Endoplasmic Reticulum Stress Signaling Pathways Regulated By Acid Sphingomyelinase Contributes Toapoptosis In Human Gastric Cancer Cells

Objective To define the SMase contributing to Evodiamine-inducedapoptosis in human gastric cancer SGC-7901cells and the effect of Endoplasmicreticulum stress signaling pathways in this apoptosis; to define the effect ofEvodiamine-induced apoptosis in human gastric cancer BGC-823cells and theprotein expression of caspase3、caspase8and caspase9. Methods Human gastriccancer SGC-7901and BGC-823cells were cultured in humidified atmosphere of5%CO2/5%air at37℃, SGC-7901were treated with1.5μM Evodiamine、1.5μMEvodiamine and100μM D609、1.5μM Evodiamine and1μM GW4869、1.5μMEvodiamine and30μM salubrinal、100μM D609、1μM GW4869、30μM salubrinal for24h；BGC-823cells were treated with Evodiamine of different concentrations for24h、36h and48h respectively.①Cell proliferation was assessed by CCK8;②cellapoptosis was analyzed by Annexin V-FITC/PI double staining associated with flowcytometry;distribution of cell cycle was detected by propidium iodide(PI) associatedwith flow cytometry;③The structural changes of Endoplasmic reticulum ofSGC-7901cells were observed by transmission electronmicroscope;④The geneexpression of JNK、CHOP and CASPASE4in SGC-7901were detected by Real-timePCR;⑤The protein expression of JNK、p-JNK、CHOP、CASPASE4in SGC-7901cells and CASPASE3、CASPASE8、CASPASE9in BGC-823cells were detected bywestern Blotting. Results.1.CCK8and Annexin V-FITC/PI double staining showedthat Evodiamine induced apoptosis in SGC-7901cells,which can be blocked by100μM D609and inhibited by30μM Salubrinal; Evodiamine induced apoptosis inBGC-823cells(IC50is20μM) and the effects were dose and time dependent; 2.Evodiamine induced G0/G1phase arrest in BGC-823cells;3.The resuts oftransmission electronmicroscope showed that1.5μM Evodiamine treatment causedER dilation；which was no significant change when co-treated with100μM D609;4.Real-time PCR indicated that the gene expression of JNK、CHOP、CASPASE4inSGC-7901cells treated with1.5μM Evodiamine was upregulated,which wasdownregulated when co-treated with100μM D609;5.Western blotting indicated thatprotein levels of p-JNK、CHOP、CASPASE4in SGC-7901cells treated with1.5μMEvodiamine were significantly expressed,which were reduced when co-treated with100μM D609; the protein levels of Caspase3、Caspase8、Caspase9in BGC-823cellstreated with20μM Evodiamine for24h、36h were upregulated,which weredownregulated when treated for48h. Conclusion By the results, it is consideredthat:1.aSMase contributed to Evodiamine-induced apoptosis in human gastric cancerSGC-7901cells；Endoplasmic reticulum stress signaling pathways can be activatedby aSMase through upregulated protain levels of p-JNK、CHOP、CASPASE4toinduce apoptosis of SGC-7901cells2. Evodiamine induced G0/G1phase arrest andupregulated protain levels of CASPASE3、CASPASE8、 CASPASE9to induceapoptosis of BGC-823cells...