HNK Induces Apoptosis Of T24 Bladder Cancer Cells Via Upregulation Of Caspase-3, -9, And Inhibition Of NF-κB, Akt And ERK Expression, Produces Synergistic Effect On Induction Of Apoptosis Of T24 Bladder Cancer Cells In Combination With HCPT

Objective:to investigate the potential molecular mechanism of the induction of apoptosis of T24 bladder cancer cells by HNK in vitro. to ascertain synergistic effect on induction of apoptosis and inhibition of proliferation of T24 bladder cancer cells by HNK and HCPT.Methods:T24 bladder cancer cells were divided into four groups:namely, treated with HNK, treated with HCPT. treated with HNK and HCPT, and untreated. IC10 of T24 bladder cancer cells treated with HNK and HCPT were detected by MTT assay, and then, the experimental concentrations spectrum of HNK and HCPT were defined according to IC10. The cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal calf serum (FCS) and 4 mM/L of glutamine. The morphological characteristics of T24 bladder cancer cells were examined microscopically at the 5th day of culture, viability of T24 bladder cancer cells was detected by MTT assay at 24h.48h and 72h of culture, respectively, the percentage of apoptotic T24 bladder cancer cells was determined by Annexin/PI staining and flow cytometric analysis. The expression of cleaved caspase-3, capase-9, phosphorylated NF-κB p65. Akt and ERK was analyzed by western blot assay.Results:Treated with experimental concentrations spectrum of HNK, T24 bladder cancer cells grew well, only slight T24 bladder cancer cells death phenomenon was observed Morphologically in 25uM HNK group. treated with 1.25ug/ml HCPT, growth of T24 bladder cancer cells was partially inhibited. treated with gradient concentrations of HNK and 1.25ug/ml HCPT. growth of T24 bladder cancer cells was significantly inhibited, in a HNK concentration-dependent manner. the viability of T24 bladder cancer cells was uninhibited by MTT assay in HNK group. the viability of T24 bladder cancer cells was also uninhibited at 24h of culture in HCPT group, whereas significantly inhibited at 48h and 72h of culture,in a time-dependent manner. treated with HNK and HCPT. the viability of T24 bladder cancer cells was significantly inhibited in both HNK concentration-dependent and time-dependent manner, inhibition of the viability of T24 bladder cancer cells in HNK and HCPT group had statistical significance as compared to HNK group and HCPT group. the percentage of apoptotic T24 bladder cancer cells determined by Annexin/PI staining and flow cytometric analysis had no statistical significance at lower concentration in HNK group as compared to control group, but apoptosis of T24 bladder cancer cells was significantly induced by higher concentration of HNK(25uM). apoptosis of T24 bladder cancer cells was significantly induced in HCPT group, and furthermore, apoptosis of T24 bladder cancer cells was even significantly induced in HNK and HCPT group in HNK concentration-dependent manner. induction of apoptosis of T24 bladder cancer cells in HNK and HCPT group had statistical significance as compared to HNK group and HCPT group. expression of Caspase-3,-9 was lower, while expression of NF-KB-p65、Akt、ERK higher in T24 bladder cancer cells. expression of cleaved Caspase-3,-9、NF-κB-p65、Akt、ERK had no statistical significance as compared to control group. expression of cleaved Caspase-3，-9 was induced and expression of phosphorylated NF-KB-p65、Akt、ERK was inhibited in HNK group. expression of cleaved Caspase-3，-9 was significantly induced and expression of phosphorylated NF-κB-p65, Akt. ERK was significantly inhibited in HNK and HCPT group in a HNK concentration-dependent manner.Conclusion:HNK induces apoptosis of T24 bladder cancer cells via upregulation of expression of cleaved caspase-3,-9，and inhibition of expression of phosphorylated NF-κB-p65,Akt and ERK, and produces synergistic effect in combination with HCPT on induction of apoptosis and inhibition of viability of T24 bladder cancer cells.