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.
Purpose: Nuclear factor (erythroid-derived 2)-like 2, also known as NFE2L2 or NRF2, a transcription factor capable of upregulating antioxidant response element (ARE)-mediated expression and cytoprotective proteins, plays critical roles in chemoprevention, inflammation and aging. NRF2 has recently been proposed as a novel target for cancer chemoprevention. The fungicide miconazole has shown promising antiproliferative effects in cancer cells. Materials and Methods: After miconazole treatment, the p62-KEAP1-NRF2 activation was analyzed by qPCR and Western blot. The nuclear translocation indicating NRF2 activation was further confirmed by immunofluorescence. Finally, the ROS production was detected by CM-H2DCFDA staining. Results: We demonstrate in this study that miconazole dramatically increases NRF2 activation in bladder cancer cells, in a dose-and time-dependent manner. Interestingly, levels of expression of p62, a noncanonical pathway that mediates NRF2 activation, appeared to increase in accordance with NRF2. We also investigated levels of the negative regulator kelch-like ECH-associated protein 1 (KEAP1), which is involved in NRF2 activation. As expected, a decrease in KEAP1 expression was found after miconazole exposure. Confirmation of NRF2 nuclear translocation was monitored by immunofluorescence. Miconazole-induced generation of reactive oxygen species (ROS) promoted NRF2 activation. Pretreatment of bladder cancer cells with ROS scavengers abolished NRF2 expression and nuclear translocation, indicating that miconazole activates the noncanonical p62-KEAP1-NRF2 pathway, which is regulated by ROS production. Conclusion: Our study elucidates the mechanisms through which miconazole stimulates NRF2 which may contribute to cancer chemopreventive effects.
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 Nterminal 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. K E Y W O R D SAP-1, BITC, bladder cancer, ERK, miR-99a
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.
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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