Abstract Chloroquine (CQ) and hydroxychloroquine (HCQ), two antimalarial drugs, are suggested to have potential anticancer properties. in the present study, we investigated the effects of CQ and HCQ ...on cell growth of bladder cancer with emphasis on autophagy inhibition and apoptosis induction in vitro . The results showed that CQ and HCQ inhibited the proliferation of multiple human bladder cell lines (including RT4, 5637, and T24) in a time- and dose-dependent fashion, especially in advanced bladder cancer cell lines (5637 and T24) compared to immortalized uroepithelial cells (SV-Huc-1) or other reference cancer cell lines (PC3 and MCF-7). We found that 24-hour treatment of CQ or HCQ significantly decreased the clonogenic formation in 5637 and T24 cells compared to SV-Huc-1. As human bladder cancer tumor exhibits high basal level of autophagic activities, we detected the autophagic flux in cells treated with CQ and HCQ, showing an alternation in LC3 flux in CQ- or HCQ-treated cells. Moreover, bladder cancer cells treated with CQ and HCQ underwent apoptosis, resulting in increased caspase 3/7 activities, increased level of cleaved poly(ADP-ribose) polymerase (PARP), caspase 3, and DNA fragmentation. Given these results, targeting autophagy with CQ and HCQ represents an effective cancer therapeutic strategy against human bladder cancer.
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FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
A high basal level of autophagic flux in bladder cancer (BC) cells prevents cell death and weakens chemotherapy efficacy. However, how autophagy influences cancer‐associated immunosuppression in BC ...remains undetermined. In this study, we observed a negative correlation between the autophagy‐related markers LC3‐II and programmed death ligand‐1 (PD‐L1) in BC cells. The autophagy inhibitors chloroquine (CQ) and bafilomycin A1 (Baf‐A1) increased PD‐L1 expression in BC cells through the ERK–JNK–c‐Jun signal‐transduction pathway. Moreover, the treatment of BC cells with CQ and Baf‐A1 inhibited hsa‐microRNA‐34a (miR‐34a) expression and miR‐34a overexpression in BC cells prevented the autophagy blockade–induced PD‐L1 expression; a negative correlation between miR‐34a and PD‐L1 expression was observed during treatment with autophagy inhibitors. Furthermore, miR‐34a overexpression induced the cytotoxic activity of natural killer cells against BC cells. Our results provide evidence that autophagy blockade and its regulatory pathway affect cancer‐associated immunosuppression through PD‐L1 elevation. Thus, the coadministration of autophagy inhibitors and a PD‐L1 immune checkpoint blockade provides a potential therapeutic approach for treating BC.
Graphical
Our model illustrates the pharmacological inhibition of autophagy could induce PD‐L1 expression in BC cells through the ERK–JNK–c‐Jun signaling transduction pathway and miR‐34a downregulation, revealing the effect of genetic and epigenetic regulation of autophagy on PD‐L1. In consequence, BC cells expressing PD‐L1 suppress NK cell cytotoxic activity.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Bladder cancer (BC) is the second most common urologic malignancy and the ninth most common malignancy worldwide. Surgical resection is the mainstay of treatment for patients with early-stage ...disease, whereas therapeutic options are limited for patients with advanced-stage or residual BC. Programmed cell death ligand-1 (PD-L1) is an important target for immunotherapy. It is known that PD-L1 is overexpressed in BC; a clinical trial involving PD-L1 immune checkpoint inhibitors in advanced BC is ongoing. In the present study, we used Western blot and quantitative real-time PCR (qPCR) to define the expression level of PD-L1 after cisplatin treatment in BC-derived cell lines. The signal activation was also evaluated by Western blot in BC-derived cell lines. We found that chemotherapeutic drug cisplatin can induce PD-L1 but not PD-L2 expression in BC-derived cell lines. Furthermore, the expression level of PD-L1 was increased in a dose- and time-dependent manner after cisplatin treatment. The cisplatin-induced PD-L1 expression is mainly mediated by ERK1/2 but not Akt/mTOR signal pathway. Moreover, we found that cisplatin activates transcription factor activator protein-1 (AP-1) to regulate PD-L1 expression. The chemotherapy drug such as cisplatin may trigger resistance of BC through PD-L1 up-regulation. The present study suggests that PD-L1 antibody should be used concomitantly with chemotherapy in the setting of advanced and metastatic BC.
Bladder cancer (BC) is the second most common urological tumour in Western countries. Approximately, 80% of patients with BC will present with non-muscle invasive bladder cancer (NMIBC), whereas a ...quarter will have muscle invasive disease (MIBC) at the time of BC diagnosis. However, patients with NMIBC are at risk of BC recurrence or progression into MIBC, and an MIBC prognosis is determined by the presence of progression and metastasis. Matrix metalloproteinase 2 (MMP2), a type of matrix metalloproteinase (MMP), plays a major role in tumour invasion and is well-characterized in BC prognosis. In BC, the mechanisms regulating MMP2 expression, and, in turn, promote cancer invasion, have hardly been explored. Thrombospondin-4 (THBS4/TSP4) is a matricellular glycoprotein that regulates multiple biological functions, including proliferation, angiogenesis, cell adhesion and extracellular matrix modelling. Based on the results of a meta-analysis in the Gene Expression Profiling Interactive Analysis 2 database, we observed that TSP4 expression levels were consistent with overall survival (OS) rate and BC progression, with the highest expression levels observed in the advanced stages of BC and associated with poor OS rate. In our pilot experiments, incubation with recombinant TSP4 promoted the migration and invasion in BC cells. Furthermore, MMP2 expression levels increased after recombinant TSP4 incubation. TSP4-induced-MMP2 expression and cell motility were regulated via the AKT signalling pathway. Our findings facilitate further investigation into TSP4 silencing-based therapeutic strategies for BC.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Cisplatin-based chemotherapy is the first line treatment for several cancers including bladder cancer (BC). Autophagy induction has been implied to contribute to cisplatin resistance in ovarian ...cancer; and a high basal level of autophagy has been demonstrated in human bladder tumors. Therefore, it is reasonable to speculate that autophagy may account for the failure of cisplatin single treatment in BC. This study investigated whether cisplatin induces autophagy and the mechanism involved using human BC cell lines.
Human BC cells (5637 and T24) were used in this study. Cell viability was detected using water soluble tetrazolium-8 reagents. Autophagy induction was detected by monitoring the levels of light chain 3 (LC3)-II and p62 by Western blot, LC3-positive puncta formation by immunofluorescence, and direct observation of the autophagolysosome (AL) formation by transmission electron microscopy. Inhibitors including bafilomycin A1 (Baf A1), chloroquine (CQ), and shRNA-based lentivirus against autophagy-related genes (ATG7 and ATG12) were utilized. Apoptosis level was detected by caspase 3/7 activity and DNA fragmentation.
Cisplatin decreased cell viability and induced apoptosis of 5637 and T24 cells in a dose-and time-dependent manner. The increased LC3-II accumulation, p62 clearance, the number of LC3-positive puncta, and ALs in cisplatin-treated cells suggested that cisplatin indeed induces autophagy. Inhibition of cisplatin-induced autophagy using Baf A1, CQ, or ATG7/ATG12 shRNAs significantly enhanced cytotoxicity of cisplatin toward BC cells. These results indicated that cisplatin induced protective autophagy which may contribute to the development of cisplatin resistance and resulted in treatment failure. Mechanistically, upregulation of beclin-1 (BECN1) was detected in cisplatin-treated cells, and knockdown of BECN1 using shRNA attenuated cisplatin-induced autophagy and subsequently enhanced cisplatin-induced apoptosis.
Collectively, the study results indicated that cisplatin-induced autophagy is mediated by BECN1 in BC cells. Therefore, combinative treatment using cisplatin and autophagy inhibitors could potentially overcome cisplatin resistance related to autophagy induction.
Bladder cancer (BC), one of the most common urological neoplastic disorders in men, has an extremely low survival rate because of its tendency to metastasize. The anticancer drugs chloroquine (CQ) ...and hydroxy CQ (HCQ) might inhibit tumor progression and invasiveness. However, the mechanism by which CQ and HCQ influence BC is undetermined. In this study, CQ and HCQ treatments inhibited the migration and invasion of two BC cell types (5637 and T24) through expression modulation of matrix metalloproteinase‐2 (MMP‐2), which belongs to the matrix MMP family and is a key mediator of cancer progression. Moreover, additional data revealed that the migrative and invasive effects of BC cells treated with CQ or HCQ were abolished after treatment with rapamycin, which induces autophagy, demonstrating that CQ and HCQ functions in BC are based on autophagy inhibition. In conclusion, our research demonstrated that CQ and HCQ regulated cell motility in BC through MMP‐2 downregulation by targeting autophagy functions, providing a novel therapeutic strategy for BC treatment.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Benzyl isothiocyanate (BITC), a bioactive natural product present in cruciferous vegetables, has been proved to prevent cancer progression through various mechanisms. In our previous report, we ...proved that BITC exhibits antitumor effects in bladder cancer by suppressing IGF1R, FGFR3, and mTOR, which is mediated by miR‐99a expression. In this study, we identified the signal pathway involved in regulating miR‐99a expression after BITC exposure in bladder cancer. Treatment with different BITC concentrations resulted in induction of miR‐99a expression in bladder cancer cell lines. Activation of extracellular signal‐regulated protein kinase (ERK) and c‐jun N‐terminal kinase was observed in bladder cancer after BITC treatment for 24 hours. Interestingly, by using a chemical inhibitor of candidate pathways, we found that only the ERK signal pathway is required for miR‐99a expression. Furthermore, we evaluated the transcription factor that may contribute to miR‐99a expression in response to BITC treatment. The results indicated that c‐Jun/AP‐1 was activated after BITC treatment. Moreover, we confirmed c‐Jun/AP‐1 activation through immunofluorescence and the luciferase reporter assay. The results showed that BITC treatment markedly improved nuclear translocation of c‐Jun/AP‐1 and luciferase activity dose dependently. Finally, pretreatment with the ERK inhibitor U0126 diminished c‐Jun phosphorylation and transcriptional activation, suggesting that BITC elicits ERK/c‐Jun signal transduction, which is responsible for miR‐99a expression in bladder cancer. The present work identifies the mechanism involved in upregulation miR‐99a after BITC treatment, which provides an explanation for BITC biological function in our previous work.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Autophagy plays a dual function in cancer progression; autophagy activation can support cancer cell survival or contribute to cell death. Miconazole, a Food and Drug Administration‐approved ...antifungal drug, has been implicated in oncology research recently. Miconazole was found to exert antitumor effects in various tumors, including bladder cancer (BC). However, whether it provokes protective autophagy has been never discussed. We provide evidence that miconazole induces protective autophagy in BC for the first time. The results indicated that 1A/1B‐light chain 3 (LC3)‐II processing and p62 expression were elevated after miconazole exposure. Also, adenosine monophosphate‐activated protein kinase phosphorylation was increased after miconazole treatment. We also confirmed the autophagy‐promoting effect of miconazole in the presence of bafilomycin A1 (Baf A1). The result indicates that a combination treatment of miconazole and Baf A1 improved LC3‐II processing, confirming that miconazole promoted autophagic flux. The acridine orange, Lysotracker, and cathepsin D staining results indicate that miconazole increased lysosome formation, revealing its autophagy‐promoting function. Finally, miconazole and autophagy inhibitor 3‐methyladenine cotreatment further reduced the cell viability and induced apoptosis in BC cells, proving that miconazole provokes protective autophagy in BC cells. Our findings approve that miconazole has an antitumor effect in promoting cell apoptosis; however, its function of protective autophagy is needed to be concerned in cancer treatment.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Bladder cancer (BC), a common urologic cancer, is the fifth most frequently diagnosed tumor worldwide. hsa‑miR‑34a displays antitumor activity in several types of cancer. However, the functional ...mechanisms underlying hsa‑miR‑34a in BC remains largely unknown. We observed that hsa‑mir‑34a levels were significantly and negatively associated with clinical disease stage as well as regional lymph node metastasis in human BC. In a series of in vitro investigations, overexpression of hsa‑miR‑34a inhibited cell migration and invasion in BC cell lines 5637 and UMUC3 as detected by Transwell assays. We further found that hsa‑miR‑34a inhibited cell migration and invasion by silencing matrix metalloproteinase‑2 (MMP‑2) expression and thus interrupting MMP‑2‑mediated cell motility. Our analysis of BC datasets from The Cancer Genome Atlas database revealed a negative correlation between hsa‑miR‑34a and MMP‑2. Moreover, higher MMP‑2 protein expression was observed in the BC tissues when compared with that noted in the normal tissue. MMP‑2 levels were also significantly associated with clinical disease stage and poor survival rate in human BC. These findings indicate that MMP‑2 plays a critical role in regulating BC progression. Therefore, hsa‑miR‑34a is a promising treatment to target MMP‑2 for the prevention and inhibition of cell migration and invasion in BC.
Summary Background Prostate cancer (PCa) is a leading cause of cancer-related death in men, which emphasizes the need for novel therapeutic approaches. Targeting microRNA (miRNA) has been considered ...as a therapeutic strategy against cancers. Human miR-204-5p potentially targeting BCL2 has been reported to be downregulated in various cancers. We hypothesized that miR-204-5p overexpression induces cancer cell apoptosis by repressing BCL2 expression. Methods A vector harboring mature miR-204-5p was constructed and delivered into human PCa cells. The expression level of miR-204-5p was determined by miRNA quantitative polymerase chain reaction (QPCR). Luciferase reporter assays were performed to verify the function of mature miR-204-5p and its direct binding to BCL2 transcripts. The expression levels of BCL-2 messenger RNA (mRNA) and protein samples were measured by QPCR and Western blot, respectively. Cell viability was detected by WST-1 assays. Induction of apoptosis was determined by increased levels of cleavage caspase 3 and caspase 3/7 activity. Results The expression levels of miR-204-5p were downregulated in PCa cells compared with normal prostate epithelial cells. Transfection of pSM-204 resulted in up to 6.2-fold higher expression of miR-204-5p when compared with pSM control. The mRNA levels of several potential target genes of miR-204-5p were decreased in pSM-204-transfected PC3 and Rv1 cells. BCL2 mRNA and protein expression decreased in miR-204-5p-transfected cells, which led to cytochrome C release from mitochondria. It subsequently increased cleaved caspase 3 and caspase 3/7 activities and reduced cell viability. Cotransfection of a reporter vector harboring the BCL2 3′-untranslated region to compete with endogenous transcripts partially rescued miR-204-5p-induced apoptosis. Conclusion Human miR-204-5p targets BCL2 in PCa cells. Restoration of miR-204-5p in PCa could therefore be considered as a novel strategy by targeting antiapoptotic BCL2.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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