The Impact Of Of Ligustrazine Combined With Cisplatin On Small Cell Lung Cancer

Purpose:Small cell lung cancer accounts for about 20 percent of lung cancer.It has high degree of malignancy,rapid development,wide range of distant metastasis and poor prognosis. Although small cell lung cancer is sensitive to chemotherapy in short-term effect,it is prone to relapse and drug resistance in long-term.Despite the continued launch of a new generation of chemotherapeutic agents further enhance its role in cancer,its side effects are inevitable. Chemotherapy can not completely solve the problem of drug resistance,which limits its clinical application.The survival rate of some patients with Small cell lung cancer can not be improved.Tetramethylpyrazine(TMP) can inhibit the growth and the metastasis of certain solid tumors.Its mechanism is related to promoting immune function and inducing apoptosis of the cell.Cisplatin(DDP) is one of the most effective single-drug in the current treatment of small cell lung cancer.It has such characteristics as a broad spectrum of anti-cancer effect,a variety of antineoplastic synergistic effect and no cross-resistance.In recent years a number of studies have shown that the combination of certain chemotherapeutic drugs and TMP can enhance the effectiveness of tumor treatment.In this study,we apply TMP and DDP on nude mice vaccinating with small cell lung cancer cell line NCI-H446 and observe the impact on nude mice tumor growth after the drug effect by measuring the tumor size and tumor weight changes of the nude mice inoculated small-cell lung cancer cells.Cytology test is to observe the proliferation and apoptosis changes of small cell lung cancer cell line NCI-H446 through the application of TMP and DDP on small cell lung cancer cell line NCI-H446 to a certain extent.Material and method:Animal experiments were divided into four groups:control group,TMP treatment group,DDP treatment group,combined TMP and DDP therapy group. We respectively injected about 1×10⁷ NCI-H446 cells in the right hip of nude mice in each group.The total liquid was 0.2ml,Control group of nude mice were conventional fed after vaccinating small cell lung cancer cells.The drugs were given one week after the nude mice were vaccinated with small cell lung cancer cell on the remaining treatment groups.The TMP treatment group:TMP 100mg/kg intraperitoneal injection,1 times per day,for 21 days.DDP treatment group were given DDP 2mg/kg intraperitoneal injection,one injection every three days,for 7 times.Combined TMP and DDP therapy group:the combination of TMP group and DDP group In accordance with the above-mentioned method.The nude mice in each group were killed through neck dislocation 5 days after finishing the treatment,dipping for 2-3 minutes in 75%alcohol.The tumors were removed and put into the dish.The peripheral blocks and connective tissue were cleared.The tumors were washed with saline containing strong antibiotics by 40,000 IU/ml penicillin and 40,000 IU/ml streptomycin.The weights of tumors were measured with precision scales.Vernier caliper measurement of the tumor length and width were taken with vernier caliper,and calculated the volume of the tumers.Cut fresh tumor of each group after treatment,put it under 4%neutral formaldehyde fixative,fixed for 72 hours.Paraffin sections were conventional produced,HE staining,observed under light microscope.We cut several pieces of 1mm3 blocks of fresh tumor after treatment and quickly added them into 4℃precooling 2.5%glutaraldehyde and 1%paraformaldehyde mixed fixative,1%osmium tetroxide post-fixation.Epoxy resin-embedded,staining with uranyl acetate and lead citrate to ultrathin sections,observed the ultrastructure of tumor tissue under electron microscopy(TMP) and photographed the record.Small cell lung cancer cell line NCI-H446 were cultured in the 1640 complete culture medium with 10%fetal bovine serum,under the conditions of 37℃and 5%CO₂ training.The solution were changed for every three days.The celles digested and subcultured with 0.25% trypsin.Cytology tests were divided into four groups:control group,TMP treatment group, DDP treatment group and combined TMP and DDP therapy group.MTT experiment, colony-forming experiment,flow cytometry detection,RT-PCR and Western blot detection were respectively cayyied out.Observed the cell proliferation and apoptosis in each group and the changes of proliferation-related genes cyclin D1,P16 and apoptosis-related gene Bcl-2, p53.Results:1 Through observing the following five indicators:the mental state,activities,response to the stimulation,weight loss and appetite,We evaluated the quality of life and toxicity in the nude.The results showed that nude TMP group had no significant toxicity of the drug,and has the best quality of life,Followed by co-treatment group,again as the control group,while the toxicity in DDP group had the worst quality of life.The nude's toxicity in Combined TMP and DDP therapy group was significantly lower than in DDP group,the quality of life was superior to DDP group,indicating that combination treatment could reduce DDP toxicity.2 Observed the size of nude mice tumor after treatment:TMP treatment group(14.36±2.54), DDP treatment group(12.12±1.57) and combined TMP and DDP therapy group(8.22±3.05). The size.of tumors in the groups above were all smaller than the size in control group(15.13±6.38).The tumor weight of nude mice:TMP treatment group(0.0948±0.0328),DDP treatment group(0.0496±0.0299) and combined TMP and DDP therapy group(0.0264±0.0257).The weight of tumors in the groups above were all lower than those in control group (0.1255±0.0412).The reduction of tumor size and weight in combined TMP and DDP therapy group were more significant,showing that combined TMP and DDP therapy group can more enhance the therapeutic effect than DDP treatment group.3 By light microscopy and electron microscopy detection,the nude mice tumors were consistent with the characteristics of small cell lung cancer cells.Through light microscopy detection,degeneration and necrosis of the tumor tissue could be seen in TMP treatment group.More lipid droplets appeared in the tumor cells.The tumor cells lined along with osteoporosis in DDP treatment group.Part of the tumor cells became smaller in size.The tumor cells in-combined TMP and DDP therapy group rared.The tumor cells became smaller in size.The cell nuclear became pyknosis.Connective tissue growthed between the tumor cells.Fibrosis was obvious.More fibroblasts could be seen in the tumor tissue.Through electron microscopy examination,in TMP treatment group we found more fatty degeneration in the tumor cells.Lipid droplets inside the cytoplasm increased.Necrotic cells and its debris could be seen more.The cell membrane was not complete.Reduction of cellular organ, structural unclear and vacuolization was like.In DDP treatment group,the tumor cells rared. The tumor cells became significantly smaller in size.More cell debris could be seen.In part of the tumor cells,mitochondria in cytoplasm swelled.The heterochromatin increased in the nucleus.Tumor cell apoptosis was common.In combined TMP and DDP therapy group,the tumor cells were scarce,and the patterns were similar to in the DDP treatment group.4 By TMP,DDP and joint treatment on small cell lung cancer cell line NCI-H446,the survival of the cells decreased.The cells became round and necrosis.MTT test showed the absorbance value in 72h and 96h:TMP treatment group(1.031±0.034)(1.129±0.049),DDP treatment group(0.986±0.028)(1.018±0.038 ),combined TMP and DDP therapy group(0.854±0.036) (0.923±0.041),all decreased compared with the control group(1.336±0.067)(1.437±0.045 ), (P＜0.05).Those indicated that the cell proliferation decreased in each treatment group and the absorbance value reduced to the extent of the largest in combined TMP and DDP therapy group.(compared with DDP treatment group P＜0.05).The colony-forming experiments showed the rate of colony-forming cells:TMP treatment group(17.9±3.7),DDP treatment group(15.8±3.5),combined TMP and DDP therapy group(11.2±2.9).Decreased significantly compared with the control group(23.8±4.2).The results also showed that the cell proliferation in each treatment group is reduced after administration of the drugs,and the combined TMP and DDP therapy group decreased to the largest extent in the number of the colony formation(compared with DDP treatment group P＜0.05).The results of RT-PCR and Western blot showed that the expression level of cyclin D1 gene in the small-cell lung cancer cell line NCI-H446 decreased,and increased the expression level of P16 gene,after treated by TMP,DDP and the combination of both.There was a significant difference(P＜0.05).5 The result of detecting apoptosis by acridine orange(AO) and olfactory bromide(EB) staining showed:TMP treatment group(11.36±2.18),DDP treatment group(15.73±3.25), combined TMP and DDP therapy group(21.32±3.27).The apoptosis rate of the cells increased compared with the control group(5.21±1.94).There was a significant difference (P＜0.05).The result of flow cytometry detecting showed:TMP treatment group (16.37±1.23),DDP treatment group(19.2±1.51),combined TMP and DDP therapy group (21.2±1.79).The apoptosis rate of the cells increased compared with the control group (5.23±1.01).There was a significant difference(P＜0.05).The apoptosis rate increased to the largest extent(compared with DDP treatment group P＜0.05).DNA electrophoresis revealed that each group could result in DNA fragmentation in addition to the control group. The level of DNA damage increased after combined application of the two drugs compared to separate application.The results of RT-PCR and Western blot showed TMP,DDP and combination therapy could reduce the level of Bcl-2 gene expression,and increase p53 gene expression level.Conclusion:1 TMP,DDP and combination therapy can inhibit tumor growth in nude mice tumors.The role of TMP is weak,a stronger role in DDR the role of joint treatment of the two is stronger than DDP alone.2 Application TMP after DDP in the treatment of nude mice can significantly improve the quality of life in nude mice.3 TMP DDP and the combination of two can induce apoptosis of small-cell lung cancer cell in nude mice tumor.4 TMP,DDP and combination therapy can significantly reduce the small-cell lung cancer cell line NCI-H446 proliferation capacity.The role of TMP is weak,a stronger role in DDP,the role of joint treatment of the two is stronger than DDP alone.The expression level of cyclin D1 gene in the small-cell lung cancer cell line NCI-H446 decreased,and increased the expression level of P16 gene.5 TMP,DDP and combination of both can induce small cell lung cancer cell line NCI-H446 cell apoptosis.The role of TMP is weak,a stronger role in DDP,the role of joint treatment of the two is stronger than DDP alone.Meanwhile the expression level of Bcl-2 gene decreased, and increased the expression level of p53 gene.