ABSTRACT
AIM: To elucidates the mechanism that disulfiram/copper complex (DSF/Cu) treatment activates chloride channels and induces apoptosis in prostate cancer cells. METHODS: Cellular membrane currents were measured by membrane clamp technique; western blot to detect protein expression; flow cytometry to detect apoptosis; immunofluorescence to detect target protein co-localization, and further validated by a combination of protein-protein interaction and mock protein molecular docking techniques. RESULTS: DSF/Cu activated chloride channels and induced apoptosis in LNCaP (a type of androgen-dependent prostate cancer cells) cells. The chloride currents activated by DSF/Cu were significantly reduced after knockdown of CLC3 with siRNA. In addition, DSF/Cu-activated chloride currents were reduced to background current levels after perfusion with genistein, a highly specific tyrosine kinase inhibitor. Conversely, DSF/Cu failed to activate chloride currents in LNCaP cells after 30 minutes of pre-incubation with genistein. When genistein was removed, and DSF/Cu was added, the activated currents were small and unstable, and gradually decreased. Immunofluorescence in LNCaP cells also showed co-localization of the CLC3 protein with tyrosine kinase 2ß (PTK2B). CONCLUSION: DSF/Cu can activate chloride channels and induce apoptosis in LNCaP cells with the involvement of tyrosine kinase. These results provide new insights into the target therapy of prostate cancer.
Subject(s)
Disulfiram , Prostatic Neoplasms , Apoptosis , Cell Line, Tumor , Chloride Channels , Chlorides , Copper/pharmacology , Disulfiram/pharmacology , Genistein/pharmacology , Humans , Male , Molecular Docking Simulation , Prostatic Neoplasms/drug therapy , Protein-Tyrosine KinasesABSTRACT
BACKGROUND: Patients with lymph node (LN) metastatic bladder cancer (BCa) present with extremely poor prognosis. BCa-derived exosomes function as crucial bioactive cargo carriers to mediate the signal transduction in tumor microenvironment triggering tumor metastasis. However, the mechanisms underlying exosome-mediated LN metastasis in BCa are unclear. METHODS: We conducted the high-throughput sequencing to explore the expression profile of long noncoding RNA (lncRNA) in urinary exosomes (urinary-EXO) from patients with BCa and further evaluated the clinical relevance of exosomal lncRNA BCYRN1 in a larger 210-case cohort. The functional role of exosomal BCYRN1 was evaluated through the migration and tube formation assays in vitro and the footpad-popliteal LN metastasis model in vivo. RNA pull-down assays, luciferase assays, and actinomycin assays were conducted to detect the regulatory mechanism of exosomal BCYRN1. RESULTS: LncRNA BCYRN1 was substantially upregulated in urinary-EXO from patients with BCa, and associated with the LN metastasis of BCa. We demonstrated that exosomal BCYRN1 markedly promoted tube formation and migration of human lymphatic endothelial cells (HLECs) in vitro and lymphangiogenesis and LN metastasis of BCa in vivo. Mechanistically, BCYRN1 epigenetically upregulated WNT5A expression by inducing hnRNPA1-associated H3K4 trimethylation in WNT5A promoter, which activated Wnt/ß-catenin signaling to facilitate the secretion of VEGF-C in BCa. Moreover, exosomal BCYRN1 was transmitted to HLECs to stabilize the VEGFR3 mRNA and thus formed an hnRNPA1/WNT5A/VEGFR3 feedforward regulatory loop, ultimately promoting the lymphatic metastasis of BCa. Importantly, blocking VEGFR3 with specific inhibitor, SAR131675 significantly impaired exosomal BCYRN1-induced the LN metastasis in vivo. Clinically, exosomal BCYRN1 was positively associated with the shorter survival of BCa patients and identified as a poor prognostic factor of patients. CONCLUSION: Our results uncover a novel mechanism by which exosomal BCYRN1 synergistically enhances VEGF-C/VEGFR3 signaling-induced lymphatic metastasis of BCa, indicating that BCYRN1 may serve as an encouraging therapeutic target for patients with BCa.
Subject(s)
Exosomes/metabolism , RNA, Long Noncoding/metabolism , Urinary Bladder Neoplasms/pathology , Vascular Endothelial Growth Factor C/metabolism , Vascular Endothelial Growth Factor Receptor-3/metabolism , Wnt-5a Protein/metabolism , Animals , Cell Line, Tumor , Endothelial Cells/cytology , Endothelial Cells/metabolism , Heterogeneous Nuclear Ribonucleoprotein A1/metabolism , Histones/metabolism , Humans , Lymphangiogenesis , Lymphatic Metastasis , Mice , Mice, Nude , Promoter Regions, Genetic , Protein Kinase Inhibitors/pharmacology , RNA, Long Noncoding/genetics , Signal Transduction/drug effects , Survival Rate , Urinary Bladder Neoplasms/metabolism , Urinary Bladder Neoplasms/mortality , Vascular Endothelial Growth Factor C/genetics , Vascular Endothelial Growth Factor Receptor-3/antagonists & inhibitors , Vascular Endothelial Growth Factor Receptor-3/genetics , Wnt-5a Protein/geneticsABSTRACT
BACKGROUND: Circular RNAs have been widely explored as potential biomarkers and therapeutic targets in bladder cancer; however, few have been functionally characterized. RESULTS: ciRs-6 is expressed at low levels in cancer tissues and advanced tumor grades and stages, and its expression correlates with better outcomes for bladder cancer patients. In vitro and in vivo, ciRs-6 was shown to suppress bladder cancer growth by sponging miR-653 to elevate March1 levels. March1 is an E3 ubiquitin ligase that has been proven to suppress bladder cancer growth; knocking down March1 in ciRs-6 overexpressed bladder cancer cells reversed the tumor suppressive effect of ciRs-6. CONCLUSIONS: Our study identifies an oncogenic role of ciRs-6 and suggests its usefulness as a novel biomarker for bladder cancer diagnosis and prognosis and as a therapeutic target for bladder cancer. METHODS: ciRs-6 was identified by RNA-seq and qPCR; CCK8 assays, clone forming assays and cell cycle analyses were performed to evaluate the in vitro effect of ciRs-6 in bladder cancer; further, a mouse subcutaneous tumor model was designed for in vivo analysis. RNA pulldown assays, miRNA capture experiments and dual luciferase assessments were applied for mechanistic studies.
Subject(s)
Gene Expression Regulation, Neoplastic/physiology , MicroRNAs/metabolism , RNA, Circular/metabolism , Ubiquitin-Protein Ligases/metabolism , Urinary Bladder Neoplasms/metabolism , Animals , Cell Line, Tumor , Down-Regulation , Female , Humans , Mice , Mice, Nude , MicroRNAs/genetics , Neoplasms, Experimental , RNA, Circular/genetics , Ubiquitin-Protein Ligases/genetics , Up-RegulationABSTRACT
BACKGROUND: Increasing evidence suggests that circular RNAs play a key role in regulating bladder cancer progression. However, this remains to be fully elucidated. RESULTS: In this study, we reanalyzed our previous RNA sequence, and circ5912 was found to downregulate significantly in bladder cancer tissues compared with normal control. Expression of circ5912 inversely correlates with bladder cancer grade, stage, metastasis, and better patient outcomes. In vitro and in vivo, circ5912 has been shown to repress transforming growth factor ß signaling, which suppresses proliferation, invasion and migration of bladder cancer induced by mesenchymal-to epithelial transition. CONCLUSIONS: Our study firstly demonstrate that circ5912 regulates mesenchymal-to epithelial transition pathway to suppress bladder cancer progression and propose new therapeutic targets and biomarkers for bladder cancer. MATERIALS AND METHODS: Clinical values of circ5912 in human bladder cancer were examined in a cohort of 58 patients by qPCR. 2 bladder cancer cell lines, T24 and SW780, were used for biological evaluation of circ5912. CCK8, clone formation, wound healing and trans-well assays were performed to determine the in vivo effect of circ5912; a mouse subcutaneous model was designed for in vivo analysis. Western blotting, RNA pulldown assays and florescent in situ hybridization were applied for mechanistic analysis.
