Human Bladder Cancer EJ Cells Research Articles

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.

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.

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.

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.

Effects of exogenous hydrogen sulfide on the proliferation and invasion of human Bladder cancer cells.

The objective of this study is to evaluate the effects of exogenous hydrogen sulfide (H2S) on the proliferation and invasion of human bladder cancer cells. Human bladder cancer EJ cells were cultured and then treated with sodium bisulfide (NaHS) (100 μmol/L for low dosage, 200 μmol/L for moderate dosage, 400 μmol/L for high dosage). The differences on proliferation and invasion of EJ cells were detected among different groups with MTT and transwell invasion assays. The differences in the expression levels of matrix metalloproteinase 2 (MMP2) and MMP9 among the groups were detected with Western blot. Cell proliferation activity was elevated by exogenous NaHS, with significantly statistical difference compared to the blank control group (P < 0.05). With the increased NaHS concentration, the EJ cell proliferation activity presented a statistically significant trend of increase, showing comparative differences among different groups (P < 0.05). Exogenous NaHS could also improve the invasion ability of bladder cancer EJ cells, which was significantly enhanced with increased NaHS dose (P < 0.001). The levels of MMP2 and MMP9 in EJ cells were significantly upregulated with the increased NaHS dose (P < 0.001). Exogenous H2S may promote cell proliferation and invasion by upregulating the expression level of MMP2 and MMP9 in human bladder cancer EJ cells.

Long non-coding RNA ANRIL is up-regulated in bladder cancer and regulates bladder cancer cell proliferation and apoptosis through the intrinsic pathway

Antisense non-coding RNA in the INK4 locus (ANRIL) is a member of long non-coding RNAs and has been reported to be dysregulated in several human cancers. However, the role of ANRIL in bladder cancer remains unclear. This present study aimed to investigate whether and how ANRIL involved in bladder cancer. Our results showed up-regulation of ANRIL in bladder cancer tissues versus the corresponding adjacent non-tumor tissues. To explore the specific mechanisms, ANRIL was silenced by small interfering RNA or short hairpin RNA transfection in human bladder cancer T24 and EJ cells. Knockdown of ANRIL repressed cell proliferation and increased cell apoptosis, along with decreased expression of Bcl-2 and increased expressions of Bax, cytoplasmic cytochrome c and Smac and cleaved caspase-9, caspase-3 and PARP. However, no change of cleaved caspase-8 level was observed. Furthermore, in vivo experiment confirmed that knockdown of ANRIL inhibited tumorigenic ability of EJ cells in nude mice. Meanwhile, in accordance with in vitro study, knockdown of ANRIL inhibited expression of Bcl-2 and up-regulated expressions of Bax and cleaved caspase-9, but did not affect cleaved caspase-8 level. In conclusion, we first report that ANRIL possibly serves as an oncogene in bladder cancer and regulates bladder cancer cell proliferation and apoptosis through the intrinsic apoptosis pathway.

Fucoidan induces G1 arrest of the cell cycle in EJ human bladder cancer cells through down-regulation of pRB phosphorylation

Fucoidan, a sulfated polysaccharide found in marine algae and brown seaweeds, has been shown to inhibit the in vitro growth of human cancer cells. This study was conducted in cultured human bladder cancer EJ cells to elucidate the possible mechanisms by which fucoidan exerts its anti-proliferative activity, which until now has remained poorly understood. Fucoidan treatment of EJ cells resulted in dose-dependent inhibition of cell growth and induced apoptotic cell death. Flow cytometric analysis revealed that fucoidan led to G1 arrest in cell cycle progression. It was associated with down-regulation of cyclin D1, cyclin E, and cyclin-dependent-kinases (Cdks) in a concentration-dependent manner, without any change in Cdk inhibitors, such as p21 and p27. Furthermore, dephosphorylation of retinoblastoma protein (pRB) by this compound was associated with enhanced binding of pRB with the transcription factors E2F-1 and E2F-4. Overall, our results demonstrate that fucoidan possesses anticancer activity potential against bladder cancer cells by inhibiting pRB phosphorylation.

Silencing ubiquitin specific peptidase 22 inhibits the cell proliferation of human bladder cancer EJ cells via down-regulated Myc

Objective To investigate the role of silencing USP22 gene by aiRNA on the cell proliferation in human bladder cancer EJ cells.Methods USP22 aiRNA and NC-aiRNA were designed and synthesized.The structure of USP22 aiRNA had 15 bp sense sequence and 21 bp antisense sequence and was named USP22 aiRNA (15/21).The EJ cells were transfected with USP22 aiRNA (15/21) or NC-aiRNA for 48 h.The mRNA and protein levels of USP22,Myc,cyclin D1 and cyclin E1 were measured by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot,respectively.The proliferation rate of EJ cells was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-Diphenyl tetrazolium bromide (MTT) assay.The transcriptional activity of Myc gene was determined by luciferase reporter gene assay.The ability of Sp1 binding to Myc promoter was tested by a ChIP assay.Results Compared with control group,the mRNA and protein levels of USP22,Myc,cyclin D1 and cyclin E1 in USP22 aiRNA (15/21) group were reduced by (87.4±5.2)％ and (91.2±7.3)％,(69.2±4.3)％ and (61.0±6.6)％,(78.5±4.1)％ and (67.4±5.5) ％,(81.0±5.5)％ and (78.3 4.0)％ (P＜0.05).MTT assay demonstrated that the cell viability at 24,48,72,96 h in the USP22 aiRNA (15/21) group was (85.4±5.7)％,(71.3±8.4)％,(52.5±6.7)％ and (45.8±6.4) ％ (P＜0.05) and luc,iferase reporter gene assay showed that the transcriptional activity of Myc in USP22 aiRNA (15/21) group was decreased by (65.5±4.2) ％ (P＜0.05).Moreover,ChIP assay confirmed that the ability of Sp1 binding to the Myc promoter in USP22 aiRNA (15/21) group was inhibited by (48.0±3.2) ％ in comparison with control group (P＜0.05).Conclusions These results suggested that silencing USP22 inhibited the EJ cells proliferation by blocking Sp1 binding to the Myc promoter. Key words: Ubiquitin specific peptidase 22; Myc gene; Transcription factor Sp1; Transcription regulation; Bladder cancer

Kaempferol Promotes Apoptosis in Human Bladder Cancer Cells by Inducing the Tumor Suppressor, PTEN

Kaempferol (Kae), a natural flavonoid, is widely distributed in fruits and vegetables. Previous studies have identified Kae as a possible cancer preventive and therapeutic agent. We found Kae to exhibit potent antiproliferation and anti-migration effects in human bladder cancer EJ cells. Kaempferol robustly induced apoptosis in EJ cells in a dose-dependent manner, as evidenced by increased cleavage of caspase-3. Furthermore, we found Kae-induced apoptosis in EJ cells to be associated with phosphatase and the tensin homolog deleted on the chromosome 10 (PTEN)/PI3K/Akt pathway. Kae significantly increased PTEN and decreased Akt phosphorylation. Kae-induced apoptosis was partially attenuated in PTEN-knockdown cells. Our findings indicate that Kae could be an alternative medicine for bladder cancer, based on a PTEN activation mechanism.

JR6, a new compound isolated from Justicia procumbens, induces apoptosis in human bladder cancer EJ cells through caspase-dependent pathway

Ethnopharmacological relevanceNumerous efforts have been conducted in searching for effective agents against cancer, in particular from herbal medicines. Justicia procumbens is a traditional herbal remedy which was produced in the south-western and southern provinces of China and Taiwan province used to treat fever, pain, and cancer. Here, we identified a new compound 6′-hydroxy justicidin A (JR6) from Justicia procumbens, which showed obvious anti-cancer effects. Materials and methodsThe cytotoxicity activity was assayed using MTT and SRB. Intracellular ROS visualization and quantification were acquired by using a laser scanning confocal microscopy. Apoptosis was measured using a propidium iodide (PI) apoptosis detection kit by flow cytometry. Activation of caspases (caspase-3, caspase-8, and caspase-9) was evaluated respectively using GloMax luminescence detector and Caspase-Glo 3,8,9 assay kits. Loss of mitochondrial membrane potential was observed by microscopy using JC-1 dye. Quantitative real-time PCR analysis was employed to detect the expression of protein associated with cell death. ResultsJR6 remarkably inhibited growth in human bladder cancer EJ cells by decreasing cell proliferation, reduced the SOD activity, increased the content of reactive oxygen species (ROS), and induced apoptosis. Activation of caspase-8, caspase-9, and the subsequent activation of caspase-3 indicated that JR6 may be inducing intrinsic and extrinsic apoptosis pathways. Caspase-3, caspase-8, and caspase-9 inhibition rendered this extract ineffective, thus JR6-induced apoptosis is caspase-dependent. JR6 also disrupted the mitochondrial membrane potential (Δψm) and unregulated the Bax and p53 expressions in EJ cells. ConclusionThese observations suggest that JR6 induce apoptosis through caspase-dependent pathway in human bladder cancer EJ cells, emphasizing the importance of this traditional medicine and thus presents a potential novel alternative to bladder cancer therapy.

Apoptin Induces Apoptosis in Human Bladder Cancer EJ and BIU-87 Cells

To investigate whether apoptin is a apoptosis-inducing protein with a potential for bladder cancer therapy. We constructed a PCDNA3/Apoptin eukaryotic expression vector, and transfected this vector into bladder cancer cell lines BIU-87 and EJ, then observed the results by RT-PCR, transmission electron microscopy, MTT assay and the flow cytometry (TUNEL method). PCDNA3/Apoptin successfully induced a high level apoptosis in both bladder cancer cell lines, compared with the controls (p<0.05). Apoptin can induce high level apoptosis in human bladder cancer EJ and BIU-87 cells, which suggests a potential for human bladder cancer therapy.

Effect of membrane toxin 12 isolated from Naja naja atra on proliferation and invasion of human bladder cancer EJ cells

We investigated the effect of membrane toxin 12 (MT-12), isolated from Naja naja atra, a species of Chinese Cobra, on the proliferation and invasion of human bladder cancer EJ cells and studied its mechanisms using MTT assay, Transwell chamber invasion assay, scanning electron microscopy and flow cytometry. The results indicated that MT-12 inhibited the proliferation of bladder cancer cells in a dose-dependent manner. The half maximal inhibitory concentration (IC50) after 72 h was 0.66 µg/ml. The invasion of cells decreased with increasing doses of MT-12 (0.125-0.5 µg/ml, P<0.001). Expression of CXCR4 decreased with the effect of MT-12 for a particular concentration range (0.125-0.5 µg/ml). To conclude, MT-12 is capable of inhibiting the proliferation and invasion of bladder cancer EJ cells. This mechanism may be associated with reduced expression of CXCR4 protein.

Phosphatidylinositol 3-Kinase Inhibitor LY294002 Suppresses Proliferation and Sensitizes Doxorubicin Chemotherapy in Bladder Cancer Cells

Background: Phosphatidylinositol 3-kinase (PI3K)-AKT signaling is a well-characterized pathway involved in the control of cell proliferation, apoptosis and oncogenesis. LY294002 is a commonly used pharmacologic inhibitor which acts at the ATP-binding site of the PI3K enzyme, thus selectively inhibiting the PI3K-AKT nexus. The purpose of the present study was to examine whether PI3K inhibited by LY294002 had an effect on human bladder cancer cells. Methods: After treatment with LY294002, MTT assay, chemosensitivity test, colony formation assay, apoptosis assay and Western blot analysis were conducted in EJ cells. Result: EJ cells treated with LY294002 showed significant AKT phosphorylation suppression in a dose-response manner. Also, PI3K/AKT signaling inhibitor LY294002 suppressed cell proliferation and enhanced the chemosensitivity of doxorubicin in human bladder cancer EJ cells. Furthermore, LY294002 increased cell apoptosis to doxorubicin. Conclusion: The augmentation of doxorubicin with PI3K inhibitor LY294002 may resolve the multidrug resistance of bladder cancer, and this may be a new strategy for achieving tolerance for chemotherapeutic agents in bladder cancer therapy.

Phosphatidylinositol 3-Kinase Inhibitor LY294002 Suppresses Proliferation and Sensitizes Doxorubicin Chemotherapy in Bladder Cancer Cells

Background: Phosphatidylinositol 3-kinase (PI3K)-AKT signaling is a well-characterized pathway involved in control of cell proliferation, apoptosis and oncogenesis. LY294002 is a commonly used pharmacologic inhibitor which acts at the ATP-binding site of the PI3K enzyme, and thus selectively inhibits the PI3K-AKT nexus. The purpose of the study was to examine whether PI3K inhibited by LY294002 had effects in human bladder cancer cells. Methods: After treatment with LY294002, MTT assay, a chemosensitivity test, colony formation assay, apoptosis assay and Western blot analysis were conducted in EJ cells. Result: EJ cells treated with LY294002 showed significant AKT phosphorylation suppressing in a dose-response manner. Additionally, the PI3K/AKT signaling inhibitor LY294002 suppressed cell proliferation and enhanced chemosensitivity to doxorubicin in human bladder cancer EJ cells. Furthermore, LY294002 increased cell apoptosis to doxorubicin. Conclusion: The augmentation of doxorubicin with the PI3K inhibitor LY294002 may resolve the multidrug resistance of bladder cancer, and this may be a new strategy for achieving tolerance for chemotherapeutic agents in bladder cancer therapy.

EBAG9 is a tumor‐promoting and prognostic factor for bladder cancer

Upregulation of EBAG9 expression has been observed in several malignant tumors such as advanced breast and prostate cancers, indicating that EBAG9 may contribute to tumor proliferation. In the present study, we assess the role of EBAG9 in bladder cancer. We generated human bladder cancer EJ cells stably expressing FLAG-tagged EBAG9 (EJ-EBAG9) or empty vector (EJ-vector), and investigated whether EBAG9 overexpression modulates cell growth and migration in vitro as well as the in vivo tumor formation of EJ transfectants in xenograft models of BALB/c nude mice. EBAG9 overexpression promoted EJ cell migration, while the effect of EBAG9 to cultured cell growth was rather minimal. Tumorigenic experiments in nude mice showed that the size of EJ-EBAG9-derived tumors was significantly larger than EJ-vector-derived tumors. Loss-of-function study for EBAG9 using small interfering RNA (siRNA) in xenografts with parental EJ cells showed that the intra-tumoral injection of EBAG9 siRNA markedly reduced the EJ tumor formation compared with control siRNA. Furthermore, immunohistochemical study for EBAG9 expression was performed in 60 pathological bladder cancer specimens. Intense and diffuse cytoplasmic immunostaining was observed in 45% of the bladder cancer cases. Positive EBAG9 immunoreactivity was closely correlated with poor prognosis of the patients (p = 0.0001) and it was an independent prognostic predictor for disease-specific survival in multivariate analysis (p = 0.003). Our results indicate that EBAG9 would be a crucial regulator of tumor progression and a potential prognostic marker for bladder cancer.

Preparation of curcumin prodrugs and theirin vitro anti-tumor activities

The curcumin prodrugs, which could be selectively activated in tumor cells, were prepared to establish a basis for the targeted chemotherapy for cancer. On the basis of the molecular structure of curcumin, the N-maleoyl-L-valine-curcumin (NVC), N-maleoyl- glycine-curcumin (NGC) were chemically synthesized and identified by IR and NMR spectroscopy. After treatment with these two prodrugs for 6 - 24 h, the rates of growth inhibition on human bladder cancer EJ cells and renal tubular epithelial (HKC) cells were detected by MTT colorimetry. Our results showed that after the treatment with 20 micromol/L - 40 micromol/L NVC and NGC for 6 - 24 h, the growth inhibitory effects on EJ cells were 6.71% - 65.13% (P < 0.05), 10.96% - 73.01% (P < 0.05), respectively, in both dose- and time-dependent manners. When compared with the curcumin of same concentrations, the growth inhibitory effects of these two prodrugs on HKC cells were significantly decreased (P < 0.01). It is concluded that activation of curcumin prodrugs via hydrolysis functions of cellular esterase could inhibit the growth activities of tumor cells, and reduce the side effects on normal diploid cells. This provided a novel strategy for further exploration of tumor-targeted chemotherapeutic drugs.

Vitamin C inhibits p53-induced replicative senescence through suppression of ROS production and p38 MAPK activity

We previously reported that tumor cells expressing p53 increase intracellular levels of reactive oxygen species (ROS). In this study, we described an inhibitory effect of vitamin C on replicative senescence. Vitamin C was found to inhibit p53-induced senescence in human bladder cancer EJ cells. The senescence-like phenotype (SLP) induced by p53, which showed a morphological change and an irreversible cell cycle arrest, was not observed in vitamin C-treated EJ cells. In addition, vitamin C did not significantly affect normal cell proliferation. We investigated the molecular mechanisms of the inhibitory effect of vitamin C on the development of replicative senescence in EJ cells. We found that vitamin C inhibited this p53-induced ROS generation. Moreover, p38 kinase which was activated during p53-induced senescence was not observed in vitamin C-treated EJ cells. SB203580, a chemical inhibitor of p38 kinase, was found to consistently inhibit p53-induced senescence. Therefore, it is suggested that vitamin C inhibits p53-induced senescence by preventing ROS generation, which in turn leads to the activation of p38 MAPKinase. These results reveal the inhibitory mechanism of vitamin C on cellular senescence.