Autophagy In Bladder Cancer Cells Research Articles

JS-K activates G2/M checkpoints through the DNA damage response and induces autophagy via CAMKKβ/AMPKα/mTOR pathway in bladder cancer cells.

The aim of this study was to investigate the effects of JS-K, a nitric oxide donor prodrug, on DNA damage and autophagy in bladder cancer (BCa) cells and to explore the potential related mechanisms. Through detecting proliferation viability, cell morphology observation and colony formation assay low concentrations of JS-K significantly inhibited BCa growth while having no effect on normal cells. JS-K induced an increase in the level of DNA damage protein γH2AX and a decrease in the level of DNA damage repair-related proteins PCNA and RAD51 in BCa cells, indicating that JS-K can induce DNA damage in BCa cells and inhibit DNA damage repair. JS-K induced G2/M phase block and calcium overload using flow cytometry analysis. Moreover, we also investigated the levels of cell G2/M cycle checkpoint-related protein and autophagy-associated protein by western blot. The results of our study demonstrated that JS-K induced BCa cells G2/M phase arrest due to upregulating proteins related to DNA damage-related G2/M checkpoint activation (p-ATM, p-ATR, p-Chk1, p-Chk2, and p-Cdc2) and down-regulation of Cyclin B1 protein. In addition, our study demonstrated that JS-K-induced autophagy in BCa cells was related to the CAMKKβ/AMPKα/mTOR pathway.

Inhibition of autophagy can promote the apoptosis of bladder cancer cells induced by SC66 through the endoplasmic reticulum stress pathway

Bladder cancer is among the ten most prevalent cancer types worldwide, and its prognosis has not improved significantly in the past three decades because of cognitive limitations in the molecular mechanisms that drive the malignant progression of bladder cancer. Therefore, there is an urgent need to identify new therapeutic drugs or molecular targets to improve the prognosis of patients with bladder cancer. SC66, a novel allosteric inhibitor of AKT, has recently been reported to exert potent anticancer effects on various cancer cells. However, the mechanisms underlying its anticancer effects in bladder cancer remain largely unknown. Consequently, this study aimed to conduct a series of molecular and cellular biology experiments to verify the anticancer effect and potential mechanism of action of SC66 in bladder cancer in vitro. A xenograft tumor model was established to confirm its anticancer role in vivo. Our results showed that SC66 inhibited cell proliferation, triggered mitochondria-mediated apoptosis, and initiated autophagy in bladder cancer cells dose-dependently. In addition, our results suggested that SC66-caused apoptosis and autophagy were endoplasmic reticulum stress-dependent. Interestingly, the activation of autophagy can partially protect bladder cancer cells from apoptosis under endoplasmic reticulum stress induced by SC66 treatment. This study shows that SC66 exerts its anticancer impact on bladder cancer by inhibiting cell proliferation and inducing apoptosis. It also reveals that inhibiting autophagy can increase the cytotoxic effects of SC66 in bladder cancer. Overall, this is the first study on the anticancer effect of SC66 mediated by the endoplasmic reticulum stress pathway and the first report on the AKT-independent anticancer mechanism of SC66 in bladder cancer. Conclusively, exploring the relationship between apoptosis, autophagy, and endoplasmic reticulum stress induced by SC66 indicates that SC66 is a promising novel agent for patients with bladder cancer.

CircHIPK3 negatively regulates autophagy by blocking VCP binding to the Beclin 1 complex in bladder cancer

Circular RNA HIPK3 (circHIPK3) mediates the progression of multiple cancers, including bladder cancer, by regulating cell migration, autophagy and epithelial mesenchymal transition. However, the mechanism by which circHIPK3 regulates autophagy in bladder cancer cells remains unclear. Autophagy is a common self-protection mechanism in eukaryotic cells and is essential for cell survival and death regulation. However, it is unclear whether circHIPK3 affects the level of autophagy in bladder cancer through binding proteins, and the potential regulatory mechanism is unknown. Here, we found that circHIPK3 levels were significantly lower and autophagy-related proteins were significantly upregulated in bladder cancer cells and tissues compared to normal controls. CircHIPK3 downregulation promoted bladder cancer cell proliferation, while circHIPK3 overexpression inhibited proliferation. CircHIPK3 overexpression significantly suppressed autophagy in bladder cancer cells. Overexpression of circHIPK3 did not affect VCP protein expression but inhibited the VCP/Beclin 1 interaction. VCP also stabilized Beclin 1 and promoted autophagy in bladder cancer cells by downregulating ataxin-3. Thus, circHIPK3 may play an important role in bladder cancer by inhibiting VCP-mediated autophagy.

Loss of Lactate/Proton Monocarboxylate Transporter 4 Induces Ferroptosis via the AMPK/ACC Pathway and Inhibition of Autophagy on Human Bladder Cancer 5637 Cell Line.

Ferroptosis and autophagy have an important role in the occurrence and development of cancer, and lactate in cells and microenvironment is one of the influencing factors of ferroptosis and autophagy. The lactate/proton monocarboxylate transporter 4 (MCT4), which is expressed in the cell membrane, regulates the transport of intracellular lactic acid and lactate. The knockout of MCT4 can affect intracellular and extracellular lactic acid levels, thereby affecting the growth, proliferation, and metastasis of tumor cells via regulation of the oxidative stress in cells. However, whether MCT4 affects ferroptosis and autophagy in bladder cancer cells remains unclear. Colony formation assay and bladder cancer xenograft animal model were used to assess the effect of MCT4 on the growth in 5637 cells. Reactive oxygen species (ROS) assay, lipid ROS assay, lipid peroxidation assay (MDA), and transmission electron microscopy were performed to assess the level of lipid peroxidation in 5637 cells. RNA-sequence, RT-PCR, and Western Blot were used to analyze the mechanism of MCT4 of ferroptosis and autophagy. AdPlus-mCherry-GFP-LC3B reporter system was used to detect the effect of MCT4 on autophagy in 5637 cells, and the effect of knockdown of MCT4 on apoptosis was analyzed by flow cytometry. The mRNA level of MCT4 was significantly upregulated in patients with bladder cancer, which was associated with a poor prognosis. In vivo and in vitro studies demonstrated that knockdown of MCT4 could inhibit the proliferation of bladder cancer cells. Furthermore, knockdown of MCT4 led to the significant increase of ROS and MDA levels in 5637 cells and ferroptosis in 5637 cells induced by ferroptosis inducers including RSL3 (APExBIO) and erastin (APExBIO) via inhibition of AMPK-related proteins. Moreover, knockdown of MCT4 inhibited autophagy in 5637 cells, while siMCT4 promoted inhibition of autophagy by CQ (an autophagy inhibitor), which increased the level of apoptosis. This study confirmed that knockdown of MCT4 could affect oxidative stress and induce ferroptosis and inhibition of autophagy, thus suggesting that MCT4 may be a potential target for the treatment of bladder cancer.

Stellettin B Induces Cell Death in Bladder Cancer Via Activating the Autophagy/DAPK2/Apoptosis Signaling Cascade

Bladder cancer (BC) is one of the most prevalent cancers worldwide. However, the recurrence rate and five-year survival rate have not been significantly improved in advanced BC, and new therapeutic strategies are urgently needed. The anticancer activity of stellettin B (SP-2), a triterpene isolated from the marine sponge Rhabdastrella sp., was evaluated with the MTT assay as well as PI and Annexin V/7-AAD staining. Detailed mechanisms were elucidated through an NGS analysis, protein arrays, and Western blotting. SP-2 suppressed the viability of BC cells without severe toxicity towards normal uroepithelial cells, and it increased apoptosis with the activation of caspase 3/8/9, PARP, and γH2AX. The phosphorylation of FGFR3 and its downstream targets were downregulated by SP-2. Meanwhile, it induced autophagy in BC cells as evidenced by LC3-II formation and p62 downregulation. The inhibition of autophagy using pharmacological inhibitors or through an ATG5-knockout protected RT-112 cells from SP-2-induced cell viability suppression and apoptosis. In addition, the upregulation of DAPK2 mRNA and protein expression also contributed to SP-2-induced cytotoxicity and apoptosis. In RT-112 cells, an FGFR3-TACC3-knockout caused the downregulation of DAPK2, autophagy, and apoptosis. In conclusion, this is the first study demonstrating that SP-2 exhibits potent anti-BC activity by suppressing the FGFR3-TACC3/Akt/mTOR pathway, which further activates a novel autophagy/DAPK2/apoptosis signaling cascade.

Ropivacaine inhibits proliferationand invasionand promotes apoptosis and autophagy in bladder cancer cells via inhibiting PI3K/AKT pathway.

Application of a certain concentration of local anesthetics during tumor resection inhibits the progression of tumor. The effects of ropivacaine in bladder cancer (BC) have never been explored. We explored the effects of ropivacaine on the progression of BC in vitro and in vivo.CCK8 assay and EDU staining was conducted to examine cell proliferation. Flow cytometry and transwell assay were performed to evaluate apoptosis and invasion, respectively. Expression of light chain 3 (LC3) was observed through immunofluorescence. Furthermore, the xenograft tumor model of BC was built to detect the effects of ropivacaine in vivo. IHCand TUNEL assaywere conducted to detect cell proliferation and apoptosis in vivo. Ropivacaine inhibited the proliferation of T24 and 5639 cells with the 50% inhibitory concentration (IC50) of 20.08 and 31.86 µM, respectively. Ropivacaine suppressed the invasion ability and induces the apoptosis of cells. Besides, ropivacaine triggers obvious autophagy in BC cells. Moreover, ropivacaine blocks the PI3K/AKT signal pathway in BC cells. The impact of ropivacaine on cell viability, motility, and autophagy was reversed by 740 Y-P, the activator of PI3K/AKT signal pathway. The in vivo experiments demonstrated that ropivacaine inhibited the proliferation and mobility of BC. Ropivacaine has anti-carcinoma effects in BC via inactivating PI3K/AKT pathway, providing a new theoretical reference for the use of local anesthetics in the treatment of BC.

Long Noncoding RNA H19 Derived from M2 Tumor-Associated Macrophages Promotes Bladder Cell Autophagy via Stabilizing ULK1.

Purpose M2-like tumor-associated macrophages (TAMs) are crucial component of immune infiltration in tumor microenvironment (TME), and exosomes derived from TAMs contributed to the regulation of tumor progression through cellular communication. However, in bladder cancer, the role of exosomal components still remains largely unknown. In the current study, we investigated the role of exosomes derived from M2-like TAMs in the regulation of autophagy in bladder cancer (BC) cells. Methods THP-1 cells were stimulated with IL-4 and IL-13 for the polarization of TAMs, and exosomes were extracted by ultracentrifugation. H19 overexpression plasmid and H19 siRNAs were used in the study. Fluorescent analysis was performed for GFP-LC3 detection. Levels of autophagy and potential target were confirmed by western blot assay and immunoprecipitation. Results We found that TAMs-exosome treatment significantly enhanced autophagy in BC cells, and the expression of lncRNA H19 was greatly upregulated in TAMs-exosome. Silencing of lncRNA H19 in TAMs-exo obviously decreased the levels of LC3-II expression whereas the p62 levels were increased. Mechanistically, silencing of exosomal H19 from TAMs alleviated ULK1 stabilization in BC cells through promoting K48-linked polyubiquitination of ULK1. At last, we found that overexpression of exosomal H19 from TAMs suppressed the interaction between ULK1 and its specific E3 ligase NEDD4L in BC cells. Conclusion We revealed the effect of TAMs-exo-contained lncRNA H19 on regulating autophagy of bladder cancer cells, which indicated that targeting TAMs-H19 is a promising therapeutic strategy for the treatment of BC.

Secretory autophagy-induced bladder tumour-derived extracellular vesicle secretion promotes angiogenesis by activating the TPX2-mediated phosphorylation of the AURKA-PI3K-AKT axis.

Tumour angiogenesis is an independent risk factor for bladder cancer (BCa) progression, but viable and promising antiangiogenic targets are understudied. Secretory autophagy has received increasing interest recently, while the roles and executing mechanisms in the tumour microenvironment (TME) remain unclear. Herein, we found that active cathepsin B (CTSB) was upregulated in tumour tissues and serum EVs of 241 BCa patients from four cohorts and was significantly associated with poor prognosis. Starving TME (STME)-induced conventional autophagy in BCa cells elevated active CTSB levels by facilitating the expression and nuclear translocation of NFATC2. In addition, STME-induced secretory autophagy simultaneously led to markedly increased secretion of LC3-conjugated EVs loaded with active CTSB (EV-CTSB) into the TME. The increased exogenous active CTSB in endothelial cells by directly ingesting EV-CTSB prominently activated the TPX2-mediated phosphorylation of the AURKA-PI3K-AKT axis, increased VEGFA expression, and promoted angiogenesis. Our findings not only verify that EV-CTSB can be a promising target for antiangiogenic strategies in bladder cancer, but also reveal a novel action pattern based on secretory autophagy-induced EV secretion which is enlightening to explore crosstalk in the TME from various perspectives.

Fin56-induced ferroptosis is supported by autophagy-mediated GPX4 degradation and functions synergistically with mTOR inhibition to kill bladder cancer cells

Ferroptosis is a form of regulated cell death that emerges to be relevant for therapy-resistant and dedifferentiating cancers. Although several lines of evidence suggest that ferroptosis is a type of autophagy-dependent cell death, the underlying molecular mechanisms remain unclear. Fin56, a type 3 ferroptosis inducer, triggers ferroptosis by promoting glutathione peroxidase 4 (GPX4) protein degradation via a not fully understood pathway. Here, we determined that Fin56 induces ferroptosis and autophagy in bladder cancer cells and that Fin56-triggered ferroptosis mechanistically depends on the autophagic machinery. Furthermore, we found that autophagy inhibition at different stages attenuates Fin56-induced oxidative stress and GPX4 degradation. Moreover, we investigated the effects of Fin56 in combination with Torin 2, a potent mTOR inhibitor used to activate autophagy, on cell viability. We found that Fin56 synergizes with Torin 2 in cytotoxicity against bladder cancer cells. Collectively, our findings not only support the concept that ferroptosis is a type of autophagy-dependent cell death but imply that the combined application of ferroptosis inducers and mTOR inhibitors is a promising approach to improve therapeutic options in the treatment of bladder cancer.

MicroRNA‑16‑5p/BIMP1/NF‑κB axis regulates autophagy to exert a tumor‑suppressive effect on bladder cancer.

Bladder cancer (BC) is the second most common urological disease worldwide. Previous studies have reported that microRNA (miR)-16-5p is associated with the development of BC, but whether miR-16-5p regulates BC cell autophagy remains unknown. Thus, the aim of the present study was to investigate this issue. miR-16-5p expression in BC cells was assessed by reverse transcription-quantitative PCR. Cell viability and apoptosis were detected via Cell Counting Kit-8 and flow cytometry assays, respectively. For cell autophagy detection, autophagic flux was detected using a mCherry-green fluorescent protein-microtubule-associated proteins 1A/1B light chain 3B (LC3) puncta formation assay, followed by determination of autophagy-related protein markers. The targeting relationship between miR-16-5p and caspase recruitment domain family member 10 (BIMP1) was confirmed using a dual-luciferase reporter assay, followed by detection of the BIMP1/NF-κB signaling pathway. The results showed that miR-16-5p overexpression inhibited cell viability, whereas miR-16-5p knockdown promoted cell viability in BC. Furthermore, miR-16-5p overexpression induced autophagy, which was accompanied by increased autophagic flux and expression of the autophagy-related proteins LC3-II and beclin 1, as well as decreased p62 expression, whereas miR-16-5p silencing led to an inhibition of autophagy in BC cells. Moreover, autophagy inhibitor 3-methyladenine treatment inhibited cell autophagy and apoptosis in miR-16-5p-overexpressing cells. Mechanistic studies demonstrated that miR-16-5p could inhibit the BIMP1/NF-κB signaling pathway and this inhibition was achieved by directly targeting BIMP1. Furthermore, it was found that blockade of the BIMP1/NF-κB signaling pathway inversed the inhibitory effects of miR-16-5p knockdown on autophagy in BC cells. In vivo experiments further verified the tumor-suppressive effect on BC of the miR-16-5p/BIMP1/NF-κB axis. Therefore, the results of the present study indicated that miR-16-5p promotes autophagy of BC cells via the BIMP1/NF-κB signaling pathway, and an improved understanding of miR-16-5p function may provide therapeutic targets for clinical intervention in this disease.

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.

Blockage of O-linked GlcNAcylation induces AMPK-dependent autophagy in bladder cancer cells

BackgroundHigh levels of the post-translational modification O-GlcNAcylation (O-GlcNAc) are found in multiple cancers, including bladder cancer. Autophagy, which can be induced by stress from post-translational modifications, plays a critical role in maintaining cellular homeostasis and regulating tumorigenesis. The impact of O-GlcNAcylation on autophagy in bladder cancer remains unclear. Here, we evaluate the change in autophagic activity in response to O-GlcNAcylation and explore the potential mechanisms.MethodsO-GlcNAcylation levels in bladder cancer cells were altered through pharmacological or genetic manipulations: treating with 6-diazo-5-oxo-norleucine (DON) or thiamet-G (TG) or up- and downregulation of O-GlcNAc transferase (OGT) or O-GlcNAcase (OGA). Autophagy was determined using fluorescence microscopy and western blotting. Co-immunoprecipitation (Co-IP) assays were performed to evaluate whether the autophagy regulator AMP-activated protein kinase (AMPK) was O-GlcNAc modified.ResultsCellular autophagic flux was strikingly enhanced as a result of O-GlcNAcylation suppression, whereas it decreased at high O-GlcNAcylation levels. Phosphorylation of AMPK increased after the suppression of O-GlcNAcylation. We found that O-GlcNAcylation of AMPK suppressed the activity of this regulator, thereby inhibiting ULK1 activity and autophagy.ConclusionWe characterized a new function of O-GlcNAcylation in the suppression of autophagy via regulation of AMPK.Graphical abstractBlockage of O-linked GlcNAcylation induces AMPK dependent autophagy in bladder cancer cells.

Suppressed OGT expression inhibits cell proliferation while inducing cell apoptosis in bladder cancer

BackgroundThis study aimed to explore hyper-O-linked N-acetylglucosaminylation (O-GlcNAcylation) with an elevation of the expression of O-linked-β-N-acetylglucosamine transferase (OGT) in human bladder cancer.MethodsImmunohistochemical staining for OGT and O-GlcNAcylation was performed in 20 paired human bladder cancer and adjacent normal tissues, as well as in human bladder cancer tissue microarrays (N = 169). The expression level of OGT and O-GlcNAcylation in cell lines were detected using the Western blot analysis. The effects of O-GlcNAcylation on the cell proliferation of bladder cancer were detected using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and clone formation assays. Cell apoptosis and cell cycle analysis were detected using flow cytometry. The autophagy of bladder cancer cells was investigated using the Western blot analysis, and GFP–LC3 plasmid was used to detect the autophagic flux. MTT assay was performed to detect the sensitivity of bladder cancer cells to cisplatin after OGT knockdown.ResultsThe expression of OGT and the O-GlcNAcylation were upregulated in bladder cancer tissues and cell lines. O-GlcNAcylation and OGT were observed in nucleus and cytoplasm and found to be higher in muscle-invasive bladder cancer (MIBC) than in non-muscle-invasive bladder cancer (NMIBC). Reducing hyper-O-GlcNAcylation by OGT knockdown inhibited the proliferation of bladder cancer cells in vitro and xenograft tumor growth in vivo, triggered apoptosis, as well as led to cell cycle arrest. It also increased autophagy in bladder cancer cells. This study demonstrated increased autophagy pro-survival, but not pro-death. Reducing hyper-O-GlcNAcylation by OGT knockdown facilitated the chemosensitivity of bladder cancer cells to cis-platinum.ConclusionsThe data indicated that hyper-O-GlcNAcylation enhanced oncogenic phenotypes and was involved in DNA damage response in bladder cancer.

Fangchinoline Induces Apoptosis, Autophagy and Energetic Impairment in Bladder Cancer

Background/Aims: Tetrandrine and Fangchinoline (Fcn) are two natural products that are found in Stephania tetrandra. Tetrandrine is a known anti-bladder cancer compound, but the effects of Fcn on bladder cancer have been previously unclear. In the present study, we focused on the anti-tumor effects of Fcn on bladder cancer. Methods and Results: We treated T24 and 5637 bladder cancer cell lines with Fcn in vitro. We observed that Fcn inhibited the viability of bladder cancer cells in a concentration-dependent manner. The expression of PCNA, a biomarker of proliferation, was down-regulated. Fcn treatment induced both apoptosis and autophagy in bladder cancer cells, as shown by the increased cleavage of caspase-3, an up-regulated LC3-II/LC3-I ratio and the down-regulated p62 level. Blocking autophagy with 3-MA (3-Methyladenine) enhanced Fcn-induced apoptosis, indicating that Fcn-induced autophagy was adaptive. Additionally, we observed that Fcn treatment inhibited mTOR and reduced the intracellular ATP levels. The exogenous addition of methyl pyruvate (MP) to compensate metabolic substrates alleviated Fcn-induced apoptosis and autophagy. Conclusions: Our data indicated that Fcn caused an impairment in energy generation, which led to apoptosis and adaptive autophagy in bladder cancer. These results demonstrated that Fcn may be a potential candidate for use in the prevention and treatment of bladder cancer.

MP45-19 BRUCE BRIDGES AUTOPHAGY AND APOPTOSIS IN SILIBININ-INDUCED CELL DEATH IN BLADDER CANCER

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology I1 Apr 2016MP45-19 BRUCE BRIDGES AUTOPHAGY AND APOPTOSIS IN SILIBININ-INDUCED CELL DEATH IN BLADDER CANCER Jin Zeng, Wei Liu, Feng Li, Yi Sun, Lei Li, Xinyang Wang, and Dalin He Jin ZengJin Zeng More articles by this author , Wei LiuWei Liu More articles by this author , Feng LiFeng Li More articles by this author , Yi SunYi Sun More articles by this author , Lei LiLei Li More articles by this author , Xinyang WangXinyang Wang More articles by this author , and Dalin HeDalin He More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.296AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Autophagy is a tightly-regulated catabolic process that involves the degradation of intracellular components via lysosomes. Although the pivotal role of autophagy in cell growth, development, and homeostasis has been well understood, its function in bladder cancer (BCa) prevention and intervention remains to be delineated. We previously identified intravesical silibinin, a natural flavonoid, as a novel and effective chemopreventive therapy against BCa, which were associated with its proapoptotic effects. However, underlying mechanisms are poorly understood. The aim of this study was to investigate the function and mechanism of autophagy induced by silibinin in BCa. METHODS Human BCa cell line 5637, RT4 and T24 were used as the model system in vitro and in vivo. The autophagy status in cells was determined by the detection of microtubule-associated light chain 3 (LC3-I/II) using immunofluorescent staining and western blot and the formation of autophagolysosome vacuoles using transmission electron microscopy. The expression of autophagy and apoptosis related molecules after silibinin treatment were examined by western blot. Detection of apoptosis level was measured by caspase 3/7/9 activation, caspase 3/7/9 cleavage and flow cytometry assays. Both nude mice xenografts and orthotopic rat BCa tissues were analyzed for apoptosis and autophagy molecular alterations after oral or intravesical silibinin treatment in vivo. RESULTS Silibinin treatment increased the expression of LC3-II, promoted autophagosome accumulation and autophagolysosome vacuoles formation and enhanced autophagic flux by clearance of p62 in BCa cells. Mechanically, the adenosine 5’-monophosphate activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) signaling pathway was involved in the regulation of silibinin-induced autophagy. Blocking autophagy with choloroquine or bafilomycin A1 or inhibition of autophagosome formation using Atg5 or Atg7 SiRNA restored p62 levels, inhibited caspase 3 processing and PARP cleavage and thus reversed silibinin-induced cell death and apoptosis. Furthermore, we firstly found that BIR repeat containing ubiquitin-conjugating enzyme (BRUCE), a member of the inhibitor of apoptosis protein family, was degraded by autophagy in BCa cells. Silibinin treatment resulted in a strong decrease in BRUCE expression and consequent caspase 9/3/7 activation. Blocking autophagy with choloroquine or bafilomycin A1 can inhibit the degradation of BRUCE by silibinin. Overexpression of exogenous BRUCE inhibited caspases processing and PARP cleavage and finally led to the inhibition of apoptotic cell death. In vivo data showed that, either oral silibinin or intravesical instillation of silibinin inhibited the growth of xenografts in nude mice or orthotopic bladder tumors in rats respectively, which were associated with autophagy induction, BRUCE downregulation and caspase 3 activation. CONCLUSIONS Our study provides new insights regarding the biological and anti-proliferative activities of silibinin against BCa, focusing on BRUCE-mediated autophagy and apoptosis interaction, which thereby further advocates its clinical effectiveness against BCa. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e615-e616 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Jin Zeng More articles by this author Wei Liu More articles by this author Feng Li More articles by this author Yi Sun More articles by this author Lei Li More articles by this author Xinyang Wang More articles by this author Dalin He More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

MiR-222 attenuates cisplatin-induced cell death by targeting the PPP2R2A/Akt/mTOR Axis in bladder cancer cells.

Increased miR-222 levels are associated with a poor prognosis in patients with bladder cancer. However, the role of miR-222 remains unclear. In the present study, we found that miR-222 enhanced the proliferation of both the T24 and the 5637 bladder cancer cell lines. Overexpression of miR-222 attenuated cisplatin-induced cell death in bladder cancer cells. miR-222 activated the Akt/mTOR pathway and inhibited cisplatin-induced autophagy in bladder cancer cells by directly targeting protein phosphatase 2A subunit B (PPP2R2A). Blocking the activation of Akt with LY294002 or mTOR with rapamycin significantly prevented miR-222-induced proliferation and restored the sensitivity of bladder cancer cells to cisplatin. These findings demonstrate that miR-222 modulates the PPP2R2A/Akt/mTOR axis and thus plays a critical role in regulating proliferation and chemotherapeutic drug resistance. Therefore, miR-222 may be a novel therapeutic target for bladder cancer.

MP36-12 CLONALITY OF BLADDER TUMORS FOLLOWING RADICAL NEPHROURETERECTOMY – AGAINST THE FIELD DEFECT HYPOTHESIS

INTRODUCTION AND OBJECTIVES: Macroautophagy, also known as type II programmed cell death, is a regulated catabolic pathway to degrade cellular organelles and macromolecules. The role of autophagy in cancer is complex and may differ depending on tumor type or context. Here, we investigated the activated basal autophagy in bladder cancer cells and its roles in maintaining cancer cell growth. METHODS: Human immortalized uroepithelium (SV-Huc-1) and bladder cancer cells including RT-4 (grade I), 5637 (grade II), HT1376 and T24 (grade III) were used. We also included human prostate cancer cells (Rv1 and PC3), breast cancer cell (MCF-7) and embryonic kidney cancer cell (293T) as reference cells. The cell viability was accessed by WST-8. Autophagy was detected by the expression level of LC3-II protein. Human bladder tumor tissue array (TMA; 12 cases, paired with normal tissue) was used to detect the LC3 expression level. Detection of apoptosis level was measured by caspase 3/7 activation, caspase 3 cleavage and TUNEL assays. The ROS generation, mitochondria membrane potential in cells treated with autophagy inhibitors was assayed by staining with H2DCFCA and JC-1 dyes, respectively, and subsequently subjected to flow cytometry analysis. Disruption of lysomome membrane (LMP) by was detected by immunofluorescent of cathepsin-D in cells treated with CQ. RESULTS: We found bladder cancer cells exhibited high basal level of autophagy compared to prostate, breast or embryonic kidney cancer cells. Immunohistochemistry (IHC) detection of LC3 in human bladder TMA showed increased level of LC3 in bladder tumor tissues compared to their paired normal tissues. In the cell-based experiment, we found that autophagy inhibitors alone inhibited bladder cancer cell growth and increased apoptosis. The ROS generation and MMP disruption were not detected in cells treated with CQ or HCQ. Immunofluorescent detection of cathepsin-D in cells treated with CQ showed dispread pattern in the cytosol, suggesting the disruption of LMP in CQ-treated bladder cancer cells. CONCLUSIONS: Our results showed that human bladder cancer exhibits high basal level of autophagy. Autophagy inhibitors showed significant effects on the growth of bladder cancer cells by disruption of lysosome function and elevation of apoptotic cell death.

MP36-11 AUTOPHAGY IS ACTIVATED IN HUMAN BLADDER CANCER AND REQUIRED FOR CANCER CELL GROWTH

INTRODUCTION AND OBJECTIVES: Macroautophagy, also known as type II programmed cell death, is a regulated catabolic pathway to degrade cellular organelles and macromolecules. The role of autophagy in cancer is complex and may differ depending on tumor type or context. Here, we investigated the activated basal autophagy in bladder cancer cells and its roles in maintaining cancer cell growth. METHODS: Human immortalized uroepithelium (SV-Huc-1) and bladder cancer cells including RT-4 (grade I), 5637 (grade II), HT1376 and T24 (grade III) were used. We also included human prostate cancer cells (Rv1 and PC3), breast cancer cell (MCF-7) and embryonic kidney cancer cell (293T) as reference cells. The cell viability was accessed by WST-8. Autophagy was detected by the expression level of LC3-II protein. Human bladder tumor tissue array (TMA; 12 cases, paired with normal tissue) was used to detect the LC3 expression level. Detection of apoptosis level was measured by caspase 3/7 activation, caspase 3 cleavage and TUNEL assays. The ROS generation, mitochondria membrane potential in cells treated with autophagy inhibitors was assayed by staining with H2DCFCA and JC-1 dyes, respectively, and subsequently subjected to flow cytometry analysis. Disruption of lysomome membrane (LMP) by was detected by immunofluorescent of cathepsin-D in cells treated with CQ. RESULTS: We found bladder cancer cells exhibited high basal level of autophagy compared to prostate, breast or embryonic kidney cancer cells. Immunohistochemistry (IHC) detection of LC3 in human bladder TMA showed increased level of LC3 in bladder tumor tissues compared to their paired normal tissues. In the cell-based experiment, we found that autophagy inhibitors alone inhibited bladder cancer cell growth and increased apoptosis. The ROS generation and MMP disruption were not detected in cells treated with CQ or HCQ. Immunofluorescent detection of cathepsin-D in cells treated with CQ showed dispread pattern in the cytosol, suggesting the disruption of LMP in CQ-treated bladder cancer cells. CONCLUSIONS: Our results showed that human bladder cancer exhibits high basal level of autophagy. Autophagy inhibitors showed significant effects on the growth of bladder cancer cells by disruption of lysosome function and elevation of apoptotic cell death.

MP36-10 MIR-99A ACTS AS TUMOR SUPPRESSOR VIA TARGETING TO MTOR IN HUMAN BLADDER CANCER CELLS

You have accessJournal of UrologyBladder Cancer: Basic Research I1 Apr 2015MP36-10 MIR-99A ACTS AS TUMOR SUPPRESSOR VIA TARGETING TO MTOR IN HUMAN BLADDER CANCER CELLS Te-Fu Tsai, Ji-Fan Lin, Yi-Chia Lin, Kuang-Yu Chou, Hung-En Chen, and Thomas I.S. Hwang Te-Fu TsaiTe-Fu Tsai More articles by this author , Ji-Fan LinJi-Fan Lin More articles by this author , Yi-Chia LinYi-Chia Lin More articles by this author , Kuang-Yu ChouKuang-Yu Chou More articles by this author , Hung-En ChenHung-En Chen More articles by this author , and Thomas I.S. HwangThomas I.S. Hwang More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2015.02.738AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES mTOR is recognized as an important target in many cancer types. The mTOR complex 2 (mTORC2) containing mTOR and rictor is thought to be insensitive to RAD001 and was shown to regulate the prosurvival kinase AKT by phosphrylation. We previously showed that the expression level of miR-99a was down-regulated in human bladder tumor tissues compared to their adjacent normal tissues. Since mTOR is predicted to be a potential target of miR-99a, we investigate the anti-cancer activity of miR-99a in bladder cancer cells. METHODS Vectors that express miR-99a were transfected into human bladder, prostate, embryonic kidney and cervical cancer cells. Expression level of miR-99a was monitored by miRNA Q-PCR. 3'-UTR of mTOR was constructed downstream of a luciferase gene, and luciferase reporter assays were performed to verify the direct binding of miR-99a to mTOR transcripts. The mRNA and protein expression level of mTOR were measured by Q-PCR and Western blot, respectively. Cell viability of miR-99a transfected cells was detected by WST-1 and colony formation assays. Inhibition of mTOR complex 1 and 2 (mTORC1 and mTORC2) signaling was monitored by detecting the phosphrylation of S6K and Akt using Western blot. Induction of autophagy was accessed by the expression of LC3-II marker protein. RESULTS Transfection of miR-99a expressing vector elevated the expression level of miR-99a up to 4.5-fold in cells compared to vector-only control. The function of matured miR-99a was confirmed by luciferase reporter assays. The level of mTOR RNA and protein were decreased in miR-99a transfected cells. Dual inhibition of mTORC1 and mTORC2 was confirmed by immunoprecipitation (IP) of mTOR associated Rictor and Raptor, and the decreased phosphrylation of S6K and Akt in miR-99a transfected cells. The LC3-II protein was accumulated in miR-99a transfected cells compared to monk transfected control, suggesting that inhibition of mTOR by miR-99a induces autophagy in bladder cancer cells. CONCLUSIONS This is the first study showed that miR-99a markedly inhibits bladder and prostate cancer cell growth via dual inhibition of mTORC1 and mTORC2. miR-99a treatment also induces autophagy through mTOR inhibition. However, the role of miR-99a induces autophagy remains further investigation. © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 193Issue 4SApril 2015Page: e432 Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information Te-Fu Tsai More articles by this author Ji-Fan Lin More articles by this author Yi-Chia Lin More articles by this author Kuang-Yu Chou More articles by this author Hung-En Chen More articles by this author Thomas I.S. Hwang More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Pirarubicin induces an autophagic cytoprotective response through suppression of the mammalian target of rapamycin signaling pathway in human bladder cancer cells

Pirarubicin is widely used in intravesical chemotherapy for bladder cancer, but its efficacy is limited due to drug resistance; the mechanism has not been well studied. Emerging evidence shows that autophagy can be a novel target for cancer therapy. This study aimed to investigate the role of autophagy in pirarubicin-treated bladder cancer cells. Bladder cancer cells EJ and J82 were treated with pirarubicin, siRNA, 3-methyladenine or hydroxychloroquine. Cell proliferation and apoptosis were tested by cell survival assay and flow cytometric analysis, respectively. Autophagy was evaluated by immunoblotting before and after the treatments. The phosphorylated mammalian target of rapamycin, serine/threonine kinase p70 S6 kinase, and eukaryotic translation initiation factor 4E binding protein 1 were also investigated by immunoblotting. We found that pirarubicin could induce autophagy in bladder cancer cells. Inhibition of autophagy by 3-methyladenine, hydroxychloroquine or knockdown of autophagy related gene 3 significantly increased apoptosis in pirarubicin-treated bladder cancer cells. Pirarubicin-induced autophagy was mediated via the mTOR/p70S6K/4E-BP1 signaling pathway. In conclusion, autophagy induced by pirarubicin plays a cytoprotective role in bladder cancer cells, suggesting that inhibition of autophagy may improve efficacy over traditional pirarubicin chemotherapy in bladder cancer patients.