Experimental Study Of PESV To Enhance The Restraint Of CTX On Lung Cancer

Research backgrounds:Lung cancer is one of the most common malignant tumor which threaten to human health. In recent years, the morbidity and mortality of lung cancer rapidly rise in the world, especially in developed industry country. Non-small cell lung cancer accounts for more than 80% of all cases of lung cancer. The current main treatments of lung cancer are surgery, radiotherapy and chemotherapy, but the radiotherapy and chemotherapy have more serious side effects. And it's also prone to chemotherapy resistance to lead to treatment failure. Many patients with advanced cancer due to loss surgery opportunities usually have very poor prognosis. And this greatly limited the treatment of patients. At present, the five-year survival rate of lung cancer is less than 15%. Cyclophosphamide (CTX) is a traditional antineoplastic chemotherapy drug which can reduce the number of regulatory T cells in vivo and reduce its function. Chemotherapy can kill tumor cells, but also can damage the normal cell which lead to the immune function declined and accelerated proliferations of tumor cells, and eventually leading to the treatment failure. Traditional Chinese medicine can antitumor growth and transfer, enhance the curative effect of chemotherapy, reduce the toxicity of chemotherapy, improve the immune function and ameliorate the patients' symptoms, and improve the quality of life. Polypeptide extract from scorpion venom (PESV) is anti-tumor active ingredient which is extracted from Buthus martensii Karsch. PESV is polypeptide mixture which contains 50～60 amino acids. PESV is heat-resistant, with stable PH and molecular weight of 6000～7000. Studies confirmed that PESV had significant anti-tumor effects in vivo and in vitro. It could improve the inhibitory state of immune function in tumor bearing body. When combined with the chemoradiation, they can produce good synergy antitumor effect. But the mechanisms have not been fully clarified. In our study, we mainly discuss and analysis the mechanisms of against with cancer by observing the effects of PESV Combined with CTX on the immune escape associated factors of Lewis lung cancer related factors.ObjectiveTo observe the effects of polypeptide extract from scorpion venom (PESV) combined with cyclophosphamide (CTX) on the expression of TGF-β1,VEGF,PCNA cytokines and the differentiation and maturation of dendritic cells (DC) in tumor microenvironment. To improve the immune tolerance status of non-small cell lung cancer patients. Initiate and maintain the immune response which is induced by DC. Enhance the patient's immune function by using the drugs. Further reveal the anti-tumor mechanisms of PESV. Provide the experimental basis for the treatment of non-small cell lung cancer patients. Thereby PESV can be combined with traditional chemotherapy drugs and DC-based immunotherapy.Methods1. Lewis lung tumor models were established by subcutaneously implanting Lewis lung cells (1×107/mL) into C57BL/6 mice in right armpit subcutaneously. The tumor-bearing mice were randomly divided into four groups: the control group, the chemotherapy treatment group (treated with CTX), the PESV group and the experimental group (treated with PESV between courses of chemotherapy). The tumor volume and tumor inhibitory rate were determined and to observe the effect of PESV on inhibition of tumor growth.2. Conventional HE staining was done, and tumor tissue pathological changes were observed with light microscopy.3. The expression levels of TGF-β1, VEGF and PCNA in tumor tissue were detected by immunohistochemistry-staining.4. The mRNA expression levels of TGF-β1, VEGF in tumor tissue were detected by RT-PCR。5. Surface costimulatory molecules B7-1 and B7-2 of tumor infiltrating dendritic cells (DC) were detected by immunofluorescence.6. Surface molecules phenotype B7-1 and MHC classⅡ, B7-2 and MHC classⅡof tumor infiltrating dendritic cells(DC) were detected by immunofluorescence technique .Results1. Effects of drug intervention on the growth of Lewis lung carcinomaIn the experimental group, the tumor weights of the mice were significantly reduced, and the inhibition effect of experimental on tumor volume was obvious. Compared with the control group, the chemotherapy group, the PESV group and the experimental group after 21d, their tumor inhibition rates were 31.57%, 17.32% and 53.63% respectively(P<0.05).2. Effects of PESV on the expression of Cytokines TGF-β1, VEGF and PCNA in tumor tissueThe results of immunohistochemistry staining showed that, positive products of TGF-β1, VEGF were mainly localized in the cytoplasm of tumor cells, and positive products of PCNA was mainly localized in the nucleus of tumor cells. The positive expression of the control group has the highest in + + + 80% above; the chemotherapy group and PESV group has higher in + + + 60% above; the experimental group has only 30% of + + +. Compared with the experimental group and the control group ,they has significant difference (P < 0.01).3. Effects of drug on the expression of B7-1 and B7-2 in Lewis lung cancer tissueThe results of fluorescence microscope showed that, positive products of B7-1 and B7-2 were expressed green fluorescent signal. Compared with the control group,the green fluorescent signal in the PESV group was strong, but the immune fluorescent signal in the chemotherapy treatment group was lower. Compared with chemotherapy treatment group, the fluorescent signal strength and expression in the experimental group were increased. 4. Effects of drug on the expression of B7-1and MHC classⅡ,B7-2 and MHC classⅡin Lewis lung cancer tissueThe results of fluorescence microscope showed that, positive products of B7-1and MHC classⅡ,B7-2 and MHC classⅡwere mainly expressed in the tumor tissue. The bright green fluorescent signal was showed B7-1 and B7-2; Bright red fluorescent signal was showed MHC classⅡ; Yellow fluorescent signal was showed that both of them were expressed. compared with the control group,the yellow signal in the PESV group was increased, but the yellow signal in the chemotherapy treatment group was lower. The yellow fluorescent signal strength and expression in the experimental group were increased.5. Effects of drug on VEGF-A,TGF-β1 expression level of mRNA in Lewis lung cancer tissue of Lewis lung carcinomaCompared with the control group, the expression of TGF-β1 and VEGF-A both in PESV group and the chemotherapy treatment group were lower (P<0.05). Compared with the PESV group and the chemotherapy treatment group, the expression of TGF-β1 and VEGF-A in the experimental group was lower (P<0.05).ConclusionsPESV can influence the expression levels of VEGF,TGF-β1 and PCNA in tumor microenvironment and promoting the mature of DC while Restoring its function of the antigen intake and the antigen presentation; Reverse the body's immune injury by the CTX. PESV has an inhibitory effect on growth of Lewis lung carcinoma, and it can inhibite the proliferation of Lewis lung carcinoma.