE markers and reduced prostate specific antigen) of PC3, NCI-H660 and NE-LNCaP cells. In addition, miR-147b reduced the proliferation rate of LNCaP cells via elevated p27kip1 and lowered cyclin D1 for promoting differentiation. In reporter assays, we have identified ribosomal protein S15A (RPS15A) is a direct target of miRNA-147b and RPS15A expression was negatively regulated by miR-147b in PCa cells. Furthermore, we also report that RPS15A is downregulated in NEPC cells and its expression is inversely correlated with NE markers. Targeting the miR-147b - RPS15A axis may overcome the progression of NEPC and serve as a novel therapeutic target to attenuate NED progression of PCa.

Abstract 4802: Identification of critical hypoxia induced factors in castrate resistant prostate cancer

Abstract Background: Prostate cancer (PCa) is the second most diagnosed cancer in men and the second cause of cancer-related death amongst men worldwide. PCa is a heterogeneous disease, and the outcome is worse for patients when the disease progresses from localized PCa to a castrate-resistant disease. Androgen receptor (AR) signaling is crucial for PCa development and has been a major therapeutic target for decades. Despite the success made with hormone therapy to block AR signaling, castration resistant disease can arise through mutations and amplifications of the androgen receptor (AR) and AR splice variants (AR-Vs). Amongst them, AR-V7 and AR-V12 can promote AR signaling independent of androgen. Furthermore, prostate tumors are highly hypoxic due to abnormal tumor vasculature. Hypoxia influences the efficacy of local and systemic treatment strategies, such as radiotherapy, hormone therapy, and chemotherapy, resulting in poorer clinical outcomes for PCa patients. Therefore, a complete understanding of AR and AR variant function, as well as how they are modulated by hypoxia and therapy is warranted for developing new strategies to treat PCa. Methods: To identify the transcriptional apparatus associated with the AR and its variants under normal and hypoxic conditions, we carried out Bio-ID proximity labeling mass spectrometry (BioID-MS). We generated stable androgen-independent human prostate cancer cell lines, PC-3 and DU145, expressing AR and the AR-V7 and AR-V12 variants fused to a MiniTurboID (MTID) enzyme. To delineate the AR proximal interaction network, we treated cells with androgen or vehicle for 3 hours and affinity-purified biotinylated proteins after the addition of biotin for the final 2 hours. Cells were grown in normoxia (20% O2) or treated with hypoxia (95% N2,5% CO2 and 0.5% O2) for 24 h. Nonspecific interactions were filtered using controls and statistical methods to identify high-confidence interactomes. Results: BioID proximity labeling in PC3 cells identified several AR-associated proteins, including many knowninteractors, validating the approach. Hypoxic conditions led to a striking alteration in proximal interactions and many metabolic proteins were highly enriched with AR upon exposure of cells to hypoxia for 24h. Among these, phosphoglycerate kinase 1 (PGK1) was a top hit. PGK1 has been reported to interact with AR to mediate the expression of genes that regulates cell proliferation and apoptosis. Additionally, PGK1 has an essential role in metabolic reprogramming induced by c-MYC and HIF-1α, leading to enhanced tumorigenesis. Conclusion: Bio-ID mass spectrometry was used to map the proximal interactome of AR and its variants, identifying novel interactions partners for further analysis. We found that hypoxia strongly alters interactors with the AR, and we identified PGK1 as a hypoxia induced AR interaction in PCA. Current work to functionally validate this interaction will be presented. Citation Format: Oloruntoba I. Osagie, Jordann Smakk, Deborah E. Citrin, Travis H. Stracker. Identification of critical hypoxia induced factors in castrate resistant prostate cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4802.

Veratramine Inhibits the Cell Cycle Progression, Migration, and Invasion via ATM/ATR Pathway in Androgen-Independent Prostate Cancer.

Prostate cancer (PC) is the second leading cause of cancer-related death among men. Treatment of PC becomes difficult after progression because PC that used to be androgen-dependent becomes androgen-independent prostate cancer (AIPC). Veratramine, an alkaloid extracted from the root of the Veratrum genus, has recently been reported to have anticancer effects that work against various cancers; however, its anticancer effects and the underlying mechanism of action in PC remain unknown. We investigated the anticancer effects of veratramine on AIPC using PC3 and DU145 cell lines, as well as a xenograft mouse model. The antitumor effects of veratramine were evaluated using the CCK-8, anchorage-independent colony formation, trans-well, wound healing assays, and flow cytometry in AIPC cell lines. Microarray and proteomics analyses were performed to investigate the differentially expressed genes and proteins induced by veratramine in AIPC cells. A xenograft mouse model was used to confirm the therapeutic response and in vivo efficacy of veratramine. Veratramine dose dependently reduced the proliferation of cancer cells both in vitro and in vivo. Moreover, veratramine treatment effectively suppressed the migration and invasion of PC cells. The immunoblot analysis revealed that veratramine significantly downregulated Cdk4/6 and cyclin D1 via the ATM/ATR and Akt pathways, both of which induce a DNA damage response that eventually leads to G1 phase arrest. In this study, we discovered that veratramine exerted antitumor effects on AIPC cells. We demonstrated that veratramine significantly inhibited the proliferation of cancer cells via G0/G1 phase arrest induced by the ATM/ATR and Akt pathways. These results suggest that veratramine is a promising natural therapeutic agent for AIPC.

Dynamics of antioxidant heme oxygenase-1 and pro-oxidant p66Shc in promoting advanced prostate cancer progression.

The castration-resistant (CR) prostate cancer (PCa) is lethal and is the second leading cause of cancer-related deaths in U.S. males. To develop effective treatments toward CR PCa, we investigated reactive oxygen species (ROS) signaling pathway for its role involving in CR PCa progression. ROS can regulate both cell growth and apoptosis: a moderate increase of ROS promotes proliferation; its substantial rise results in cell death. p66Shc protein can increase oxidant species production and its elevated level is associated with the androgen-independent (AI) phenotype of CR PCa cells; while heme oxygenase-1 (HO-1) is an antioxidant enzyme and elevated in a sub-group of metastatic PCa cells. In this study, our data revealed that HO-1 and p66Shc protein levels are co-elevated in various AI PCa cell lines as well as p66Shc cDNA-transfected cells. Knockdown and/or inhibition of either p66Shc or HO-1 protein leads to reduced tumorigenicity as well as a reduction of counterpart protein. Knockdown of HO-1 alone results in increased ROS levels, nucleotide and protein oxidation and induction of cell death. Together, our data indicate that elevated HO-1 protein levels protect PCa cells from otherwise apoptotic conditions induced by aberrant p66Shc/ROS production, which thereby promotes PCa progression to the CR phenotype. p66Shc and HO-1 can serve as functional targets for treating CR PCa.

Abstract 5471: PRT2527, a novel highly selective cyclin-dependent kinase 9 (CDK9) inhibitor, is active in preclinical models of prostate cancer

Abstract CDK9 is a serine/threonine kinase belonging to the subclass of the transcription associated CDKs. CDK9 complexes with cyclin T and cyclin K, the positive transcription elongation factor b (P-TEFb), and phosphorylates Serine 2 of RNA polymerase II (pSer2 RNAPII) to activate transcription. Consequently, targeting CDK9 could effectively interfere with epigenetic and transcriptional reprogramming and prevent disease progression and treatment resistance in human cancers. Androgen receptor (AR)-dependence in prostate cancer is linked to CDK9 function. CDK9 stabilizes AR-associated proteins, and pharmacological inhibition of CDK9 can inhibit AR, AR variants, and their downstream transcription programs. We evaluated the novel CDK9 inhibitor, PRT2527, in prostate cancer models to evaluate its effects on cell proliferation, stem-like tumor cells, and tumor growth. PRT2527 is a potent inhibitor of CDK9/CyclinT1 complex, and when evaluated at concentration 200 times the biochemical IC50, PRT2527 was highly selective for CDK9 inhibition. We verified in biochemical assays the ability of PRT2527 to suppress pSer2 RNAPII and reduce expression of c-Myc, a common target of CDK9, in a concentration-dependent manner in multiple human prostate cancer cell lines. PRT2527 also inhibited c-Myc-dependent transcription in vitro in luciferase reporter assays. Furthermore, RNA sequencing showed altered expression of several genes with significant enrichment of c-Myc and E2F targets and RNAPII dependent transcription among downregulated genes in PRT2527-treated VCaP cells. In vitro, PRT2527 inhibited the proliferation of androgen-dependent and androgen-independent prostate cancer cell lines (IC50, ≤50 nM). PRT2527 was highly effective (IC50, ≤10 nM) in tumor-spheroid assays in blocking the growth of stem-like tumor cells and significantly suppressed the in vitro growth of tumor organoids from both human cell lines and patient-derived xenografts (PDXs). In mice, PRT2527 IV administration reduced pSer2 RNAPII in tumor xenografts and c-Myc-dependent transcriptional activity in a DU145 luminescence reporter model. PRT2527 administration in mice significantly reduced growth of PDX LuCaP 35 (castration-sensitive, adenocarcinoma) and LuCaP 145.2 (castration-resistant, neuroendocrine) along with the fraction of tumor-initiating stem-like cells in ex vivo assays. PRT2527 reduced pSer2 RNAPII in both PDXs, whereas c-Myc decreased in LuCaP 35 and Sox2 in LuCaP 145.2, relative to basal expression levels. Collectively, our data demonstrate that PRT2527 has potent pharmacodynamic and antitumor activity in multiple models of castration-sensitive and castration-resistant prostate cancer. PRT2527 is advancing into phase 1 studies in solid tumors. Citation Format: Elisa Federici, Gianluca Civenni, Aleksandra Kokanovic, Giada Sandrini, Luca Guarrera, Simone Mosole, Alessia Cacciatore, Valeria Uboldi, Manuel Lessi, Giovanni Papa, Domenico Albino, Elisa Storelli, Jessica Merulla, Andrea Rinaldi, Marco Bolis, Yang Zhang, Kris Vaddi, Peggy Scherle, Bruce Ruggeri, Giuseppina M. Carbone, Carlo V. Catapano. PRT2527, a novel highly selective cyclin-dependent kinase 9 (CDK9) inhibitor, is active in preclinical models of prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5471.

PLK4 is upregulated in prostate cancer and its inhibition reduces centrosome amplification and causes senescence.

BackgroundIdentification of novel molecular target(s) is important for designing newer mechanistically driven approaches for the treatment of prostate cancer (PCa), which is one of the main causes of morbidity and mortality in men. In this study, we determined the role of polo‐like kinase 4 (PLK4), which regulates centriole duplication and centrosome amplification (CA), in PCa.Materials and MethodsEmploying human PCa tissue microarrays, we assessed the prevalence of CA, correlated with Gleason score, and estimated major causes of CA in PCa (cell doubling vs. centriole overduplication) by staining for mother/mature centrioles. We also assessed PLK4 expression and correlated it with CA in human PCa tissues and cell lines. Further, we determined the effects of PLK4 inhibition in human PCa cells.ResultsCompared to benign prostate, human PCa demonstrated significantly higher CA, which was also positively correlated with the Gleason score. Further, most cases of CA were found to arise by centriole overduplication rather than cell doubling events (e.g., cytokinesis failure) in PCa. In addition, PLK4 was overexpressed in human PCa cell lines and tumors. Moreover, PLK4 inhibitors CFI‐400945 and centrinone‐B inhibited cell growth, viability, and colony formation of both androgen‐responsive and androgen‐independent PCa cell lines. PLK4 inhibition also induced cell cycle arrest and senescence in human PCa cells.ConclusionsCA is prevalent in PCa and arises predominantly by centriole overduplication as opposed to cell doubling events. Loss of centrioles is cellular stress that can promote senescence and suggests that PLK4 inhibition may be a viable therapeutic strategy in PCa.

CYP1B1-catalyzed 4-OHE2 promotes the castration resistance of prostate cancer stem cells by estrogen receptor \u03b1-mediated IL6 activation

BackgroundResistance to androgen deprivation therapy remains a major challenge for the clinical treatment of patients with castration-resistant prostate cancer (CRPC). CYP1B1, a critical enzyme that catalyzes the conversion of estradiol to 4-Hydroxy-17β-estradiol (4-OHE2), has been reported to promote the development and progression of hormone-related cancer, but its role in CRPC is unclear.MethodsTo explore the underlying mechanism which CYP1B1 promotes the prostate cancer stem cells (PCSCs) characteristics, bioinformatics analyses of human clinical prostate cancer (PCa) datasets were performed. CYP1B1, IL6, and estrogen receptor-α (ERα) expression levels were evaluated in PCa and CRPC tissues via immunohistochemistry. The high-performance liquid chromatography-mass spectrometry assay was carried out to examine intracellular 4-OHE2 levels. Serum-free suspension culture and flow cytometry assays were performed to evaluate PCSCs. Chromatin immunoprecipitation was used to validate that 4-OHE2 recruited ERα to the IL6 promoter.ResultsCYP1B1 expression was significantly increased in CRPC tissues and androgen-independent PCa cell lines. CYP1B1+ PCa cells were significantly enriched in bicalutamide-treated LNCaP cells, and CYP1B1 knockdown reduced the cell viability under bicalutamide treatment. In addition, CYP1B1 knockdown decreased the intracellular 4-OHE2 concentration, accompanied by reduced PCSC characteristics. In PCa cells, 4-OHE2 stimulated ERα transcriptional activity and upregulated the expression of IL6 and downstream genes of the IL6-STAT3 signaling. 4-OHE2 increased cell viability under bicalutamide treatment and promoted PCSC characteristics, while IL6 neutralizing antibody reversed these effects. Mechanistically, siERα and the ER antagonist ICI182780 significantly attenuated 4-OHE2-induced IL6 expression, and 4-OHE2 promoted the binding of ERα to the estrogen response element of the IL6 promoter.ConclusionsOur findings indicate that CYP1B1-catalyzed 4-OHE2 enhanced PCSC characteristics and attenuated bicalutamide sensitivity by ERα-mediated the IL6-STAT3 pathway activation. Our study further emphasizes the role of CYP1B1 in castration resistance and illustrates a novel mechanism of CRPC development.Graphical 54dyAth8NatsYXqV4_5FMeVideo .

Targeting IKKε in Androgen-Independent Prostate Cancer Causes Phenotypic Senescence and Genomic Instability.

Advanced prostate cancer will often progress to a lethal, castration-resistant state. We previously demonstrated that IKKε expression correlated with the aggressiveness of prostate cancer disease. Here, we address the potential of IKKε as a therapeutic target in prostate cancer. We examined cell fate decisions (proliferation, cell death, and senescence) in IKKε-depleted PC-3 cells, which exhibited delayed cell proliferation and a senescent phenotype, but did not undergo cell death. Using IKKε/TBK1 inhibitors, BX795 and Amlexanox, we measured their effects on cell fate decisions in androgen-sensitive prostate cancer and androgen-independent prostate cancer cell lines. Cell-cycle analyses revealed a G2-M cell-cycle arrest and a higher proportion of cells with 8N DNA content in androgen-independent prostate cancer cells only. Androgen-independent prostate cancer cells also displayed increased senescence-associated (SA)-β-galactosidase activity; increased γH2AX foci; genomic instability; and altered p15, p16, and p21 expression. In our mouse model, IKKε inhibitors also decreased tumor growth of androgen-independent prostate cancer xenografts but not 22Rv1 androgen-sensitive prostate cancer xenografts. Our study suggests that targeting IKKε with BX795 or Amlexanox in androgen-independent prostate cancer cells induces a senescence phenotype and demonstrates in vivo antitumor activity. These results strengthen the potential of exploiting IKKε as a therapeutic target.

Molecular mechanism of inhibitory effects of melatonin on prostate cancer cell proliferation, migration and invasion.

The increasing incidence of prostate cancer (PCa) indicates an urgent need for the development of new effective drug therapy. There are limited options to treat the PCa, this study tried to determine a new therapy option for this acute cancer. Androgen-independent PCa cell lines PC3 and DU145 were treated with different melatonin concentrations (0.1~3.5 mM) for 1~3 days and assessed cell migration, cell invasion, cycle arrest in G0/G1 phase as well as apoptosis. We utilized RNA-seq technology to analyze the transcriptional misregulation pathways in DU145 prostate cancer cell line with melatonin (0.5 mM) treatment. Data revealed 20031 genes were up and down-regulated, there were 271 genes that differentially expressed: 97 up-regulated (P<0.05) and 174 down-regulated (P<0.05) genes. Furthermore, RNA-seq results manifested that the melatonin treatment led to a significant increase in the expression levels of HPGD, IL2Rβ, NGFR, however, IGFBP3 and IL6 (P <0.05) had decreased expression levels. The immunoblot assay revealed the expression of IL2Rβ and NGFR genes was up-regulated, qPCR confirmed the gene expression of HPGD and IL2RB were also up-regulated in Du145 cells. Consequently, we probed mechanisms that generate kinetic patterns of NF-κB-dependent gene expression in PCa cells responding to a NF-κB-activation, the significant results were indicated by the inhibition of the NF-kB pathway via IL2Rβ actions. Based on our investigation, it could be concluded that melatonin is a chemotherapeutic molecule against PCa and provides a new idea for clinical therapy of PCa.

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.

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.

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.

α-Trifluoromethyl Chalcones as Potent Anticancer Agents for Androgen Receptor-Independent Prostate Cancer.

α-Trifluoromethyl chalcones were prepared and evaluated for their antiproliferative activities against androgen-independent prostate cancer cell lines as well as five additional types of human tumor cell lines. The most potent chalcone 5 showed superior antitumor activity in vivo with both oral and intraperitoneal administration at 3 mg/kg. Cell-based mechanism of action studies demonstrated that 5 induced cell accumulation at sub-G1 and G2/M phases without interfering with microtubule polymerization. Furthermore, several cancer cell growth-related proteins were identified by using chalcone 5 as a bait for the affinity purification of binding proteins.

Receptor Tyrosine Kinase‐like Orphan Receptor 1 (ROR1) as a Selective Molecular Target in Androgen‐Dependent and Androgen‐Independent Prostate Cancer

Prostate cancer is a disease that has a high prevalence and mortality amongst men. The severity of the cancer is stratified by risk, which is based on the Gleason grade, prostate-specific antigen (PSA) level, and clinical staging. Unlike breast cancer which can be classified into subtypes based on molecular heterogeneity, prostate cancer has no such classification and is therefore treated with non-specific chemotherapy and radiation if local, and androgen deprivation therapy (ADT) if metastatic. It is of the utmost importance to investigate molecular targets that can specifically eradicate prostate cancer while retaining the functions of normal tissue. Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is a receptor that is highly expressed in embryogenesis and cancer, while being minimally expressed in normal tissue. In particular, prostate cancer exhibits moderate to high levels of ROR1 expression. The goal of this study is to elucidate the role of ROR1 in aggressive prostate cancer and selectively target it for treatment. Our laboratory had previously characterized Strictinin, a ROR1 inhibitor, as being selectively cytotoxic against TNBC via inhibition of the ROR1 receptor. We hypothesize that Strictinin can reduce the proliferation and invasion of prostate cancer and may potentially serve as a specific therapeutic agent. Preliminary results indicate that Strictinin is selectively lethal to androgen-dependent and androgen-independent prostate cancer cell lines. Strictinin treatment reduced expression of proteins downstream of ROR1 implicated in promoting migration, invasion, and anti-apoptosis. Furthermore, Strictinin treatment decreased migration and invasion while inducing apoptosis in PC3 and LnCAP cell lines, providing evidence that inhibition of the ROR1 molecular pathway is linked to reduction of cancerous phenotypes. These preliminary results indicate that ROR1 could be a novel molecular target that can be utilized for subtyping and treating prostate cancer. The data from this study will establish Strictinin as a potential therapeutic that targets ROR1 to reduce migration, invasion, and survival of prostate cancer.

Periprostatic adipose tissue promotes prostate cancer resistance to docetaxel by paracrine IGF‐1 upregulation of TUBB2B beta‐tubulin isoform

Growing evidence supports the pivotal role played by periprostatic adipose tissue (PPAT) in prostate cancer (PCa) microenvironment. We investigated whether PPAT can affect response to Docetaxel (DCTX) and the mechanisms associated. Conditioned medium was collected from the in vitro differentiated adipocytes isolated from PPAT which was isolated from PCa patients, during radical prostatectomy. Drug efficacy was studied by 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide citotoxicity assay. Culture with CM of human PPAT (AdipoCM) promotes DCTX resistance in two different human prostate cancer cell lines (DU145 and PC3) and upregulated the expression of BCL‐xL, BCL‐2, and TUBB2B. AG1024, a well‐known IGF‐1 receptor inhibitor, counteracts the decreased response to DCTX observed in presence of AdipoCM and decreased TUBB2B expression, suggesting that a paracrine secretion of IGF‐1 by PPAT affect DCTX response of PCa cell. Collectively, our study showed that factors secreted by PPAT elicits DCTX resistance through antiapoptotic proteins and TUBB2B upregulation in androgen independent PCa cell lines. These findings reveal the potential of novel therapeutic strategies targeting adipocyte‐released factors and IGF‐1 axis to overcome DCTX resistance in patients with PCa.

Follicle-stimulating hormone promotes growth of human prostate cancer cell line-derived tumor xenografts.

Chemical castration in prostate cancer can be achieved with gonadotropin-releasing hormone (GnRH) agonists or antagonists. Their effects differ by the initial flare of gonadotropin and testosterone secretion with agonists and the immediate pituitary-testicular suppression by antagonists. While both suppress luteinizing hormone (LH) and follicle-stimulating hormone (FSH) initially, a rebound in FSH levels occurs during agonist treatment. This rebound is potentially harmful, taken the expression of FSH receptors (R) in prostate cancer tissue. We herein assessed the role of FSH in promoting the growth of androgen-independent (PC-3, DU145) and androgen-dependent (VCaP) human prostate cancer cell line xenografts in nude mice. Gonadotropins were suppressed with the GnRH antagonist degarelix, and effects of add-back human recombinant FSH were assessed on tumor growth. All tumors expressed GnRHR and FSHR, and degarelix treatment suppressed their growth. FSH supplementation reversed the degarelix-evoked suppression of PC-3 tumors, both in preventive (degarelix and FSH treatment started upon cell inoculation) and therapeutic (treatments initiated 3weeks after cell inoculation) setting. A less marked, though significant FSH effect occurred in DU145, but not in VCaP xenografts. FSHR expression in the xenografts supports direct FSH stimulation of tumor growth. Testosterone supplementation, to maintain the VCaP xenografts, apparently masked the FSH effect on their growth. Treatment with the LH analogue hCG did not affect PC-3 tumor growth despite their expression of luteinizing hormone/choriongonadotropin receptor. In conclusion, FSH, but not LH, may directly stimulate the growth of androgen-independent prostate cancer, suggesting that persistent FSH suppression upon GnRH antagonist treatment offers a therapeutic advantage over agonist.

Membrane Interactome of a Recombinant Fragment of Human Surfactant Protein D Reveals GRP78 as a Novel Binding Partner in PC3, a Metastatic Prostate Cancer Cell Line.

Surfactant protein-D (SP-D), a member of the collectin family has been shown to induce apoptosis in cancer cells. SP-D is composed of an N-terminal collagen-like domain and a calcium-dependent carbohydrate recognition domain (CRD). Recently, we reported that a recombinant fragment of human SP-D (rfhSP-D), composed of homotrimeric CRD region, induced intrinsic apoptotic pathway in prostate cancer cells. Here, we analyzed the membrane interactome of rfhSP-D in an androgen-independent prostate cancer cell line, PC3, by high resolution mass spectrometry and identified 347 proteins. Computational analysis of PPI network of this interactome in the context of prostate cancer metastasis and apoptosis revealed Glucose Regulated Protein of 78 kDa (GRP78) as an important binding partner of rfhSP-D. Docking studies suggested that rfhSP-D (CRD) bound to the substrate-binding domain of glycosylated GRP78. This was further supported by the observations that human recombinant GRP78 interfered with the binding of rfhSP-D to anti-SP-D polyclonal antibodies; GRP78 also significantly inhibited the binding of recombinant full-length human SP-D with a monoclonal antibody specific to the CRD in a dose-dependent manner. We conclude that the interaction with rfhSP-D is likely to interfere with the pro-survival signaling of GRP78.