Study On Inhibition Mechanism Of Pesv Combined With Rapamycin On Murine S180 Sarcoma By Promoting Autophagy

Objective:This study utilized murine S180 sarcoma model and focused on the effect of polypeptide extract from scorpion venom(PESV) combined with RAPA on the S180 sarcoma growth and the protein level of Beclin1、MAP1LC3A、CD133 and CD90 in order to discuss further reveal the anti-tumor mechanisms of PESV, which will provide theoretical basis for PESV combined with traditional drugs being used in clinical anti-tumor therapy. Methods:1. Establish subcutaneous S180 sarcoma model, then tumor-bearing mice were divided into five groups randomly: the model group、the high-dose PESV group、the low-dose PESV group、the RAPA group、the combination group(PESV+RAPA),10 mice in each group and 21 days doing tests. Tumor volumes were measured once every other day, the tumor volume growth curves were drawn and the tumor inhibitory rates were calculated to observe the inhibit effect of each groups with drugs.2. Tumor pathological changes were observed by light microscopy after routine HE staining.3. Laser scanning confocal microscope were performed to detect the expression level of Beclin1、MAP1LC3A、CD133 and CD90.4. Immunohistochemistry were performed to detect the expression level of Beclin1、MAP1LC3A、CD133 and CD90.5. The expression of Beclin1、MAP1LC3A、CD133 and CD90 was confirmed and quantified by western blotting. Results: 1. The effect of each medicated groups on the growth of S180 sarcoma transplantation tumorRAPA alone, PESV either high-dose or low-dose alone, and combination of PESV and RAPA resulted in inhibition of S180 sarcoma transplantation tumor growth compared to control group. The inhibitory rate was 25.4%、15.3%、34.0%�'� 42.5%, respectively(P<0.05, P<0.01); Compared with RAPA group, the tumor weight and tumor volume in the combination group were also obviously reduced(P<0.05);Compared with low-dose of PESV group, the tumor volume in the high-dose of PESV group were reduced and the speed of tumor growth were more slowly(P<0.05)。 2. Pathologic and morphometric analysis of sarcoma 180 after treatment in each groupsThere were less necrosis area and less pyknosis morphological changes in the control group. We can see a plurality of necrosis area in the combination group、RAPA group, PESV either high-dose and low-dose group with pyknosis and karyorrhexis. The necrosis area in the combination group is the largest. There were the morphology of autophagy in the each groups with drugs likecytoplasm amorphous state and broken nuclear. There were some monocyte around thecancer nest. 3.The effects of PESV combined with RAPA in protein expression level of Beclin1、MAP1LC3A、CD133 �'� CD90 in sarcoma 180 tumor tissue.We can see from the Laser scanning confocal microscope, Beclin1、MAP1LC3A、CD133 and CD90 were expressed in the cytoplasm or cytomembrane of tumor cells, CD133 and CD90 were primary expressed in cytomembrane of tumor cells with green fluorescence. Compared with the control group, the protein expressions of Beclin1 and MAP1LC3 A in other medicated groups were all increased and the the protein expressions of CD133 and CD90 in other medicated groups were all increased.The protein expressions of Beclin1 and MAP1LC3 A in the combination group were increased significantly and CD133 and CD90 in the combination group were decreased significantly.By immunohistochemistry staining, Compared with control group, the protein expression of Beclin1、MAP1LC3A were all increased in the groups with drug(P<0.05,P<0.01)and the protein expression of CD13 and CD90 all decreased( P<0.05,P<0.01). The protein expression of Beclin1 and MAP1LC3 A in the combination group were all increased significantly(P<0.01) and the protein expression of CD133 and CD90 in the combination group were all decreased significantly(P<0.01).Compared with low-dose PESV group, there were more expression in Beclin1 and MAP1LC3 A and less expression CD133 and CD90 in the high-dose PESV group(P<0.05).The effect of PESV treatment on Beclin1、MAP1LC3A、CD133 and CD90 protein expression was also assessed by western blotting analysis. Beclin1 and MAP1LC3 A expression were increased and CD133 and CD90 were reduced in high and low dose of PESV 、 RAPA and the combination groups. The protein expression of Beclin1 and MAP1LC3 A in the combination group were all increased significantly(P<0.01),and the protein expression of CD133 and CD90 in the combination group were all decreased significantly(P<0.01). The results of Western blot were generally consistent with the results of immunohistochemistry and immunofluorescence staining. Conclusions:1. PESV combined with RAPA can inhibit the growth of S180 sarcoma transplantation tumor.2. PESV can raise the protein expression of Beclin1 、 MAP1LC3 A and increase autophagy death in tumor cells.3.PESV can reduce the protein expression of CD133、CD90 in tumor tissue and inhibit the growth of tumor stem cells.4.PESV combined with RAPAcan play an antitumor role byincreasingautophagy of tumor cells and inhibiting tumor stem cells.