Experimental Study On Anti - Tumor Effect And Autophagic Effect Of Feijun Prescription On Lewis Lung Cancer

Purpose:The anti-cancer mechanism of Feijining Decoction and its mechanism of action were explored by in vivo and in vitro experiments. In vitro, we observed the effect of Feijining Decoction containing serum on cell proliferation and apoptosis of Lewis lung cancer cell. And we also detect the expression of protein and RNA level of apoptosis related autophagy genes, such as Beclin1, LC3B-Ⅱ, LC3B-Ⅰ, Atg5, the anti-cancer effect of Feijining Decoction and its effect on autophagy were delineated. In vivo, Nude-mouse transplanted tumor model of Lewis lung cancer were administered Feijining Decoction, Lewis lung cancer cells form and autophagy-related genes Beclin 1, LC3B-Ⅱ, LC3B-Ⅰ, Atg5 protein level in tumor tissue were tested to analysis the anti-cancer mechanism of Feijining Decoction.Material and method:In vitro: Preparation of drug containing serum, and treat Lewis lung cancer cells with the drug containing serum. 40 rats were randomly divided into 5 groups, Feijining Decoction super-high dose group, Feijining Decoction high dose group, Feijining Decoction medium dose group, Feijining Decoction low dose group and control group. All animal were given medicines or normal saline via intragastric administration, 1 time per day for 3 days. The control group were given normal saline via intragastric administration and forbid food but not water for 12 h at 3rd day, the heads of rats were cut down for blood next day. Then prepare drug containing serum with the blood. We detect the inhibitory effect of Feijining Decoction containing serum on cell proliferation and apoptosis of Lewis lung cancer cell with MTT method in each group at 24 h, 48 h and 72 h, find out the optimal serum dose group for next step. Then we divided the cell for 4 groups, Feijining Decoction group(best volum), cisplatin group, combination group and control group, cell detected by flow cytometry after 48 h to find out the apoptosis rate. The cells were transfected with GFP-LC3 to detect cell autophagy: Lewis lung cancer cells were planted into 6-well culture plate at 6 × 105/ m L, divided into 4 groups coated with different treatment, Feijining Decoction group(20%containing serum in DMEM), cisplatin group(1μg/ml IN DMEM), combination group(20%containing serum + 1μg/ml cisplatin in DMEM) and control group(control serum in DMEM). All 4 groups cultured for 24 h, observed by fluorescence microscope, photograph and count cell number. Collect cell from 4 groups, to find out the expression of protein and RNA level of apoptosis related autophagy genes, Beclin1, LC3B-Ⅱ, LC3B-Ⅰ, Atg5 by western-blot and RT-PCR.Results:1. effect of Feijining Decoction containing serum on cell proliferation of Lewis lung cancer cellFeijining Decoction containing serum has Significant inhibition effect on cell proliferation of Lewis lung cancer cell, at the same time, with the increase of the concentration or on the contrary, the inhibition effect on Lewis lung cancer cell increased significantly(P<0.01). Detect the inhibition effect on Lewis lung cancer cell in 24 h, 48 h and 72 h, results showed that compared with the control group, Feijining Decoction containing serum inhibit the proliferation of Lewis lung cancer cells, and the inhibitory effect increased with increasing concentration and time. And the effect of inhibition was enhanced with the concentration increased and time prolonged. We find out the optimal serum dose was the super-high dose group, we use this dose for the next step.2. Combination group(Feijining Decoction containing serum and cisplatin combined) has inhibition effect on Lewis lung cancer cell. Feijining Decoction group:The 24 h, 48 h, 72 h inhibition rate was 28%, 32% and 33%, cisplatin group: The 24 h, 48 h, 72 h inhibition rate was 42%, 66% and 75%, Combination group Feijining Decoction containing serum and cisplatin combined): The 24 h, 48 h, 72 h inhibition rate was 58%, 86% and 91%.3. Flow cytometry instrument detection cell apoptosis rate in 4 groups, control group, Feijining Decoction group, cisplatin group and the combined group at 48 h, the cell apoptosis rate was 3.20% and 10.36%, 14.31%, 30.18%, the highest apoptosis rate was the combined group. Compared with the control group, the Feijining Decoction group had more effect on induce the cell apoptosis, P<0.01. Compared with cisplatin group, the apoptosis rate increased significantly in combined group, P<0.01.When cell in G0/G1 phase the Feijining Decoction group had much number of the cell in this phase, the is 38.69%.In the period of G0/G1, the Feijining Decoction group cell number was 38.69%, the cisplatin group was 56.12%, and the combined group was the highest, 68.35%.4. Autophagy GFP-LC3 transfection and countingFluorescent spots of autophagy body was rarely observed in the control group. Green gathered fluorescent was more observed in Feijining Decoction group than control group(P<0.05). Fluorescent spots of autophagy body was more observed in Cisplatin group than combining group(P<0.05).5. Protein expression of Beclin1、LC3B-Ⅱ/ LC3B-Ⅰ、Atg5 in tumor tissue were tested by Western blottingProtein expression of Beclin1、LC3B-Ⅱ/ LC3B-Ⅰ、Atg5 in tumor tissue of Feijining group is more than control group, P<0.05.As well as, these protein expression in Cisplatin group is more than than combining group, P<0.05.6.m RNA expression of Beclin1、LC3B-Ⅱ Atg5 in tumor tissue were tested by real time PCR Primers validation: each primer amplification curve is normal, dissolve curve without impurity peak, unimodal, dissolve the average temperature is consistent, is unique. m RNA expression of Beclin1、LC3B-Ⅱ、Atg5 in tumor tissue of Feijining group is more than control group, P<0.05.As well as, these m RNA expression in Cisplatin group is more than than combining group,P<0.05.7. Spirit, diet, activities, and urine of nude mice were observed and recorded. And the tumor weight were tested in each groups.Autonomous activity, and weight of B group and C group were better than A group, p<0.05.Compared with C group, the autonomous activity, and weight of B group is only slightly better, but there is no significant difference between the two groups. Compared with D group, the autonomous activity, and weight of E group is better, and there is significant difference between the two groups,P<0.05.8.Growth of nude mouse Lewis transplantation tumor and rate of inhibiting tumor were measured.Tumor weight of B group and C group were less than A group,P<0.05.Tumor weight of B group is lightly less than C group,P>0.05. Tumor weight of E group and Dgroup were less than A group,P<0.05.The rate of inhibiting tumor of Bgroup, C group,Dgroup and E group respectively are 33.7%、40.6%、56.6%、66.9%.Feijining Decoction restrain the growth of the Lewis transplanted tumor and has synergy with cisplatin.9.HE staining results of each tumor bearing nude mice group:Tumor tissue is more regular, more easy to peel off observed by naked eye. Partial tumor necrosis in vitro. A lots of large tumor cells, messy arrangement more closely, and a large stained nucleus cytoplasm ratio imbalance in A group were observed; More regular tumor size,lots of necrotic cells, less cell atypia in B group and C group;more necrotic cells, less fibrous tissue in D group; lots of necrotic cells; less cell atypia; Lots of lymphocytes showed in E group.10. Electron microscopic observation of autophagosome on each tumor bearing nude mice groupAutophagosome were observed in each group, autophagosome was encapsulated in a double membrane, typical autophagosome is visible and was encapsulated in its membrane. The B group and C group showed more scattered autophagosome, D group also showed a large number of vacuoles within the cytoplasm, but E group got the most autophagosome number. In order to increase: A group, B group, C group, D group, E group.11. Detect the expression of protein level of apoptosis related autophagy genes, Beclin1, LC3B-Ⅱ, LC3B-Ⅰ, Atg5 by western-blotDetect the expression of protein level of apoptosis related autophagy genes, Beclin1, LC3B-Ⅱ, LC3B-Ⅰ, Atg5 by western-blot for each group. Compared with the control group, Beclin1, LC3B- II/ LC3-Ⅰ, Atg5 protein expression was significantly increased in the B group, compared with the control group, D group and C group, Beclin1, LC3B- II and Ag5 were also enhanced. Relative gray value comparison, there are significant differences(P<0.05).Conclusion:1. Feijining Decoction medicated serum can inhibit Lewis lung cancer cell growth, and promote its apoptosis.2. Feijining Decoction medicated serum induce Lewis lung cancer cell autophagy, enhanced the expression of protein and RNA level of apoptosis related autophagy genes, Beclin1, LC3B-Ⅱ and Atg5, improve the proportion of LC3B-Ⅱ/LC3B-Ⅰ.3. Feijining Decoction could improve the general state and increase the number of independent activities of tumor bearing nude mice, which can inhibit tumor growth of tumor bearing nude mice.4. The expression of Beclin1, LC3B-Ⅱ/LC3B-Ⅰand Atg5 protein level was raised by Feijining Decoction, and it also improve the proportion of LC3B-Ⅱ/LC3B-Ⅰ.5. Feijining Decoction can inhibit tumor, which may be related to the induction of autophagy.