Autophagy blockade potentiates cancer-associated immunosuppression through programmed death ligand-1 upregulation in bladder cancer.

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.

Thrombospondin-4 promotes bladder cancer cell migration and invasion via MMP2 production.

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.

Attenuation of chloroquine and hydroxychloroquine on the invasive potential of bladder cancer through targeting matrix metalloproteinase 2 expression.

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.

Miconazole induces protective autophagy in bladder cancer cells.

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.

Benzyl isothiocyanate promotes miR-99a expression through ERK/AP-1-dependent pathway in bladder cancer cells.

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.

Inhibition of High Basal Level of Autophagy Induces Apoptosis in Human Bladder Cancer Cells.

PURPOSE: Cancer cells adapt to stress by activation of the autophagy pathway primed for survival. A high basal level of autophagic activity was found in human bladder cancer cell lines. We studied the significance of the phenomenon on cancer cell survival. MATERIALS AND METHODS: The immortalized human bladder epithelial cell line SV-HUC-1 and the human bladder cancer cell lines RT-4 and 5637 together with human bladder cancer specimens collected from patients were used. A commercially available bladder cancer microarray was applied to confirm the findings. LC3 (light chain-3) II protein detection was done to determine the presence of autophagy. Caspase 3 and DNA fragmentation was performed to detect apoptosis. RESULTS: Bladder cancer cell lines showed activated autophagic flux compared to SV-HUC-1 cells, prostate cancer cells and breast cancer cells. Results were confirmed in human bladder cancer specimens. Autophagy inhibition by Baf (bafilomycin) A1, or by knockdown of ATG (autophagy related protein) 7 or 12 induced cytotoxicity in multiple human bladder cell lines. Induction of apoptosis was found in cells with autophagy inhibition. Although the disruption of mitochondria membrane potential or the generation of reactive oxygen species was detected in Baf A1 treated cells, intensity was mild and not thought to be related to apoptosis of bladder cancer cells. CONCLUSIONS: Our results indicate that autophagy is required for the growth and survival of human bladder cancer cells.

Induction of testicular damage by daily methamphetamine administration in rats.

Methamphetamine (METH)-induced brain damage and apoptosis within the central nervous system are well documented. This study was conducted to investigate the toxic effects of daily METH administration on the testes in a rat model. Male Sprague-Dawley rats (5 weeks old, ~100 g, n = 64) were divided into two groups and treated with vehicle (saline, control) or METH (10 mg/kg) for 15, 30, 60 and 90 days. The results showed that daily administration of METH decreased the body, testicular and epididymis weights as well as the serum levels of total testosterone. The increased apoptotic index (Bad/Bcl2 expression ratio) and levels of cleaved caspase-3 indicated that apoptosis had occurred in the testes of the METH-treated rats. The oxidative stress levels increased as the reduced and oxidized glutathione (GSH/GSSG) ratio decreased. The overall sperm counts decreased at 15 and 90 days, where- as morphologically abnormal sperm counts increased at 30, 60 and 90 days in the METH-treated rats. This study demonstrates that daily exposure to METH significantly reduced the number and quality of sperm in rats. The underlying pathophysiological mechanisms likely include the reduction of serum testosterone levels and the increase of oxidative stress and apoptosis in the rat testes.

Benzyl isothiocyanate induces protective autophagy in human prostate cancer cells via inhibition of mTOR signaling.

Benzyl isothiocyanate (BITC) is a dietary chemopreventive agent that inhibits the growth of various human cancer cells by causing apoptotic cell death. In this study, we demonstrate that BITC not only induces apoptosis but also induces autophagy in human hormone-sensitive (Rv1) and -refractory (PC3) prostate cancer cells. In BITC-treated cells, the induction of autophagy was detected by monitoring the processing of an autophagy marker protein, microtubule-associated protein 1 light chain 3 (LC3), the aggregation of LC3 into granular structures and the formation of acidic organelles. Inhibition of autophagy using 3-methyladenine increased BITC-induced apoptosis, whereas the administration of caspase inhibitor suppressed BITC-induced cell death. Our data also showed that BITC inhibits mammalian target of rapamycin (mTOR) kinase activity in a dose-dependent manner. The expression of phospho-mTOR (Ser2481), an indicator of mTOR intrinsic catalytic activity, and phospho-UNC-51-like kinase 1 (Ser757), a direct substrate of mTOR, were decreased in BITC-treated cells. However, the increased expression of phospho-mTOR (Ser2448), phospho-AKT (Ser473) and antiapoptotic Bcl-2 were detected only in PC3 cells at later stages of BITC treatment. Collectively, our results show that BITC induces a protective autophagy response in Rv1 and PC3 cells through inhibition of the mTOR signaling pathway. Activation of the AKT survival pathway was only observed in PC3 cells, representing a resistance mechanism of advanced prostate cancer upon BITC treatment. These findings could potentially contribute to the beneficial effect of BITC in prostate cancer treatments.

Tumor suppressive functions of hsa­miR­34a on cell cycle, migration and protective autophagy in bladder cancer.

Bladder cancer (BC) cells exhibit a high basal level of autophagy activity, which contributes to the development of a protective mechanism for cellular survival against current treatments. Hsa­microRNA­34a (miR­34a) presents anti­tumor function in several types of cancer. However, the functional mechanism of miR­34a in regulating tumor aggressiveness and protective autophagy of BC remains largely unknown. First, transfected BC cells with miR­34a mimic exhibited LC3­II and p62 accumulation through immunofluorescence staining. It was demonstrated that syntaxin 17 (STX17), which is required for autophagosome­lysosome fusion, was downregulated upon miR­34a mimic treatment. Mechanistically, miR­34a reduced the expression of STX17 proteins that directly bind on STX17 3'­untranslated regions and thus suppressed STX17 mRNA translation to eventually inhibit protective autophagy in BC. Cell viability and colony formation assays revealed that overexpression of miR­34a in BC cells enhances the chemosensitivity of cisplatin, doxorubicin, epirubicin and mitomycin C. Furthermore, miR­34a inhibited cell proliferation and triggered G0/G1 cell cycle arrest by inhibiting cyclin D1 and cyclin E2 protein expression. Moreover, miR­34a suppressed cell motility through the downregulation of epithelial­mesenchymal transition. In summary, miR­34a inhibits cell proliferation, motility and autophagy activity in BC, which can benefit BC treatment.

Hsa-miR-30a-3p overcomes the acquired protective autophagy of bladder cancer in chemotherapy and suppresses tumor growth and muscle invasion.

Bladder cancer (BC) is the second most common urologic cancer in western countries. New strategies for managing high-grade muscle-invasive bladder cancer (MIBC) are urgently required because MIBC has a high risk of recurrence and poor survival. A growing body of evidence indicates that microRNA has potent antitumorigenic properties in various cancers, and thus, therapeutic strategies based on microRNA may show promising results in cancer therapy. Analysis of The Cancer Genome Atlas (TCGA) database indicated that hsa-miR-30a-3p is downregulated in human BC. Our in vitro investigation demonstrated that hsa-miR-30a-3p suppresses the expression of matrix metalloproteinase-2 (MMP-2) and MMP-9 and reduces the cell invasive potential of BC cells. Furthermore, hsa-miR-30a-3p directly targets ATG5, ATG12, and Beclin 1; this in turn improves the chemosensitivity of BC cells to cisplatin through the repression of protective autophagy. In a tumor-xenograft mice model, hsa-miR-30a-3p suppressed muscle invasion. Cotreatment with hsa-miR-30a-3p enhanced the antitumor effect of cisplatin in reducing tumor growth in BC. The current study provides a novel strategy of using hsa-miR-30a-3p as an adjuvant or replacement therapy in future BC treatment.

Zoledronic acid induces autophagic cell death in human prostate cancer cells.

PURPOSE: Bisphosphonates are potent inhibitors of bone resorption. In vitro studies show that zolendronic acid inhibits prostate cancer cell growth by activating apoptosis. We investigated whether zolendronic acid also inhibits prostate cancer cell growth by autophagy (type II programmed cell death). MATERIALS AND METHODS: We investigated the induction of autophagy in the PC-3, DU-145, LNCaP and CRW22Rv1 cell lines upon zolendronic acid treatment. LC3-II protein formation was detected by Western blot. LC3-II incorporation into autophagosomes was detected by immunofluorescence staining. Acidic organelle formation was detected by acridine orange staining. Rescue experiments using an apoptosis inhibitor and/or an autophagy inhibitor were performed by MTT assay. RESULTS: Autophagy induction was detected by formation of the LC3-II protein after exposure to 100 µM zolendronic acid. LC3-II and caspase-3 processing was detected 6 days after treatment. Acidic organelles were detectable by acridine orange staining and immunofluorescence showed round-up and condensed staining of LC3-II, suggesting autophagosome formation in the cytoplasm during autophagic cell death. Cell growth was rescued only by administering an apoptosis and autophagy inhibitor during zolendronic acid treatment, indicating that zolendronic acid induces prostate cancer death by apoptotic and autophagic cell death. CONCLUSIONS: To our knowledge we report the first study showing that zolendronic acid markedly inhibits human prostate cancer cell growth through autophagic cell death. Zolendronic acid shows its anticancer activity via apoptosis and autophagy. These findings can potentially contribute to the beneficial use of zolendronic acid for prostate cancer treatment.

MicroRNA­34a­5p serves as a tumor suppressor by regulating the cell motility of bladder cancer cells through matrix metalloproteinase­2 silencing.

Bladder cancer (BC), a common urologic cancer, is the fifth most frequently diagnosed tumor worldwide. hsa­miR­34a displays antitumor activity in several types of cancer. However, the functional mechanisms underlying hsa­miR­34a in BC remains largely unknown. We observed that hsa­mir­34a 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 hsa­miR­34a inhibited cell migration and invasion in BC cell lines 5637 and UMUC3 as detected by Transwell assays. We further found that hsa­miR­34a inhibited cell migration and invasion by silencing matrix metalloproteinase­2 (MMP­2) expression and thus interrupting MMP­2­mediated cell motility. Our analysis of BC datasets from The Cancer Genome Atlas database revealed a negative correlation between hsa­miR­34a and MMP­2. Moreover, higher MMP­2 protein expression was observed in the BC tissues when compared with that noted in the normal tissue. MMP­2 levels were also significantly associated with clinical disease stage and poor survival rate in human BC. These findings indicate that MMP­2 plays a critical role in regulating BC progression. Therefore, hsa­miR­34a is a promising treatment to target MMP­2 for the prevention and inhibition of cell migration and invasion in BC.

Miconazole Contributes to NRF2 Activation by Noncanonical P62-KEAP1 Pathway in Bladder Cancer Cells.

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.

Simultaneous determination of residual thiamphenicol and florfenicol in foods of animal origin by HPLC/electrospray ionization-MS/MS.

A simple and reliable method using HPLC/MS/MS has been developed for determination of thiamphenicol and florfenicol in foods of animal origin, such as pork, porcine liver, porcine kidney, beef, bovine liver, fish, and chicken. The HPLC separation was performed on a Waters XTerra phenyl column (100 x 2.1 mm, 3.5 microm) with a gradient mobile phase system of 0.1% formic acid-methanol at a flow rate 0.35 ml/min. Negative ionization produced the molecular ions at m/z 354 and 356 for thiamphenicol and florfenicol, respectively. Two characteristic transition reactions (m/z 354-->185, 290 and m/z 356-->336, 185) in the selected reaction monitoring mode were tested for detection and confirmation of thiamphenicol and florfenicol simultaneously. The analytes were extracted with ethyl acetate and defatted with n-hexane by liquid-liquid extraction. The recoveries of thiamphenicol and florfenicol by the developed method were 72.5-97.6%. The limits of determination were 1.0 ng/g (ppb) for both compounds.

Benzyl isothiocyanate suppresses IGF1R, FGFR3 and mTOR expression by upregulation of miR-99a-5p in human bladder cancer cells.

Benzyl isothiocyanate (BITC) is known for its pharmacological properties against malignant neoplasm, including bladder cancer (BC). The current study investigated microRNAs (miRNA or miR) expression profiles with an emphasis on the role of miR­99a­5p in BITC­treated BC cells. A quantitative polymerase chain reaction (qPCR) microarray containing 79 aberrantly expressed miRNAs in BC was used to detect miRNA expression in BITC­treated cells. Several dysregulated miRNAs were identified and further confirmed using miRNA stem­loop reverse transcription (RT)­qPCR in 5637 cells. Insulin­like growth factor 1 receptor (IGF1R), fibroblast growth factor receptor 3 (FGFR3) and mammalian target of rapamycin (mTOR) expression were determined by RT­qPCR and western blotting. Cell viability was evaluated using WST­1 reagent and apoptosis was monitored by determining the levels of cleaved­poly ADP­ribose polymerase and cleaved­caspase­3. BITC treatment significantly upregulated miR­99a­5p levels in a dose­dependent manner. miR­99a­5p overexpression decreased IGF1R, mTOR and FGFR3 expression, predicted targets of miR­99a­5p. In addition, antisense miR­99a­5p sequences inhibited BITC­induced miR­99a­5p overexpression, resulting in the restoration of protein expression and decreased cell viability. The current study identified multiple miRNAs responsive to BITC treatment, including miR­99a­5p. In addition, the induction of miR­99a­5p decreased IGF1R, mTOR and FGFR3 expression in BITC­treated BC cells. The current study provided novel insight into the antitumor mechanism by which BITC restores miR­99a­5p expression and decreases cancer cell survival.

Cisplatin contributes to programmed death-ligand 1 expression in bladder cancer through ERK1/2-AP-1 signaling pathway.

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.

Allyl Isothiocyanate Induces Autophagy through the Up-Regulation of Beclin-1 in Human Prostate Cancer Cells.

Allyl isothiocyanate (AITC), one of the most widely studied phytochemicals, inhibits the survival of human prostate cancer cells while minimally affecting normal prostate epithelial cells. Our study demonstrates the mechanism of AITC-induced cell death in prostate cancer cells. AITC induces autophagy in RV1 and PC3 cells, judging from the increased level of LC3-II protein in a dose- and time-dependent manner, but not in the normal prostate epithelial cell (PrEC). Inhibition of autophagy in AITC-treated cells decreased cell viability and enhanced apoptosis, suggesting that the autophagy played a protective role. There are several pathways activated in ATIC-treated cells. We detected the phosphorylation forms of mTOR, ERK, AMPK, JNK and p38, and ERK AMPK and JNK activation were also detected. However, inhibition of AITC-activated ERK, AMPK and JNK by pre-treatment of specific inhibitors did not alter autophagy induction. Finally, increased beclin-1 expression was detected in AITC-treated cells, and inhibition of AITC-induced beclin-1 attanuated autophagy induction, indicating that AITC-induced autophagy occurs through upregulating beclin-1. Overall, our data show for the first time that AITC induces protective autophagy in Rv1 and PC3 cells through upregulation of beclin-1. Our results could potentially contribute to a therapeutic application of AITC in prostate cancer patients.

miR-99a-5p acts as tumor suppressor via targeting to mTOR and enhances RAD001-induced apoptosis in human urinary bladder urothelial carcinoma cells.

INTRODUCTION: miR-99a-5p, known to play an important role in mammalian target of rapamycin (mTOR) regulation, is downregulated in human bladder cancer. The study aimed to investigate the anticancer activity of miR-99a-5p and the possible mechanism associated with mTOR in bladder cancer cells. MATERIALS AND METHODS: Vectors expressing miR-99a-5p were transfected into human urinary bladder urothelial carcinoma (5637 and T24) cells. The level of miR-99a-5p was monitored by microRNA (miRNA) quantitative polymerase chain reaction (QPCR). Luciferase reporter assays were performed to verify the direct binding of miR-99a-5p to mTOR transcripts. The mTOR transcripts and protein levels were measured by QPCR and Western blot, respectively. Cell viability of miR-99a-5p-transfected cells was detected by tetrazolium salt (WST-1). Inhibition of mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) signaling was detected by the phosphorylation of mTOR and AKT using Western blot. The ability of miR-99a-5p to enhance RAD001-induced apoptosis was determined as the expression of cleaved caspase 3 and levels of DNA fragmentation. RESULTS: Transfection of miR-99a-5p-expressing vector elevated the expression level of miR-99a-5p up to sixfold compared to vector-only controls. The results from luciferase assay verified that miR-99a-5p directly binds to the predicted sequence in the 3' untranslated region (3'-UTR) of mTOR. The levels of mTOR RNA and protein were decreased in miR-99a-5p-transfected cells. Dual inhibition of mTORC1 and mTORC2 by miR-99a-5p was confirmed by the decreased phosphorylation of mTOR (at Ser2448 and Ser2481), phospho-rpS6 and phospho-4EBP1. The phosphorylation of AKT was significantly inhibited in miR-99a-5p-transfected cells upon RAD001 treatment. Enforced expression of miR-99a-5p potentiated RAD001-induced apoptosis in these cells. CONCLUSION: This is the first study showing that miR-99a-5p markedly inhibits the growth of bladder cancer cells via dual inhibition of mTORC1 and mTORC2. Our data demonstrated that forced expression of miR-99a-5p inhibits the feedback of AKT survival pathway and enhances the induction of apoptosis in RAD001-treated bladder cancer cells.

Acridine orange exhibits photodamage in human bladder cancer cells under blue light exposure.

Human bladder cancer (BC) cells exhibit a high basal level of autophagic activity with accumulation of acridine-orange(AO)-stained acidic vesicular organelles. The rapid AO relocalization was observed in treated BC cells under blue-light emission. To investigate the cytotoxic effects of AO on human BC cell lines under blue-light exposure, human immortalized uroepithelial (SV-Huc-1) and BC cell lines (5637 and T24) were treated with indicated concentrations of AO or blue-light exposure alone and in combination. The cell viability was then determined using WST-1, time-lapse imaging with a Cytosmart System and continuous quantification with a multi-mode image-based reader. Treatment of AO or blue-light exposure alone did not cause a significant loss of viability in BC cells. However, AO exhibited a dose-dependent increment of cytotoxicity toward BC cells under blue-light exposure. Furthermore, the tumor formation of BC cells with treatment was significantly reduced when evaluated in a mouse xenograft model. The photodamage caused by AO was nearly neglected in SV-Huc-1 cells, suggesting a differential effect of this treatment between cancer and normal cells. In summary, AO, as a photosensitizer, disrupts acidic organelles and induces cancer cell death in BC cells under blue-light irradiation. Our findings may serve as a novel therapeutic strategy against human BC.