LNCaP Human Prostate Cancer Cells Research Articles

Utilizing mixture design response surface methodology to determine effective combinations of plant derived compounds as prostate cancer treatments.

Prostate cancer (PC) is estimated to cause 13.1% of all new cancer cases in the United States in 2021. Natural bioactive compounds have drawn the interest of researchers worldwide in their efforts to find novel treatments for PC. Many of these bioactive compounds have been identified from traditional Chinese medicine (TCM) remedies often containing multiple bioactive compounds. However, in vitro studies frequently focus on the compounds in isolation. We used mixture design response surface methodology (MDRSM) to assess changes in PC cell viability after 48 h of treatment to identify the optimal mixture of all 35 three-compound combinations of seven bioactive compounds from TCM. We used berberine, wogonin, shikonin, curcumin, triptolide, emodin, and silybin to treat PC3 and LNCaP human PC cells at their IC50 concentrations that we calculated. These compounds modulate many chemotherapeutic pathways including intrinsic and extrinsic apoptosis, increasing reactive oxygen species, decreasing metastatic pathways, inhibiting cell cycle progression. We hypothesize that because these compounds bind to unique molecular targets to activate different chemotherapeutic pathways, they will act synergistically to decrease tumor cell viability. Results from MDRSM showed that two-way combinations were more effective than three-way or single compounds. Most notably wogonin, silybin, emodin and berberine responded well in two-compound combinations with each other in PC3 and LNCaP cells. We then conducted cell viability tests combining two bioactive compound ratios with docetaxel (Doc) and found significant results within the LNCaP cell line. In particular, mixtures of berberine and wogonin, berberine and silybin, emodin and berberine, and emodin and silybin reduced LNCaP cell viability up to an average of 90.02%. The two-compound combinations were significantly better than docetaxel treatment of LNCaP cells. Within the PC3 cells, we show that a combination of berberine, wogonin and docetaxel is just as effective as docetaxel alone. Thus, we provide new combination treatments that are highly effective in vitro for treating androgen-dependent and androgen-independent PC.

Deoxynortryptoquivaline: A unique antiprostate cancer agent.

The androgen receptor (AR) is a critical target in all the clinical stages of prostate cancer. To identify a new AR inhibitor, we constructed a new screening system using the androgen-dependent growth of prostate cancer cell lines as a screening indicator. We screened 50,000 culture broths of microorganisms using this screening system and found that the fermentation broth produced by a fungus inhibited androgen-dependent growth of human prostate cancer LNCaP cells without cytotoxicity. Purification of this culture medium was performed, and this resulted in deoxynortryptoquivaline (DNT) being identified as a novel inhibitor of AR function. DNT showed potent inhibition of androgen-dependent growth of human prostate cancer LNCaP cells. The AR competitor assay was performed, and DNT did not act as an AR antagonist. However, DNT inhibited AR-dependent transcriptional activity and AR nuclear translocation, it suggested that the suppression of AR function leads to inhibition activity against androgen-dependent growth.

1,25(OH)2 D3 induced vitamin D receptor signaling negatively regulates endoplasmic reticulum-associated degradation (ERAD) and androgen receptor signaling in human prostate cancer cells

Steroid hormone signaling is critical in the tumor progression and the regulation of physiological mechanisms such as endoplasmic reticulum-associated degradation (ERAD) and unfolded protein response (UPR) in prostate cancer. 1,25(OH)2 D3 is an active metabolite of vitamin D classified as a steroid hormone. It exhibits anti-tumor effects, including angiogenesis and suppression of cell cycle progression. Moreover, progressively reducing expression levels of vitamin D receptor (VDR) are observed in many cancer types, including the prostate. In the present study, we investigated the molecular action of 1,25(OH)2 D3 on ERAD, UPR and androgenic signaling. We found that 1,25(OH)2 D3 negatively regulated the expression level of ERAD components and divergently controlled the inositol-requiring enzyme 1⍺ (IRE1⍺) and protein kinase RNA-like ER kinase (PERK) branches of UPR in LNCaP human prostate cancer cells. Also, similar results were obtained with another human prostate cancer cell line, 22Rv1. More strikingly, we found that androgenic signaling is negatively regulated by VDR signaling. Also, molecular docking supported the inhibitory effect of 1,25(OH)2 D3 on AR signaling. Moreover, we found VDR signaling suppressed tumor progression by decreasing c-Myc expression and reducing the epithelial-mesenchymal transition (EMT). Additionally, 1,25(OH)2 D3 treatment significantly inhibited the 3D-tumor formation of LNCaP cells. Our results suggest that further molecular characterization of the action of VDR signaling in other cancer types such as estrogenic signal in breast cancer will provide important contributions to a better understanding of the roles of steroid hormone receptors in carcinogenesis processes.

Unveiling Antioxidant and Antiproliferative Effects of Prosopis juliflora Leaves against Human Prostate Cancer LNCaP Cells.

Herbal medications or formulations are regularly recommended by clinicians as a potential therapeutic method for a variety of human ailments, including cancer. Although Prosopis juliflora extracts have shown promise in anticancer activity, the effects on prostate cancer and the accompanying molecular mechanisms of action are still unexplored. This research aims at the antioxidant, antiproliferative, and apoptosis-inducing properties of Prosopis juliflora methanolic leaves extract in human prostate cancer LNCaP cells. The antioxidant ability of the extract was assessed using the DPPH (2, 2-diphenyl-2-picrylhydrazyl) and two additional reducing power tests. Antitumor activity was determined using MTT cell viability tests and LDH cytotoxicity assays. The probable mechanism of apoptotic cell death was further investigated utilizing a caspase-3 activation assay and qRT-PCR mRNA expression investigations of apoptotic-related genes. The results revealed that the methanol extract of Prosopis juliflora leaves contains alkaloids, flavonoids, tannins, glycosides, and phenols, all of which have substantial antioxidant activity. In vitro anticancer tests demonstrated that extract therapy resulted in a dose-dependent reduction in cell viability of LNCaP prostate cancer cells, but normal HaCaT cells showed no cytotoxic effects. Furthermore, plant extract therapy increased caspase-3 activation and mRNA expression of apoptotic-related genes, suggesting that this could be a mechanism for cancer cell growth suppression. The significance of Prosopis juliflora as a source of new antioxidant compounds against prostate cancer was emphasized in the current study. However, more study is needed to demonstrate the efficacy of Prosopis juliflora leaves extract in the treatment of prostate cancer.

Antrodia salmonea Extracts Regulate p53-AR Signaling and Apoptosis in Human Prostate Cancer LNCaP Cells.

Antrodia salmonea (AS) is a genus of Antrodia, an epiphyte of Cunninghamia konishii in Taiwan. AS has been reported to have potential therapeutic effects on different diseases, including diarrhea, abdominal pain, and hypertension. AS has been reported to have anticancer effects on numerous cancer types, such as ovarian carcinoma and triple-negative breast cancer. Our previous studies demonstrated that antrocins and triterpenoids are possibly bioactive compositions. However, the effects of AS on prostate cancer remain unknown. Therefore, we investigated the role of AS in prostate cancer growth, apoptosis, and cell cycle regulation. The results showed that AS extracts significantly inhibited the proliferation of prostate cancer LNCaP cells in a dose-dependent manner and increased the levels of apoptotic markers (cleaved PARP and cleaved caspase 3/8/9). In addition, the cell cycle-related proteins CDK1, CDK2, CDK4, and their respective specific regulators Cyclin B1, Cyclin A, and Cyclin D were also affected. Besides, AS treatment increased p53 protein levels and slowed its degradation in LNCaP cells. Interestingly, we found that AS treatment reduced both total protein and Ser-81 phosphorylation levels of the androgen receptor (AR). Notably, the increase of nuclear p53 was accompanied by the down-regulation of AR, suggesting a reverse regulation between p53 and AR in LNCaP cells was triggered by AS treatment. These findings suggest that AS extracts trigger the apoptosis of prostate cancer cells through the reverse regulation of p53 and AR and elucidate that AS extracts might be a potential treatment for androgen-dependent prostate cancer in the near future.

Abiraterone Acetate, in Combination with Apigenin, Attenuates the Survival of Human Castration-Sensitive Prostate Cancer Cells.

Abiraterone acetate (AA) is a selective inhibitor of CYP17 α-hydroxylase, which is crucial for androgen biosynthesis. Apigenin (Api) is a natural plant-derived flavonoid with potent antiproliferative and antimigration effects. We aimed to investigate the possible role of Api in combination with the androgen receptor inhibitor AA in the treatment of androgen-sensitive human prostate cancer LNCaP cells. The cells were either exposed to 10 μM AA, 25 μM Api, or in combination for 48 hours, then the viability rate was determined by the MTT test, whilst apoptosis and cell cycle phases were assessed by image-based cytometry. The expression of selected mRNA and proteins were evaluated by RT-qPCR and Western blot, respectively. The combination of AA and Api significantly inhibited LNCaP as well as androgen-insensitive PC3 cell survival in a manner more marked than observed with either single treatment. Co-administration of Api with AA triggered apoptosis. This effect was demonstrated by Hoechst staining, and up-regulation of Bax, cytochrome c, caspase -3, and - 8 and down-regulation of Bcl-2 expression confirmed the effect. AA and Api each individually arrested the cell cycle in the G1 phase, with dual applications, leading to no further increase in the effect produced. The expression of NF-κB p105/p50 and the phosphorylation of AKT markedly decreased after apigenin treatment, with combination treatment leading to a favourable effect in terms of further augmenting the reduction. The co-administration of Api with AA strongly enhanced the efficacy of AA therapy in the treatment of prostate cancer cells. These data suggested that the combination of AA and Api would be a potential chemotherapeutic strategy against prostate cancer.

In Vivo MRI-CEST Tumor pH Imaging Detects Resistance to Proton Pump Inhibitors in Human Prostate Cancer Murine Models.

Simple SummaryTumor acidosis plays a major role in tumor aggressiveness, invasion and resistance, and it is considered an important target for novel anticancer strategies. In this work, we investigated the therapeutic efficacy of several proton pump inhibitors (Esomeprazole, Lansoprazole, Amiloride and Cariporide) to alter tumor acidity in prostate murine cancer models. The in vitro results showed a moderate toxicity for Esomeprazole that was selected for the successive in vivo studies. However, the in vivo studies highlighted the lack of response to Esomeprazole treatment in both subcutaneous and orthotopic PC3 prostate cancer models. Overall, MRI-based tumor pH imaging is a powerful tool for monitoring the in vivo response to treatment.The tumor microenvironment acidification confers treatment resistance; therefore, the interference with pH regulating systems is considered a new therapeutic strategy. In this study, two human prostate cancer cell lines, PC3 and LNCaP, have been treated in vitro with proton pump inhibitors (PPIs), namely Lansoprazole, Esomeprazole (V-ATPases-inhibitors), Cariporide, and Amiloride (NHE1-inhibitors). The cell viability and pH were assessed at several drug concentrations either at normoxic or hypoxic conditions. Since Esomeprazole showed the highest toxicity towards the PC3 cancer cells compared to LNCaP ones, athymic nude mice bearing subcutaneous or orthotopic PC3 tumors were treated with Esomeprazole (dose: 2.5 mg/kg body weight) for a period of three weeks—and tumor growth was monitored. MRI-CEST tumor pH imaging with Iopamidol was performed upon treatment at 3 h, 1 week (in combination with FDG-PET), and after 2 weeks for evaluating acute, early, and late responses. Although acute tumor pH changes were observed in vivo, long-term studies on both PC3 prostate cancer models did not provide any significant change in tumor acidosis or tumor growth. In conclusion, this work shows that MRI-CEST tumor pH imaging is a valuable tool for assessing the in vivo treatment response to PPIs.

Picropodophyllin Inhibits the Proliferation of Human Prostate Cancer DU145 and LNCaP Cells via ROS Production and PI3K/AKT Pathway Inhibition.

The reactive oxygen species (ROS) and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway play critical roles in the pathogenesis of prostate cancer by modulating cell proliferation. Picropodophyllin (PPP), an inhibitor of the insulin-like growth factor 1 receptor (IGF-1R), exerts significant antitumor effects via the PI3K/AKT signaling pathway. However, the effects of PPP on prostate cancer via ROS production and the PI3K/AKT signaling pathway remain elusive. Herein, we focused on examining the antitumor effects of PPP on DU145 and LNCaP human prostate cancer cells to determine the possible molecular mechanism. Our data indicated that the inhibitory effect of PPP on the proliferation of DU145 and LNCaP human prostate cancer cells was mediated by apoptosis induction and cell cycle blockade. Furthermore, PPP significantly influenced the expression of apoptosis-related, cell cycle, ROS production, and PI3K/AKT signaling proteins. These findings suggest that PPP can induce cell cycle arrest and apoptosis via the production of ROS and inhibition of PI3K/AKT signaling pathway, thereby suppressing the proliferation of prostate cancer cells.

Abstract 690: An anti-leprosy drug, clofazimine, targets Wnt/β-catenin pathway in cadmium-transformed benign prostatic hyperplasia cells

Abstract Prostate cancer (PCa) is one of the most prevalent cancers among men in America, being second only to skin cancer. Every year 1.6 million men are diagnosed with PCa and 366,000 men die of PCa. PCa is associated to various risk factors that include old age, ethnicity, family history, lifestyle, diet, environmental and occupational exposures. Cadmium (Cd), a well-known metal carcinogen is one of the most copious occupational and environmental pollutant found in air, soil, dietary products, and tobacco. Cd is implicated in the carcinogenesis of PCa, but the mechanism remains elusive. To explore new targets for Cd induced PCa, chronic exposure of 10µM of Cd for over a year induced malignant transformation of benign prostatic hyperplasia (BPH1) cells which was used as a cellular model in this study. The Cd transformed BPH1 cells named as BPH-Cd were confirmed for tumorigenic characteristics by performing clonogenic assay, spheroid growth assay and wound healing experiments. BPH-Cd cells were further subjected to genomic analysis to identify signaling networks that are deregulated in the carcinogenesis of Cd. Our data suggested that one of the potential underlying mechanism of Cd carcinogenesis may be due to the upregulation of Wnt-3A and β-catenin pathway. In addition, Cd also activated stemness-related genes such as ALDHA1, NANOG and Oct1. Next, to determine whether targeting Wnt/β-catenin pathway in BPH-Cd could disrupt the oncogenic potential of these cells, Clofazimine, a FDA-approved anti-leprosy drug known to inhibit broad range of Wnt-dependent cancers were explored in this study. Cell viability assay indicated the inhibition of cell growth of BPH-Cd cells by Clofazimine in a dose-dependent manner with an IC50 value of 5µM. Furthermore, Clofazimine robustly inhibited the tumorigenesis of BPH-Cd as observed in colony formation assay. Of note, In-vitro 3D spheroid assay suggested that Clofazimine decreased the size of spheroids with increasing doses. Clofazimine also showed cell cycle arrest by flow cytometry in G2/M phase in BPH-Cd cells. Confocal imaging of Wnt-3A suggested a decrease in the intensity of expression with increasing doses of clofazimine in BPH-Cd treated cells. Western blot analysis of Clofazimine treated BPH-Cd cells showed downregulation of CDK2, increase in cleaved PARP-1 and upregulation of p21. Importantly, Clofazimine also displayed potent anti-cancer effects on hormone driven (androgen-dependent) LNCaP human prostate cancer cells by inhibiting the cell viability, clonogenic ability and inducing G2/M cell cycle arrest in this cell line. Taken together, our study suggests that Clofazimine could be a potential chemopreventive and anti-prostate cancer agent which warrants further evaluation for its clinical application. Citation Format: Rifika Jain, Gnanasekar Munirathinam. An anti-leprosy drug, clofazimine, targets Wnt/β-catenin pathway in cadmium-transformed benign prostatic hyperplasia cells [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 690.

Abstract 1570: Macrophage mediated cell-kill evasion by human prostate cancer cells

Abstract Macrophages can adopt a cytotoxic M1 phenotype which can eliminate cancer cells. However, despite the presence of macrophages in the prostate, prostate cancer (PCa) is a prevalent cancer in men. This suggests that PCa cells can evade M1 macrophage mediated cell-kill. We have shown that human THP-1 derived M1-like macrophages readily kill human prostate cancer LNCap cells, while in testosterone proficient conditions (Fraction Growth over Mono-culture (FGM): 0.1). However, in testosterone deprived conditions LNCap cells survive (FGM: 1.1). In addition, when exposed to M1-like macrophage conditioned medium, LNCap cells are killed in testosterone proficient conditions and not in depleted conditions (FGM: 0.2 and 0.7, respectively). This suggests that M1 macrophage mediated PCa cell-kill depends on Androgen Receptor (AR) signaling and the release of soluble factors by macrophages. This is supported by cell-kill of castration-resistant LNCap-abl cells with an AR amplification (FGM: 0.1) in testosterone deprived conditions and no cell-kill in both testosterone deprived and proficient conditions of castration resistant PC3 cells without AR expression when co-cultured with M1-like macrophages (FGM: 0.7 and 0.7. respectively). To dissect the mechanism of M1 macrophage mediated cell-kill evasion, a genome wide CRISPR knock-out screen was performed in LNCap cells in co-culture with M1-like macrophages. LNCap cells were transfected with the Brunello library and cultured in testosterone proficient conditions. After 72 hours of co-culture, cells were harvested and DNA was isolated and submitted for sequencing. After quality check analysis, five gene knock-outs with a significant (log2FcMedian >1.5) survival advantage were identified, AR, PRKCD (Protein Kinase C Delta), IKBKA (Inhibitor of nuclear factor kappa-B kinase subunit α:), IKBKB (Inhibitor of nuclear factor kappa-B kinase subunit β) and IKBKG (Inhibitor of nuclear factor kappa-B kinase subunit γ). The latter three genes are components of the IkB kinase (IKK) complex, which is essential for activation of members of the nuclear factor-kB (NFkB) family of transcription factors. The hits as identified in the CRISPR knock-out screen, were validated in single guide knock-out LNCap cells in co-culture with M1-like macrophages. Additionally, we have shown that AR signaling enhances PRKCD expression, both at the RNA and protein level. Moreover, expression of PRKCD is significantly lower in prostatectomy specimen of 56 patients treated for 3 months with the AR signalling inhibitor enzalutamide prior to surgery, than untreated matched controls. Furthermore, LNCap cells lacking PRKCD, IKBKB and IKBKG were significantly less affected in their growth by reactive oxygen species (H2O2) than non-targeting LNCap cells. Ongoing studies are aimed to dissect how these critical factors are interconnected in LNCap cells, M1 macrophage mediated cell-kill evasion as a result. Citation Format: Anniek Zaalberg, Emma Minnee, Wilbert Zwart, Andries M. Bergman. Macrophage mediated cell-kill evasion by human prostate cancer cells [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 1570.

Abstract 5999: TBX2 promotes prostate cancer bone-metastatic phenotype through exosomal microRNA-375-3p

Abstract Introduction: Bone is the preferred site of metastasis in about 80% of advanced prostate cancer (PCa) patients. The molecular mechanisms that drive bone metastatic PCa are not well understood. Recent reports have increasingly shed light on the role of cancer-cell-derived extracellular vesicles (exosomes) in driving the metastatic cascade including priming the secondary site for metastatic manifestation. We have previously reported that TBX2 promotes multiple facets of the PCa bone metastatic cascade via a cell-intrinsic manner. In this study, we sought to determine if the bone metastasis promoted by TBX2 is driven by microRNAs (miRs) in an exosome-mediated paracrine manner. Methods: Two converse approaches were utilized for the genetic modulation of TBX2: a) TBX2 was blocked in the metastatic PC3 and C4-2B human PCa cells using a dominant negative (DN) approach, and b) TBX2 was over-expressed in the low-TBX2 expressing LNCaP human PCa cells. Exosomes isolated from TBX2-modulated human PCa cells were added to MC3T3 pre-osteoblastic cells or mouse calvaria bone cells to examine the differential effects of TBX2 modulation on osteoblast or osteoclast differentiation. Next, unbiased next generation sequencing (NGS) was performed to identify the differential miR expression in exosomes derived from the TBX2-blocked PC3 cells. Results: Our results demonstrate that compared with control exosomes, exosomes derived from TBX2-blocked PC3 cells slowed the proliferation and migration/wound healing when added to 22Rv1 cells. Further, compared with control exosomes, exosomes derived from TBX2-blocked C4-2B cells resulted in reduced osteoblast differentiation when added to: a) MC3T3 pre-osteoblastic cells, or b) mouse calvaria bone cells. Along similar lines, when compared with the control exosomes, exosomes derived from TBX2-blocked PC3 and C4-2B cells resulted in reduced osteoclast differentiation when added to bone-marrow derived cells. These results were confirmed by q RT-PCR analysis of the osteoblastic/osteolytic bone markers. In order to identify exosomal miRs downstream of TBX2 signaling, NGS analysis of exosomal miRs obtained from TBX2-blocked PC3 human PCa cells identified miR-375-3p as the top-most differentially-expressed (over-expressed miR) when compared with control exosomes. In silico analysis revealed that miR-375-3p could potentially bind and target the 3’UTR of RBPJ, an established driver of PCa bone metastasis. In agreement, q RT-PCR analysis revealed that rescue of miR-375-3p in TBX2 modulated human PCa cells rescued RBPJ expression. Conclusions: Our studies have identified the exosome-mediated TBX2/miR-375-3p/RBPJ signaling axis as a promoter of the bone-metastatic phenotype in human PCa. Citation Format: Girijesh Kumar Patel, Sayanika Dutta, Hamed Khedmatgozar, Manisha Tripathi, Srinivas Nandana. TBX2 promotes prostate cancer bone-metastatic phenotype through exosomal microRNA-375-3p [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 5999.

Antiandrogenic activity of Riboflavin 5′-phosphate (FMN) in 22Rv1 and LNCaP human prostate cancer cell lines

The androgen receptor is a hormone activated transcription factor that regulates the development and maintenance of male characteristics and represents one of the most well-established drug targets, being implicated not only in prostate cancer but also in many non-cancerous human diseases including androgenetic alopecia, acne vulgaris, and hirsutism. In this study, the antiandrogenic effects of FMN were investigated in 22Rv1 and LNCaP prostate cancer cells. FMN inhibited dihydrotestosterone (DHT)-induced protein expression of androgen receptor in 22Rv1cells. In another prostate cancer LNCaP cells, FMN decreased the protein level of DHT-induced prostate specific antigen (PSA). In addition, FMN downregulated DHT-induced mRNA expression of androgen regulated genes in both cell lines, showing less prominent inhibition in 22Rv1cells where androgen receptor had been significantly decreased by FMN. FMN was found to bind androgen receptor, demonstrating that it acted as a competitive androgen receptor antagonist. FMN increased the phosphorylation of Akt in 22Rv1 cells and this increment was abrogated by PI3K inhibitor wortmannin, resulting in a rescued androgen receptor protein level which was decreased by FMN. Additionally, FMN was found to increase the mRNA and protein level of E3 ligase mouse double minute 2. Our data suggest that the androgen receptor signaling is regulated through PI3K-Akt-MDM2 pathway in 22Rv1 cells. Together, our results indicate that FMN facilitated the degradation of androgen receptor in 22Rv1 cells and inhibited the expression of androgen regulated genes by competing the binding of DHT to androgen receptor in LNCaP cells, demonstrating its therapeutic potential as an antiandrogen.

EEFSEC knockdown inhibits proliferation, migration and invasion of prostate cancer cells in vitro

To investigate the role of selenocysteine-tRNA specific eukaryotic elongation factor (EEFSEC) in regulating the proliferation, migration, and invasion of human prostate cancer 22Rv1 cells. We detected EEFSEC mRNA expression levels in human normal prostate cell line RWPE1 and human prostate cancer cell lines 22Rv1, LNCaP, Vcap and PC-3 using qRT-PCR and EEFSEC protein expression in surgical specimens of prostate cancer and adjacent tissues using Western blotting. 22Rv1 cells were infected with a lentiviral vector carrying EEFSEC shRNA or a control lentivirus and the interference efficiency was determined using Western blotting. XTT assay was used to assess the changes in the viability of the infected cells, and Transwell chamber assay was used to examine the changes in cell migration and invasion. The effect of EEFSEC knockdown on cell cycle progression was determined with flow cytometry and by detecting the expressions of cell cycle proteins using qRT-PCR. EEFSEC was significantly upregulated in prostate cancer cells (P < 0.05), and a high expression of EEFSEC was associated with a poor prognosis of the patients with prostate cancer. In 22Rv1 cells, EEFSEC knockdown significantly suppressed the proliferation (P < 0.001), migration (P < 0.001) and invasion (P < 0.001) of the cells, resulted in cell cycle arrest in G0/G1 phase, obviously inhibited the expression of C-myc and CCNB1, and significantly increased the expression of p15. EEFSEC knockdown can inhibit the proliferation, migration, and invasion of prostate cancer cells in vitro possibly by down-regulating the expression of C-myc.

KCa1.1 K+ Channel Inhibition Overcomes Resistance to Antiandrogens and Doxorubicin in a Human Prostate Cancer LNCaP Spheroid Model.

Several types of K+ channels play crucial roles in tumorigenicity, stemness, invasiveness, and drug resistance in cancer. Spheroid formation of human prostate cancer (PC) LNCaP cells with ultra-low attachment surface cultureware induced the up-regulation of cancer stem cell markers, such as NANOG, and decreased the protein degradation of the Ca2+-activated K+ channel KCa1.1 by down-regulating the E3 ubiquitin ligase, FBXW7, compared with LNCaP monolayers. Accordingly, KCa1.1 activator-induced hyperpolarizing responses were larger in isolated cells from LNCaP spheroids. The pharmacological inhibition of KCa1.1 overcame the resistance of LNCaP spheroids to antiandrogens and doxorubicin (DOX). The protein expression of androgen receptors (AR) was significantly decreased by LNCaP spheroid formation and reversed by KCa1.1 inhibition. The pharmacological and genetic inhibition of MDM2, which may be related to AR protein degradation in PC stem cells, revealed that MDM2 was responsible for the acquisition of antiandrogen resistance in LNCaP spheroids, which was overcome by KCa1.1 inhibition. Furthermore, a member of the multidrug resistance-associated protein subfamily of ABC transporters, MRP5 was responsible for the acquisition of DOX resistance in LNCaP spheroids, which was also overcome by KCa1.1 inhibition. Collectively, the present results suggest the potential of KCa1.1 in LNCaP spheroids, which mimic PC stem cells, as a therapeutic target for overcoming antiandrogen- and DOX-resistance in PC cells.

The Anti-Proliferative Activity of Secondary Metabolite from the Marine Streptomyces sp. against Prostate Cancer Cells.

Many active substances from marine organisms are produced by symbiotic microorganisms such as bacteria, fungi, and algae. Secondary metabolites from marine actinomycetes exhibited several biological activities and provided interesting drug leads. This study reported the isolation of Lu01-M, a secondary metabolite from the marine actinomycetes Streptomyces sp., with potent anti-proliferative activity against prostate cancers. Lu01-M blocked cell proliferation with IC50 values of 1.03 ± 0.31, 2.12 ± 0.38, 1.27 ± 0.25 μg/mL in human prostate cancer PC3, DU145, and LNCaP cells, respectively. Lu01-M induced cytotoxic activity through multiple mechanisms including cell apoptosis, necroptosis, autophagy, ER stress, and inhibiting colony formation and cell migration. Lu01-M induced cell cycle arrest at the G2/M phase and DNA damage. However, the activity of autophagy induced survival response in cancer cells. Our findings suggested that Lu01-M holds the potential to be developed as an anti-cancer agent against prostate cancers.

Towards a Precision Medicine Approach and In Situ Vaccination against Prostate Cancer by PSMA-Retargeted oHSV.

Prostate specific membrane antigen (PSMA) is a specific high frequency cell surface marker of prostate cancers. Theranostic approaches targeting PSMA show no major adverse effects and rule out off-tumor toxicity. A PSMA-retargeted oHSV (R-405) was generated which both infected and was cytotoxic exclusively for PSMA-positive cells, including human prostate cancer LNCaP and 22Rv1 cells, and spared PSMA-negative cells. R-405 in vivo efficacy against LLC1-PSMA and Renca-PSMA tumors consisted of inhibiting primary tumor growth, establishing long-term T immune response, immune heating of the microenvironment, de-repression of the anti-tumor immune phenotype, and sensitization to checkpoint blockade. The in situ vaccination protected from distant challenge tumors, both PSMA-positive and PSMA-negative, implying that it was addressed also to LLC1 tumor antigens. PSMA-retargeted oHSVs are a precision medicine tool worth being additionally investigated in the immunotherapeutic and in situ vaccination landscape against prostate cancers.

Knocking down claudin receptors leads to a decrease in prostate cancer cell migration, cell growth, cell viability and clonogenic cell survival

Prostate cancer is the most common solid organ malignancy in the United States, and has the highest probability of all cancers in becoming invasive. New molecular targets are needed to define and impede the growth and progression of advanced prostate cancers. Claudins (Cldns) are transmembrane proteins that regulate paracellular permeability and cell polarity, and their levels are elevated in many human cancers such as breast, ovarian, pancreatic, and prostatic cancers. Previously, we found that Cldn3 and Cldn4 are expressed in aggressive high-grade human prostate cancer specimens. We and others have shown that there are higher levels of Cldn3 and Cldn4 in metastatic human prostate cancer cells than in normal human prostate cells. The result of targeting Cldn3 and Cldn4 expression on the growth and viability of prostate cancer cells has not been elucidated. Human prostate cancer PC3 and LNCaP cells were transfected with Cldn3 or -4 small interfering RNAs (siRNAs). Cldn3/Cldn4 siRNA treatment resulted in a greater than 85% decrease in the protein levels of Cldn3 and Cldn4, which was accompanied by a 30–40% decrease in prostate cancer cell growth and a 60–65% reduction in cell viability. There was decreased cell migration with Cldn3 and Cldn4 siRNA in both PC3 and LNCaP cells and a 60–75% decrease in the number of clones when treated with siCldn3 or siCldn4 compared to control. Knocking down Cldn3/Cldn4 affects prostate cancer cell growth and survival and may have therapeutic implications.

Profiling of Androgen-Dependent Enhancer RNAs Expression in Human Prostate Tumors: Search for Malignancy Transition Markers.

IntroductionAlthough the ability of androgens to promote prostate cancer development has been known for decades, the molecular mechanisms of androgen receptor (AR) signaling in the tumorigenesis remain unclear. Enhancer RNAs (eRNAs) transcribed from strong enhancers, or super-enhancers (SEs), have recently emerged as a novel class of regulatory non-coding RNAs (ncRNAs) that facilitate transcription, including that of androgen target genes, through chromatin looping to position enhancers proximate to the promoters. The aim of this study was to assess androgen-dependent transcription in prostate tumors of eRNAs (designated as KLK3eRNAs) from the SE of the KLK3 gene encoding the prostate-specific antigen (PSA) protein, a clinical marker of prostate carcinogenesis.Materials and MethodsThe androgen-induced KLK3eRNAs were identified in the LNCaP human prostate cancer cell line. The expressions of these KLK3eRNAs together with KLK3 and AR mRNA transcripts were assessed by qRT-PCR in prostate tumor samples from five prostate cancer patients.ResultsAndrogen-induced KLK3eRNAs have been identified in the LNCaP cells, and their expression was further analyzed in tumors of prostate cancer patients. Transcripts of the tested KLK3eRNAs have been detected in all clinical samples, but their expression patterns differed between individual tumor specimens. We found a statistically significant correlation between the levels of the KLK3 and AR mRNAs with those of the previously reported KLK3eRNAs, while such correlation was not observed for novel KLK3eRNAs described in our recent report.ConclusionPresented data suggest that prostate tumor development may associate with epigenetic reorganization in the KLK3 genomic regulatory elements reflected by changes of the KLK3eRNA expression. Our findings support a potential of eRNAs profiling to be used as diagnostic marker.

Effect of HDAC9 inhibition on epithelial-mesenchymal transition in CD133+ prostate cancer cell lines

A small fraction of cancer cells known as cancer stem cells (CSCs) are considered to give rise to differentiated cancer cells and have been proposed to predict cancer recurrence and metastasis. There is further evidence that CSCs may act as metastatic precursors of epithelial-mesenchymal transition (EMT). In the present study, we investigated the key molecules involved in maintaining the stability of CSCs by inducing ectopic overexpression of CD133 to characterize EMT in human prostate cancer cell lines, including PC-3, DU145, and LnCaP cells. Additionally, we investigated whether a specific inhibitor of concomitantly expressed metastasis-related genes could alleviate EMT properties in CD133-overexpressing prostate cancer cells. Ectopic overexpression of CD133 in PC-3, DU145, and LnCaP cells led to an increase in the expression of HDAC9. Moreover, HDAC9 inhibition led to a decrease in EMT properties along with increased E-cadherin expression, a narrower wound gap distance, and enhanced cell invasiveness through the suppression of β-catenin activation and its translocation to the nucleus. Overall, these results suggest that HDAC9 inhibition plays a functional role in the modulation of EMT properties in CSC-like prostate cancer cells. Therefore, these findings could facilitate the development of therapeutic strategies for controlling prostate cancer metastasis.

Androprostamine A: a unique antiprostate cancer agent.

The androgen receptor (AR) is an important therapeutic target for all clinical states of prostate cancer. We screened cultured broths of microorganisms for their ability to suppress androgen-dependent growth of human prostate cancer LNCaP and VCaP cells without cytotoxicity. We have already identified androprostamine A (APA) from a Streptomyces culture broth as a functional inhibitor of AR. APA repressed R1881 (the synthetic androgen methyltrienolone)-induced androgen-regulated gene expression and dramatically inhibited R1881-induced prostate-specific antigen levels. However, APA did not act as an AR antagonist and did not inhibit AR transcriptional activity. Moreover, AS2405, an APA derivative, significantly inhibited the growth of VCaP cells in SCID mice upon oral administration.