Viability Of LNCaP Cells Research Articles

Abstract A052: Combining metabolic starvation and PI3K/AKT/mTOR pathway inhibition to chemosensitize prostate cancer cells

Abstract Prostate cancer is characterized by resistance to chemotherapy and is often associated with loss of the tumor suppressor PTEN, a negative regulator of the PI3K/AKT/mTOR pathway. Currently, taxanes are the first-line chemotherapy for advanced prostate cancer; however, effective therapeutic strategies are still needed that extend survival or alter the outcome of the disease. The inhibition of PI3K/AKT/mTOR pathway has been reported to enhance the efficacy of chemotherapeutic agents in different experimental models. Moreover, it has been reported that the overexpression of oncogenes under nutrient stress can activate a death program. Therefore, metabolic stress can be a useful strategy to sensitize AKT upregulated prostate cancer cells to antitumor drugs. We have previously demonstrated that: • mTOR is involved not only in PI3K/AKT survival pathway, but also in apoptotic pathway, induced by anti-microtubule agents such as taxanes. • AKT downregulation enhances the antitumor effects of taxanes. • Metabolic starvation increases the sensitivity to death induced by taxanes. • Downregulating Bcl-2 levels raises the sensitization to cell death induced by association of starvation plus taxanes. Here different cancers’ capabilities, such as sustained proliferative signaling, deregulated cellular energetics, cell death resistance, have been targeted by various mechanism-directed treatments in cellular model systems of human prostate cancer. The specific aim of our research project is to overcome therapeutic resistance and restore the sensitivity to anticancer drugs in prostate cancer cells, through selective inhibition of different components of the PI3K/AKT/mTOR cascade, in association with treatment with taxanes and starvation. The PI3K/AKT/mTOR pathway has been targeted using either rapamycin or small interference RNAs (siRNAs). LNCaP, PC3 (both PTEN-null) and DU145 (wild-type PTEN) cell lines have been treated with rapamycin as a single agent or in combination with metabolic starvation and/or taxanes. Cell viability of LNCaP and PC3 prostate cells, with high levels of phosphorylated AKT, was not significantly affected by rapamycin treatment alone, but the percentage of growth inhibition significantly increased in combined treatments with starvation and taxanes. DU145 cells, with lower levels of activated AKT, showed high inhibition of cell growth following rapamycin treatment alone (45%), which increased to 75% when rapamycin was associated with starvation and/or taxanes. PTEN-null and PTEN-wild type prostate cancer cells were also treated with siRNAs directed towards mTOR (simTOR), as a single agent or in association with starvation and taxanes. The combination of treatments caused a dramatic reduction of viable cell number in DU145 (up to 90%) and, to a lesser extent, in LNCaP cells (about 50%). These results have been confirmed by Annexin V assays analyzing cell death in the same samples. Therefore, we have established that acting at the same time on different cancers’ capabilities is a promising strategy to induce the apoptotic program, initiated by anti-microtubule drugs, in prostate cancer cells. We demonstrated that it is possible to induce prostate cancer cell death through selective inhibition of specific components of PI3K/AKT/mTOR signaling cascade, in association with treatment with taxanes and metabolic starvation. The identification of the most effective and safe combination of targets to achieve a specific and maximal sensitization to antiproliferative drugs might provide new therapeutic models in clinical applications. Citation Format: Annamaria Bevilacqua, Angela Calastretti, Giuliana Gatti, Gianfranco Canti. Combining metabolic starvation and PI3K/AKT/mTOR pathway inhibition to chemosensitize prostate cancer cells [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A052.

The Impact of PLGA Nanoparticle Delivered 3‐Bromopyruvate and SC‐514 on ABC Transporter Mediated Multidrug Resistance in Prostate Cancer Treatment

BackgroundAccording to the World Health Organization, prostate cancer is the second most diagnosed type of cancer in men. Stage IV prostate cancer spreads to the lymph nodes and the bones, often leading to a non‐curable bone metastasis in 65–75% of prostate cancer cases. Current research focuses on preventing ABC mediated multidrug resistance (MDR) in prostate cancer treatment by using nano‐carriers to deliver chemotherapeutic agents. Even though prostate cancer heterogeneity has limited the efficacy of therapies reversing ABC transporter‐mediated MDR. 3‐Bromopyruvate (3‐BPA) and SC‐514 separately deplete ATP source of prostate cancer cells regardless of prostate cancer heterogeneity problem.ObjectiveThis goal of this study is to overcome ABC transporter‐mediated MDR during treatment of prostate cancer. This will be achieved by depleting the ATP energy source of ABC transporter proteins with the double action of 3‐BPA and SC‐514. Further draining of ATP energy will be achieved by controlled and prolonged release of 3‐BPA and/or SC‐514 with Poly Lactic Co‐Glycolic (PLGA) acid nanoparticle.MethodTrypan blue assay was used to determine the percentage cell viability of LNCaP cells (79.5%) and the number of cells per ml (84,000 cells per ml). 5000 cells were seeded in each well of the 96‐well microtiter plate. NBT assay was used to estimate Reactive Oxygen species(ROS) modulation of LNCaP cells after treatment. LDH assay was used to assess the cytotoxicity of treatment group. MTT assay was used to assess cell viability of LNCaP cells.ResultsOne‐way ANOVA analysis of percentage cell viability in LNCaP cells treated for 24 hrs with 3‐BPA (R2= 0.51, p=0.01), SC‐514 (R2= 0.83, p=0.001 IC‐50=70 μM) and 3‐BPA + SC‐514 (R2= 0.87, p=0.001, IC‐50=68 μM) indicated a probability level of 0.03. Treatment for 48 hrs with 3‐BPA (R2=0.84, r=−0.91, p=0.00061), SC‐514 (R2= 0.47, r=−0.7, p=0.07), and 3‐BPA + SC‐514 (R2=0.59, r= −0.95, p=0.00024) indicated a probability level of 0. 00003. Time dependent treatment (24 hrs, 48 hrs, 72 hrs and 96 hrs) shows that cell viability of treated LNCaP cells decrease with time. When 24 hrs was compared with 48 hrs, p=0.00000015 and when 72 hrs was compared with 96 hrs, p=0.000000049. Probability levels between treatment groups (3‐BPA, SC‐514 and 3‐BPA + SC‐514) decreases with time: 24 hrs (p= 0.00023) > 48 hrs (p= 0.00003) > 72 hrs (p=0.000000152) > 96 hrs(p=0.000000049). One‐way ANOVA analysis comparing percentage ROS level of LNCaP cells treated with 3‐BPA (R2 = 0.30, r=−0.5, p=0.11), SC‐ 514 (R2 =0.47, p=0.04, r=− 0.72) and 3‐BPA + SC‐514(R2 = 0.36, r=−0.58, p=0.04) indicated a probability level of p=0.54.ConclusionIn conclusion, 3‐BPA and SC‐514 separately inhibit proliferation of prostate cancer cells. There might be other mechanism influencing the interaction between 3‐BPA and SC‐514 other than ROS modulation. Combination treatment had a lower IC‐50 compared to mono‐treatment of the drug. Further findings from this study will give insight on how inhibition of survival pathways, can impact carcinogenesis and ABC transporter‐mediated MDR in prostate cancer treatment.Support or Funding InformationFlorida Atlantic University Fellowships and ScholarshipsThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

CO suppresses prostate cancer cell growth by directly targeting LKB1/AMPK/mTOR pathway in vitro and in vivo

BackgroundCO is a freely diffusible gas that acts as a physiological mediator of many biological and cellular processes, which has been shown to possess anticancer effect in many kinds of cancers. However, the effect of CO on prostate cancer has not been demonstrated. Therefore, we analyzed the antitumor activities and related mechanisms of CO on prostate cancer in vitro and in vivo. MethodsCell viability of LNCaP and PC-3 cells after CORM-2 treatment was measured by CCK-8 assay, whereas the ATP production were detected by ATP detection assay. The early apoptosis induced by CO was evaluated by flow cytometry, and the expression level of apoptosis-related molecules (Caspases 3, 8, 9 and cleaved-Caspases 3, 8, 9) was detected using Western blot. Matrigel in vitro invasion assay was used to evaluate the effect of CO on cell invasion. We then evaluated the impact of CO on the expression of several key regulators involved in the LKB1 signaling pathway. At last, xenograft tumor in nude mice was used to further investigate the antitumor effect of CO in vivo. ResultsOur results showed that CO could significantly inhibit proliferation and invasion, and induce apoptosis in human prostate cancer cell lines. The expression of LKB1 could be up-regulated after CO treatment, and CO also could increase p-AMPK levels and decrease p-mTOR. Furthermore, LKB1 knockdown could weaken the effect of CO on prostate cancer cells. In vivo, CO treatment significantly suppressed tumor growth and induced apoptosis in xenografts tumor in nude mice. ConclusionsCO possesses striking anticancer effect in human prostate cancer cells in vitro and in vivo, which is largely mediated by LKB1-AMPK-mTOR axis.

Knockdown of STEAP1 inhibits cell growth and induces apoptosis in LNCaP prostate cancer cells counteracting the effect of androgens.

Six transmembrane epithelial antigen of the prostate 1 (STEAP1) is overexpressed in numerous types of tumors, especially in prostate cancer. STEAP1 is located in the plasma membrane of epithelial cells and may play an important role in inter- and intracellular communication. Several studies suggest STEAP1 as a potential biomarker and an immunotherapeutic target for prostate cancer. However, the role of STEAP1 in cell proliferation and apoptosis remains unclear. Therefore, the role of STEAP1 in prostate cancer cells proliferation and apoptosis was determined by inducing STEAP1 gene knockdown in LNCaP cells. In addition, the effect of DHT on the proliferation of LNCaP cells knocked down for STEAP1 gene was evaluated. Our results demonstrated that silencing the STEAP1 gene reduces LNCaP cell viability and proliferation, while inducing apoptosis. In addition, we showed that the cellular and molecular effects of STEAP1 gene knockdown may be independent of DHT treatment, and blocking STEAP1 may reveal to be an appropriate strategy to activate apoptosis in cancer cells, as well as to prevent the proliferative and anti-apoptotic effects of DHT in prostate cancer.

Aryl Hydrocarbon Receptor Suppresses the Prostate Cancer LNCaP Cell Growth and Invasion by Promoting DNA Damage Response Under Oxidative Stress.

Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that interacts with multiple signaling pathways during prostate development. In the present study, LNCaP cells were knocked down of AhR by siRNA, or treated with the AhR agonist 3-methylcholanthrene (3MC). The effects of AhR on LNCaP cells and the associated mechanisms were studied both under normal condition and under hydrogen peroxide (H2O2)-induced oxidative stress. MTT, transwell chamber assays and flow cytometry were employed to investigate cell proliferation, invasion, and apoptosis, respectively, whereas the DNA damage response (DDR) signaling (phosphorylation of ataxia-telangiectasia mutated [ATM], check-point kinase 2 [Chk2], histone H2AX, p53, and cleaved poly-ADP-ribose polymerase [PARP]) was detected by western blotting. Exposure of LNCaP cells to H2O2 inhibited their viability and migration, and induced apoptosis, at a greater extent compared with the culture under normal conditions. In addition, the oxidative stress increased p-ATM, p-Chk2, p-p53, and p-H2AX expression levels significantly. Knockdown of AhR attenuated the aforementioned effects caused by H2O2-induced oxidative stress. Activation of AhR by 3MC treatment, further aggravated these changes of LNCaP cells on oxidative stress. The findings indicated that AhR suppresses the viability and migration of LNCaP cells notably under oxidative stress, and this process is associated with positive regulation of the responses to oxidative DNA damage.

In vitro cytotoxicity of L-amino acid oxidase from the venom of Crotalus mitchellii pyrrhus

L-amino acid oxidase isolated from Crotalus mitchellii pyrrhus (Cmp-LAAO) exhibited cytotoxicity against LNCaP prostate adenocarcinoma cells. The viability of LNCaP cells decreased in a concentration- and time-dependent manner upon administration of Cmp-LAAO. Cmp-LAAO induced apoptosis as evidenced by AnnexinV/PI staining using flow cytometry. An increase in caspase-9 and caspase-3 activity were also observed. The damaging effect of LAAO appears to be due to its enzymatic activity, that produces hydrogen peroxide which can then induce oxidative stress within the cells. As expected, the level of oxidative stress in LNCaP cells increased with Cmp-LAAO treatment as confirmed by 2′, 7′-dichlorofluorescin diacetate (DCFDA) fluorescence assay. Co-treatment with catalase significantly reduced the cytotoxic effect of Cmp-LAAO, thereby affirming that hydrogen peroxide is probably the main mediator of Cmp-LAAO cytotoxicity. Hence, Cmp-LAAO may be a potential cancer therapeutic for prostate cancer.

Effect of synthetic small molecule RNA on cell cycle and proliferation of prostate cancer cell

Objective To investigate the effect of dsP21-555 on cell cycle and proliferation of prostate cancer cell line PC-3 and LNCaP. Methods dsP21-555 (experimental group) and dsControl (negative control group) were transfected into PC-3 and LNCaP, respectively. Real-time PCR and Western Blotting were used to detect the expression of p21 mRNA and p21 protein in prostate cancer cells after transfection. The cell cycle distribution was detected by flow cytometry. Cell viability and proliferation were measured by MTT assay and colony forming assay. Results The level of p21 mRNA in PC-3 and LNCaP cells was increased to 2.90-fold (P<0.01) and 2.05 - fold (P<0.01) respectively after transfection with dsP21-555. Western Blotting results were consistent with this trend. Flow cytometry showed that the percentage of cells in S phase and G2 / M phase decreased after transfection with dsP21-555, and the proportion of cells in G0 / G1 phase increased. MTT assay showed that the viability of PC-3 and LNCaP cells significantly decreased after dsP21-555 transfection compared with dsControl group. The colony formation assay showed that the number of colonies in the dsP21-555 group was fewer and the cell proliferation ability decreased. Conclusions Synthesized dsP21-555 can significantly activate the expression of p21 gene in prostate cancer cells and significantly inhibit the progression of cell cycle and proliferation ability of prostate cancer cells. Key words: Prostatic Neoplasms; RNA; Cell Cycle; Cell Proliferation

Abstract 4414: Arginine metabolism is modulated by androgen signalling and prostate cancer progression

Abstract Background: Metabolic reprogramming has been described as one of the hallmarks of cancer. A pathway of promise in prostate cancer management is arginine (Arg) metabolism. Phase 2 clinical trials are underway to study arginine deprivation as a treatment of prostate cancer. However, heterogeneous response has been noted and further studies are needed to better identify prostate tumors most reliant on Arg metabolism. The aim of this study was to investigate if Arg metabolism was associated with androgen signalling and prostate cancer progression from androgen dependent (AD) to androgen independent (AI) phenotype. Methods: The LNCaP prostate cancer cell line was used. Four sets of conditions were tested: LNCaP control, LNCaP in the presence of dihydrotestosterone (DHT), LNCaP with MDV3100 (an AR inhibitor), and a subline of LNCaP (CSS90) which has become AI. MTT viability assay was used to assess cell viability. The Seahorse XF bioenergetics analyzer was used to measure metabolic oxygen consumption. Gas Chromatography and Mass Spectroscopy (GC/MS) was used for metabolomics characterization. Results: LNCaP cells at a baseline are Arg-dependent. Arg starvation led to a 63% decrease in LNCaP cell viability (p&amp;lt;0.0001) in 24hrs. By contrast, the AI CSS90 only demonstrated a 23% decrease in cell viability when depleted of Arg (p&amp;lt;0.01 with Arg control, p&amp;lt;0.001 between LNCaP and CSS90) demonstrating AD cells were much more dependent on exogenous Arg. Enhanced AR signaling with DHT further sensitized cells to arginine depletion with a 73% decrease, and low level AR inhibition decreased response to arginine depletion with only a 21% decrease in cell viability (p&amp;lt;0.001 for all changes). Metabolic characterization by GC/MS demonstrated a 33.8 fold increase in buildup of an arginine metabolism intermediate, ornithine in the CSS90 cells vs LNCaP (p&amp;lt;0.0001) reflecting compensatory ability of the CSS90 cells to endogenously manage Arg metabolism. Modulation of androgen signaling with DHT and MDV3100 demonstrated similar increased reliance of exogenous Arg for AD cells with increased androgen signaling with a 2.2 fold increase in Arg dependent oxygen consumption with DHT and 95% drop with MDV (p&amp;lt;0.0001 for both). Conclusions: Arginine is potentially an important aspect of prostate cancer metabolism. Arg targeted therapies may best be suited towards earlier stage tumors and Arg metabolism characterization may have potential as a biomarker of progression to AI prostate cancer. Citation Format: De-Xue Fu, Hubert Huang, Jee-Hoon Song, Min Xu, Lucy Liu, Mohamad Afnan Khan, Krish Chandrasekaran, Arif Hussain, Ganesh Sriram, Mohummad M. Siddiqui. Arginine metabolism is modulated by androgen signalling and prostate cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4414. doi:10.1158/1538-7445.AM2017-4414

Abstract 1579: Bicalutamide-induced expression of T-type Ca2+ channels in prostate cancer cells undergoing neuroendocrine differentiation in vitro

Abstract The growth of prostate cancer cells depends on the activation of androgen receptors. Anti-androgen therapies that inhibit androgen synthesis or receptor activation are effective in limiting tumor growth. However, prolonged treatment with anti-androgen therapies result in the transition of prostate cancers into an androgen refractory state. Neuroendocrine differentiation (NED) has been associated with the progression of prostate cancers to an androgen resistant phenotype. In this work we investigated the effect of the androgen receptor blocker bicalutamide in promoting NED of LNCaP cells and whether it is accompanied by increased T-type Ca2+ channel expression. The ability of bicalutamide to evoke morphological and biochemical changes associated with NED was assessed by PCR, western blot and immuno-histochemical analysis. Changes in the Cav3.2 T-type Ca2+ channel subunit expression was studied using PCR analysis, western blot and whole cell recordings. The role of T-type Ca2+ channels in promoting the differentiation of LNCaP cells was assessed by a cell viability assay and changes in cell morphology, specifically the development of neurite-like processes. PCR analysis of bicalutamide-stimulated cells indicates no significant changes in Cav3.2 mRNA, yet results in Cav3.2 protein expression and functional channels. Western blot and immuno-histochemical analysis of LNCaP cells simulated with bicalutamide for 4-10 day reveals biochemical changes consistent with NED including the expression of tubulin IIIbeta and neuron-specific enolase. Pharmacological inhibition of T-type Ca2+ channel function with nickel ions and NNC 33-0936 disrupts the morphological differentiation and cell viability of LNCaP cells treated with bicalutamide. These results suggest that bicalutamide treatment of LNCaP cells evokes significant morphological and biochemical changes associated with NED and results in increased expression of T-type Ca2+ channels, which can significantly alter Ca2+ homeostasis. Citation Format: Miguel Martin-Caraballo, Megan Hall, Bryan Todd, Yoon Jung Kwon, Vu Nguyen, Jennifer L. Hearne. Bicalutamide-induced expression of T-type Ca2+ channels in prostate cancer cells undergoing neuroendocrine differentiation in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1579. doi:10.1158/1538-7445.AM2017-1579

Role of 20-Hydroxyeicosatetraenoic Acid (20-HETE) in Androgen-Mediated Cell Viability in Prostate Cancer Cells.

20-Hydroxyeicosatetraenoic acid (20-HETE) is generated intracellularly through the ω-hydroxylation of arachidonic acid by the cytochrome P450 (in humans, CYP4A11 and CYP4F2). 20-HETE induces mitogenic responses in different cancer cells. The aim of this study was to analyze how 20-HETE impacts cell survival, proliferation, and apoptosis in prostate cancer cells. Incubation of the human androgen-sensitive cells (LNCaP) with 1-10μM HET0016 (a selective inhibitor of 20-HETE synthesis) reduced cell viability by 49*-64%* (*p<0.05 vs. control). This was explained by a reduction in cell proliferation (vehicle, 46±3%; 1μM, 23±3%*; 10μM, 28±3%*) and by an increase in apoptosis (vehicle, 2.1±0%; 1μM, 16±4%*; 10μM, 31±3%*). Furthermore, the increase in LNCaP cell viability induced by dihydrotestosterone (DHT, 0.1nM) was abrogated by 30*-42%* by 1-10μM HET0016. Incubation with 20-HETE (5-1000nM) increased LNCaP cell viability up to 50%*, together with a 70%* reduction in apoptosis. PC-3 (androgen-insensitive) cell viability was not affected by either HET0016 or 20-HETE. In LNCaP cells, HET0016 (10μM) diminished the expression of androgen receptors (AR): messenger RNA (mRNA) (40%*) and protein (50%*). DHT (10nM) augmented CYP4F2 protein expression (1.9-fold*) and 20-HETE levels (50%*). Oppositely, enzalutamide (AR antagonist) reduced CYP4F2 mRNA and protein expressions by 30 and 25%, respectively. Thus, intracellular availability of 20-HETE is necessary to sustain LNCaP cell viability. 20-HETE may act as a signaling molecule in the pathways involved in LNCaP cell viability upon stimulation of the AR. This effect may be partially attributed to its role on securing normal AR expression levels that in turn contribute to maintain intracellular levels of 20-HETE.

Differential expression of AP-1 transcription factors in human prostate LNCaP and PC-3 cells: role of Fra-1 in transition to CRPC status.

Androgen receptor (AR) signaling axis plays a vital role in the development of prostate and critical in the progression of prostate cancer. Androgen withdrawal initially regresses tumors but eventually develops into aggressive castration-resistant prostate cancer (CRPC). Activator Protein-1 (AP-1) transcription factors are most likely to be associated with malignant transformation in prostate cancer. Hence, to determine the implication of AR and AP-1 in promoting the transition of prostate cancer to the androgen-independent state, we used AR-positive LNCaP and AR-negative PC-3 cells as an in vitro model system. The effect of dihydrotestosterone or anti-androgen bicalutamide on the cell proliferation and viability was assessed by MTT assay. Expression studies on AR, marker genes-PSA, TMPRSS2, and different AP-1 factors were analyzed by semi-quantitative RT-PCR and expressions of AR and Fra-1 proteins were analyzed by Western blotting. Dihydrotestosterone induced the cell proliferation in LNCaP with no effect on PC-3 cells. Bicalutamide decreased the viability of both LNCaP and PC-3 cells. Dihydrotestosterone induced the expression of AR, PSA, c-Jun, and Fra-1 in LNCaP cells, and it was c-Jun and c-Fos in case of PC-3 cells, while bicalutamide decreased their expression. In addition, constitutive activation and non-regulation of Fra-1 by bicalutamide in PC-3 cells suggested that Fra-1, probably a key component, involved in transition of aggressive androgen-independent PC-3 cells with poor prognosis.

Artesunate suppresses the viability and mobility of prostate cancer cells through UCA1, the sponge of miR-184.

Artesunate (ART) is a sesquiterpene lactone isolated from the leafy portions of the Chinese herb Artemisia annua. Here, we evaluated the effect of ART on the prostate cancer (PCa) cell lines DU145 and LNCaP and explored its potential mechanisms. ART inhibited the viability and mobility of DU145 and LNCaP cells. Mechanistically, we found that UCA1, one of the most important lncRNAs in malignancies of the urinary system, may be a potential mediator contributing to the tumor suppressor function of ART. First, the UCA1 level was reduced significantly after being exposed to ART. In addition, UCA1 was up-regulated in prostate cancer tissues compared to hyperplastic prostatic tissues, and a higher UCA1 level predicted poor prognosis in PCa patients. Furthermore, reintroduction of UCA1 into PCa cells reversed the effect of ART on apoptosis and metastatic ability. Then we determined that the miR-184/Bcl-2 axis might be the downstream signaling pathway of UCA1 upon ART treatment. UCA1 binds to miR-184 through its seed sequences and may function as a sponge for miR-184.

Naringenin-Induced Apoptotic Cell Death in Prostate Cancer Cells Is Mediated via the PI3K/AKT and MAPK Signaling Pathways.

Prostate cancer is the most common cancer in men and the second most common cause of cancer-related deaths in men. Although, various drugs targeting the androgen receptor are normally used, the patients frequently undergo recurrence of the disease. To overcome these limitations, natural compounds have been researched for evidence that they suppress progression and metastasis of various cancer cells. In the present study, we investigated effects of naringenin, a natural anti-oxidant flavonoid derived from citrus, on prostate cancer cells (PC3 and LNCaP). Results of present study with PC3 and LNCaP cells revealed that naringenin inhibited proliferation and migration, while inducing apoptosis and ROS production by those cells. In addition, naringenin-induced loss of mitochondrial membrane potential and increased Bax and decreased Bcl-2 proteins in PC3 cells, but not LNCaP cells. In a dose-dependent manner, naringenin decreased phosphorylation of ERK1/2, P70S6K, S6, and P38 in PC3 cells, and reduced phosphorylation of ERK1/2, P53, P38, and JNK proteins in LNCaP cells. However, naringenin activated phosphorylation of AKT in both PC3 and LNCaP cells. Then, targeted signaling proteins associated with viability of PC3 and LNCaP cells were analyzed using pharmacological inhibitors of AKT and ERK1/2 cell signaling pathways. Moreover, we compared the apoptotic effects of naringenin and paclitaxel alone and in combination to find that naringenin enhanced the efficiency of paclitaxel to suppress progression of prostate cancer cell lines. Collectively, these results indicate that naringenin is a potential chemotherapeutic agent for treatment of prostate cancer. J. Cell. Biochem. 118: 1118-1131, 2017. © 2016 Wiley Periodicals, Inc.

MED15 overexpression in prostate cancer arises during androgen deprivation therapy via PI3K/mTOR signaling.

Androgen deprivation therapy (ADT) is the main therapeutic option for advanced prostate cancer (PCa). After initial regression, most tumors develop into castration-resistant PCa (CRPC). Previously, we found the Mediator complex subunit MED15 to be overexpressed in CRPC and to correlate with clinical outcome. Therefore, we investigated whether MED15 is implicated in the signaling changes taking place during progression to CRPC. Immunohistochemistry (IHC) for MED15 on matched samples from the same patients before and after ADT reveals significantly increased MED15 expression after ADT in 72%. A validation cohort comprising samples before and after therapy confirmed our observations. Protein analysis for pAKT and pSMAD3 shows that MED15 correlates with PI3K and TGFß activities, respectively, and that hyper-activation of both pathways simultaneously correlates with highest levels of MED15. We further show that MED15 protein expression increases in LNCaP cells under androgen deprivation, and via EGF mediated PI3K activation. PI3K/mTOR and TGFß-receptor inhibition results in decreased MED15 expression. MED15 knockdown reduces LNCaP cell viability and induces apoptosis during androgen deprivation, while cell cycle is not affected. Collectively, MED15 overexpression arises during ADT via hyper-activation of PI3K/mTOR signaling, thus MED15 may serve as a predictive marker for response to PI3K/mTOR inhibitors. Furthermore, MED15 is potentially a therapeutic target for the treatment of CRPC.

FOXO3a Expression Regulated by ERK Signaling is Inversely Correlated With Y-Box Binding Protein-1 Expression in Prostate Cancer.

FOXO3a is a member of the forkhead O transcription factors. FOXO3a induces the factors that contribute to cell cycle arrest and is considered a tumor suppressor in several malignant tumors. Y-box binding protein-1 (YB-1) is a multifunctional protein whose high expression is correlated with poor prognoses in various malignant tumors. In the current study, we investigated the relationship between FOXO3a and YB-1 to validate their functional roles in prostate cancer. Western blotting and cytotoxicity assays were conducted in prostate cancer cells, LNCaP, and 22Rv1 cells. We also evaluated the protein expressions of FOXO3a and YB-1 in human prostate cancer tissues, using radical prostatectomy specimens. Then, we investigated the correlations between protein expressions and clinicopathologic parameters. We found that both FOXO3a and YB-1 proteins were phosphorylated by ERK signaling, resulting in FOXO3a inactivation and YB-1 activation in LNCaP and 22Rv1 cells. Inversely, inhibition of MEK or treatment with metformin activated FOXO3a through inactivation of ERK signaling and suppressed the viability of LNCaP and 22Rv1 cells in a dose-dependent manner. In immunohistochemical analysis, FOXO3a nuclear expression was inversely correlated with YB-1 nuclear expression (P < 0.0001). Furthermore, high FOXO3a nuclear expression was inversely correlated with a higher Gleason grade (P < 0.0001) and higher preoperative PSA (P = 0.0437). These results showed that in prostate cancer, FOXO3a, and YB-1 play inverse reciprocal roles as a tumor-suppressor gene and oncogene, respectively, through their master regulator ERK. Prostate 77:145-153, 2017. © 2016 Wiley Periodicals, Inc.

Red Maca (Lepidium meyenii) did not affect cell viability despite increased androgen receptor and prostate-specific antigen gene expression in the human prostate cancer cell line LNCaP.

We examined whether aqueous extract of Lepidium meyenii (red Maca) could inhibit growth, potentiate apoptotic activity of two anticancer drugs Taxol and 2-methoxyestradiol (2ME) or change mRNA expression for the androgen target genes, androgen receptor (Ar) and prostate-specific antigen (Psa) in the human prostate cancer cell line LNCaP. Red Maca aqueous extract at 0, 10, 20, 40 or 80μg/ml was added to LNCaP cells, and viability was evaluated by the MTS assay at 24 or 48hr after treatment. Furthermore, LNCaP cells were treated with 80μg/ml of red Maca plus Taxol or 2ME 5μM and viability was assessed 48hr later. Finally, LNCaP cells were treated with red Maca 0, 20, 40 or 80μg/ml, and 12hr later, mRNA level for Ar or Psa was assessed by real-time PCR. Treatment with red Maca did not affect viability of LNCaP cells. Apoptotic activity induced by Taxol and 2ME in LNCaP cells was not altered with red Maca treatment. Relative expression of the mRNA for Ar and Psa increased with red Maca 20 and 40μg/ml, but not at 80μg/ml. We conclude that red Maca aqueous extract does not have toxic effects, but stimulates androgen signalling in LNCaP cells.

Nephrotoxicity of epigenetic inhibitors used for the treatment of cancer

This study determined the anti-neoplastic activity and nephrotoxicity of epigenetic inhibitors in vitro. The therapeutic efficacy of epigenetic inhibitors was determined in human prostate cancer cells (PC-3 and LNCaP) using the DNA methyltransferase inhibitor 5-aza-2ʹ-deoxycytidine (5-Aza) and the histone deacetylase inhibitor trichostatin A (TSA). Cells were also treated with carbamazepine (CBZ), an anti-convulsant with histone deacetylase inhibitor-like properties. 5-Aza, TSA or CBZ alone did not decrease MTT staining in PC-3 or LNCaP cells after 48 h. In contrast, docetaxel, a frontline chemotherapeutic induced concentration-dependent decreases in MTT staining. Pretreatment with 5-Aza or TSA increased docetaxel-induced cytotoxicity in LNCaP cells, but not PC-3 cells. TSA pretreatment also increased cisplatin-induced toxicity in LNCaP cells. Carfilzomib (CFZ), a protease inhibitor approved for the treatment of multiple myeloma had minimal effect on LNCaP cell viability, but reduced MTT staining 50% in PC-3 cells compared to control, and pretreatment with 5-Aza further enhanced toxicity. Treatment of normal rat kidney (NRK) and human embryonic kidney 293 (HEK293) cells with the same concentrations of epigenetic inhibitors used in prostate cancer cells significantly decreased MTT staining in all cell lines after 48 h. Interestingly, we found that the toxicity of epigenetic inhibitors to kidney cells was dependent on both the compound and the stage of cell growth. The effect of 5-Aza and TSA on DNA methyltransferase and histone deacetylase activity, respectively, was confirmed by assessing the methylation and acetylation of the CDK inhibitor p21. Collectively, these data show that combinatorial treatment with epigenetic inhibitors alters the efficacy of chemotherapeutics in cancer cells in a compound- and cell-specific manner; however, this treatment also has the potential to induce nephrotoxic cell injury.

The reverse-mode NCX1 activity inhibitor KB-R7943 promotes prostate cancer cell death by activating the JNK pathway and blocking autophagic flux.

We explored the effects of KB-R7943, an inhibitor of reverse-mode NCX1 activity, in prostate cancer (PCa). NCX1 was overexpressed in PCa tissues and cell lines, and higher NCX1 levels were associated higher PCa grades. At concentrations greater than 10 μM, KB-R7943 dose-dependently decreased PC3 and LNCaP cell viability. KB-R7943 also increased cell cycle G1/S phase arrest and induced apoptosis in PC3 cells. KB-R7943 increased autophagosome accumulation in PCa cells as indicated by increases in LC3-II levels and eGFP-LC3 puncta. Combined treatment with chloroquine (CQ) and KB-R7943 decreased P62 and increased LC3-II protein levels in PC3 cells, indicating that KB-R7943 blocked autophagic flux. KB-R7943 induced autophagosome accumulation mainly by downregulating the PI3K/AKT/m-TOR pathway and upregulating the JNK pathway. In xenograft experiments, KB-R7943 inhibited tumor growth. Combined treatment with KB-R7943 and an autophagy inhibitor inhibited growth and increased apoptosis. These results indicate that KB-R7943 promotes cell death in PCa by activating the JNK signaling pathway and blocking autophagic flux.

MP66-16 REGENERATING ISLET-DERIVED RELATED PROTEIN 4 AS A CANDIDATE OF A NOVEL BIOMARKER IN CASTRATION-RESISTANT PROSTATE CANCER PATIENTS

You have accessJournal of UrologyProstate Cancer: Basic Research & Pathophysiology II1 Apr 2016MP66-16 REGENERATING ISLET-DERIVED RELATED PROTEIN 4 AS A CANDIDATE OF A NOVEL BIOMARKER IN CASTRATION-RESISTANT PROSTATE CANCER PATIENTS Jun Teishima, Hirotaka Nagamatsu, Koichi Shoji, Ryoken Yamanaka, Kohei Kobatake, Hiroyuki Kitano, Keisuke Goto, Tetsutaro Hayashi, Norihide Oue, Wataru Yasui, and Akio Matsubara Jun TeishimaJun Teishima More articles by this author , Hirotaka NagamatsuHirotaka Nagamatsu More articles by this author , Koichi ShojiKoichi Shoji More articles by this author , Ryoken YamanakaRyoken Yamanaka More articles by this author , Kohei KobatakeKohei Kobatake More articles by this author , Hiroyuki KitanoHiroyuki Kitano More articles by this author , Keisuke GotoKeisuke Goto More articles by this author , Tetsutaro HayashiTetsutaro Hayashi More articles by this author , Norihide OueNorihide Oue More articles by this author , Wataru YasuiWataru Yasui More articles by this author , and Akio MatsubaraAkio Matsubara More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.1290AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The aim of this study was to investigate the implication of regenerating islet-derived related protein 4 (Reg IV) as a biomarker of castration-resistant prostate cancer (CRPC). METHODS The serum Reg IV level in 39 prostate cancer (PC) patients, 36 renal cell cancer (RCC) patients, 12 urothelial cancer (UC) patients, and 14 non-cancerous subjects was measured using an enzyme-linked immunosorbent (ELISA) assay. The cell viability of PC3 and LNCaP stably transfected with Reg IV cDNA (PC3-reg and LN-reg, respectively) was examined using an MTT assay. The expression of molecules related to epithelial-mesenchymal transition (EMT), neuroendocrine differentiation, and apoptosis in these cells was examined using western immunoblot analysis. RESULTS Serum concentration levels of Reg IV in patients with PC, RCC, or UC were significantly higher than those in non-cancerous subjects (p=0.0131, p=0.0122 and p=0.0002, respectively). The levels with metastatic PC and metastatic RCC were significantly higher than those with early stages in each of the diseases (p=0.0051 and p=0.0006, respectively). A significant correlation was found between the serum Reg IV and PSA level in androgen-naive prostate cancer patients (p=0.0059, R=0.519). The level in CRPC patients was significantly higher than that in the androgen-naive prostate cancer patients (p=0.0202) and in the non-cancerous subjects (p=0.001). In all CRPC patients, it was higher than 2 ng/ml, which has been defined as the cut-off value in gastrointestinal cancer in previous studies. An EMT was induced in LN-reg. The expression of synaptophysin, NSE, and chromogranin A was upregulated in both LN-reg and PC3-reg compared with in vector control cells. The cell viability under androgen deprivation was enhanced in both LN-reg and PC3-reg, and the resistance of 5-fluorouracil and docetaxel was shown in PC3-reg. CONCLUSIONS Reg IV is a candidate for a novel biomarker of CRPC, and it might represent the effect of therapeutic agents. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e878-e879 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Jun Teishima More articles by this author Hirotaka Nagamatsu More articles by this author Koichi Shoji More articles by this author Ryoken Yamanaka More articles by this author Kohei Kobatake More articles by this author Hiroyuki Kitano More articles by this author Keisuke Goto More articles by this author Tetsutaro Hayashi More articles by this author Norihide Oue More articles by this author Wataru Yasui More articles by this author Akio Matsubara More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

In vitro and in vivo effects of a recombinant anti-PSMA immunotoxin in combination with docetaxel against prostate cancer.

Docetaxel (DOC) is used for the first-line treatment of castration resistant prostate cancer (CPRC). However, the therapeutic effects are limited, only about one half of patients respond to the therapy and severe side effects possibly lead to discontinuation of treatment. Therefore, actual research is focused on the development of new DOC-based combination treatments.In this study we investigated the antitumor effects of a recombinant immunotoxin targeting the prostate specific membrane antigen (PSMA) in combination with DOC in vitro and in vivo. The immunotoxin consists of an anti-PSMA single chain antibody fragment (scFv) as binding and a truncated form of Pseudomonas aeruginosa Exotoxin A (PE40) as toxin domain. The immunotoxin induced apoptosis and specifically reduced the viability of androgen-dependent LNCaP and androgen-independent C4-2 prostate cancer cells. A synergistic cytotoxic activity was observed in combination with DOC with IC50 values in the low picomolar or even femtomolar range. Moreover, combination treatment resulted in an enhanced antitumor activity in a C4-2 SCID mouse xenograft model. This highlights the immunotoxin as a promising therapeutic agent for a future DOC-based combination therapy of CPRC.