Effects Of Artesunate On Toumor Growth Inhibition And Drug Resistance Revsion In A Carcinoma Drug Resistant Cell Line Nude Mice Parotid Gland Xenograft Model

Objective:The aim of this study is to establish a xenograft model of drug resistant carcinoma in nude mice,and to observe the effects of Artesunate(ART)has the same effect on the proliferation of transplanted tumor cells in vivo and to explore the relevant reverse mechanisms of reverse of cisplatin(DDP)drug resistance.Methods:HELA cells were induced by low concentration and periodic cisplatin(DDP)interference in vitro to establish a HELA-DDP cell line.5-week-old BALB/C-nu male nude mice were chosen to establish the parotid gland xenograft model of drug resistant carcinoma in nude mice by inoculating the drug-resistant cells in their parotid glands.Round or oval induration formation was considered as the success of model establishment.And then all animals were randomly divided into 7groups:normal saline control group,DDP 1.0 mg / kg group,DDP 2.0 mg/ kg group,ART 50.0 mg / kg group,ART 100.0 mg / kg group,ART200.0 mg / kg group and ART 100 mg / kg + DDP 1.0 mg / kg groupaccording to the size of the transplanted tumor.The DDP and ART were injected by intraperitoneal administration.The weight of nude mice and tumor diameter length were measured to observe changes in body weights and tumor growth of nude mice.Then the nude mice were sacrificed after 4 weeks,and the inhibitory rate was calculated.The tumors and livers tissues of the mice were stained by 10% formalin solution,paraffin embedding,sectioning.Hematoxylin and eosin(H&E)and pathological changes of each treatment group were observed with light microscopy.The change of transplantation tumor in different groups of carcinoma drug resistant cell ultrastructure was observed by transmission electron microscopy(TEM).The expression of Survivin and MRP2 in transplantation tumor tissue of nude mice were detected by Immunohistochemistry(IHC),Real-time fluorescent quantitative(RT-qPCR)and Western blot(WB).Using statistical software SPSS19.0 statistical analysis was carried out on the experimental results,the measurement data results with mean±standard deviation(-X ±S)said,two sample data to compare the two independent samples t test,multiple comparison between the data set with completely random design and single factor variance analysis,P < 0.05 showed statistically significant difference.Results:1.After 7 days of inoculation,all the inoculated side of the parotid gland of mice were found indurated and the diameter of tumors reached 5mm,which indicated that a stable parotid gland xenograft model of drug resistant carcinoma in nude mice was successfully established and thetumor formation rate was 100%,After 4 weeks of intervention of DDP and ART,the the transplantation tumors size of saline group were bigger than other groups.Among which the inhibitory rate of tumors in 2.0mg/kg DDP group was as high as 59.86%(P < 0.01).This group of nude mice were obviously thin and in poor condition.The inhibitory rate of ART 200.0 mg/kg group was 43.54%(P < 0.05),and nude mice in this group were in good mental state,and a steady increase of mice weight was observed.The inhibitory rate of ART 100 mg / kg + DDP 1.0 mg / kg group was 41.16%(P < 0.05),the weight of mice in this group was slightly reduce.2.H&E staining results indicated that cells of transplanted tumor in treatment groups showed various degree of nuclear hyperchromatism and tumor shrinkage,the phenomenon of nuclear chromatin condensing and c flake cracking were observed,and red dyeing glass like area with no structure could be observed intercellularly.3.The transmission electron microscopy results showed that the treatment group of carcinoma drug resistant cells transplantation tumor slightly smaller than normal saline group,the microvilli in cell surface decreased significantly and various degree of swelling of mitochondria and vacuolar degeneration of cytoplasm appeared..4.The Immunohistochemistry results showed that the expression of Survivin and MRP2 in the drug resistant nude mice transplantation tumor tissue decreased with the increase of ART concentration.5.The RT-q PCR results showed that with the increase of concentration of ART,the mRNA expression of Survivin and MRP2 was lower than normal saline control group(P < 0.01).6.The Western blot results showed that with the increase of concentration of ART,the protein expression levels of Survivin and MRP2 were significantly lower than normal saline control group(P <0.001).Conclusion:1.The HELA-DDP cells with multidrug resistance have strong cell activity and tumorigenicity,and the model of parotid gland xenograft can be established in nude mice.2.ART can effectively reverse the resistance of HELA-DDP cells to DDP and the mechanism may be related to the downregulation of expression of Survivin and MRP2.3.ART can inhibit the proliferation and promote the apoptosis of and the side effects of artesunate on nude mice with cisplatin.