Bladder Cancer T24 Research Articles

Reduction of mitomycin C resistance in human bladder cancer T24 cells by knocking-down ras oncogene

Reduction of mitomycin C resistance in human bladder cancer T24 cells by knocking-down ras oncogene

Cinobufacini promotes apoptosis of bladder cancer cells by influencing the expression of autophagy-related genes.

The effects of cinobufacini on apoptosis of human bladder cancer T24 cells and the expression of autophagy-related genes and proteins were studied. The human bladder cancer T24 cells were selected, and the inhibitory effect of cinobufacini on the proliferation of human bladder cancer cells was detected by cell viability assay. Flow cytometry and Hoechst staining were used to detect the changes in the apoptosis of bladder cancer cells after being treated with cinobufacini; the changes in the expression levels of human bladder cancer cell apoptosis-related genes and proteins, cleaved caspase-3, Bax, B-cell lymphoma-2 (Bcl-2) and autophagy-related genes and proteins, p62, light chain 3 (LC3) and autophagy-related protein 7 (Atg7) after treatment with cinobufacini were detected by western blot analysis and reverse transcription polymerase chain reaction (RT-PCR). 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay. The results showed that 0.1 mg/ml cinobufacini significantly inhibited the proliferation of human bladder cancer cells cultured in vitro (P<0.01), and it was dose- and time-dependent. Both flow cytometry and Hoechst staining showed that cinobufacini promoted the apoptosis of cells (P<0.01), and the level of cell apoptosis increased with the increase of drug concentration. Both western blot analysis and RT-PCR showed that cinobufacini could decrease the expression level of Bcl-2 in T24 cells (P<0.01), increase the expression levels of Bax and cleaved caspase-3 (P<0.01), increase the ratio of Bax/Bcl-2 (P<0.01), upregulate the expression level of the angiotensin-related protein p62 (P<0.01), reduce the ratio of LC3-II/I (P<0.01) and decrease the expression level of Atg7 (P<0.01). After treatment with rapamycin, the expression levels of Bcl-2, Bax, cleaved caspase-3, autophagy-related genes and proteins, p62, LC3-II/I and Atg7 were similar to those in the control group. Cinobufacini can inhibit the autophagy activation of bladder cancer cells, thus promoting apoptosis of bladder cancer T24 cells and inhibiting the proliferation of T24 cells, which may provide a theoretical basis for the development of new anti-bladder cancer drugs.

Highly Fluorescent Chiral N‐S‐Doped Carbon Dots from Cysteine: Affecting Cellular Energy Metabolism

AbstractCysteine‐based chiral optically active carbon dots (CDs) and their effects on cellular energy metabolism, which is vital for essential cellular functions, have been barely reported. A green and effective synthesis strategy for chiral N‐S‐doped CDs (fluorescence quantum yield ca. 41.26 %) based on hydrothermal treatment of l‐ or d‐cysteine at as low as 60 °C has been developed. This suggested that cysteine was instable in aqueous solutions and acts as a warning for high‐temperature synthesis of nanomaterials using cysteine as stabilizer. Human bladder cancer T24 cells treated with l‐CDs showed up‐regulated glycolysis, while d‐CDs had no similar effects. In contrast, no disturbance to the basal mitochondrial aerobic respiration of T24 cells was caused by either chiral CD.

Highly Fluorescent Chiral N-S-Doped Carbon Dots from Cysteine: Affecting Cellular Energy Metabolism.

Cysteine-based chiral optically active carbon dots (CDs) and their effects on cellular energy metabolism, which is vital for essential cellular functions, have been barely reported. A green and effective synthesis strategy for chiral N-S-doped CDs (fluorescence quantum yield ca. 41.26 %) based on hydrothermal treatment of l- or d-cysteine at as low as 60 °C has been developed. This suggested that cysteine was instable in aqueous solutions and acts as a warning for high-temperature synthesis of nanomaterials using cysteine as stabilizer. Human bladder cancer T24 cells treated with l-CDs showed up-regulated glycolysis, while d-CDs had no similar effects. In contrast, no disturbance to the basal mitochondrial aerobic respiration of T24 cells was caused by either chiral CD.

Knockdown of long noncoding RNA FGFR3- AS1 induces cell proliferation inhibition, apoptosis and motility reduction in bladder cancer.

To study the expression pattern of long non-coding RNA FGFR3 antisense transcript 1(FGFR3-AS1) and the cell proliferation inhibition, apoptosis, and motility changes induced by silencing FGFR3-AS1 in bladder cancer. The differential expression levels of FGFR3-AS1 and FGFR3 in tumor tissues and paired normal tissues were determined using Real-Time qPCR in a total of 36 patients diagnosed with bladder cancer (urothelial carcinoma). Pearson's coefficient correlation was used for expression correlation assay. Expression differences of FGFR3-AS1 were analyzed according to grading and staging. FGFR3 protein was detected by western blot assay. Human bladder cancer T24 and 5637 cell lines were transiently transfected with FGFR3-AS1-specific siRNA or negative control siRNA. The cell proliferation changes of transfected bladder cancer cells were determined using CCK-8 assay. Apoptosis caused by knockdown of FGFR3-AS1 was evaluated using ELISA assay. Motility changes induced by knockdown of FGFR3-AS1 were measured using wound healing assay and transwell assay. Both FGFR3-AS1 and FGFR3 were overexpressed in bladder cancer tissues compared to matched normal tissues. They were also positively expressed in bladder cancer. FGFR3-AS1 expression levels were higher in high grade tumors than those in low grade tumors. FGFR3-AS1 expression levels were higher in invasive tumors than those in non-invasive tumors. Cell proliferation inhibition, increased apoptosis, and decreased motility were observed in FGFR3-AS1 siRNA-transfected T24 and 5637 cell lines. FGFR3-AS1 plays an oncogenic role in human bladder cancer. Knockdown of FGFR3-AS1 may provide a potential new therapeutic approach to this disease.

Long non-coding RNA HNF1A-AS1 promotes cell viability and migration in human bladder cancer.

Bladder cancer is among the most frequent types of genitourinary malignancies and results in high morbidity and mortality. Despite considerable progress in methods of bladder cancer diagnosis and treatment, the detailed underlying molecular mechanisms of bladder cancer remain unclear, and the prognosis of patients remains poor. In the present study, the role of long non-coding (lnc)RNA hepatocyte nuclear factor 1A (HNF1A)-antisense RNA (AS)1 in bladder cancer progression was examined in vitro. HNF1A-AS1 was overexpressed in clinical bladder cancer tissues and cultured bladder cancer cells. Specific short hairpin RNAs against HNF1A-AS1 knocked down the expression of HNF1A-AS1, and thus suppressed the viability and migration/invasion abilities of the cells. Additionally, the depletion of HNF1A-AS1 in bladder cancer T24 and 5637 cell lines also induced cell accumulation in G0/G1 phase with the cell cycle analysis. Overall, these data suggest that lncRNA HNF1A-AS1 may be a potential regulator of bladder cancer tumorigenesis, and provide novel insight into the diagnosis and treatment of bladder cancer.

EGCG inhibited bladder cancer T24 and 5637 cell proliferation and migration via PI3K/AKT pathway.

Epigallocatechin-3-gallate (EGCG), the bioactive polyphenol in green tea, has been demonstrated to have various biological activities. We previously found that EGCG inhibited SW780 tumor growth by down-regulation of NF-κB and MMP-9. This study demonstrated that EGCG inhibited bladder cancer T24 and 5637 cell proliferation and migration via PI3K/AKT pathway, without modulation of NF-κB. Our results showed that treatment of EGCG resulted in significant inhibition of cell proliferation by induction of apoptosis, without obvious toxicity to normal bladder SV-HUC-1 cells. EGCG also inhibited 5637 and T24 cell migration and invasion at 25–100 μM. Western blot confirmed that EGCG induced apoptosis in T24 and 5637cells by activation of caspases-3 and PARP. Besides, EGCG up-regulated PTEN and decreased the expression of phosphorylated PI3K, AKT in both T24 and 5637 cells. In addition, animal study demonstrated that EGCG (100 mg/kg, i.p. injected daily for 4 weeks) decreased the tumor weight in mice bearing T24 tumors by 51.2%, as compared with the untreated control. EGCG also decreased the expression of phosphorylated PI3K and AKT in tumor, indicating the important role of PI3K/AKT in EGCG inhibited tumor growth. When AKT was inhibited, EGCG showed no obvious effect in cell migration in T24 and 5637 cells. In conclusion, our study elucidated that EGCG was effective in inhibition of T24 and 5637 cell proliferation and migration, and presented evidence that EGCG inhibited cell proliferation and tumor growth by modulation of PI3K/AKT pathway.

Targeting bladder cancer using activated Tcells armed with bispecific antibodies.

In the present study, we aimed to investigate whether EGFR or HER2 may serve as a target for Tcell-mediated immunotherapy against human bladder cancer. Expression of EGFR and HER2 was detected on the surface of bladder cancer cells, including Pumc-91 and T24cells, and their chemotherapeutic drug-resistant counterparts. Activated Tcells (ATCs) were generated from healthy PBMCs that were stimulated by the combination of anti-CD3 monoclonal antibody and anti‑CD28 monoclonal antibody in the presence of interleukin-2 for 14days. The ATCs were then armed with chemically hetero-conjugated anti-CD3xanti-EGFR (EGFRBi-Ab) or anti-CD3xanti-HER2 (HER2Bi-Ab). The specific cytolytic activity of ATCs armed with EGFRBi-Ab or HER2Bi-Ab against human bladder cancer cells was evaluated by lactate dehydrogenase activity assays invitro. In contrast to unarmed ATCs, EGFRBi-Ab-armed ATCs and HER2Bi-Ab-armed ATCs showed increased cytotoxic activity against bladder cancer cells. Moreover, Bi-Ab-armed ATCs expressed higher levels of activating marker CD69 and secreted more IFN-γ, TNF-α and IL-2 than did unarmed ATCs. EGFRBi-Ab- or HER2Bi-Ab-armed ATCs may provide a promising immunotherapy for bladder cancer.

Anticancer effect of miR-96 inhibitor in bladder cancer cell lines

The present study aimed to investigate the role of microRNA-96 (miR-96) in the proliferation, invasion and apoptosis of bladder cancer cell lines, and the associated mechanisms. The expression of miR-96 and human ether-à-go-go-related (HERG1) potassium channel in the normal uroepithelium SV-HUC-1 cell line, and bladder cancer T24 and 5637 cell lines were examined using reverse transcription-polymerase chain reaction or/and western blotting. Transfection with miR-96 inhibitor or scrambled control (SC) was used to study the biological activities of miR-96 in bladder cancer cell lines. MTT, flow cytometric and Transwell assays were applied to detect cell viability, apoptosis and invasion, respectively. A dual-luciferase reporter assay was applied to determine the association between miR-96 and HERG1 expression. As demonstrated, miR-96 was highly expressed in the two bladder cancer cell lines, particularly in T24 cells. Following transfection with miR-96 inhibitor, miR-96 expression was significantly reduced in the T24 cell line, compared with SC. The miR-96 inhibitor suppressed cell proliferation and invasion, promoted apoptosis and arrested the cell cycle at the G1 phase. Consistently, HERG1 was also highly expressed in the two bladder cancer cell lines at the mRNA and protein level, but not in the normal uroepithelium cell line. The miR-96 inhibitor also significantly decreased HERG1 expression compared with SC. The results of the dual-luciferase reporter assay indicated that miR-96 directly targeted wild-type HERG1. In conclusion, miR-96 inhibitor exhibited anticancer effects on bladder cancer cells by inhibiting proliferation and invasion of cells, and promoting their apoptosis. HERG1 was an important target of miR-96. These results provided experimental evidence supporting miR-96 as a therapeutic target for patients with bladder cancer.

Enhanced anticancer effect by combination of proteoglucan and Vitamin K3 on bladder cancer cells

Aim: Mushroom extract, PDF, is a bioactive proteoglucan with anticancer/antitumor activity, and Vitamin K3 (VK3) is a synthetic VK derivative with antitumor activity as well. An unconventional approach using these two agents was tested to see their anticancer effects on bladder cancer cells in vitro. Methods: Human bladder cancer T24 cells were treated with PDF, VK3, or their combination, and cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay. To explore the anticancer mechanism, cell cycle and epigenetic alterations were specifically studied. Results: PDF ≥ 500 μg/mL led to a ~ 35% reduction in cell viability while VK3 had little effects. However, when PDF (300 μg/mL) was combined with VK3 (5 μM), a ~ 75% cell viability reduction was attained. This specific combination induced a G1 cell cycle arrest with the downregulation of G1-specific regulators. In addition, histone deacetylase was inactivated while histones 3 and 4 were highly acetylated. Two apoptotic regulators were significantly activated with PDF/VK3 combination as well. Conclusion: The specific combination of PDF and VK3 appears to potentiate anticancer effect on T24 cells. This is primarily attributed to a G1 cell cycle arrest with chromatin modifications, ultimately leading to apoptosis. Thus, the PDF/VK3 combination may offer a potential therapeutic option for bladder cancer.

BTG2 is a tumor suppressor gene upregulated by p53 and PTEN in human bladder carcinoma cells.

Although widely deemed as a tumor suppressor gene, the role of B‐cell translocation gene 2 (BTG2) in bladder cancer is still inconclusive. We investigated the role and regulatory mechanism of BTG2 in bladder cancer. BTG2 expression in human bladder tissues was determined by RT‐qPCR and immunoblotting assays. Expressions of BTG2 and PTEN in bladder carcinoma cells were determined by immunoblotting, RT‐qPCR, or reporter assays. The 3H‐thymidine incorporation assay, flow cytometry, and the xenograft animal model were used to determine the cell growth. BTG2 expression was lower in human bladder cancer tissues than normal bladder tissues. Highly differentiated bladder cancer cells, RT4, expressed higher BTG2 than the less‐differentiated bladder cancer cells, HT1376 and T24. Overexpression of BTG2 in T24 cells inhibited cell growth in vitro and in vivo. Camptothecin and doxorubicin treatments in RT‐4 cells or transient overexpression of p53 into p53‐mutant HT1376 cells induced p53 and BTG2 expression. Further reporter assays with site‐mutation of p53 response element from GGGAAAGTCC to GGAGTCC within BTG2 promoter area showed that p53‐induced BTG2 gene expression was dependent on the p53 response element. Ectopic PTEN overexpression in T24 cells blocked the Akt signal pathway which attenuated cell growth via upregualtion of BTG2 gene expression, while reverse effect was found in PTEN‐knockdown RT‐4 cells. PTEN activity inhibitor (VO‐OHpic) treatment decreased BTG2 expression in RT‐4 and PTEN‐overexpressed T24 cells. Our results suggested that BTG2 functioned as a bladder cancer tumor suppressor gene, and was induced by p53 and PTEN. Modulation of BTG2 expression seems a promising way to treat human bladder cancer.

Modulating autophagy to influence apoptosis induced by dihydroartemisinin in bladder cancer cell lines in vitro

Objective To investigate whether combined treatment of dihydroartemisinin (DHA) and autophagy inhibitors results in enhancing the cyotoxic effects of DHA upon human bladder cells, T24, in vitro, and to explore the mechanism of autophagy on DHA-induced T24 bladder cancer cell death. Methods DHA with or without autophagy inhibitor (3-MA, 5 mmol/L) was added to treat human bladder cell, T24, at varying concentrations (0, 12.5, 25.0, 50.0, 100.0 μmol/L)for 24, 48 and 72 h. To measure cell viability, we performed methyl thiazol tetrazolium (MTT) assay.We examined apoptosis by flow cytometry.After administration of the lower concentration of DHA for 24 h, the morphologic analysis of T24 cells autophagy after monodansylcadaverine staining was performed using fluorescence microscopy.Transmission electron microscope was employed to observed the subcellular structure of T24 cells.Western blotting analysis was used to detect the expression levels of autophagy-related proteins [microtubule-associated protein 1 light chain 3 (LC3), Beclin-1 and p62] and apoptosis marker proteins [procaspase-3, total poly adenosine diphosphate-ribose polymerase (PARP) and cleaved-PARP]. Results The results showed that DHA significantly inhibited the proliferation and induced apoptosis of T24 bladder cancer cells in a dose-and time-dependent manner as compared with control group (P=0.003) (The inhibition rate of 12.5 μmol/L concentration of DHA were 5.79%, 6.61%, 11.09% at the time of 24, 48 and 72 h; the inhibition rate of 25.0 μmol/L concentration of DHA were 11.25%, 15.32%, 21.35% at the time of 24, 48 and 72 h; the inhibition rate of 50.0 μmol/L concentration of DHA were 27.16%, 30.66%, 47.58%, at the time of 24, 48 and 72 h; the inhibition rate of 100.0 μmol/L concentration of DHA were 41.52%, 57.52%, 74.29%, at the time of 24, 48 and 72 h. The apoptosis rate of 12.5 μmol/L concentration of DHA were 3.66%, 6.44%, 9.78%, at the time of 24, 48 and 72 h; the apoptosis rate of 25.0 μmol/L concentration of DHA were 7.02%, 13.4%, 17.55%, at the time of 24, 48 and 72 h; the apoptosis rate of 50.0 μmol/L concentration of DHA were 20.53%, 26.97%, 38.69%, at the time of 24, 48 h and 72 h; the apoptosis rate of 100.0 μmol/L concentration of DHA were 29.82%, 39.26%, 44.92%, at the time of 24, 48 and 72 h). The combination of the autophagy inhibitor 3-MA and DHA resulted in enhancing DHA-induced apoptosis cell death in T24 bladder cancer cells.Under the fluorescence microscopy, we found that condensed punctate autophagic vacuoles stained by monodansylcadaverin (MDC) were located in the cytoplasm.Electron microscopy demonstrated formation of multiple autophagic vacuoles in T24 cells after DHA treatment.Western blotting analysis showed that the expression levels of both autophagy-related proteins (LC3, beclin-1) and apoptosis marker proteins (cleaved-PARP) were increased compared with the control group.On the contrary, the levels of p62, procaspase-3 and total PARP were decreased.Moreover, the combined treatment with 3-MA increased the detection of cleaved-PARP, while decreasing procaspase-3 and total PARP, compared to either group treated with a single agent. Conclusion DHA has great anti-cancer efficacy on human bladder cancer T24 cells.Autophagy was induced and a crucial survival mechanism for cancer cells during DHA-induced apoptotic cell death in human bladder cancer cells. Key words: Urinary bladder neoplasms; Apoptosis; Autophagy; Dihydroartemisinin

Anti-oncogenic activities of cyclin D1b siRNA on human bladder cancer cells via induction of apoptosis and suppression of cancer cell stemness and invasiveness.

The human cyclinD1 gene generates two major isoforms, cyclinD1a and cyclinD1b, by alternative splicing. Although cyclin D1b mRNA is hardly expressed in normal human tissues, it is detected in approximately 60% of human bladder cancer tissues and cell lines. In the present study, to assess the therapeutic ability of cyclinD1b siRNA, we investigated the anti-oncogenic effects of cyclinD1b siRNA on human bladder cancer cell lines, SBT31A and T24, which express cyclinD1b mRNA. Knockdown of cyclinD1b by specific siRNA significantly suppressed cell proliferation, invitro cell invasiveness and three-dimensional (3D) spheroid formation in these cell lines. Cell cycle analyses revealed that cyclinD1b siRNA inhibited G1-S transition in T24 cells. The increase in the sub-G1 fraction, morphological aberrant nuclei with nuclear fragmentation and caspase-3 activity in SBA31A cells treated with cyclinD1b siRNA showed that cyclinD1b siRNA induced apoptosis. In T24 cells, knockdown of cyclinD1b suppressed the expression of the stem cell marker CD44. Knockdown of cyclinD1b or CD44 suppressed the invasiveness under 3D spheroid culture conditions and expression of N-cadherin. Tumor growth of SBT31A cells in nude mice was significantly inhibited by cyclinD1b siRNA. Taken together, these results indicate that knockdown of cyclinD1b suppresses the malignant phenotypes of human bladder cancer cells via induction of apoptosis and suppression of cancer cell stemness and epithelial-mesenchymal transition. Applying cyclinD1b siRNA will be a novel therapy for cyclinD1b-expressing bladder cancers.

Effects of microRNA-200c on the proliferation and cell cycle of bladder cancer T24 cells

Objective To investigate the influence of microRNA (miRNA, miR)-200c on cell proliferation and cell cycle in bladder cancer T24 cells and the preliminary mechanism. Methods The expression of miR-200c in bladder cancer cell lines was detected using real-time quantitative polymerase chain reaction (Real-time PCR). T24 cells were transfected with miR-200c mimics (MU group) or nonsense control (NC group) individually to generate negative control group and over-expression group. Then cell proliferation was assessed using cell counting kit-8 (CCK-8) kits at 24, 48 and 72 h while cell cycle distribution was detected by flow cytometry. Finally, Western blotting was applied to detect the levels of cell cycle-related proteins. Results MiR-200c was up-regulated in bladder cancer cell lines EJ (13.204±0.946), T24 (18.259±0.776), 253J (8.333±0.543), 5637 (6.594±0.326), RT4 (11.348±0.513) as compared with normal urothelial cell line SV-Huc (1.053±0.078, P=0.000). MiR-200c mimics promoted cell proliferation at 48 and 72 h after transfection [(1.485±0.131 and 2.396±0.110) vs. (1.158±0.182 and 1.893±0.216), P=0.050, P=0.023] and induced G1/S checkpoint release (P=0.013). Meanwhile, the protein levels of cyclin-dependent kinase (CDK)2 (1.44±0.10 vs. 0.86±0.15, P=0.005), CDK4 (3.05±0.17 vs. 1.24±0.07, P=0.001), E2F3 (1.81±0.22 vs. 1.18±0.20, P=0.021) were upregulated after transfection. Conclusion MiR-200c promotes cell proliferation and induces G1/S transition in bladder cancer T24 cell lines via regulating cell cycle-related proteins. Key words: Bladder cancer; MicroRNA-200c; Cell cycle; Proliferation

Autophagy induction enhances tetrandrine-induced apoptosis via the AMPK/mTOR pathway in human bladder cancer cells.

Tetrandrine, a bisbenzylisoquinoline alkaloid isolated from the roots of Stephania tetrandra is a traditional Chinese medicine and exerts anticancer capacity in various types of cancers. Previous studies have shown that tetrandrine induces apoptosis in bladder cancer cells via activation of the caspase cascade. However, the underlying mechanism has not yet been reported. Autophagy is a cellular process involved in the degradation of broken proteins and aging organelles to maintain homeostasis. Recent studies indicate that autophagy is implicated in cancer therapy. Thus, we focused on the correlation between autophagy and apoptosis upon tetrandrine treatment in human bladder cancer cells. Firstly, our results observed a marked increase in autophagic double-membrane vacuoles and fluorescent puncta of red fluorescence protein-green fluorescence protein-LC3 (GRP-RFP-LC3) upon tetrandrine treatment, as evidenced by transmission electron microscopy and confocal fluorescence microscopy. Secondly, the expression of LC3-II was increased in tetrandrine-treated T24 and 5637cells in a time-and concentration-dependent manner. Subsequently, downregulation of p62 and LC3 turnover assay further confirmed that tetrandrine induced autophagic flux in bladder cancer T24 and 5637 cells. Thirdly, the protein levels of phosphorylated-AMP-activated protein kinase (AMPK) and phosphorylated-acetyl-coenzymeA carboxylase (ACC) were upregulated in the tetrandrine-treated cells, while the mammalian target of rapamycin (mTOR)-related proteins were downregulated. Moreover, AICAR, a common AMPK activator, further increased the expression the LC3-II, while AMPK inhibitor compoundC partially reversed the LC3-II protein levels in bladder cancer T24 cells. Finally, AICAR significantly reinforced the growth inhibition and apoptosis induction of tetrandrine in T24 and 5637 cells, while compoundC had an opposite effect, suggesting that AMPK-mediated autophagy enhanced the cytotoxic and pro-apoptosis effect of tetrandrine in human bladder cancer cells. Taken together, the present study showed that tetrandrine induced autophagy in human bladder cancer cells by regulating the AMPK/mTOR signaling pathway, which contributed to the apoptosis induction by tetrandrine, indicating that tetrandrine may be a potential anticancer candidate for the treatment of bladder cancer, and autophagy may be a possible mechanism for cancer therapy.

Isoliquiritigenin pretreatment attenuates cisplatin induced proximal tubular cells (LLC-PK1) death and enhances the toxicity induced by this drug in bladder cancer T24 cell line

Cisplatin is an effective antineoplastic agent widely used in the treatment of solid tumors, however, it induces nephrotoxicity. Cisplatin-induced nephrotoxicity is associated with increased reactive oxygen species (ROS) production and decreased antioxidant system defense in kidney. Isoliquiritigenin (IsoLQ) is a chalcone, which is characterized by its antioxidant and antiinflammatory properties. Herein, we investigated the protective effect of IsoLQ on LLC-PK1 proximal tubular cells against cisplatin-induced death and its effect on the antineoplastic activity of cisplatin on bladder cancer T24 cell line. It was found that pretreatment with IsoLQ attenuates cisplatin-induced cell death, ROS production, and activation of caspase-3. IsoLQ also induced heme oxygenase-1 (HO-1) expression that may be associated with nuclear factor (erythroid-derived 2)-like 2 (Nrf2) nuclear translocation. The protective effect of IsoLQ was abrogated by tin mesoporphyrin (SnMP), an HO inhibitor. Further, bilirubin and carbon monoxide releasing molecule-2 also showed a protective effect against cisplatin-induced cell death. In addition, IsoLQ induced in a dose-dependent way, death of T24 cells and exacerbated cisplatin-induced cell death. These results suggest that IsoLQ has a protective effect on cisplatin-induced toxicity in LLC-PK1 cells, in part through induction of HO-1, without interfering with the antineoplastic activity of this agent in T24 cells.

Role of 5-Aza-CdR in mitomycin-C chemosensitivity of T24 bladder cancer cells.

Chemotherapeutic insensitivity is one of key obstacles to effectively treating muscle invasive bladder cancer. 5-Aza-2'-deoxycytidine (5-Aza-CdR) has been identified as a tumor suppressive agent in various types of cancer. The aim of the present study was to identify the effects of 5-Aza-CdR on the mitomycin-C (MMC) chemosensitivity of T24 bladder cancer cells and investigate the underlying mechanisms. T24 cells were treated with a combination of MMC and 5-Aza-CdR at various concentrations. The rates of proliferation and apoptosis were assessed by an MTT assay and flow cytometry, respectively. The expression of drug resistance-associated proteins, including P-glycoprotein (P-gp) and multidrug resistance-associated protein 1 (MRP1), and autophagy-associated proteins, including beclin 1, nucleoporin 62 (p62) and autophagy protein 5 (ATG5) were detected with western blotting. Treatment with 5-Aza-CdR significantly promoted the MMC chemosensitivity of T24 cells. The proliferation of T24 cells was significantly inhibited with increasing 5-Aza-CdR concentration, whereas apoptosis was significantly increased. This was associated with the decreased expression of P-gp, MRP1, beclin 1, p62 and ATG5. In conclusion, 5-Aza-CdR enhanced MMC chemosensitivity in bladder cancer T24 cells, which may be caused by the suppression of drug resistance- and autophagy-associated proteins.

Epigallocatechin gallate inhibits the growth and promotes the apoptosis of bladder cancer cells.

Epigallocatechin gallate (EGCG) has been revealed to inhibit the proliferation and induce the apoptosis of several types of tumor, in addition to inhibiting DNA methyltransferase activity, leading to CpG demethylation. Tissue factor pathway inhibitor 2 (TFPI-2) expression is downregulated in bladder cancer. The present study revealed that this downregulation was partly due to hypermethylation of the TFPI-2 gene promoter, which was decreased by EGCG treatment. In addition, the present study demonstrated that EGCG could inhibit the viability and invasion, and induce the apoptosis, of bladder cancer T24 cells. Furthermore, western blot analysis and reverse transcription-quantitative polymerase chain reaction analyses demonstrated that EGCG could upregulate the expression of TFPI-2. These results suggest that EGCG inhibits the growth and induces the apoptosis of bladder cancer cells through restoring TFPI-2 expression. Thus, EGCG is a potential therapeutic candidate for the treatment of bladder cancer.

Hsa-miR-429 promotes bladder cancer cell proliferation via inhibiting CDKN2B.

Background and ObjectivesHsa-miR-429 is increased in bladder cancer. Its roles in bladder cancer are poorly understood.MethodsThe expression levels of hsa-miR-429 and cyclin-dependent kinase inhibitor 2B (CDKN2B) were determined using Real-Time qPCR in a total of 50 patients with bladder cancer. Bladder cancer T24 and 5637 cells were transfected CDKN2B siRNA or hsa-miR-429 mimic. CDKN2B expression levels after transfection were detected by Real-Time qPCR and Western blot assay respectively. Binding sites between hsa-miR-429 and 3’-untranslated region of CDKN2B were confirmed by Dual luciferase reporter assay. Cell proliferation was evaluated using MTT and EdU assays. Cell apoptosis was determined using ELISA assay.ResultsHigher hsa-miR-429 expression levels were associated with higher tumor grade and stage. All patients with low hsa-miR-429 expression survived 5 years, while with high hsa-miR-429 expression, only 58% survived. Hsa-miR-429 and CDKN2B were inversely expressed in bladder cancer. Hsa-miR-429 mimic decreased the expression of CDKN2B at both mRNA and protein levels. The binding site was confirmed between hsa-miR-429 and 3’-untranslated region of CDKN2B. Up-regulation of hsa-miR-429 or down-regulation of CDKN2B promoted cell growth and decreased apoptosis.ConclusionsOur data suggest a mechanism for hsa-miR-429 to play oncogenic roles via inhibiting CDKN2B.

Effects of miR-1236-3p and miR-370-5p on activation of p21 in various tumors and its inhibition on the growth of lung cancer cells.

The mechanism of dsRNA-induced gene activation (RNAa) is being gradually unveiled. The plentiful evidence that it existed in mammalian species other than human demonstrated that dsRNA-mediated RNAa is a conservative phenomenon. Simultaneously, accumulating evidence suggested that microRNAs could activate gene expression by targeting promoter. Nevertheless, it is ambiguous whether microRNA-induced gene activation in different human cells is a common phenomenon. The study we performed verified that miR-1236-3p (miR-1236) and miR-370-5p can activate p21 expression in bladder cancer (BCa) T24, EJ cells, and non-small-cell lung carcinoma A549 cells, while in hepatocellular HepG2 cells both microRNAs cannot effectively induce the expression of P21WAF1/CIP1 (p21). In pancreatic cancer PANC-1 cells, only miR-370-5p had the potent abilities to induce p21 expression rather than miR-1236-3p. Unlike microRNA-mediated RNA activation, we can observe that dsP21-322 significantly activated p21 in above cells. Besides, we demonstrated that miR-1236 and miR-370 inhibited cyclin D1-CDK4/CDK6 pathway while upregulated E-cadherin expression by upregulation of p21. Overexpression of these two microRNAs in A549 induced cell-cycle arrest and cell senescence, delayed cell proliferation and colony formation, and inhibited migration and invasion. In conclusion, microRNA-mediated RNAa depends on the cell context, and miR-1236 and miR-370 can inhibit non-small-cell lung carcinoma cell growth by upregulating p21 expression in vitro.