Study On Induction Of Apoptosis And Molecular Mechanisms By CsA In Gastric Adenocarcinoma MGC80-3Cells

Gastric carcinoma(GC) is one of the most common malignant tumors andseriously threaten human health. The mortality rate of GC ranks second in the worldand the incidence of GC also showed obvious geographic differences with higherrates occurring in Asian countries especially including Japan, Korea and China. In ourcountry，more than60%of GC are diagnosed at Stage III or IV. Surgery treatmentmay be the only curative method. However，the postoperative recurrence rate of GC ishigh and the survival rate is still low. Therefore，systematic and effective treatment isvery necessary to the patients with GC，and most chemotherapy regimens achieveonly a low clinical complete response rate and do not make any progress in improvingsurvival. In our study，we research about CsA inhibiting effects on proliferation andinducing apoptosis in human gastric adenocarcinoma MGC80-3cells and investigateits possible mechanism of anti-tumor，in order to support the clinical application ofCsA.Methods: MTT was used to detect the growth and proliferation of CsA on MGC80-3cells；PI staining examined cell cycle via flow cytometry（FCM）；Acridineorange/ethidium bromide (AO/EB) fluorescent staining detected cell apoptosis;Transmission electron microscopy deserved mitochondria after treatment with CsA onMGC80-3cells. Annexin V-FITC/PI double staining determined the apoptosis rateafter treated with CsA on MGC80-3cells via FCM. FCM detected change of theproduction of reactive oxygen species（ROS）in MGC80-3cells; Western blottingmeasured the expression of P-glyprotein（P-gp）and NF-κB p65.Results：(1) MTT results showed significant inhibited proliferation on MGC80-3cellsafter treated with25.0μM CsA for24h or15.0~25.0μM CsA for48h,72h.The IC50value for MGC80-3cells was20.7μM at72h after CsA treatment.Flow cytometry showed that the percentages of G0/G1phase of all dose groups of CsA were increasedcompared with the control group, cell cycle was blocked at G0/G1phase.(2) Morphological changes of MGC80-3cells were also observed by fluorescencemicroscope after CsA treatment by AO/EB staining. It showed that normal cellsstained with normal cell morphology and green fluorescence, while apoptotic cells ordamaged cells showed orange fluorescence, with cell debris and nuclear chromatincondensation, cell morphological also changed and cell density condensateddecreased. CsA treated cells showed apparent apoptotic situation.(3) Transmission electron microscopy（TEM）showed that MGC80-3cells wasshrinked and round，mitochondrial crista became less and short or disappear, nucleusoccurred typical nuclear chromatin condensation in morphological changes ofapoptosis after different CsA concentration treatment.(4)After CsA treated for48h，the intracellular ROS levels in MGC80-3cells increaseddramatically compared with the control groups（p<0.01）.(5) Flow cytometry（FCM）results showed that the apoptosis rate induced by CsAagainst MGC80-3cells significantly increased in a time-and concentration-dependentmanner compared with control groups（p<0.05， p<0.01）.(6) Western blotting also showed that CsA significantly reduced the proteinexpression of P-gp and NF-κB p65in a concentration-dependent manner.Conclusion: CsA could inhibit human gastric adenocarcinoma MGC80-3cellsproliferation, and also induced apoptosis within a certain time and the concentration.The mechanism of CsA-induced apoptosis may be related to mitochondrial oxidativestress, reducing the expression of P-gp and NF-κB p65；through the NF-κB signalingpathway and mitochondrial pathway, and then generating inhibition effect of tumorcell proliferation.