The Effect And Mechanisms In Growth Inhibition And Apoptosis Of HMBA And CINN To CNE2Cells

Objective:To study the effect and mechanisms in growth inhibition and apoptosis of CNE2cells treated by HMBA/CINN in cellular and molecular level.Methods:After being treated by HMBA, cell growth and morphology of CNE2cells were observed under microscope. The cell growth and cell cycles were detected by MTT and flow cytometry. Cell morphology of apoptosis were stained by Hoechst33258and Acridine orange (AO) respectively. Early apoptosis of CNE2cells was assessed by flow cytometry by using annexin V-FITC/PI. The expression of KLF6mRNA was detected by RT-PCR. The expression of KLF6, p53, cyclinD1and c-Jun proteins were detected by immunohistochemistry after being treated by CINN.Results:After being treated by HMBA, the number of adherent cells decreased obviously with the increasing concentration of HMBA. Proliferation of CNE2cells inhibited by HMBA could be observed in a dose-and time-dependent manner. The inhibition rate increased with HMBA concentration increased and time extended. The cell morphology converged:in control groups, cells were in such shapes as oval and round;while they turned into irregular shapes after being treated by HMBA, cytoplasm abundant and nucleoplasm proportion became larger. The concentration of5.0mmol/L was chosen as the best concentration to do the follow-up research work. Results of flow cytometry are as the following:in control group, after being cultured24h, the rate of G1phase was59.5%, while S phase was27.5%and12.9%in G2phase; G1phase increased to64.9%, S phase to28.3%and6.7%for G2phase after being treated by5.0mmol/L HMBA in24h. With time growth, the number of G1phase cells increased to67.5%,28.5%for S phase and G2phase cells decreased to4.04%<0.05) in48h. Cells were arrested at G1phase after72h. When came to96h, G1phase was60.2%, S phase was32.2%and G2phase was7.63%, which showed a more obvious effect of G1phase arrested. Three methods were conducted to detect apoptosis:stained Hoechst33258to get apoptosis cells in heavy-blue color, nuclear fragmentation could be seen in some cells. Apoptosis bodies could be seen by AO. Early stage cell apoptosis appeared after being treated for12h tested by Annexin V-FITC/PI (p<0.05). The expression of KLF6mRNA increased after being treated by HMBA for72h(p<0.05).The expression of KLF6, p53, cyclin D1and c-Jun proteins were detected by immunocytochemistry after being treated by CINN, the results showed that the proteins expression of KLF6and p53increased (p<0.05), while the expression of c-Jun and cyclin D1decreased (p<0.05)Conclusions:Treated with HMBA, cell growth of CNE2cells was inhibited, cells showed differentiation morphology, the cell cycles were arrested which caused cell apoptosis. In this process, HMBA/CINN could upregulate the expression of KLF6gene then activated the p53passway to induce cell apoptosis; on the other hand, KLF6could remove the inhibition of c-Jun to p21, decrease the expression of cyclin D1, inhibit complex cdk4/cyclin Dl, arrest cell cycles at G0/G1phase and consequently cause cell apoptosis.