Study On Recombinant Mutant Human Tumor Necrosis Factor Enhancing The Chemotherapeutic Effect Of Cisplatin In The Mice With Lewis Lung Carcinoma And The Mechanism

Objective: Lung cancer is the leading cause of cancer deaths around our world with high morbidity, and its five year survival is only about 10%.Although new anticancer drugs and chemotherapeutic schemes turn out one after another,the therapeutic efficacy still remains unsatisfied. The problem that is urgent to solve at present is to seek an effective measure to enhance the chemotherapeutic effect. Biochemotherapy which combines biotherapy and chemotherapy becomes the new trend of tumor's combined therapy. It can not only plus the effect but also decrease the adverse reaction in contrast to chemotherapy alone.Tumor necrosis factor is belong to the category of cytokines in biological agents. The application of cytokines in clinic is the maturest in biotherapy.This experiment aimed to observe rmhTNF enhancing the chemotherapeutic effect of cisplatin in the mice with Lewis lung carcinoma and improving the immunological function in animals to further investigate that the possible mechanism of its role as angiogenesis inhibitor might one of the cause of enhancing the chemotherapeutic effect and feasibility to cure lung cancer combinating cisplatin. Methods: 60 healthy C57BL/6 mice were randomized into 4 groups:control group,Recombinant Mutant Human Tumor Necrosis Factor group(rmhTNF),cisplatin group(DDP), and Recombinant Mutant Human Tumor Necrosis Factor plus cisplatin group(rmhTNF+DDP,combination group). Then a sus- pension of 2×106 murine Lewis lung carcinoma cells (LLC) was inoculated into each mouse by right armpit to establish model. After the inoculation,we needed to examine the growth of each tumor every other day. The 5th day after inoculation, tumors could be touched in half of the mice and the 7th day in the 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 mice in control group was injected with Sodium Chloride of 0.2ml,and rmhTNF(1500000 IU/kg) of 0.2ml in rmhTNF group, DDP(6.15mg/kg) of 0.2ml in DDP group, rmhTNF(1500000 IU/kg) of 0.2ml plus DDP(6.15mg/kg) of 0.2ml in the combination group.The four groups were injected for three days and once a day. After the first drug injection,the animation of the mice in each group was observed. After 24 hours of the last injection,all mice were killed.After that all tumorous tissues were decoherenced and weighted. The body weights of mice without tumor in the groups were also weighted. The volumes of tumors were measured and the inhibitions ratio of tumor growth were calculated.At the same time,the level of HIF-1αmRNA expression was examined by semiquantitative RT-PCR in mice tumor.The expression of VEGF,VEGFR-2 (KDR) protein in tumor tissue was qualitationly studied by immunohistochemistry staining and MMP-2 was semi-quantitat- ely examined by flow cytometry.Results: 1 The animation of the mice in each group,median body weight of mice without tumor:depilation was found in each group in some degree,but the animation of the mice in the rmhTNF group was the best,and the combination group was more better than the DDP group.Median body weight of mice without tumor in the rmhTNF group was higher than in the control group(P<0.05),but in the other groups were lower than in the control group(P<0.05). Median body weight of mice without tumor in the combination group was higher than in the DDP group (P<0.05). 2 Tumor weight,tumor volume,inhibition ratio of tumor growth: The single therapy groups brought about lower tumor weight and lower tumor volume than did the control group(P<0.05).The combination group brought about higher antitumor rate, lower tumor volume, and lower tumor weight than did the single groups(P<0.05). Between the single groups,there was no significant difference(P>0.05). 3 HIF-1αmRNA: The expression of HIF-1αmRNA in the single groups was lower than in the control group significantly(P<0.05).The combination group brought about lower HIF-1αmRNA expression than did the single groups significantly(P<0.05). Between the single groups,there was no significant difference (P>0.05).4 VEGF,KDR:The expression of VEGF and KDR in the single groups was lower than in the control group(P<0.05). The combination group brought about lower VEGF and KDR expression than did the single groups(P<0.05). Between the single groups,there was no significant difference (P>0.05).5 The detection result of associativity:The expression of VEGF and KDR protein had significant positive associativity and r-values were 0.703,0.538,0.644,0.608,and p-values were 0.003, 0.039, 0.010,0.016. 6 MMP-2: The expression of MMP-2 in the single groups was lower than in the control group significantly (P<0.05).The combination group brought about lower MMP-2 expression than did the single groups significantly (P<0.05). Between the single groups,there was no significant difference (P>0.05).Conclusions:1 Both rmhTNF and DDP used alone could make the tumor growth depressed,but when they were used in combination, the effect was enhanced significantly. rmhTNF could enhance the chemotherapeutic effect of cisplatin and improve the immunological function in the mice with Lewis lung carcinoma.2 Both rmhTNF and DDP used alone could make the angiogenesis depressed,but when they were used in combination, the effect of antiangiogenesis could be enhanced. This phenomenon further investigated that the possible mechan- ism of its role as angiogenesis inhibitor might be one of the cause of enhancing the chemotherapeutic effect.3 The reason that rmhTNF and DDP could depress the angiogenesis and inhibit the tumor growth might be that they could decrease the expression of HIF-1αin the level of gene,which could make the lower expression of HIF-1αin the level of protein .Then the HIF-1αprotein could decrease the VEGF protein which could decrease the KDR protein. rmhTNF and DDP could also decrease the expression of MMP-2 protein.The effect of antitu- mor would be produced when the indexes above-mentioned interacted.4 HIF-1αmRNA,VEGF,KDR and MMP-2 could be expressed in the tumor of mice with Lewis lung carcinoma. The expression of VEGF and KDR protein had significant positive associativity.5 Our research provides basic theory to the application of rmhTNF and DDP in combination so as to improve the chemotherapeutic effect in the treatment of lung cancer,and provides a new choice for combined therapy in lung cancer.