Bladder Cancer EJ Cells Research Articles

Biological function and regulatory mechanism of mta2 expression in bladder cancerous cells

Purpose: To study the function, regulation, and potential clinical use of mta2 gene in carcinoma of the bladder.
 Methods: Viral vector and si-mta2 were transfected into both T24 and EJ bladder cancer cells, for induction of overexpression and knockdown of metastasis-associated gene 2 (mta2), respectively. In vitro invasiveness and migratory potential of cell lines were assayed using Matrigel and Transwell procedures, respectively, under the two mta2 expression conditions.
 Results: Transwell migration data revealed that overexpressed mta2 protein significantly enhanced the migratory capacity of T24 bladder cancer cell line, while knockdown of mta2 significantly reduced the migration of EJ bladder cancer cell line (p < 0.01). Matrigel invasion assay showed that overexpression of mta2 protein significantly enhanced the invasive potential of T24 bladder cancer cell line, but knockdown of mta2 significantly (p < 0.01) reduced the invasive capacity of EJ bladder cancer cell line. There was a lower amount of E-cadherin protein in T24 bladder cancer cell line overexpressing mta2 than in cd511b-transfected and un-transfected cells, but N-cadherin protein level was significantly higher. Furthermore, there was a significantly higher amount of E-cadherin protein expression in EJ bladder cancer cell line with mta2 knockdown than in si-NC transfection and un-transfected cells (p < 0.01).
 Conclusion: Knockdown of mta2 inhibits the proliferation, migration and invasion of bladder cancer cell lines through a mechanism related to the inhibition of epithelial-mesenchymal transition-related process proteins.

Pro-oxidant response and accelerated ferroptosis caused by synergetic Au(I) release in hypercarbon-centered gold(I) cluster prodrugs

Medicinal applications of gold complexes have recently attracted attention due to their innovative antitumor mechanisms. In this work, two hypercoordinated carbon-centered gold clusters PAA4 and PAA5 are quantitatively synthesized by an intramolecular 6-exo-dig cyclization of polymetalated precursors. The on-bench and in vitro experimental studies demonstrate that the characteristic hypercarbon-tetragold(I) multi-center bonding in PAA4 and PAA5 not only guarantees their stability under common physiological conditions, but also facilitates a glutathione (GSH)-triggered prompt and synergetic release of active Au(I) ions in the GSH-overexpressed and acidic microenvironment of human bladder cancer EJ cells. The instantly massive release of coordination unsaturated Au(I) ions causes the efficient inhibition of thioredoxin reductases and then induces a rapid pro-oxidant response, consequently causing the occurrence of accelerated ferroptosis of EJ cells. As a result, these hypercarbon-centered gold(I) cluster prodrugs show high cytotoxicity to bladder cancer cell lines and thus exhibit a significant inhibition effect towards bladder tumors in vivo. Correlation of the synergetic domino dissociation of carbon-polymetal multi-center bonding in metal clusters with the accelerated ferroptosis of cancer cells provides a strategy for metallo-prodrugs and opens a broader prospect for the biological application of metal cluster compounds.

Betulinic acid induces autophagy-dependent apoptosis via Bmi-1/ROS/AMPK-mTOR-ULK1 axis in human bladder cancer cells.

Betulinic acid (BA), a pentacyclic triterpenoid isolated from tree bark, exhibits antitumor effects against solid malignancies and triggers autophagy and/or apoptosis in human cancer cells. Nonetheless, the relationship between autophagy and apoptosis and the potential modulatory actions of BA on autophagy-dependent bladder cancer cell death remain unclear. The present study showed that BA exposure significantly suppressed viability, proliferation, and migration of EJ and T24 human bladder cancer cells. These effects reflected caspase 3-mediated apoptosis and could be attenuated or abolished by inhibiting ROS production with N-acetyl-L-cysteine, inhibiting autophagy with chloroquine, or silencing ATG7 with targeted siRNA. BA-induced autophagy was evidenced by epifluorescence imaging of lentivirus-induced expression of mCherry-GFP-LC3B and increased expression of two autophagy-related proteins, LC3B-II and TEM. Moreover, enhanced AMPK phosphorylation and decreased mTOR and ULK-1 phosphorylation suggested BA activates autophagy via the AMPK/mTOR/ULK1 pathway. Accordingly, exposure to dorsomorphin (Compound C), an AMPK inhibitor, and AICAR, an AMPK activator, respectively inhibited and stimulated BA-induced autophagy in EJ and T24 cells. The effects of Bmi-1 overexpression in vitro and decreased Bmi-1 expression in BA-treated T24 cell xenografts in nude mice suggested that downregulation of Bmi-1 is the underlying mechanism in BA-mediated, autophagy-dependent apoptosis.

Long non-coding RNA PCAT6 regulates bladder cancer progression via the microRNA-143-3p/PDIA6 axis.

Although long non-coding (lnc)RNAs have been reported to be involved in the pathological development of bladder cancer, the functions of lncRNA prostate cancer-associated transcript 6 (PCAT6) and its underlying mechanism of action in bladder cancer remain unknown. The present study aimed to investigate the effect of PCAT6 in bladder cancer progression and explore its potential application as a novel treatment target. The expression of PCAT6 and miR-143-3p in bladder cancer tissues, adjacent normal tissues and cell lines was measured using reverse transcription-quantitative PCR. Fluorescence in situ hybridization assay was used to detect the subcellular localization of PCAT6. MTT, EdU, Transwell and wound healing assays were conducted to assess the biological function of PCAT6 on cell proliferation, migration and invasion. Putative binding sites between miR-143-3p and PCAT6 or PDIA6 were predicted using starBase, Lncbase and TargetScan analyzes. Dual-luciferase reporter assay was also used to confirm the potential binding between PCAT6 and miR-143-3p. RNA immunoprecipitation assay was performed to verify the possible interaction between PCAT6 and miR-143-3p. Western blotting was used to measure the expression of PDIA6. The results demonstrated that the expression levels of PCAT6 were upregulated in bladder cancer tissues relative to those in adjacent normal bladder tissues. Knockdown of PCAT6 served a role in suppressing the proliferation, migration and invasion of T24T and EJ bladder cancer cells. PCAT6 knockdown contributed to a reduction of PDIA6 expression at the mRNA and protein levels compared with that in negative control-transfected cells, whilst the miR-143-3p inhibitor partially mitigated this reduction effect. In addition, rescue experiments revealed that the miR-143-3p inhibitors reversed the effects of PCAT6 silencing on the malignant phenotypes of bladder cancer. Collectively, the results of the present study demonstrated that PCAT6 may serve an oncogenic role in bladder cancer via the miR-143-3p/PDIA6 axis. These results may provide a potential therapeutic target for the treatment of bladder cancer.

Aloperine Exerts Antitumor Effects on Bladder Cancer in vitro.

BackgroundHuman bladder cancer is the most common malignant tumor of the urinary system and one of the 10 most common tumors of the whole body. Although most patients with bladder cancer exhibit a good prognosis with standard treatment, effective therapies for patients with a recurrent or advanced bladder cancer are unavailable. Therefore, highly effective drugs to treat such patients need to be developed. Aloperine (ALO), a natural compound isolated from Sophora alopecuroides, has antitumor properties. However, the role of ALO in human bladder cancer remains unclear.MethodsIn the present study, MTT was used to detect the cytotoxic effect of ALO on human BC cell line EJ and human urothelium cell line SV-HUC-1cells. Meanwhile, in order to investigate the effects of ALO on the proliferation, apoptosis, migration, and invasion of BC EJ cells and its mechanism by Cell Counting Kit-8 (CCK-8) assay, immunofluorescence, Hoechst 33342 staining, wound scratch assay, transwell migration and invasion assay, Western blot analysis.ResultsALO can inhibit the proliferation and invasion of human bladder cancer EJ cells, and is low-toxic to human urothelium cells. Moreover, it can promote cellular apoptosis in vitro. Further analysis demonstrated the involvement of Caspase-dependent apoptosis following ALO treatment. ALO also downregulated the protein expression levels of Ras, p-Raf1 and p-Erk1/2.ConclusionALO is a potential drug for human bladder cancer therapy.

In Vitro and In Vivo Antitumor Efficacy of Hizikia fusiforme Celluclast Extract against Bladder Cancer

Various physiological benefits have been linked to Hizikia fusiforme (HF), an edible brown seaweed. Here, fucose-containing sulfated polysaccharides were extracted from celluclast-processed HF (SPHF) and their antitumor efficacy against bladder cancer was evaluated in vitro and in vivo. SPHF possesses high sulfated polysaccharide and fucose contents and free radical scavenging activities compared to those of celluclast-processed HF extracts (CHF). SPHF inhibited bladder cancer EJ cell proliferation via G1-phase cell cycle arrest. This was due to the induction of p21WAF1 expression associated with the downregulation of CDKs and cyclins. Moreover, JNK phosphorylation was identified as an SPHF-mediated signaling molecule. SPHF treatment also hindered the migration and invasion of EJ cells by inhibiting MMP-9 expression, which was attributed to the repression of transcriptional binding to NF-κB, AP-1, and Sp-1 in the MMP-9 promoter region. In an animal study, SPHF treatment suppressed EJ tumor growth in xenograft mice similarly to cisplatin. Furthermore, no toxicity signs were found after weight loss assessment, biochemical tests, and organ tissue immunostaining during oral administration of 20–200 mg/kg SPHF for 20 days. Therefore, our study demonstrates the antitumor efficacy of SPHF in vitro and in vivo, thus highlighting its potential for bladder cancer treatment development.

Desloratadine, a Novel Antigrowth Reagent for Bladder Cancer.

Desloratadine, a potent antagonist for human histamine H1 receptor, has been revealed to exhibit antihistaminic activity and anti-inflammatory activity. However, it is not yet known whether desloratadine has any effect on the biological behaviors of tumor cells. In this study, we aimed to investigate the effects of desloratadine on cell growth and invasion in bladder cancer EJ and SW780 cells in vitro. We observed that desloratadine inhibited cell viability of EJ and SW780 cells in a dose- and time-dependent manner. Desloratadine treatment was also revealed to suppress colony-formation ability and induce cell cycle arrest at G1 phase in EJ cells. Desloratadine promoted cell apoptosis via modulating the expression of Bcl-2, Bax, cleaved caspase 3, and cleaved caspase 9 in EJ and SW780 cells. Western blot resulted showed that desloratadine also impaired the expression of autophagy-related proteins, such as Beclin 1, P62, and LC3I/II in EJ and SW780 cells; while autophagy inhibitor LY294002 reversed the effects of desloratadine on these proteins. Moreover, desloratadine remarkably attenuated cell migration and invasion. Furthermore, we illustrated that desloratadine downregulated the expression of N-cadherin, Vimentin, Snail1, and Snail2, while upregulated the expression of E-cadherin in EJ and SW780 cells in vitro. The level of interleukin 6 was reduced in desloratadine-treated cells, while upregulation of interleukin 6 significantly abolished the anticancer activity of desloratadine in cell invasion and Bcl-2, Bax, Beclin1, LC3-I/II, N-cadherin, and E-cadherin expression in EJ cells. Taken together, our data suggest a potential anticancer activity of desloratadine on cell growth and invasion for bladder cancer, which may be mediated by diminishing the epithelial-to-mesenchymal transition and interleukin 6.

Carnosine exerts antitumor activity against bladder cancers in vitro and in vivo via suppression of angiogenesis

Carnosine, a naturally occurring dipeptide, was recently reported to exhibit anticancer activity; however, the molecular mechanisms and regulators underlying its activity against tumor-associated angiogenesis remain unidentified. In this study, we evaluated the in vitro and in vivo antitumor effects of carnosine in EJ bladder cancer cells and EJ-xenografted BALB/c nude mice, respectively. In addition, in vitro capillary tube formation of HUVECs, ex vivo aortic ring and in vivo Matrigel plug assays were employed to examine the antiangiogenic potential of carnosine. Carnosine significantly inhibited EJ cell proliferation. Flow cytometric and immunoblot analyses indicated that carnosine modulated regulators of the G1 cell cycle phase, including cyclin D1, CDK4 and p21WAF1. The mitogen-activated protein kinases, ERK and p38, but not JNK or AKT, responded to carnosine. Carnosine inhibited the migratory and invasive potential of EJ cells by inhibiting MMP-9 activity, which was associated with suppression of binding activity of NF-κB, SP-1 and AP-1. In xenograft tumors, carnosine exhibited antitumor activity equivalent to cisplatin, but no weight loss occurred in carnosine-treated mice. In HUVECs, carnosine inhibited VEGF-mediated proliferation, colony tube formation, migration and invasion. The antiangiogenic activity of carnosine was partially due to the suppression of VEGFR-2-mediated ERK/AKT/eNOS signaling and MMP-2. Furthermore, using aortic ring and Matrigel plug assays, we confirmed the antiangiogenic activity of carnosine. Given that targeting tumor-associated angiogenesis is a proven effective therapeutic strategy, our results may provide valuable information for the development of preventive or therapeutic agents for bladder cancer patients.

Synergistic antitumour effects of triptolide plus 10-hydroxycamptothecin onbladder cancer

BackgroundHydroxycamptothecin (HCPT) is used as a clinical chemotherapy regimen to treat bladder cancer, but more efficacious novel combination treatments are needed. MethodsCultured bladder cancer cell lines EJ and UMUC3 were treated with triptolide (TPL) and/or HCPT. A flow cytometry approach was used to detect cell cycle phase, apoptosis and reactive oxygen species. Western blotting was used to measure CDK4, CDK6, CyclinD1, catalase, Caspase8 and Bcl-xl protein levels in control, TPL treatment, HCPT treatment and TPL plus HCPT treatment bladder cancer cells. AKT pathway proteins, including AKT and p-AKT, were also detected by western blotting. UMUC3 cells treated with DMSO, HCPT, TPL and HCPT plus TPL were injected subcutaneously into mice (n = 3 per group). ResultsThe flow cytometry and western blot results indicated that compared to TPL or HCPT treatment alone, combination treatment of HCPT and TPL significantly induced cell cycle arrest at the G1 phase via suppressing CDK4, CDK6 and CyclinD1 in the EJ and UMUC3 bladder cancer cell lines. HCPT and TPL combination treatment also significantly increased the apoptosis rate and apoptosis-related protein levels (Caspase8 and Bcl-xl). Levels of the AKT pathway-related proteins AKT/p-AKT were significantly lower in EJ and UMUC3 cells treated with TPL and UMUC3 than in those cells treated with TPL or HCPT alone. TPL plus HCPT treatment significantly reduced bladder tumour sizes in vivo on the seventh and tenth days. ConclusionsCompared to TPL or HCPT treatment alone, TPL plus HCPT combination treatment had significantly better anticancer effects.

Hydrangenol suppresses VEGF-stimulated angiogenesis by targeting p27KIP1-dependent G1-cell cycle arrest, VEGFR-2-mediated signaling, and MMP-2 expression

ABSTRACTWe previously reported that hydrangenol has potent antitumor activity against human bladder cancer EJ cells. Here, we investigated the antiangiogenic activity of hydrangenol using in vitro and ex vivo models. Treatment with hydrangenol significantly inhibited the proliferation of vascular endothelial growth factor (VEGF)-induced HUVECs in a concentration-dependent manner (EC50 = 10 μM). Flow cytometry analysis revealed that hydrangenol suppressed the VEGF-induced inhibition of G1-cell cycle phase and also decreased cyclin D1, cyclin E, CDK2, and CDK4 levels. Hydrangenol-mediated arrest in the G1-cell cycle phase was associated with p27KIP1 level, but not p21WAF1 or p53 level. Hydrangenol also significantly inhibited VEGFR-2-mediated signaling pathways including ERK1/2, AKT, and endothelial nitric oxide synthase. Interestingly, immunoprecipitation assay demonstrated that the inhibition of VEGFR-2 activation was independent of VEGF binding, thereby suggesting an allosteric regulation of hydrangenol against VEGFR-2. Additionally, hydrangenol inhibited migration, invasion, and capillary-like tubular formation in VEGF-stimulated HUVECs. Zymography and immunoblot analyses revealed that these inhibitory activities were partially owing to the VEGF-induced inhibition of matrix metalloproteinase-2 activity. Finally, VEGF-mediated microvessel sprouting was inhibited in the presence of hydrangenol in ex vivo aortic ring assay. Taken together, hydrangenol possesses a potent antiangiogenesis potential; thus we believe that hydrangenol may be developed as a therapeutic reagent to treat angiogenesis-mediated diseases.

Kaempferol Attenuates ROS-Induced Hemolysis and the Molecular Mechanism of Its Induction of Apoptosis on Bladder Cancer.

Bladder cancer has become the most common malignant urinary carcinoma. Studies have shown that significant antioxidant and bladder cancer-fighting properties of several plant-based diets like Psidium guajava, ginger and amomum, are associated with their high kaempferol content. In this paper, we evaluated the antioxidant and anticancer activities of kaempferol and its mechanism of induction to apoptosis on bladder cancer cells. Our findings demonstrated that kaempferol showed an obvious radical scavenging activity in erythrocytes damaged by oxygen. Kaempferol promoted antioxidant enzymes, inhibited ROS generation and lipid peroxidation and finally prevented the occurrence of hemolysis. Additionally, kaempferol exhibited a strong inhibitory effect on bladder cancer cells and high safety on normal bladder cells. At the molecular level, kaempferol suppressed EJ bladder cancer cell proliferation by inhibiting the function of phosphorylated AKT (p-AKT), CyclinD1, CDK4, Bid, Mcl-1 and Bcl-xL, and promoting p-BRCA1, p-ATM, p53, p21, p38, Bax and Bid expression, and finally triggering apoptosis and S phase arrest. We found that Kaempferol exhibited strong anti-oxidant activity on erythrocyte and inhibitory effects on the growth of cancerous bladder cells through inducing apoptosis and S phase arrest. These findings suggested that kaempferol might be regarded as a bioactive food ingredient to prevent oxidative damage and treat bladder cancer.

Effect of microRNA-497-5p on epithelial-to-mesenchymal transition in bladder cancer

Objective To observe the expression of microRNA-497 (miR-497) in bladder cancer cells and investigate its possible functions in epithelial-mesenchymal transition (EMT) of bladder cancer. Methods The expression of miR-497 in bladder cells and human normal bladder tissue were measured by real-time quantitative polymerase chain reaction (RT-qPCR). MiR-497 mimics and miR-497 inhibitor were transfected into bladder cancer EJ cells and RT-qPCR was used to analyze the effect of transfection. Then, morphological changes of cells were observed under the inverted microscope and the wound healing assay, transwell invasion assay were used to detect the cell migration and invasion of bladder cancer EJ cells transfected by miR-497 mimics and miR-497 inhibitor. cytometry. Finally, Western blotting was applied to detect the levels of EMT-related proteins. Results MiR-497 levels were obviously down-regulated in bladder cancer cells (5637, EJ, BIU-87, TCCSUP and T24) in comparison to human bladder pillon biochemical cell (SV-HUC-1), the difference was statistically significant (P=0.000). Compared with negative control, overexpression of miR-497 significantly suppressed EJ cells migration [ (74.00±3.61)% vs. (25.33±6.11)%] and invasion [ (219±17) cells vs. (109±3) cells], and EJ cells turned into epithelioid cells morphology, when compared with the control group. Moreover, opposite results were obtained when miR-497 inhibitor was transfected into EJ cells. Compared with mimics NC, the protein expression of E-cadherin was upregulated (P=0.000), while N-cadherin and vimentin downregulated (P=0.001, P=0.000)in miR-497 mimics transfection group. Conclusion MiR-497 may play an important role of EMT in bladder cancer. Key words: MicroRNA-497; Bladder cancer; Migration; Invasion; Epithelial-to-mesenchymal transition

Hydrangenol inhibits the proliferation, migration, and invasion of EJ bladder cancer cells via p21WAF1-mediated G1-phase cell cycle arrest, p38 MAPK activation, and reduction in Sp-1-induced MMP-9 expression.

Hydrangenol is a dihydroisocoumarin that is mainly obtained from Hydrangea macrophylla. Recently, hydrangenol has garnered attention since several studies have reported that it has anti-inflammatory, anti-allergic, anti-diabetic, and anti-malarial activities. However, there have been few studies on the effect of hydrangenol on oncogenesis. In this study, we evaluated the anti-cancer activity of hydrangenol against the EJ bladder cancer cell line. Hydrangenol significantly inhibited the proliferation of EJ cells in a dose-dependent manner with an IC50 of 100 µM. Flow cytometry and immunoblotting experiments indicated that EJ cells were arrested in the G1-phase of the cell cycle and showed reduced expression of CDK2, CDK4, cyclin D1, and cyclin E mediated via the upregulation of p21WAF1. Hydrangenol increased the phosphorylation of p38 MAPK without affecting the phosphorylation of ERK and JNK. In addition, hydrangenol significantly inhibited the migratory and invasive activities of EJ cells by suppressing the enzymatic activity of MMP-9. Electrophoretic mobility shift assays suggested that the inhibition of MMP-9 activity by hydrangenol was attributable to its suppression of the Sp-1 transcription factor binding activity. This study is the first report on the mode of action of hydrangenol as an inhibitor of bladder cancer. We believe that these results provide novel insights that could aid the development of hydrangenol-based chemotherapeutic agents.

Effects of paclitaxel combined with miR-448 on growth and proliferation of bladder cancer EJ cells.

Bladder cancer is one of the most common malignant tumors of the urinary system characterized by a high recurrence rate after treatment. Paclitaxel is a new type of anti-neoplastic agent used for treatment by inhibiting the proliferation of bladder cancer cells. This study aimed to explore the mechanism of paclitaxel on the inhibition of bladder cancer cell proliferation by applying paclitaxel combined with miR-448 on bladder cancer cells. Bladder cancer EJ cells were divided into 5 groups, including control group, paclitaxel group, negative control (NC) group, microRNA-448 (miR-448) mimic group, and paclitaxel combined with miR-448 mimic group. Cell apoptosis was tested by flow cytometry. Cell proliferation was determined by cell counting kit 8 (CCK8) assay. The regulation of miR-448 on B cell lymphoma 2 (Bcl-2) gene was assessed by dual luciferase reporter assay. Bcl-2 protein expression was detected by Western blot. The apoptotic rate of cells was significantly increased, while the cell proliferation ability was significantly reduced in paclitaxel group and miR-448 mimic group compared with control (p<0.05). Cell apoptotic rate in paclitaxel combined with miR-448 mimic group was markedly higher than that of paclitaxel group (p<0.05). MiR-448 can bind to the 3'UTR region of Bcl-2 mRNA and regulate the expression of Bcl-2. The expression of Bcl-2 protein in paclitaxel group and miR-448 mimic group was significantly lower than that in control group and higher than paclitaxel combined with miR-448 mimic group (p<0.05). Paclitaxel combined with miR-448 promoted EJ cell apoptosis and inhibited cell proliferation by suppressing Bcl-2 gene expression.

Inhibitory effect of Au@Pt-NSs on proliferation, migration, and invasion of EJ bladder carcinoma cells: involvement of cell cycle regulators, signaling pathways, and transcription factor-mediated MMP-9 expression.

BackgroundAlthough the diverse biological properties of nanoparticles have been studied intensively, research into their mechanism of action is relatively rare. In this study, we investigated the molecular mechanisms of the anticancer activity of heterometallic Au@Pt-nanoseeds (NSs) against bladder cancers.Materials and methodsMode of action of Au@Pt-NSs was investigated through MTT assay, flow cytometry analysis, Western immunoblots, real-time qPCR, wound-healing migration and invasion assays, zymography, and electrophoretic mobility shift assay (EMSA).ResultsTreatment with Au@Pt-NSs significantly inhibited the proliferation of EJ cells in a dose-dependent manner by inducing G1 phase cell cycle arrest. Among the regulators associated with the G1 cell cycle phase, CDK2, CDK4, cyclin D1, cyclin E, and p21WAF1 were shown to participate in the inhibitory pathways of Au@Pt-NSs. In addition, treatment with Au@Pt-NSs led to upregulation of phospho-p38 MAPK and downregulation of phospho-AKT in EJ cells. Interestingly, Au@Pt-NSs inhibited the migratory and invasive potential of the cells, which was attributed to the suppression of the enzymatic activity of matrix metalloproteinase-9 (MMP-9). Using MMP-9-specific oligonucleotides, we showed that transcription factors such as NF-κB and Sp-1 were responsible for the MMP-9-mediated metastatic potential of EJ cells.ConclusionAu@Pt-NSs significantly limited the progression, migration, and invasion of bladder cancer EJ cells. Our data represent a novel insight into developing cisplatin-like chemotherapeutic reagents with fewer side effects and provide useful information on molecular markers to monitor patients under Au@Pt-NSs-based chemotherapy.

Anti-proliferative benefit of curcumol on human bladder cancer cells via inactivating EZH2 effector

We investigated the molecular mechanism of curcumol-induced apoptosis in bladder cancer cells. The mitochondrial membrane potential was measured using JC-1 staining. ROS generation of bladder cancer cells was determined using the DCFH staining method. The apoptosis of bladder cancer cells was examined using the Annexin V-FITC and PI double-staining method. Enforced expression of EZH2 in bladder cancer cells was accomplished by transfecting an EZH2 expression plasmidinto EJ and T24 cells. siRNAs targeting EZH2 were used to inhibit endogenous expression of EZH2. Curcumol dose-dependently inhibited proliferation and colony formation and induced apoptosis in EJ and T24 bladder cancer cells. These effects correlated with decreased accumulation of EZH2. In addition, suppression of EZH2 enhanced the inhibitory effects of curcumol on cell growth and colony formation and increased curcumol-induced apoptosis. Conversely, enforced expression of EZH2 ameliorated the inhibitory effects of curcumol on cell growth and colony formation and decreased curcumol-induced apoptosis in EJ and T24 cells. We also found that suppression of EZH2 induced ROS generation and MMP loss in both EJ and T24 cells. Conversely, up-regulation of EZH2 suppressed ROS generation and MMP loss. Our data indicate that curcumol inhibits proliferation and induces apoptosis by targeting EZH2 and modulating the mitochondrial apoptosis pathway.

Cell-penetrating peptide conjugates of gambogic acid enhance the antitumor effect on human bladder cancer EJ cells through ROS-mediated apoptosis.

BackgroundGambogic acid (GA) is the main active ingredient of resin gamboges and possesses anti-cancer activity toward various human cancer cells. However, clinical application of GA has been limited by its poor aqueous solubility and dose-limiting toxicities. Cell-penetrating peptides (CPPs) are widely used to deliver anti-cancer drugs into cancer cells and to enhance the water solubility of drugs.PurposeThe object of this study was to synthesize peptide-drug conjugates in which the cell-penetrating peptide TAT (trans-activator of transcription) was conjugated to GA and evaluated the anti-cancer activity of this GA-CPP conjugate (GA-TAT) in EJ bladder cancer cells.MethodsGA is built onto the TAT, and the GA-TAT conjugates are cleaved from the solid support and purified via HPLC. The equilibrium solubility of GA-TAT was measured using the shake-flask method. The effects of GA-TAT on EJ cell viability and proliferation were determined by MTT assay, Edu assay and colony formation assay, respectively. After treated with 1.0 μM GA-TAT for 24 h, the apoptosis rate of EJ cells were detected by Acridine orange/ethidium bromide (AO/EB) assay and flow cytometry assay. The proteins of caspase-3 (processing), caspase-9 (processing), Bcl-2 and Bax were analyzed by Western blotting, and the intracellular reactive oxygen species (ROS) production was evaluated by a reactive oxygen species assay.ResultsIn contrast to free GA, the solubility of GA-TAT in water was significantly improved. Meanwhile, GA-TAT significantly increased EJ cellular uptake, toxicity and apoptosis. Mechanistic analysis revealed that GA-TAT enhanced the anti-cancer effect of GA against EJ cells through ROS-mediated apoptosis. The results were demonstrated that GA-TAT increased the ROS level in EJ cells, and N-acetyl-L-cysteine (NAC; a well-known ROS scavenger) inhibited GA-TAT-induced ROS generation and apoptosis. Additionally, GA-TAT activated caspase-3 and caspase-9 and down-regulated the Bcl-2/Bax ratio, but these effects were largely rescued by NAC.ConclusionGA-TAT has outstanding potential for promoting tumor apoptosis and exhibits promise for use in bladder cancer therapy.

Long noncoding RNA GAS5 promotes bladder cancer cells apoptosis through inhibiting EZH2 transcription

Aberrant expression of long noncoding RNA GAS5 in bladder cancer (BC) cells was identified in recent studies. However, the regulatory functions and underlying molecular mechanisms of GAS5 in BC development remain unclear. Here, we confirmed that there was a negative correlation between GAS5 level and bladder tumor clinical stage. Functionally, overexpression of GAS5 reduced cell viability and induced cell apoptosis in T24 and EJ bladder cancer cells. Mechanistically, GAS5 effectively repressed EZH2 transcription by directly interacting with E2F4 and recruiting E2F4 to EZH2 promoter. We previously reported that miR-101 induced the apoptosis of BC cells by inhibiting the expression of EZH2. Interestingly, the present study showed that downregulation of EZH2 by GAS5 resulted in overexpression of miR-101 in T24 and EJ cells. Furthermore, the level of GAS5 was increased under the treatment of Gambogic acid (GA), a promising natural anti-cancer compound, whereas knockdown of GAS5 suppressed the inhibitory effect of GA on cell viability and abolished GA-induced apoptosis in T24 and EJ cells. Taken together, our findings demonstrated a tumor-suppressor role of GAS5 by inhibiting EZH2 on transcriptional level, and additionally provided a novel therapeutic strategy for treating human bladder cancer.

Effect of curcumol on proliferation, apoptosis and enhancer of zeste homolog 2 expression in bladder cancer EJ cells

Objective To observe the effect of curcumol on proliferation, apoptosis and enhancer of zeste homolog 2 (EZH2) expression in human bladder cancer line cell EJ, and to explore its molecular mechanism. Methods The bladder cancer EJ cells were treated with different concentrations of curcumol (12.5, 25.0, 50.0, and 100.0 mg/L) and the control group was given 1% anhydrous ethanol. The inhibitory effect of curcumol on the growth of EJ cells was evaluated by cell counting kit-8 (CCK-8) assay. Special morphological changes of apoptosis were observed by Hoechst 33258 flourescence staning. Flow cytometry technique was used to detect the apoptosis rate of each experimental group. The expression of EZH2 mRNA and protein was examined with reverse transcriptase-polymerase chain reaction (RT-PCR) and Western boltting. Results EJ cells proliferation was obviously inhibited by curcumol in a time- and concentration-dependent manner. The inhibiton rate was (32.94±0.93)%, (46.75±1.28)%, (58.16±0.76)% and (69.13±1.94)%, and the apoptosis rate was (3.60±0.46)%, (6.70±1.83)%, (16.20±1.61)% and (28.70±2.65)% in curcumol-treated (12.5, 25.0, 50.0, and 100.0 mg/L) groups respectively. A typical form of apoptosis was detected in the high concentration of curcumol by fluorescent inverted phase contrast microscope. As compared with the control group, with the increases in concentrations of curcumenol, the expression of EZH2 mRNA and protein was gradually declined. Conclusion Curcumol could significantly inhibit the growth of EJ cells and induce the apoptosis, which may be related to the down-regulation of EZH2. Key words: Curcumol; Bladder cancer; Enhancer of zeste homolog 2

HSPA6 augments garlic extract-induced inhibition of proliferation, migration, and invasion of bladder cancer EJ cells; Implication for cell cycle dysregulation, signaling pathway alteration, and transcription factor-associated MMP-9 regulation.

Although recent studies have demonstrated the anti-tumor effects of garlic extract (GE), the exact molecular mechanism is still unclear. In this study, we investigated the molecular mechanism associated with the inhibitory action of GE against bladder cancer EJ cell responses. Treatment with GE significantly inhibited proliferation of EJ cells dose-dependently through G2/M-phase cell cycle arrest. This G2/M-phase cell cycle arrest by GE was due to the activation of ATM and CHK2, which appears to inhibit phosphorylation of Cdc25C (Ser216) and Cdc2 (Thr14/Tyr15), this in turn was accompanied by down-regulation of cyclin B1 and up-regulation of p21WAF1. Furthermore, GE treatment was also found to induce phosphorylation of MAPK (ERK1/2, p38MAPK, and JNK) and AKT. In addition, GE impeded the migration and invasion of EJ cells via inhibition of MMP-9 expression followed by decreased binding activities of AP-1, Sp-1, and NF-κB motifs. Based on microarray datasets, we selected Heat shock protein A6 (HSPA6) as the most up-regulated gene responsible for the inhibitory effects of GE. Interestingly, overexpression of HSPA6 gene resulted in an augmentation effect with GE inhibiting proliferation, migration, and invasion of EJ cells. The augmentation effect of HSPA6 was verified by enhancing the induction of G2/M-phase-mediated ATM-CHK2-Cdc25C-p21WAF1-Cdc2 cascade, phosphorylation of MAPK and AKT signaling, and suppression of transcription factor-associated MMP-9 regulation in response to GE in EJ cells. Overall, our novel results indicate that HSPA6 reinforces the GE-mediated inhibitory effects of proliferation, migration, and invasion of EJ cells and may provide a new approach for therapeutic treatment of malignancies.