Inhibiting Effects Of Recombinant Mutant Human Tumor Necrosis Factor(rmhTNF) Combined With Cisplatin (DDP) On Lewis Lung Carcinoma And Its Mechanisms

Objective: Lung cancer is a one of the common cancer around our country and the world.Alough normalized therapy has been enforce in clinical practice. Its five year survival is only about 20%.It is increasing important to decrease drug adverse reactions.It is one of the research direction to improve use of the killed-cancer drug. Established an experimental Lewis lung carcinoma model and therapies with sodium chloride,DDP, rmhTNF,rmhTNF and DDP,to observe tumor weight and tumor necrosis rate of each group were calculated,detect cell apoptosis and cell cycle and the expression of Survivin and PCNA in tumor tissue.The study is to reveal the inhibition action induced by recombinant mutant human tumor necrosis factor(rmhTNF) combined with cisplatin (DDP) on Lewis lung carcinoma in mice as well as to explore it's mechanism.Methods: 1,Animals and treatment:40 healthy male C57BL/6 mice were randomized into 4 groups:sodium chloride (control group),DDP group(6.15mg/kg),rmhTNF group(150万U/kg),rmhTNF(150万U/kg) combined with DDP(6.15mg/kg) group. was inoculated into each mouse by right armpit to establish model.2,Evaluation of tumor inhibiting effects: Then examined the growth of each tumor every other day. The 7th day after inoculation,tumor could be touched in whole mice. When in the 12th day ,the tumors became to 1.0×1.0×1.0cm3 on average,the drugs began to be injected in tumors. The four groups was injected for three days and once a day.All mice were slaughted 24 hours later, subcutaneous tumors were processed for tumor weight and tumor necrosis rate of each group were calculated.3,Flow cytometric analysis was used to detect cell apoptosis and cell cycle.4,The expression of Surviv- in and PCNA in tumor tissue was detected by immunohistoche- mical method.The differences in all indicators of each group were compared and contrasted.5,Expression of Survivin mRNA was examined by reverse transcriptionpolymerase chain reaction (RT-PCR).The differences in the expression of Survivin mRNA of each group were compared and contrasted.6,Statistic analys- is:ALL data were showed by ( x±s) and analyzed with the spss 13.0 software, including T-test, One-Way analysis of variance (One-Way ANOVA).Results: 1 The average weights of the tumors and inhibiti- on rate of tumor:The average weights of the tumors in DDP group and in rmhTNF group were (2.964±0.091)g and (3.010±0.089)g,lower than that of the sodium chloride(P<0.05),but between the single therapy groups,there was no significant difference(P>0.05).rmhTNF combined with DDP group were (2.267±0.050)g lower than that of the sodium chloride(P<0.01). The average weights of the tumors in rmhTNF combined with DDP group was lower than that of in DDP group and in rmhTNF group(P<0.05).the ration of inhibition in DDP group and in rmhTNF group and rmhTNF combined with DDP group were 17.115%,15.828% 36.605% .2 FCM analysis:Compared with the sodium chloride, DDP group and rmhTNF group the ratio of cancer cells in"G0/G1"stage was increased, decreased in the"S"stage. (P<0.05).In rmhTNF combine with DDPgroup the ratio of cancer cells in"G0/G1"stage was increased, decreased in the"S"stage. So percent of the proliferation index(PI) was decreased (P<0.01). rmhTNF combine with DDPgroup Compared with DDP group and rmhTNF group (P<0.05).The apoptotic percentage of DDP group,rmhTNF group were 21.599%,20.269% compared with control group 16.103% ,there was statistically difference(P<0.05). but between the sing- le therapy groups,there was no significant difference(P>0.05). rmhTNF combined with DDP group were 28.175%.there was statistically significant difference between control group(P<0.01 ). rmhTNF combined with DDP group compared with DDP group,rmhTNF group there was statistically significant differe- nce (P<0.05).3 Immunohistochemical studies: Lewis lung carci- noma were treated with DDP,rmhTNF and rmhTNF combined with DDP the expression of PCNA,Survivin protein weakened gradually, LI(Labeling index)value of PCNA and Survivin in the tumor tissue treated with DDP,rmhTNF lower than control group(P<0.05). but between the single therapy groups,there was no significant difference(P>0.05). rmhTNF combined with DDP was statistically significant difference between control group(P< 0.01).However The PCNA and Survivin LI value of tumor tissue in DDP group and in rmhTNF group compared with the combined group was difference(P < 0.05).4 RT-PCR was significantly down-egulated after treatment with rmhTNF and DDP group,than that of the control(P<0.01).But was lower than that in DDP group and in rmhTNF group(P<0.05).DDP group and rmhTNF group compared with the control group,there was difference(P<0.05).but between the single therapy groups,there was no significant difference(P>0.05).Conclusions:1 The study had established an experimental lung carcinoma model successfully that was very similar human's. It provides great contribution for further studying lung carcinoma.2 The combination of rmhTNF and DDP have synergic effect in the treatment of tumor,it can effectively inhibition the growth and induce apoptosis of the tumor cells apparently,regulate substancmetabolism,down-regulate the level of cytokines etc in lung carcinoma model.In rmhTNF combine with DDP group the ratio of cancer cells in"G0/G1"stage was increased,but decreased in the"S"stage.3 DDP group, rmhTNF group,rmhTNF combined with DDP group all can down-regulate the expression of Survivin and PCNA in tumor tissue of lewis lung carcinoma.Moreover the expression of Survivin can be affected significantly by rmhTNF combined with DDP therapied.4 rmhTNF combined with DDP Significantl inhibited the growth of implanted lewis lung carcinoma in mice,and down regulated Survivin and PCNA expression of lewis lung carcinoma.The mechanism may be two kinds of ways. First way is the mechanism depending on inhibition of PCNA expression.It can induce cancer cell to apoptosis and necrosis through down-regulating the expression of DNA,can also inhibit the proliferation of cancer cell through regulating the expression of cyclic protein and the secondary distribution of cell cycle. Secondly,the mechanism depending on inhibition of Survivin expression,the cativation of apoptosis signaling pathway mediat- ed by Caspase gene protein to induce apoptosis of cells.It is inhibited the growth of implanted lewis lung carcinoma in mice.