| Background:Lung cancer continues to be a major global health problem because of the highest morbidity and mortality rates.Presently,chemotherapy is still the main therapy method in all lung cancer therapies.However,with the clinical use of chemotherapy drugs,some problems appeared such as the side effects,drug resistance and so on.Lots of studies have found that thiazolidine compound is a novel anticancer compound which has low toxic and side effects,and has strong anti-tumor activity to human lung cancer cells especially.Our study has found that as a novel thiazolidine compound,YFZ-22 A has a strong anti-tumor effect.In this paper,we mainly study on its anti-tumor mechanism.Method:The effect of YFZ-22 A on the growth of lung cancer established subcutaneously in nude mice was evaluated by measuring the volume mass with digimatic caliper.To analyze cytotoxicity of A549,if any,by YFZ-22 A,we adopted three different in vitro methods,including SRB assay,CCK-8 assay and RTCA.Flow cytometry was used to test the effects of YFZ-22 A on the cell cycle.The changes of A549 cell surface structure was detected by using scanning electron microscope.The ratio of apoptosis was determined by Nucleosome ELISA assay.RNA-Seq and Western blot were used to test the expression changes of cell cycle and endoplasmic reticulum stress related genes and proteins.Result:The result of YFZ-22 A on anti-tumor in vivo showed that YFZ-22 A could inhibit the growth of transplanted tumor in nude mice in a time-dependent manner.YFZ-22 A was effective in inhibiting the proliferation of A549 cells in a dose and time dependent manner.Flow cytometry analysis demonstrated that after exposing to YFZ-22 A for 24-48 h,most of A549 cells were blocked in G2/M phase in a time dependent manner.Morphological change of apoptosis such as cell shrinkage,membrane blebbing and apoptotic bodies were observed in A549 cells following YFZ-22 A treatment for 24 h.The proportion of DNA fragmentation cells in YFZ-22A-treated cells was increased in a dose and time dependent manner.Compared to the control,treatment with YFZ-22 A for indicated time of A549 cells resulted in an increase in the activities of caspase-4,-8,-9 and-3,but not of caspase-6.To further examine the relationship between caspase activation and YFZ-22 A –induced cytotxicity in A549 cells six caspase inhibitor were applied.Obviously,the activities of caspase-4,-8,-9 and-3,were significantly up-regulated by being treated 0.36 ?M YFZ-22 A,which verified that caspase-4,-8,-9 and-3 were activated and participated in the cell death pathway induced by YFZ-22 A.According to the results of RNA-seq,we selected 49 differentially expressed Genes associated with cell cycle,including 10 up-regulated genes and 39 down regulated genes,and selected 48 differentially expressed Genes associated with ERS-mediated apoptosis,including 44up-regulated genes and 4 down regulated genes.The results of Western blot showed that with the extension of time and the increase of concentration by YFZ-22 A,it was gradually decreased to expression level of the proteins associated with cell cycle such as p53,CDK1,CDK2,Cdc25 c,Cyclin A2,Cyclin B1.And it was gradually increased to expression level of the proteins associated with ERS-mediated apoptosis such as GRP78,PERK,ATF4,CHOP,IRE1?,ASK1,p-JNK,Calpain2 and Caspase-4.Conclusions:1.YFZ-22 A could significantly inhibit the growth of A549 cells in a dose and time-dependent manner.2.YFZ-22 A induced A549 cells arrest at G2/M phase.3.YFZ-22 A could induce apoptosis in A549 cells.4.The YFZ-22A-induced apoptosis in A549 cells was involved in the pathway of ERS-mediated apoptosis,and might also be related to mitochondria. |
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