In Vitro And In Vivo Study On The Correlation Between Per2 Gene Expression And Apoptosis Induced By Paclitaxel-Loaded Polymeric Nanoparticles Combined With Chronomodulated Chemotherapy

Objective:Paclitaxel(PTX)nanoparticles(PTX-NPs)loaded with poly(caprolactone)-poly(ethyleneglycol)-poly(pcl-peg-pcl,PCEC)have been shown to have a strong anticancer effect on lung cancer cells.Previous studies have shown that PTX-NPs exhibits the greatest anti-tumor effect on lung cancer cells After 15 Hours After Light Onset(HALO),but the mechanism of its action in chronomodulated chemotherapy remains unclear.In our study,In our study,we try to through the exploration of the regulation of lung cancer cells Per2 gene expression,to observe Per2 gene expression level at day and night of the day if there is a difference.Whether there is a certain correlation between the two kinds of differences,further to answer the reasons for the differences in the anticancer effect of lung cancer cells after chemotherapy.Methods:In vitro experiments,cultured A549 cells were treated in groups(control group,PTX group,PTX-NPs group).The three groups were treated with chemotherapy drugs at 0HALO,5HALO,10HALO,15HALO and20HALO,respectively(the control group was treated with normal saline).Cells in each group were collected 48 hours later,and the proportion of cells in G2phase and the proportion of apoptosis were detected by flow cytometry.The expression of Per2 gene was detected by western blot(WB)and Real-time fluorescence polymerase chain reaction experiment(RT-PCR).In vivo experiments,the purchased nude mice were first divided into three groups(the same grouping method as in vitro experiments),and then each group was divided into five subgroups according to the treatment time of 0HALO,5HALO,10HALO,15HALO and 20HALO.Feeding in the same light environment for 3 weeks established the same biological rhythm.Then,a nude mouse model of subcutaneous xenotransplantation of A549 lung cancer was constructed.After the tumor volume grew to about 100mm~3,the mice in each subgroup were given drug treatment at 5 time points,Every four days,a total of3 times.The length and diameter of tumors in nude mice were measured every other day after administration,tumor volume was calculated,tumor growth curve was drawn.Twelve days after the end of the drug intervention,the nude mice were sacrificed and the tumor tissue was stripped,weighed,and the tumor growth inhibition rate was calculated.the nude mice were sacrificed and their hearts,liver,spleen,lungs and kidneys were stripped.The internal organs of the nude mice after chemotherapy were observed by HE staining.The expression of Per2,Bax and Bcl-2 genes in tumor cells in each group was determined by WB and RT-PCR.Results:In vitro experiments,15HALO group had the best anticancer effect and 0HALO group had the worst chemotherapy effect(P﹤0.01).The expression of Per2 gene in A549 cells was the strongest at 15HALO and the weakest at 0HALO(P﹤0.01).In the animal experiment,the 15HALO group showed the slowest tumor growth and the highest tumor growth inhibition rate(P﹤0.05).The expression of Per2 and Bax gene was strongest in 15HALO group and weakest in 0HALO group(P﹤0.05).The expression of Bcl-2 gene was the strongest in the 0HALO group and the weakest in the15HALO group(P﹤0.05),showing a 24-hour cycle.The results of HE staining showed that liver was the main visceral lesion of PTX and PTX-NPs,and the lesion was the least in the 15HALO group and the most in the 0HALO group.Conclusions:The anticancer effect of chronomodulated chemotherapy on lung cancer cells was correlated with the expression of circadian clock gene Per2.Per2 gene may be a reference for individualized chronomodulated chemotherapy of lung cancer.