Viability Of PC-3 Cells Research Articles

MiR-92a promotes proliferation and inhibits apoptosis of prostate cancer cells through the PTEN/Akt signaling pathway

ABSTRACT MicroRNAs (miRNAs) play an important role in the development of prostate cancer (PCa). Recent studies have shown that miR-92a expression is significantly increased in various cancers including PCa. However, its specific mechanism in PCa remains unknown. The goal of this study was to investigate the effect of miR-92a expression on the function and mechanism of PCa. PCa cell lines PC-3 and LNCap were transfected with miR-92a inhibitor to reduce the expression of miR-92a, respectively. The cell proliferation, cell viability, apoptosis, cell invasion and migration ability of PCa cells were examined by CCK8 assay, cell cloning, flow cytometry, Transwell assay and scratch assay, respectively. The effects of miR-92a on PTEN/Akt signaling pathway-related factors (PI3k, Akt, p-PI3k, p-Akt, PTEN) were also observed by RT-qPCR and Western blot. Compared with the control group and NC inhibitor group, the viability, cell migration and invasion ability of PC-3 and LNCap cells were decreased and apoptosis was significantly increased after interference with miR-92a expression. In addition, the mRNA and protein levels of PTEN in PC-3 and LNCap cells in the miR-92a inhibitor group were significantly increased, while the phosphorylation levels of PI3K and AKT were significantly decreased. MiR-92a might play a key role in regulating the proliferation, migration and invasion of PCa cells through the PTEN/Akt signaling pathway. Inhibition of miR-92a expression has practical value against PCa and provides ideas for further clinical treatment of patients with PCa.

Regulation of circGOLPH3 and its binding protein CBX7 on the proliferation and apoptosis of prostate cancer cells.

To clarify the mechanism of circGOLPH3 regulation on prostate cancer cells, we performed an overexpression and interference circGOLPH3 assay in prostate cancer cells PC-3 and then evaluated cellular viability, proliferation, cell cycle, and apoptosis of prostate cancer cells by MTT, CCK8, Edu stain, TUNEL stain, and flow cytometry. Binding proteins of CircGOLPH3 were identified by RNA pull-down, mass spectrometry, and RNA-binding protein immunoprecipitation (RIP) assays. The expressions of CircGOLPH3 and CBX7 were measured by qRT-PCR. The results showed that after overexpression of circGOLPH3, the proliferative capacity and the viability of PC-3cells were significantly improved, whereas apoptosis was inhibited. CircGOLPH3 could bind to the CBX7 protein that was highly expressed in the PC-3 cell. Additionally, a functional test on CBX7 showed that the CBX7 overexpression notably improved the proliferative capacity and the viability of PC-3 cells and decreased cellular apoptosis, which was consistent with the effects of circGOLPH3. The validated the present study that circGOLPH3 and its binding protein CBX7 can promote prostate cancer cell proliferation and inhibit apoptosis.

Imipramine Inhibits Migration and Invasion in Metastatic Castration-Resistant Prostate Cancer PC-3 Cells via AKT-Mediated NF-κB Signaling Pathway.

Imipramine (IMI) is a tricyclic synthetic antidepressant that is used to treat chronic psychiatric disorders, including depression and neuropathic pain. IMI also has inhibitory effects against various cancer types, including prostate cancer; however, the mechanism of its anticancer activity is not well understood. In the present study, we investigated the antimetastatic and anti-invasive effects of IMI in metastatic castration-resistant prostate cancer PC-3 cells, with an emphasis on the serine/threonine protein kinase AKT-mediated nuclear factor kappa B (NF-κB) signaling pathway. While IMI did not induce cell death, it attenuated PC-3 cell proliferation. According to the wound healing assay and invasion assay, migration and invasion in PC-3 cells were significantly inhibited by IMI in a dose-dependent manner. IMI significantly downregulated p-AKT protein expression but upregulated phospho-extracellular signal-regulated kinase (ERK1)/2 protein expression levels. Furthermore, IMI treatment resulted in decreased AKT-mediated downstream signaling, including p-inhibitor of κB kinase (IKK)α/β, p-inhibitor of κB (IκBα), and p-p65. Inhibited NF-κB signaling reduced the secretion of several proinflammatory cytokines and chemokine by PC-3 cells. Overall, our study explored the negative correlation between the use of antidepressants and prostate cancer progression, showing that IMI attenuated cell viability, migration, and invasion of PC-3 cells by suppressing the expression of AKT and NF-κB-related signaling proteins and secretion of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and monocyte chemoattractant protein-1 (MCP-1).

Anticancer Properties of Asian Water Monitor Lizard (Varanus salvator), Python (Malayopython reticulatus) and Tortoise (Cuora kamaroma amboinensis).

Cancer contributes to significant morbidity and mortality despite advances in treatment and supportive care. There is a need for the identification of effective anticancer agents. Reptiles such as tortoise, python, and water monitor lizards are exposed to heavy metals, tolerate high levels of radiation, feed on rotten/germ-infested feed, thrive in unsanitary habitat and yet have prolonged lifespans. Such species are rarely reported to develop cancer, suggesting the presence of anticancer molecules/mechanisms. Here, we tested effects from sera of Asian water monitor lizard (Varanus salvator), python (Malayopython reticulatus) and tortoise (Cuora kamaroma amboinensis) against cancer cells. Sera were collected and cytotoxicity assays were performed using prostate cancer cells (PC3), Henrietta Lacks cervical adenocarcinoma cells (HeLa) and human breast adenocarcinoma cells (MCF7), as well as human keratinized skin cells (Hacat), by measuring lactate dehydrogenase release as an indicator for cell death. Growth inhibition assays were performed to determine the effects on cancer cell proliferation. Liquid chromatography mass spectrometry was performed for molecular identification. The findings revealed that reptilian sera, but not bovine serum, abolished viability of Hela, PC3 and MCF7 cells. Samples were subjected to liquid chromatography mass spectrometry, which detected 57 molecules from V. salvator, 81 molecules from Malayopython reticulatus and 33 molecules from C. kamaroma amboinensis and putatively identified 9 molecules from V. salvator, 20 molecules from Malayopython reticulatus and 9 molecules from C. kamaroma amboinensis when matched against METLIN database. Based on peptide amino acid composition, binary profile, dipeptide composition and pseudo-amino acid composition, 123 potential Anticancer Peptides (ACPs) were identified from 883 peptides from V. salvator, 306 potential ACPs from 1074 peptides from Malayopython reticulatus and 235 potential ACPs from 885 peptides from C. kamaroma amboinensis. To our knowledge, for the first time, we reported comprehensive analyses of selected reptiles' sera using liquid chromatography mass spectrometry, leading to the identification of potentially novel anticancer agents. We hope that the discovery of molecules from these animals will pave the way for the rational development of new anticancer agents.

Zingerone suppresses cell proliferation via inducing cellular apoptosis and inhibition of the PI3K/AKT/mTOR signaling pathway inhuman prostate cancer PC-3 cells.

Prostate cancer (PCa) is both the foremost and second cause of cancer death in the male population. Patients with hormone-dependent PCa are initially sensitive to androgen-deprivation therapy, later the cancer progress to a hormone-independent state and fails to respond and progress to the metastatic stage, where the cells gain the ability to escape cell death and develop resistance to current therapies, thereby leadingto migration, invasion, and metastasis of cancer. Many clinical trialsusing nutraceuticals on cancer using human subjects have also been extensively studied, these studies confirm the efficacy of drugs tested in in vitro and in vivo preclinical models. Among various dietary phytochemicals, ginger is commonly used in the diet and possesses manyactive principles that act against cancer. Among various active principles, zingerone isa key active phenolic compound present in Zingiber officinale (Ginger), it has potent antioxidant property and it acts against carcinogens. The present study evaluated the efficacy of zingerone at different doses on the PCa cell line regarding apoptosis, upstream signing molecules such as Akt/mTOR, and migration metastasis. A cell viability assay using MTT wasperformed to estimate the percentage of viability of zingerone-treated PC-3 cells. The mitochondrial membrane potential, intracellular reactive oxygen species, and apoptosis induction in the zingerone-treated PC-3 cells were studied by using different fluorescencestaining techniques. The expression patterns of PI3K, AKT, p-AKT, mTOR, and p-mTOR were investigated through the Western blot analysis assay. Zingerone induces apoptosis and alters Akt/mTOR molecules; it also inhibits cell adhesion andmigration of PCa cells. From the present study, it is concluded thatzingerone effectively induces apoptosis and inhibits cancer signaling, thereby acting as a potent drug against PCa.

Characterization of Dopamine Receptor Associated Drugs on the Proliferation and Apoptosis of Prostate Cancer Cell Lines.

Dopamine Receptor (DR) gene family play an essential role in the regulation of Interleukin- 6 (IL-6) production. Our prior analysis of human prostate biopsy samples demonstrated the increased expression of IL-6 and a downregulating trend for dopamine receptor gene family. The objective was to investigate the expression of dopamine receptors, their catabolizing enzyme and IL-6 in prostate cancer cell lines and assess pharmacological effect of dopamine receptor modulators as a novel class of drugs repurposed for the treatment of prostate cancer. The therapeutic effect of dopamine, DR agonists, and DR antagonist were examined using LNCaP and PC3 cell lines. Cell viability and proliferation were assessed by MTT assay and proliferating cell nuclear antigen expression analysis, respectively. Furthermore, bax/bcl2 ratio, immunofluorescence assay and flow cytometric assay were performed for apoptosis analysis. RT- qPCR analysis was used to characterize the relative expression of dopamine-related genes, catabolic enzyme Catechol-o-Methyl-Transferase (COMT) and IL-6 before and after treatment to assess the therapeutic effects of drugs. LNCaP cells express DRD1, DRD2, DRD5 and COMT genes and PC3 cells only express IL-6 gene. In-vitro, dopamine receptor agonists reduced cell viability of LNCaP and PC3 cells. In contrast, dopamine and dopamine receptor antagonist significantly increased tumor growth in PC3 cells. Our results offer novel suggestion for a pathogenic role of dopamine receptor signaling in prostate cancer adenocarcinoma and indicates that modulators of DR- IL-6 pathway, including FDA-approved drug bromocriptine, might be utilized as novel drug repurposing strategy.

Heterofunctional ruthenium(II) carbosilane dendrons, a new class of dendritic molecules to fight against prostate cancer.

A family of heterofunctional Schiff base carbosilane metallodendrons with [Ru(η5-C5H5)(PTA)Cl] (PTA=1,3,5-triaza-7-phosphatricyclo-[3.3.1.1]decane) at the focal point and dimethylamino groups on the periphery are described. The new systems have proved their ability to interact with biological molecules such as Human Serum Albumin (HSA) without affecting its secondary structure and erythrocytes membranes, causing haemolysis in a dose and generation dependent way. The combination of two active functional groups in one single dendritic platform has shown a cooperative effect in the viability of HeLa and PC-3cells, with the second generation derivative standing out as the most promising with the lowest IC50. Experiments focused on advanced prostate cancer have shown an antimetastasic activity for those metallodendrons, hindering the adhesion of cells in one of the main targets of metastasis, bones, and inhibiting cell migration. Finally, the second generation metallodendron with one single metal centre and four dimethylamino groups on the dendritic wedge, was selected for an exvivo experiment in nude mice with advanced prostate cancer inhibiting the tumour growth in a 40% compared to control mice.

Enhanced inhibition of tumor growth using TRAIL-overexpressing adipose-derived stem cells in combination with the chemotherapeutic agent CPT-11 in castration-resistant prostate cancer.

BackgroundThis study investigated the inhibition of tumor growth in castrate-resistant prostate cancer (CRPC)-bearing mice by tumor necrosis factor–related apoptosis-inducing ligand (TRAIL)-overexpressing adipose-derived stem cells (ADSCs) (hTERT-ADSC.sTRAIL), which was enhanced by combined treatment with CPT-11.Materials and methodsAn hTERT-ADSC.sTRAIL cell line was established by transfection with a lentiviral vector (CLV-Ubic) encoding the human sTRAIL gene. Quantitative polymerase chain reaction and Western blots were performed to confirm gene overexpression. An invasion study for the selective migration ability toward PC3 cells was performed. In the in vivo study, the tumor volume in mice treated with ADSC. sTRAIL and CPT-11 was measured.ResultsCarboxylesterase was generated from hTERT-ADSCs. The gene expression of sTRAIL from hTERT-ADSC.sTRAIL was shown. The directional migration of ADSC.sTRAIL cells toward PC3 cells was significantly stimulated by PC3 cells in vitro (P < 0.05). In the in vitro study, the viability of PC3 cells significantly decreased in the presence of ADSC.sTRAIL (62.7 ± 2.0%) and CPT-11 compared with that of CPT-11 alone (83.0 ± 1.0%) at a cell ratio as low as 0.05 (PC3: ADSC.sTRAIL) (P < 0.05). The proportion of apoptotic PC3 cells significantly increased in the presence of ADSC.sTRAIL (37.2 ± 2.1%) and CPT-11 compared with that of CPT-11 alone (16.5 ± 1.0%) (P < 0.05). In the in vivo study, the inhibition of tumor growth in CRPC-bearing mice by TRAIL-overexpressing adipose stem cells was enhanced by combined treatment with the chemotherapeutic agent CPT-11 compared with that in the treatment with cpt-11 alone. Immunohistochemical staining of the removed tumors showed anti-TRAIL–positive cells and apoptotic bodies after hTERT-ADSC.sTRAIL treatment or combined treatment with hTERT-ADSC.sTRAIL and CPT-11.ConclusionsTherapeutic stem cells expressing sTRAIL genes combined with CPT-11 can provide a new strategy for treating CRPC in clinical trials using the patients’ own ADSCs.

Increased phospholipase D activity contributes to tumorigenesis in prostate cancer cell models.

Prostate cancer (PCa) is the most frequent cancer among men and the first cause of death over 65. Approximately 90% of patients with advanced disease will develop bone metastasis, which dramatically reduces long-term survival. Therefore, effective therapies need to be developed, especially when disease is still well-localized. Phospholipase D (PLD), an enzyme that hydrolyzes phosphatidylcholine to yield phosphatidic acid, regulates several cellular functions as proliferation, survival, migration or vesicular trafficking. PLD is implicated in numerous diseases such as neurodegenerative, cardiovascular, autoimmune disorders or cancer. Indeed, PLD controls different aspects of oncogenesis including tumor progression and resistance to targeted therapies such as radiotherapy. PLD1 and PLD2 are the only isoforms with catalytic activity involved in cancer. Surprisingly, studies deciphering the role of PLD in the pathophysiology of PCa are scarce. Here we describe the correlation between PLD activity and PLD1 and PLD2 expression in PCa bone metastasis-derived cell lines C4-2B and PC-3. Next, by using PLD pharmacological inhibitors and RNA interference strategy, we validate the implication of PLD1 and PLD2 in cell viability, clonogenicity and proliferation of C4-2B and PC-3 cells and in migration capacity of PC-3 cells. Last, we show an increase in PLD activity as well as PLD2 protein expression during controlled starvation of PC-3 cells, concomitant with an augmentation of its migration capacity. Specifically, upregulation of PLD activity appears to be PKC-independent. Taken together, our results indicate that PLD, and in particular PLD2, could be considered as a potential therapeutic target for the treatment of PCa-derived bone metastasis.

Modulating tumor reactive stroma by extracorporeal shock wavesto control prostate cancer progression.

Prostate cancer is the second most common cancer worldwide. Tumor microenvironment is composed of activated fibroblasts, the so called carcinoma-associated fibroblasts (CAFs). They express high levels of α-smooth muscle actin (α-SMA) and type I collagen (COL1), and support proliferation and migration of tumor epithelial cells. Extracorporeal shock waves (ESWs), acoustic waves, are effective in the treatment of hypertrophic scars, due to their ability to modulate fibrosis. Based on this rationale, the study evaluated the effects of ESWs on CAF activation and the influence of ESW-treated CAFs on the growth and migration of epithelial prostatic carcinoma cells. Primary cultures of CAFs (n = 10) were prepared from tumors of patients undergoing surgery for high-risk prostate carcinoma. CAFs were treated with ESWs (energy levels: 0.32 mJ/mm2 , 1000 pulses; 0.59 mJ/mm2 , 250 pulses). After treatment, the messenger RNA and protein levels of the stromal activation markers α-SMA and COL1 were determined. Subsequently, two different stabilized cell lines (PC3 and DU145) of androgen-resistant prostate cancer were treated with the conditioned media produced by ESW-treated CAFs. At different times, viability and migration of PC3 and DU145 cells were evaluated. Viability was also assessed by coculture system using CAFs and PC3 or DU145 cells. ESWs reduced gene expression and protein level of α-SMA and COL1 in CAFs. The treatment of PC3 and DU145 with conditioned media of ESW-treated CAFs determined a reduction of their growth and invasive potential. Coculture systems between ESW-treated CAFs and PC3 or DU145 cells confirmed the epithelial cell number reduction. This in vitro study demonstrates for the first time that ESWs are able to modulate the activation of prostate CAFs in favor of a less "reactive" stroma, with consequent slowing of the growth and migration of prostate cancer epithelial cells. However, only further studies to be performed in vivo will confirm the possibility of using this new therapy in patients with prostate cancer.

Diversity-oriented generation and biological evaluation of new chemical scaffolds bearing a 2,2-dimethyl-2H-chromene unit: Discovery of novel potent ANO1 inhibitors

Chemical territory bearing a 2,2-dimethyl-2H-chromene motif was expanded by utilizing an o-hydroxy aldehyde group of 5-hydroxy-2,2-dimethyl-2H-chromene-6-carbaldehyde as a synthetic handle to install distinctive morphology and functionality of each scaffold. Cell based assays and in silico docking analysis led us to discover that these new compounds exhibit inhibitory effect on anoctamin1 (ANO1). ANO1 is amplified and highly expressed in various carcinomas including prostate cancer, esophageal cancer, breast cancer, and pancreatic cancer. Biological assays revealed that (E)-1-(7,7-dimethyl-7H-furo[2,3-f]chromen-2-yl)-3-(1H-pyrrol-2-yl)prop-2-en-1-one (3n, Ani-FCC) is a novel, potent and selective ANO1 inhibitor with an IC50 value of 1.23 μM. 3n showed 144 times stronger activity on ANO1 inhibition than ANO2 inhibition and did not alter the chloride channel activity of CFTR and the intracellular calcium signaling. Notably, 3n strongly decreased cell viability of PC-3 and FaDu cells expressing high levels of ANO1 with a decrease in ANO1 protein levels. In addition, 3n significantly enhanced apoptosis via activation of caspase 3 and cleavage of PARP in PC-3 and FaDu cells. This study shows that a novel ANO1 inhibitor, 3n, can be a potential candidate for the treatment of cancers overexpressing ANO1, such as prostate cancer and esophageal cancer.

Eprinomectin, a novel semi-synthetic macrocylic lactone is cytotoxic to PC3 metastatic prostate cancer cells via inducing apoptosis

Prostate Cancer (PCa) is the second most common cancer among men in United States after skin cancer. Conventional chemotherapeutic drugs available for PCa treatment are limited due to toxicity and resistance issues. Therefore, there is an urgent need to develop more effective treatment for advanced PCa. In this current study, we focused on evaluating the anti-cancer efficacy of Eprinomectin (EP), a novel avermectin analog against PC3 metastatic PCa cells. EP displayed robust inhibition of cell viability of PC3 cells in addition to suppressing the colony formation and wound healing capabilities. Our study showed that EP targets PC3 cells via inducing ROS and apoptosis activation. EP treatment enforces cell cycle arrest at G0/G1 phase via targeting cyclin-dependent kinase 4 (CDK4) and subsequent induction of apoptosis in PC3 cells. At the molecular level, EP effectively inhibited the expression of various cancer stem cell markers such as ALDH1, Sox-2, Nanog, Oct3/4 and CD44. Interestingly, EP also inhibited the activity of alkaline phosphatase, a maker of pluripotent stem cells. Of note, EP treatment resulted in the translocation of β-catenin from the nucleus to the cytoplasm indicating that EP antagonizes Wnt/β-catenin signaling pathway. Western blotting analysis revealed that EP downregulated the expression of key cell cycle markers such as cyclin D1, cyclin D3, CDK4, and c-Myc. In addition, EP inhibited the anti-apoptotic markers such as Mcl-1, XIAP, c-IAP1 and survivin in PC3 cells. On the other hand, EP treatment resulted in the activation of pH2A.X, Bad, caspase-9, caspase-3 and cleavage of PARP1. Taken together, our data suggests that EP is a potential agent to treat advanced PCa cells via modulating apoptosis signaling.

Formononetin induces apoptosis of PC-3 human prostate cancer cells via regulating long noncoding RNA H19 and the mitochondrial apoptosis pathway

Background: Prostate cancer is a life-threating disease with high incidence and mortality in male. Formononetin, the main active component of some natural products, has been hypothesized as a promising anticancer agent in previous studies. Objectives: We investigated the toxic effects and potential molecular mechanism of formononetin in PC-3 prostate cancer cells to further understand the pharmacological effects of formononetin and provide more references for intensive research. Materials and Methods: PC-3 cells were incubated with different doses of formononetin for 24 h or 48 h. After that, cell viability was measured by Cell Counting Kit-8, and apoptosis was analyzed by Hoechst 33258 stains. The expression levels of tumor-related factors such as long noncoding RNA (LncRNA) H19, Bax, and Bcl-2 were determined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blot methods. Subsequently, PC-3 cells were infected with a lentiviral vector to overexpress or knock down H19, and then, the expression of insulin-like growth factor-1 receptor (IGF-1R) mRNA was measured by RT-qPCR. Results: Formononetin significantly inhibited the viability of PC-3 cells and promoted apoptosis in a time-dose-dependent manner. We observed that the expressions of lncRNA H19 and Bcl-2 were significantly downregulated compared with the untreated group, while an opposite pattern was observed for Bax. According to the results of gene interaction experiments, IGF-1R may be a downstream target of H19 in PC-3 cells. Conclusion: Our results present evidence that formononetin induced apoptosis of PC-3 cells by regulating lncRNA H19 and the mitochondrial apoptosis pathway. Furthermore, we put forward the hypothesis that formononetin has an interference effect on the H19/IGF-1R pathway, which remains to be further confirmed.

MP16-04 IN SILICO SCREENING FOR IDENTIFICATION OF NOVEL DRUGS FOR OVERCOMING CABAZITAXEL-RESISTANT PROSTATE CANCER

You have accessJournal of UrologyProstate Cancer: Basic Research & Pathophysiology I (MP16)1 Apr 2020MP16-04 IN SILICO SCREENING FOR IDENTIFICATION OF NOVEL DRUGS FOR OVERCOMING CABAZITAXEL-RESISTANT PROSTATE CANCER Hiroshi Hongo*, Takeo Kosaka, Yota Yasumizu, Nobuyuki Tanaka, Toshikazu Takeda, Kazuhiro Matsumoto, Ryuichi Mizuno, and Mototsugu Oya Hiroshi Hongo*Hiroshi Hongo* More articles by this author , Takeo KosakaTakeo Kosaka More articles by this author , Yota YasumizuYota Yasumizu More articles by this author , Nobuyuki TanakaNobuyuki Tanaka More articles by this author , Toshikazu TakedaToshikazu Takeda More articles by this author , Kazuhiro MatsumotoKazuhiro Matsumoto More articles by this author , Ryuichi MizunoRyuichi Mizuno More articles by this author , and Mototsugu OyaMototsugu Oya More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000841.04AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Cabazitaxel (CBZ) has been identified as the most effective cytotoxic agent for improving survival in men with docetaxel-refractory castration-resistant prostate cancer (CRPC). However, the development of acquired resistance to CBZ was inevitable. The study aimed to use bioinformatic analysis to screen for candidate drugs that could overcome CBZ-resistance of human whole genome array data. METHODS: Two CRPC cell lines, DU145 and PC3, were used in this study. We incubated the cell lines with gradually increasing concentrations of CBZ to establish CBZ-resistant cell lines, DU145CR and PC3CR. In addition, we analyzed the gene expression profile of CBZ-resistant prostate cancer biopsy tissue collected at our institution and performed screening tests for candidate drugs that could overcome CBZ-resistance. A cell viability assay was used to determine the combined cytotoxic effects of CBZ and a candidate drug on CBZ-resistant cell lines. RESULTS: We compared the cytotoxic effect of CBZ on CBZ-resistant cells using a cell viability assay. The half maximal inhibitory concentration (IC50) of CBZ in DU145CR and PC3CR were three times higher than that in parental cells. The Broad Institute’s connectivity map analysis of human whole genome array data identified a candidate drug, DCN176. The relative cell viability of DU145 and PC3 cells treated with 10 μM DCN176 were 73.1% ± 1.0% and 68.0% ± 1.3% and that of DU145CR and PC3CR cells treated with 10 μM DCN176 were 61.9% ± 0.6% and 58.7% ± 1.2%.DCN176 appeared to have an anti-tumor effect on CBZ-resistant prostate cancer. CBZ and DCN176 showed a synergic effect in DU145CR and PC3CR cells (Combination Index: 0.76 and 0.81). DCN176 also had an anti-tumor effect in DU145CR and PC3CR xenograft mice models in vivo. To explore the mode of action of DCN176, we analyzed genomic and phosphorylation changes by exon sequencing and liquid chromatography-mass spectrometry (LC-MS). Exon sequencing revealed chromosomal instability in CBZ-resistant cell lines. LC-MS demonstrated an inhibitory effect of DCN176 on the DNA mismatch repair (MMR) pathway. Survival analysis using The Cancer Genome Atlas cohort data showed that loss of MMR genes was a factor for poor prognosis. We tested the sensitivity for DNA damage in CBZ-resistant models by radiation exposure and found that 4-Gy radiation significantly induced more γH2AX accumulation in CBZ-resistant cells than in CBZ-sensitive cells. CONCLUSIONS: Targeting DNA repair pathways with DCN176 can overcome CBZ-resistance in CRPC. Source of Funding: This study was supported by Grants-in-Aid for Scientific Research (#17K16813 to H. Hongo and #17K11158 to T. Kosaka) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e215-e215 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Hiroshi Hongo* More articles by this author Takeo Kosaka More articles by this author Yota Yasumizu More articles by this author Nobuyuki Tanaka More articles by this author Toshikazu Takeda More articles by this author Kazuhiro Matsumoto More articles by this author Ryuichi Mizuno More articles by this author Mototsugu Oya More articles by this author Expand All Advertisement PDF downloadLoading ...

Identification and characterization of a novel Anoctamin 1 inhibitor and its anticancer effects

Anoctamin 1 (ANO1 also known as TMEM16A) is a calcium‐activated chloride channel that is amplified in various carcinomas including prostate cancer, esophageal cancer, breast cancer, and pancreatic cancer, and its overexpression promotes cell proliferation and cell migration in various cancer cells. A cell base‐screening revealed that (E)‐1‐(7,7‐dimethyl‐7Hfuro[2,3‐f]chromen‐2‐yl)‐3‐(1H‐pyrrol‐2‐yl)prop‐2‐en‐1‐one (Ani‐E1) is a novel, potent and selective ANO1 inhibitor with an IC50 value of 1.23 μM. Ani‐E1 showed 144 times stronger activity on ANO1 inhibition than ANO2 inhibition and did not alter the chloride channel activity of CFTR and the intracellular calcium signaling. Notably, Ani‐E1 strongly decreased cell viability of PC‐3 and FaDu cells expressing high levels of ANO1 with a decrease in ANO1 protein levels. In addition, Ani‐E1 significantly enhanced apoptosis via activation of caspase 3 and cleavage of PARP in PC‐3 and FaDu cells. This study shows that a novel ANO1 inhibitor, Ani‐E1, can be a potential candidate for the treatment of cancers overexpressing ANO1, such as prostate cancer and esophageal cancer.Support or Funding InformationWe thank the National Research Foundation of Korea (NRF‐2017R1A2A2A05069364 and 53 NRF‐2018R1A6A1A03023718, NRF‐2019R1I1A1A01061117) for generous financial support. This research was supported (in part) by Yonsei University Research Fund (Post Doc. Researcher Supporting Program) of 2019 (project no.: 2019‐12‐0013).

ANNONA MURICATA NON‐SELECTIVELY SUPPRESS PC3 CELLS by INHIBITNG CELL PROLIFERATION and INDUCING CELL CYCLE ARREST

Annona muricata (AM) also known as graviola or soursop is a tropical evergreen tree that belongs to family Annonaceae. The leaves of the tree have various therapeutic benefits against numerous diseases including various multi drug resistant cancers. The major phytochemical constituents associated with its anticancer activity are annonaceous acetogenis (AAGs). It was hypothesized that AAGs exhibit their anticancer activity by possibly inhibiting mitochondrial NADH‐ubiquinone complex which limits the ATP supply to the cancer cells. However, the exact mechanism is unknown. The purpose of the study was to determine whether ethyl acetate fraction (EAF) is selectively kills prostate cancer cells and the mechanism by which the EAF induces cell death or reduces cell proliferation.3‐(4,5‐dimethylthiazol‐2‐y1)‐5‐(3‐carboxymethoxyphenyl)‐2‐(4‐sulfopheny1)‐2H‐tetrazolium (MTS) cell viability assay was performed on both cancer and healthy cells and it was observed that EAF non‐selectively reduces cell viability of PC3 cells and non‐cancer cells. To further examine the effect of EAF on PC3 cells, cell proliferation assay was performed, and it was observed that EAF inhibits cell proliferation in PC3 cells. Western blot analysis for cyclin D1 expression level was performed and EAF exposed cells showed reduced cyclin D1 levels suggesting that AM induces cell cycle arrest in PC3 cells. Immunoblotting analysis suggested that there was no significant effect on poly (ADP ribose) polymerase 1 (PARP‐1) expression levels however EAF significantly increases c‐PARP expression levels indicating that EAF does not induce apoptosis in prostate cancer cells. These results suggest that EAF reduces cell viability of PC3 cells by inhibiting cell proliferation via inducing cell cycle arrest.

3,6-diazabicyclo[3.3.1]heptanes Induces Apoptosis and Arrests Cell Cycle in Prostate Cancer Cells.

BackgroundProstate cancer, non-cutaneous malignant tumor, is the second common cause of cancer related mortalities in American men and is responsible for 13% of deaths related to cancer. The present study investigated the anti-cancer effects of 3,6-diazabicyclo[3.3.1]heptane on LNCaP and PC3 prostate cancer cells in vitro and on tumor growth in vivo in BALB/C nude mice.Material/MethodsReduction of cell viability by 3,6-diazabicyclo[3.3.1]heptane was evaluated by sulphorhodamine-B staining and apoptosis onset using annexin V and propidium iodide (PI) staining. The 2′,7′-dichlorofluorescein-diacetate stain was used for assessment of reactive oxygen species (ROS) formation while as western blotting for analysis of protein expression.ResultsThe viability of LNCaP and PC3 cells was reduced significantly (P<0.05) by 3,6-diazabicyclo[3.3.1]heptane in dose-based manner. At 30 μM of 3,6-diazabicyclo[3.3.1]heptane the viability of LNCaP and PC3 cells was reduced to 32 and 28%, respectively. The 3,6-diazabicyclo[3.3.1]heptane treatment increased apoptosis in LNCaP cells to 43.31% at 30 μM. The cell cycle in LNCaP cells was arrested in G1 phase on treatment with 3,6-diazabicyclo[3.3.1]heptane. The expression of cyclin D1 and p21 proteins was significantly increased by 3,6-diazabicyclo[3.3.1]heptane in LNCaP and PC3 cells. The growth of prostate tumor was also suppressed in vivo in mice by 3,6-diazabicyclo[3.3.1]heptane treatment.ConclusionsIn summary, the study demonstrated that LNCaP and PC3 prostate cancer cell viability is suppressed by 3,6-diazabicyclo[3.3.1]heptane treatment. The suppression of prostate cancer cell viability by 3,6-diazabicyclo[3.3.1]heptane involves apoptosis induction, cell cycle arrest and upregulation of p21 expression. Therefore, 3,6-diazabicyclo[3.3.1]heptane can be a potential chemotherapeutic agent for prostate cancer.

Carvacrol Induced Program Cell Death and Cell Cycle Arrest in Androgen-Independent Human Prostate Cancer Cells via Inhibition of Notch Signaling.

Several studies have revealed that abnormal activation of Notch signaling is closely related with the development and progression of prostate cancer. Although there are numerous therapeutic strategies, a more effective modality with least side effects is urgently required for the treatment of prostate cancer. Carvacrol is a monoterpenoid phenol and majorly present in the essential oils of Lamiaceae family plants. Many previous reports have shown various biological activities of carvacrol like antioxidant, antiinflammatory and anticancer properties. Recently, we have shown potent anticancer property of carvacrol against prostate cancer cell line DU145. In the current study, we report the chemopreventive and therapeutic potential of carvacrol against another prostate cancer cell line PC-3 with its detailed mechanism of action. To determine the effect of the carvacrol on prostate cancer cells, the cell viability was estimated by MTT assay and cell death was estimated by LDH release assay. The apoptotic assay was performed by DAPI staining and FITC-Annexin V assay. Reactive Oxygen Species (ROS) was estimated by DCFDA method. Cell cycle analysis was performed by flow cytometry. Gene expression analysis was performed by quantitative real time PCR. Our results suggested that the carvacrol treatment significantly reduced the cell viability of PC-3 cells in a dose- and time-dependent manner. The antiproliferative action of carvacrol was correlated with apoptosis which was confirmed by nuclear condensation, FITC-Annexin V assay, modulation in expression of Bax, Bcl-2 and caspase activation. The mechanistic insight into carvacrol-induced apoptosis leads to finding of elevated level of Reactive Oxygen Species (ROS) and mitochondrial membrane potential disruption. Cell cycle analysis revealed that carvacrol prevented cell cycle in G0/G1 that was associated with decline in expression of cyclin D1 and Cyclin-Dependent Kinase 4 (CDK4) and augmented expression of CDK inhibitor p21. Having been said the role of hyperactivation of Notch signaling in prostate cancer, we also deciphered that carvacrol could inhibit Notch signaling in PC-3 cells via downregulation of Notch-1, and Jagged-1. Thus, our previous and current findings have established the strong potential of carvacrol as a chemopreventive agent against androgen-independent human prostate cancer cells.

LncRNA MEG3 inhibits the progression of prostate cancer by facilitating H3K27 trimethylation of EN2 through binding to EZH2.

This study aims to study the effects of intra-nuclear lncRNA MEG3 on the progression of prostate cancer and the underlying mechanisms. Expressions of relative molecules were detected by Quantitative real time PCR (qRT-PCR) and western blot. Chromatin immunoprecipitation and RNA immunoprecipitation (RIP) assays were used to evaluate the interaction between intra-nuclear MEG3, histone methyltransferase EZH2 and Engrailed-2 (EN2). The impacts of MEG3 on the viability, proliferation and invasion of prostate cancer cells (PC3) were evaluated by methyl thiazolyl tetrazolium, colony formation and transwell assays, respectively. PC3 cells were transfected with MEG3 and transplanted into nude mice to analyse the effect of MEG3 on tumourigenesis of PC3 cells in vivo. EN2 expression was inversely proportional to MEG3 in the prostate cancer tissues and PC3 cells. RIP results showed that intra-nuclear MEG3 could bind to EZH2. Knockdown of MEG3 and/or EZH2 up-regulated EN2 expression and reduced the recruitment of EZH2 and H3K27me3 to EN2, while over-expressed MEG3 caused opposite effects. MEG3 over-expression suppressed cell viability, colony formation, cell invasion and migration of PC3 cells in vitro and inhibited tumourigenesis of PC3 cells in vivo, while EN2 over-expression diminished the effects. These findings indicated that MEG3 facilitated H3K27 trimethylation of EN2 via binding to EZH2, thus suppressed the development of prostate cancer.

Anticancer Activity of Earthworm Powder (Lumbricus terrestris) Against MCF-7 and PC-3 Cancer Cell Lines.

The importance of earthworm in treatment of various diseases has been proven in ancient literatures. Nowadays, with advances in biotechnology, earthworm is considered a rich natural source of many biomolecules that possesses antioxidant and antitumor activities. The present study aimed to evaluate the antitumor activity of earthworm powder (Lumbricus terrestris) against two cell lines, breast cancer cells (MCF-7) and prostate cancer cells (PC-3). Fully matured earthworms (L. terrestris) were collected from soil in Baghdad, Iraq. To assess the cytotoxicity of earthworm powder, the MTT assay was conducted on cancerous (MCF-7 and PC-3 cells) and normal cell line (WRL68 cells) lines. It was revealed that earthworm powder exerts cytotoxic effects against two cancer cell lines. The viability of MCF-7 and PC-3 cells decreased with increasing the concentration. The IC50 values for PC-3 and MCF-7 cell lines were 265.5 and 965.9μg/ml, respectively, while the earthworm powder exhibited no cytotoxicity against the WRL68 cells. According to the analysis of the results of the multiple cytotoxicity assay (HCS), the treatment of PC-3 cells with 100, 200, and 400μg/ml of earthworm powder for 24h at 37°C led to cell death by changing the permeability of mitochondrial membrane resulting in cytochrome C release and inducing apoptosis. The results of the present study contribute additional evidence for the antitumor activity of earthworm extracts. Therefore, further research should concentrate on isolating and identifying the earthworm's active biomolecules that have antitumor activity by investigating the molecular mechanism, genetics, and pathways responsible for the antitumor activity of these biomolecules.