Treatment Of Bladder Cancer Cells Research Articles

Synergistic Combination of Quercetin and Mafosfamide in Treatment of Bladder Cancer Cells.

Bladder cancer, which has a rising incidence, is the 10th most common cancer. The transitional cell carcinoma histotype is aggressive and often current therapies are ineffective. We investigated the anti-proliferative effect of quercetin, a natural flavonoid, in combination with the alkylating agent mafosfamide (MFA) on two human bladder cancer cell lines, namely RT112 and J82, representing the progression from low-grade to high-grade tumors, respectively. In both cell types, the combined treatment led to a synergic reduction in cell viability confirmed by a combination index of less than one, though different biological responses were noted. In J82 cells, MFA alone and, to a lesser extent, with quercetin caused cell cycle arrest in the G2/M phase, but only the combined treatment triggered apoptotic cell death. In contrast, in RT112 cells, quercetin induced autophagy, evidenced by the autophagosome formation and the increase in LC-3 lipidation. Interestingly, the synergistic effect was observed only when cells were pre-treated with MFA for 24 h before adding quercetin, not in the reverse order. This suggests that quercetin may help overcome MFA resistance to apoptosis. Although further studies are needed, investigating the combined effects of quercetin and MFA could help elucidate the mechanisms of drug resistance in bladder cancer treatment.

USP5 promotes tumor progression by stabilizing SLUG in bladder cancer.

Bladder cancer ranks as the second most prevalent urology malignancy globally. Invasive metastasis is a significant contributor to mortality among patients with bladder cancer, yet the underlying mechanisms remain elusive. Deubiquitinases are pivotal in carcinogenesis, with USP5 implicated in the malignant progression of hepatocellular carcinoma, colorectal cancer and non-small cell lung cancer. The present study assessed the role and mechanism of ubiquitin-specific proteinase 5 (USP5) in the malignant progression of bladder cancer. The association between USP5 expression and bladder cancer prognosis and stage was analyzed using The Cancer Genome Atlas database. Moreover, to elucidate the role of USP5 in bladder cancer, USP5 overexpression and knockdown cell lines were established using T24 cells. Cell viability, proliferation and migration were assessed using Cell Counting Kit-8, Transwell and scratch assays, respectively. Cyclohexanamide was used to evaluate the effect of USP5 expression on Snail family zinc finger 2 (SLUG) stability. Immunoprecipitation and immunofluorescence co-localization were utilized to probe the interaction between USP5 and SLUG. Changes in mRNA and protein levels were assessed using reverse transcription-quantitative PCR and western blotting, respectively. The results revealed that patients with bladder cancer with high USP5 expression had significantly shorter survival (P<0.05) and a higher clinicopathologic stage (P<0.05) than those with low USP5 expression. T24 cells overexpressing USP5 demonstrated significantly increased proliferation (P<0.05), invasion (P<0.05) and expression of epithelial-mesenchymal transition markers (P<0.05); whereas T24 cells with knocked-down USP5 expression revealed significantly reduced proliferation (P<0.05), invasion (P<0.05) and epithelial-mesenchymal transition markers (P<0.05). Immunoprecipitation experiments demonstrated the binding of USP5 to SLUG in bladder cancer cells, with further analysis revealing that USP5 upregulated protein levels of SLUG by inhibiting its ubiquitination. Furthermore, the treatment of bladder cancer cells with Degrasyn, a USP5 inhibitor, was associated with a significant inhibition of the proliferation (P<0.05) and invasion (P<0.05) of T24 cells. In conclusion, the findings of the present study underscore the role of USP5 in promoting the malignant progression of bladder cancer through the stabilization of SLUG. Targeting USP5 holds promise as a strategy for inhibiting bladder cancer progression.

Evaluating a combination treatment of NK cells and reovirus against bladder cancer cells using an in vitro assay to simulate intravesical therapy

Intravesical treatment using either reovirus or natural killer (NK) cells serves as an efficient strategy for the treatment of bladder cancer cells (BCCs); however, corresponding monotherapies have often shown modest cytotoxicity. The potential of a locoregional combination using high-dose reovirus and NK cell therapy in an intravesical approach has not yet been studied. In this study, we evaluated the effectiveness of reoviruses and expanded NK cells (eNK) as potential strategies for the treatment of bladder cancer. The anti-tumor effects of mono-treatment with reovirus type 3 Dearing strain (RC402 and RP116) and in combination with interleukin (IL)-18/-21-pretreated eNK cells were investigated on BCC lines (5637, HT-1376, and 253J-BV) using intravesical therapy to simulate in vitro model. RP116 and IL-18/-21-pretreated eNK cells exhibited effective cytotoxicity against grade 1 carcinoma (5637 cells) when used alone, but not against HT-1376 (grade 2 carcinoma) and 253J-BV cells (derived from a metastatic site). Notably, combining RP116 with IL-18/-21-pretreated eNK cells displayed effective cytotoxicity against both HT-1376 and 253J-BV cells. Our findings underscore the potential of a combination therapy using reoviruses and NK cells as a promising strategy for treating bladder cancer.

Expression of TMEFF2 gene and significance of initiation methylation in bladder cancer

Objective: To analyze the expression and significance of TMEFF2 gene methylation in bladder cancer. Methods: The promoter region methylation level of the MEFF 2 gene was analyzed by applying the expression database and gene methylation.64 urine samples were detected by demethylated drug-nitrogen-2 deoxycytidine to observe the changes of cell invasion and apoptotic valence, and its application value in bladder cancer was analyzed by applying high expression of TMEFF2 gene. Results: Drug treatment of bladder cancer cells showed that the appreciation and migration of bladder cancer were inhibited, but the number of apoptosis increased. Conclusion: TMEFF2 gene promoter methylation is one of the markers of bladder cancer and plays an important role in the diagnosis of bladder cancer.

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.

FOXC1 Binds Enhancers and Promotes Cisplatin Resistance in Bladder Cancer

Simple SummaryIn bladder cancer, cisplatin remains the front-line therapy, but drug resistance is common. Previously, we showed that cancer cells can spontaneously convert to an aggressive drug-resistant phenotype without mutational events. In the current work, we explored the epigenetic mechanism behind the conversion to the drug-resistant phenotype. We discovered that drug-resistant cells have differentially accessible enhancers, which are bound by FOXC1, a transcription factor that is overexpressed in these cells. Accordingly, FOXC1 knockout significantly attenuates the emergence of the drug-resistant phenotype and reduces cell survival upon cisplatin treatment. These findings suggest that FOXC1 binding at accessible enhancers promotes cisplatin drug resistance in bladder cancer cells. Therefore, FOXC1 targeting may be a new therapeutic avenue to mitigate cisplatin resistance and improve treatment efficacy in bladder cancer.Chemotherapy resistance is traditionally attributed to DNA mutations that confer a survival advantage under drug selection pressure. However, in bladder cancer and other malignancies, we and others have previously reported that cancer cells can convert spontaneously to an aggressive drug-resistant phenotype without prior drug selection or mutational events. In the current work, we explored possible epigenetic mechanisms behind this phenotypic plasticity. Using Hoechst dye exclusion and flow cytometry, we isolated the aggressive drug-resistant cells and analyzed their chromatin accessibility at regulatory elements. Compared to the rest of the cancer cell population, the aggressive drug-resistant cells exhibited enhancer accessibility changes. In particular, we found that differentially accessible enhancers were enriched for the FOXC1 transcription factor motif, and that FOXC1 was the most significantly overexpressed gene in aggressive drug-resistant cells. ChIP-seq analysis revealed that differentially accessible enhancers in aggressive drug-resistant cells had a higher FOXC1 binding, which regulated the expression of adjacent cancer-relevant genes like ABCB1 and ID3. Accordingly, cisplatin treatment of bladder cancer cells led to an increased FOXC1 expression, which mediated cell survival and conversion to a drug-resistant phenotype. Collectively, these findings suggest that FOXC1 contributes to phenotypic plasticity by binding enhancers and promoting a mutation-independent shift towards cisplatin resistance in bladder cancer.

A Novel Cyclic Pentadepsipeptide, N-Methylsansalvamide, Suppresses Angiogenic Responses and Exhibits Antitumor Efficacy against Bladder Cancer.

Simple SummaryWe found a novel cyclic pentadepsipeptide, N-methylsansalvamide (MSSV), and evaluated its anti-tumor action against bladder cancer using in vitro and in vivo model systems. Additionally, we report its anti-angiogenic responses both in vitro and in vivo. Moreover, acute toxicity test and tissue staining for liver function revealed that orally administered MSSV (2000 mg/kg for 14 days) exerted no harmful effects as it did not cause animal death, undesirable weigh alteration, adverse clinical symptoms, and abnormal biochemical marker levels (AST, ALT).Here, we explored the anti-tumor efficacy of a cyclic pentadepsipeptide, N-methylsansalvamide (MSSV), in bladder cancer. MSSV inhibited the proliferation of both bladder cancer 5637 and T24 cells, which was attributed to the G1-phase cell cycle arrest, apoptosis induction, and alteration of mitogen-activated protein kinases (MAPKs) and protein kinase b (AKT) signaling pathways. Additionally, the treatment of bladder cancer cells with MSSV suppressed migratory and invasive potential via the transcription factor-mediated expression of matrix metalloproteinase 9 (MMP-9). MSSV abrogated vascular endothelial growth factor (VEGF)-induced angiogenic responses in vitro and in vivo. Furthermore, our result showed the potent anti-tumor efficacy of MSSV in a xenograft mouse model implanted with bladder cancer 5637 cells. Finally, acute toxicity test data obtained from blood biochemical test and liver staining indicated that the oral administration of MSSV at 2000 mg/kg caused no adverse cytotoxic effects. Our preclinical data described the potent anti-angiogenic and anti-tumor efficacy of MSSV and showed no signs of acute toxicity, thereby suggesting the putative potential of oral MSSV as a novel anti-tumor agent in bladder cancer treatment.

Overexpression of GAS6 Promotes Cell Proliferation and Invasion in Bladder Cancer by Activation of the PI3K/AKT Pathway.

BackgroundGrowth arrest-specific 6 (GAS6) is a secreted vitamin K-dependent protein abnormally expressed in various human tumor tissues and can activate the receptor Tyro3, Axl, and Mer to promote cancer cell proliferation and invasion. Until now, the role of GAS6 has been poorly understood in bladder cancer (BCa).Materials and MethodsUsing bioinformatics analysis, we screened genes significantly associated with overall survival in BCa. The association between GAS6 and survival was evaluated by tissue microarray and IHC staining. We investigated the effect of GAS6 on the development of BCa through in vitro and in vivo experiments.ResultsHere, we report that GAS6 is highly expressed in bladder cancer and is significantly associated with tumor grade, T stage, and worse prognosis. We found that GAS6 depletion inhibited proliferation, migration, and invasion of BCa cells. In addition, bioinformatics analysis revealed that GAS6 may be involved in the regulation of PI3K-AKT signaling pathway by binding to receptor TAM and has a significant positive correlation with PI3K family gene expression. Furthermore, Western blot experiments have shown that GAS6 might modulate the PI3K-AKT signaling to regulate proliferation and invasion of BCa cells. Treatment of BCa cells with SC79, an AKT activator, partially restored the effect of GAS6 silencing on cell proliferation and invasion.ConclusionThe present study suggests that GAS6 may play a pivotal role in the development of BCa and may be a potential target for its treatment.

Antitumor Activity and Mechanistic Characterization of APE1/Ref-1 Inhibitors in Bladder Cancer.

Bladder cancer is the ninth most common cause of cancer-related deaths worldwide. Although cisplatin is used routinely in treating bladder cancer, refractory disease remains lethal for many patients. The recent addition of immunotherapy has improved patient outcomes; however, a large cohort of patients does not respond to these treatments. Therefore, identification of innovative molecular targets for bladder cancer is crucial. Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1) is a multifunctional protein involved in both DNA repair and activation of transcription factors through reduction-oxidation (redox) regulation. High APE1/Ref-1 expression is associated with shorter patient survival time in many cancer types. In this study, we found high APE1/Ref-1 expression in human bladder cancer tissue relative to benign urothelium. Inhibition of APE1/Ref-1 redox signaling using APE1/Ref-1-specific inhibitors attenuates bladder cancer cell proliferation in monolayer, in three-dimensional cultures, and in vivo. This inhibition corresponds with an increase in apoptosis and decreased transcriptional activity of NF-κB and STAT3, transcription factors known to be regulated by APE1/Ref-1, resulting in decreased expression of downstream effectors survivin and Cyclin D1 in vitro and in vivo. We also demonstrate that in vitro treatment of bladder cancer cells with APE1/Ref-1 redox inhibitors in combination with standard-of-care chemotherapy cisplatin is more effective than cisplatin alone at inhibiting cell proliferation. Collectively, our data demonstrate that APE1/Ref-1 is a viable drug target for the treatment of bladder cancer, provide a mechanism of APE1/Ref-1 action in bladder cancer cells, and support the use of novel redox-selective APE1/Ref-1 inhibitors in clinical studies. SIGNIFICANCE: This work identifies a critical mechanism for APE1/Ref-1 in bladder cancer growth and provides compelling preclinical data using selective redox activity inhibitors of APE1/Ref-1 in vitro and in vivo.

Impact of Methadone on Cisplatin Treatment of Bladder Cancer Cells.

Cisplatin-based chemotherapy is the treatment of choice for advanced bladder cancer. Since many tumor cells show inherent or acquired cisplatin resistance, research is needed to improve the therapeutic efficacy. Since the analgesic methadone is discussed as being a sensitizer for chemotherapy, we tested its effects on the cisplatin treatment of bladder cancer cells. T24 and HT-1376 bladder cancer cells were incubated with cisplatin in combination with methadone. Cytotoxicity was examined using the WST-1 viability assay and induction of apoptosis was analyzed via phase-contrast microscopy, flow cytometry, and western blot analysis. Methadone was shown to enhance the cytotoxic effects of cisplatin on T24 cells based on the induction of apoptosis. In contrast, HT-1376 cells were identified as non-responders to methadone. Methadone could act as a chemosensitizer in the future treatment of advanced bladder cancer. Further research is needed to identify the underlying molecular mechanisms.

Down-regulation of LncRNA TUG1 enhances radiosensitivity in bladder cancer via suppressing HMGB1 expression

BackgroundLong non-coding RNAs (lncRNAs) have been reported to regulate the sensitivity of different cancer cells to chemoradiotherapy. Aberrant expression of lncRNA Taurine-upregulated gene 1 (TUG1) has been found to be involved in the development of bladder cancer, however, its function and underlying mechanism in the radioresistance of bladder cancer remains unclear.MethodsQuantitative real-time PCR (qRT-PCR) was conducted to measure the expression of TUG1 and HMGB1 mRNA in bladder cancer tissues and cell lines. HMGB1 protein levels were tested by western blot assays. Different doses of X-ray were used for radiation treatment of bladder cancer cells. Colony survival and cell viability were detected by clonogenic assay and CCK-8 Kit, respectively. Cell apoptosis was determined by flow cytometry. A xenograft mouse model was constructed to observe the effect of TUG1 on tumor growth in vivo.ResultsThe levels of TUG1 and HMGB1 were remarkably increased in bladder cancer tissues and cell lines. Radiation treatment markedly elevated the expression of TUG1 and HMGB1. TUG1 knockdown inhibited cell proliferation, promoted cell apoptosis and decreased colony survival in SW780 and BIU87 cells under radiation. Moreover, TUG1 depletion suppressed the HMGB1 mRNA and protein levels. Furthermore, overexpression of HMGB1 reversed TUG1 knockdown-induced effect in bladder cancer cells. Radiation treatment dramatically reduced the tumor volume and weight in xenograft model, and this effect was more obvious when combined with TUG1 silencing.ConclusionLncRNA TUG1 knockdown enhances radiosensitivity of bladder cancer by suppressing HMGB1 expression. TUG1 acts as a potential regulator of radioresistance of bladder cancer, and it may represent a promising therapeutic target for bladder cancer patients.

The SMAD2/3 pathway is involved in hepaCAM-induced apoptosis by inhibiting the nuclear translocation of SMAD2/3 in bladder cancer cells.

The aim of this study was to explore the correlation between hepatocyte cell adhesion molecule (hepaCAM) and SMAD family member 2/3 (SMAD2/3) in bladder carcinoma, and the involvement of the SMAD2/3 pathway in hepaCAM-induced tumor apoptosis. Immunohistochemistry was used to measure hepaCAM and p-SMAD2/3 protein levels in bladder cancer tissues. Flow cytometry and Hoechst staining were used to study the effect of hepaCAM on cellular apoptosis. Western blot was employed to determine the expression of hepaCAM and SMAD2/3/caspase pathway molecules using a hepaCAM overexpression adenovirus, a caspase inhibitor (Z-VAD-FMK), and a SMAD2/3 activator (transforming growth factor (TGF)-β1), respectively. Translocation of p-SMAD2/3 was measured by immunofluorescence and western blot. HepaCAM proteins were significantly decreased (P < 0.05), while p-SMAD2/3 proteins were remarkably increased (P < 0.05) in bladder carcinoma compared to adjacent tissues. However, the low hepaCAM and high p-SMAD2/3 were not statistically associated with clinicopathological characteristics of the patients. A negative linear correlation between hepaCAM and p-SMAD2/3 was observed according to Pearson analysis (r = -0.712/-0.724, P = 0.008/0.011). Overexpression of hepaCAM activated caspase 3/8/9 and downregulated poly-ADP ribose polymerase (PARP) and p-SMAD2/3. Treatment of bladder cancer cells with Z-VAD-FMK + hepaCAM significantly downregulated procaspase 3/8/9 and PARP and induced cellular apoptosis, compared with that using Z-VAD-FMK alone. Similarly, combined treatment of TGF-β1 + hepaCAM significantly downregulated p-SMAD2/3, procaspase 3/8/9, and PARP and induced apoptosis of bladder cancer cells, compared with TGF-β1 alone. Overexpression of hepaCAM prevented the p-SMAD2/3 translocation from the cytoplasm to the nucleus in bladder cancer cells BIU-87 and T24. Our findings uncover that the p-SMAD2/3 pathway is critical for hepaCAM-induced cancer cell apoptosis and provide valuable insights for current and future Ad-hepaCAM and p-SMAD2/3 clinical trials.

MP45-10 CORRELATION BETWEEN FIBRONECTIN GENE EXPRESSION AND LOCAL TOXICITY INDUCED BY ADJUVANT INTRAVESICAL THERAPY

performed using nuclear extracts from treated cells and antibodies directed against HIF1a, HIF2a, and HIF1b. RESULTS: Treatment of bladder cancer cells with emetine decreased expression of HIF1a and HIF2a, but not HIF1b in a dose dependent manner. Using reverse transcriptase quantitative PCR, we demonstrated that emetine did not modulate HIFa at the transcriptional level. Instead, HIFa was modulated primarily at the level of protein synthesis with proteasome degradation playing a minor role as demonstrated by western blot of bladder cancer cells treated with cycloheximide or MG132 and emetine. CONCLUSIONS: Emetine is a known inhibitor of protein synthesis at micromolar concentrations. However, we found low nanomolar concentrations of emetine completely inhibit expression of HIF1a and HIF2a, but not HIF1b. The decrease in HIFa expression was due in part to protein synthesis inhibition, but proteasomal degradation also contributed significantly to the results. Given the important role of HIF proteins and hypoxia signaling in promoting tumor growth and progression, patients may benefit from treatment with a HIFa inhibitor, like emetine, in addition to their standard therapeutic regimen.

Methyl jasmonate sensitizes human bladder cancer cells to gambogic acid‐induced apoptosis through down‐regulation of EZH2 expression by miR‐101

Gambogic acid (GA) and methyl jasmonate (MJ) are increasingly being recognized as novel natural anticancer compounds. Here, we investigated the antitumour effects of GA in combination with MJ on human bladder cancer cells. Cell viability was detected by cell counting kit-8 assay. Cell apoptosis was assessed by Hoechst 33258 staining and flow cytometry. Protein levels were determined by immunoblotting and expressions of mRNA and miRNAs by RT-PCR. Differential expressions of a group of downstream genes were identified using microarray analysis. MJ significantly sensitized bladder cancer cells to GA-induced growth inhibition and apoptosis while sparing normal fibroblasts. MJ enhanced GA-induced activation of caspase-3 and caspase-9, and down-regulated the expression of XIAP. Furthermore, treatment of bladder cancer cells with a combination of GA and MJ induced synergistic inhibition of the enhancer of zeste homologue 2 (EZH2) expression, whereas miR-101 expression was up-regulated. Conversely, knockdown of miR-101 restored this decreased expression of EZH2 and suppressed the inhibitory effect of GA and MJ on the growth of bladder cancer cells. Microarray analysis showed that genes closely associated with bladder cancer development were significantly down-regulated by GA and MJ. In a s.c. xenograft mouse model of human bladder carcinoma, the combination of GA and MJ exerted an increased antitumour effect compared with GA alone. MJ sensitizes bladder cancer cells to GA-induced apoptosis by down-regulating the expression of EZH2 induced by miR-101. Thus, the combination of selective anti-cancer agents MJ and GA could provide a novel strategy for treating human bladder cancer.

Meloxicam synergistically enhances the in vitro effects of sunitinib malate on bladder-cancer cells

To evaluate the in vitro effects of sunitinib malate and meloxicam in isolation, and to analyse the ability of meloxicam to enhance the cytotoxicity of sunitinib malate in three human bladder-cancer cell lines. Cell lines were treated with sunitinib malate and meloxicam, either in isolation or combined. Leishman staining, MTT method, comet assay, MDC staining and M30 CytoDEATH antibody were performed. The Chou and Talalay method was applied. Sunitinib malate and meloxicam supressed cell proliferation in bladder-cancer cells in isolation, in a concentration-dependent manner. Treatment of bladder-cancer cells with a combination of sunitinib malate and meloxicam showed a synergistic effect. When exploring the mechanism of this combination by means of comet assay, there is the suggestion that meloxicam increases sunitinib malate cytotoxicity through DNA damage. Autophagic and apoptotic studies show a greater incidence of autophagic vacuoles and early apoptotic cells when the combined treatment was put into use. In isolation, sunitinib malate and meloxicam demonstrated anti-tumour effects in our study. Furthermore, simultaneous exposure of cells to sunitinib malate and meloxicam provided a combinatorial beneficial effect. This hints at the possibility of a new combined therapeutic regimen, which could lead to improvements in the treatment of patients with bladder cancer.

Effect of sunitinib and pazopanib on necrosis and autophagic cell death in cancer cells: Role of cathepsin B.

e15513 Background: Tyrosine kinase inhibitors (TKI), such as sunitinib, sorafenib and pazopanib, have replaced immunotherapy as the standard of care for metastatic renal cell carcinoma (mRCC). However, their use in sequential or combined strategies is limited by the lack of evidences on TKI-induced cell death in cancer cells. Aim of our study was to evaluate the different mechanisms responsible of the anti-proliferative and cytotoxic effects induced in vitro by µM doses of sunitinib, sorafenib and pazopanib in 5637 and J82 bladder cancer (BC) cell lines. Methods: The viability of BC cell lines were tested by MTT assay. Autophagy was evaluated by western blot analysis with anti-LC3 and anti-p62 antibodies, acridine orange staining and cytofluorimetric analysis. Necrotic cell death was evaluated by Annexin-V/PI staining and FACS analysis. The cathepsin B activation was evaluated by western blot using an anti-cathepsin B antibody; the cathepsin B proteolytic activity was determined using the fluorogenic Z-Arg-Arg-AMC peptide and the fluorescence of the hydrolyzed 7-amino-4-methyl-coumarin was detected by a SpectraMax Gemini XPS microplate reader. Results: We found that sunitinib and pazopanib markedly reduced at mM dose the viability of BC cells. Treatment for 24h with 20µM of sunitinib, by triggering “Incomplete autophagy”, induced necrosis of BC cells. In addition, sunitinib as a lysosomotropic agent, entered free within the lysosomes, where by increasing lysosomal pH and impairing cathepsin B activity, induced lysosomal-dependent necrosis. By contrast, treatment of BC cells for 72h with 20µM of pazopanib induced autophagic cell death, which was markedly reversed in a dose-dependent manner by the autophagic inhibitor 3-MA. The pazopanib-induced autophagic cell death was associated with increased procathepsin B cleavage and enhanced cathepsin B activity. Conclusions: Overall, our results show different cathepsin B-dependent cancer cell death mechanisms induced by sunitinib or pazopanib, providing the biological basis for novel molecularly targeted approaches.

Different effects of sunitinib, sorafenib, and pazopanib on inducing cancer cell death: The role of autophagy.

270 Background: Tyrosine kinase inhibitors (TKI), such as sunitinib, sorafenib and pazopanib, have replaced immunotherapy as the standard of care for metastatic renal cell carcinoma (mRCC). However, their use in sequential or combined strategies is limited by the lack of evidences on the ability of TKIs to induce cell death in cancer cells. Aim of our study was to evaluate the different mechanisms responsible of the cytotoxic effects induced in vitro by µM doses of sunitinib, sorafenib and pazopanib in 5637 and J82 bladder cancer (BC) cell lines. Methods: The viability of BC cell lines were tested by MTT assay. Autophagy was evaluated by western blot analysis with the anti-LC3 and anti-p62 antibodies, acridine orange staining and cytofluorimetric analysis. Necrosis and apoptosis, (ΔΨm) dissipation and ROS generation were determined by Annexin-V/PI, JC-1 and DCFDA staining, respectively and cytofluorimetric analysis. The cathepsin B activity was evaluated by ELISA. Finally, by mRNA estraction and RT-PCR array the pazopanib-induced gene profile expression was evaluated. Results: We found that treatment of 5637 and J82 BC cells with the three TKI agents markedly reduced cell viability. Treatment for 24 h with sunitinib and sorafenib at 20 µM dose, triggers an incomplete autophagy of BC cells. In addition, inhibition of autophagy induced by sunitinib and sorafenib triggers cell death of BC cells. Thus, sunitinib by imparing the cathepsin B activity induces lysosomal-dependent necrosis. Similarly, sorafenib by defective lysosomial degradation triggers ROS- and mitochondrial-dependent apoptosis. As regard to pazopanib, we first demonstrate that treatment of BC cells for 72 hrs (20 µM) induces autophagic Type II cell death, which was markedly reversed in a dose-dependent manner by 3MA and chloroquine autophagic inhibitors. Finally, pazopanib upregulates the mRNA expression of α-glucosidase (GAA) and TP73 belonging to the p53 tumor suppressor genes. Conclusions: Overall, our results showing different TKI-induced cell death mechanisms provide the rationale for the sequential use of these agents and the biological basis for novel molecularly targeted approaches.

Silencing of mutant p53 by siRNA induces cell cycle arrest and apoptosis in human bladder cancer cells

Backgroundp53 is the most frequently mutated tumor-suppressor gene in human cancers. It has been reported that mutations in p53 result not only in the loss of its ability as a tumor suppressor, but also in the gain of novel cancer-related functions that contribute to oncogenesis. The present study evaluated the potential of silencing of mutant p53 by small interfering RNA in the treatment of bladder cancer cells in vitro.MethodsWe used the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay to assess cell viability and flow cytometry to detect cell cycle alterations and apoptosis. The related molecular mechanisms were assessed by western blotting. We also used the MTT assay and flow cytometry to investigate if silencing of mutant p53 by knockdown with small interfering (si)RNA would change the sensitivity to cisplatin treatment.ResultsUsing 5637 and T24 human bladder cancer cell lines characterized by mutations in p53, we found that silencing of the mutant p53 by RNA interference induced evident inhibition of cell proliferation and viability, which was related to the induction of G2 phase cell cycle arrest and apoptosis. Moreover, our study also showed that the p53-targeting siRNA cooperated with cisplatin in the inhibition of bladder cancer cells.ConclusionsThese findings suggest that RNA interference targeting mutant p53 may be a promising therapeutic strategy for the treatment of bladder cancer.

Abstract 598: Grape seed extract targets bladder cancer cells via initiating both apoptotic and autophagic machineries

Abstract Bladder cancer is the fifth most common malignancy in the US; statistical reports for 2011 estimate ∼69,250 new bladder cancer cases and 14,990 associated deaths in the US alone. Of all types of cancers, bladder cancer has an unusually high propensity for recurring after treatment, making it the most expensive cancer to treat on a per patient basis. Thus, discovery and development of agents, especially natural, non-toxic, dietary- agents, which target growth and development of bladder cancer cells, might provide opportunities to reduce the incidence of bladder cancer pre-neoplastic lesions and/or prevent the progression to carcinoma. In this regard, several pre-clinical studies conducted by us and others have established strong anti-cancer efficacy of one such phytochemical agent: grape seed extract (GSE) in various malignancies including prostate, colon, skin, lung etc. Based on these previous reports, in the present study, we sought to establish the efficacy of GSE in bladder cancer and to delineate the mechanistic effects involved in its protective effect. The cell culture studies revealed that indeed GSE has the potential to target bladder cancer as indicated by a significant (P&amp;lt;0.05-0.001) dose and time dependant inhibitory effect (20-60% cell death, 24-48 h) of GSE (25-100 µg/ml) on the viability of two different urinary bladder cancer cell lines viz., HT-B9 and T-24, representing stage I and stage II bladder cancer, respectively. The observed cell death was apoptotic in nature as determined by AnnexinV/PI staining, which was also confirmed by increase in the expression levels of cleaved caspase-3 and -9. Further, we also observed that exposure of urinary bladder cancer cells to GSE causes a dose and time dependant increase in intense cytoplasmic vacuolization, which under electron microscopy was confirmed to be due to an enlargement of mitochondrial organelle as well as due to the presence of autophagosomes. Through detailed in vitro studies; we also examined and defined that GSE targets expression of various molecules regulating apoptosis, as indicated by decreased levels of anti-apoptotic molecules Bcl-2 and Mcl-1. Interestingly, the increase in apoptotic response co-related with increased autophagic events as evidenced by tracking the dynamics of LC3-II within the cells. Together, these results indicate that autophagy might be the major event initiated during GSE treatment of bladder cancer cells, driving the cells towards an apoptotic death pathway; however, more mechanistic studies are warranted in future to confirm our hypothesis. Nevertheless, the findings of the present study are highly significant in establishing, for the first time, that GSE could suppress bladder cancer growth and progression, by targeting both apoptotic and autophagic machineries. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 598. doi:1538-7445.AM2012-598

891 VITAMIN D AND BCG IMMUNOTHERAPY

You have accessJournal of UrologyBladder Cancer: Basic Research II1 Apr 2012891 VITAMIN D AND BCG IMMUNOTHERAPY Jong-Wei Hsu, Edward Messing, and Yi-Fen Lee Jong-Wei HsuJong-Wei Hsu Rochester, NY More articles by this author , Edward MessingEdward Messing Rochester, NY More articles by this author , and Yi-Fen LeeYi-Fen Lee Rochester, NY More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2012.02.986AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Bacillus Calmette-Guérin (BCG), a vaccine against tuberculosis (TB), has been used and proved to be one of the most effective treatments for superficial bladder cancer (BCa). However, the mechanisms of BCG action have not been completely understood, which limits the improvement of BCG therapy. Vitamin D deficiency has been associated with the high risk of TB infection, and the beneficial effect of UV exposure in TB patients was proven to be mediated via activation of vitamin D signals of innate immune cells. Thus, vitamin D signals might be involved in mediating BCG immunotherapy for BCa. METHODS We applied a transwell migration assay to examine vitamin D signal¡ߣs impact on the BCG-mediated innate immune cells¡ߣ migration. The corresponding pro-inflammatory cytokines induced by vitamin D/vitamin D receptor were screened by cytokine array and confirmed by ELISA. Preclinical evaluation of efficacy of intravesical BCG with 1£\,25-dihydroxyvitamin D3 (1,25-VD) therapy was performed using Nbutyl- N-(4-hydroxybutyl)-nitrosamine (BBN)-induced BCa mouse model. RESULTS ELISA assay revealed that treatment of BCa cells with 1,25-VD promotes BCG-induced interleukin 8 (IL-8) production, consequently inducing the migration of neutrophil-like cells, HL-60, and monocyte-like cells, THP-1. This 1,25-VD-mediated IL-8-induced innate immune cells migration was blocked by IL-8 neutralized antibody. These in vitro studies suggest that 1,25-VD could improve BCG responsiveness by enhancing recruitment of innate immune cells to promote cytotoxic killing of BCa cells. More importantly, our in vivo data revealed that intravesical treatment of 1,25-VD improves BCG efficacy where 1,25-VD+BCG treatment increased the survival rate of mice with BCa induced by BBN compared to BCG treatment alone. CONCLUSIONS The results of these studies indicate that 1,25-VD signaling is critical for success of BCG immunotherapy and co-treatment with 1,25-VD will enhance BCG's anti-tumor effects in BCa. © 2012 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 187Issue 4SApril 2012Page: e362-e363 Advertisement Copyright & Permissions© 2012 by American Urological Association Education and Research, Inc.MetricsAuthor Information Jong-Wei Hsu Rochester, NY More articles by this author Edward Messing Rochester, NY More articles by this author Yi-Fen Lee Rochester, NY More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...