A Mechanisam Study Of Quxie Capsule On Treating Colon Cancer And Treating Method First Exploration

Background:Quxie capsule is improved and optimized from ancient prescription of Yinyanggongjiwan, after clinical application for more than ten years, we found that Quxie capsules can reduce colorectal cancer mortality in the late prolong survival, median survival time and improve survival quality. It can reduce the recurrence of II-III colorectal cancer. However, the molecular mechanism of action research is unknown. This study is based on the results of past clinical studies to explore possible mechanism of Quxie capsules on advanced colorectalMethods:In this study, we use cell culture techniques, APA, MTS assay to study proliferation inhibition rates of Quxie capsules containing serum on tumor cells line HCT-116colon and other tissue sources cancer cell lines.we also study the proliferation inhibition rates of combined prescription,broken prescription and combined with SijunziTang; We use flow cytometry Anexin V-FITC+/IP double staining to study Quxie capsules and Sijunzitang combined or single treating HCT-116cells with which to study apoptosis rate and late apoptosis rate. Western bloting assay was used to detect the apoptosis and autophagy related protein Caspase-3and LC3.We also use CT-26tumor-bearing Balb/c mouse model to study Quxie capsules demolition parties and party together with Sijunzitang survival inhibition rate, body weight, tumor weight, prognosis related colorectal proteins PI3K, PTEN, KRAS expression,as well as anti-tumor-associated cytokines IFN-γ, IL-4.We als study the changes of Thl/Th2balance. Confocal microscopy prognosis of colorectal cancer related proteins LC3, PI3K, PTEN, KRAS expression were also studied.Results:In this study, Quxie Capsule drug-containing serum inhabits human colon cancer HCT-116cells in vitro,the inhibition rate was (71.28±5.22),while we also study drug-containing serum of Quxie Capsule on other cell lines:MDA-MBA-231(22.94±2.87), hepatoma Bel-7402(26.74±3.34), cervical cancer Hela (45.80±4.21), compared with a significant difference (P<0.05). But we found a proliferation effect on the health human PBMC(-10.38±1.13)(P<0.01). Compared other time points (6h,12h,48h) and concentration (5%,20%),we found the best working concentration and timing of Quxie capsule on colorectal cancer HCT-116cells was10%serum containing acting drugs work for24h. Compared with Quxie capsules group, Quxie capsules without key toxic ingredients Croton and fructus gleditsiae was found decreased in vitro inhibition respectively:Quxie capsules without Croton (63.29±4.24), Quxie capsules without Croton and fructus gleditsiae (61.34±2.34)(P<0.05). Quxie capsules combined with Sijunzitang antagonisted inhibitive rate (20.01±3.83)(P<0.05). Anexin V-FITC+/IP double staining method for detecting early apoptosis(13.3±3.21) and late apoptosis rate (35.38±5.23) of Quxie Capsule. From Western bloting,we observed Quxie capsules can induce apoptosis effect by increasing cleaved caspase-3pathway.Quxie capsule.In Quxie capsules combined with Sijunzitang group/we observed both increasing of cleaved LC3and apoptosis. However, this inhibitive effect can be antagonisted when combined with Sijunzitang.Adding autophagy inhibitor3-MA can increase inhibitive effect of Quxie Capsule combined Sijunzitang group (34.21±3.92). An acute toxicity in vivo experiments show that the maximum dose of capsules Quxie20g/kg. By the establishment of CT-26colon cancer cells grown babl/c mouse model, this study found that tumor Quxie capsules with reduced tumor weight compared with the control group, but decreased survival in mice, weight loss, Thl/Th2.The tumor inhibitive rate and the survival rates.were:Quxie Capsule group (16.92%±1.53,21.34%±1.50), Quxie Capsule without Croton (-8.72%±3.87,39.32%±3.27), Quxie Capsule without croton honeylocust (21.26%±3.21,54.21%±9.85), Quxie Capsule combined with Si Junzi tang (19.14%±7.84,58.51%±4.92), Quxie Capsule alternated taking with Si Junzi tang(21.23%±4.90,72.98%±5.27),5-FU (22.44%±4.23,60.29%±3.32), negative control (0,49.28%±3.29). Quxie Capsule without Croton and Fructus Gleditsiae group had a beter survival rate than Quxie Capsule group,but Thl/Th2decreased (P<0.05). Compared with control group,Quxie Capsule combined with Si Junzi tang and Quxie Capsule alternated with Si Junzi tang were observed increased survival rate, weight, mouse Thl/Th2(P <0.05). LC3、PI3K and PTEN expression were observed increased expression of different degree under laser scanning confocal microscope on Quxie Capsule group, Quxie Capsule without Croton group,Quxie Capsule without croton honeylocust group. Quxie capsules (Low toxicity) was found has higher survival rate than,the mechanism of may exist in increasing IFN-γ and decreasing IL-6(P<0.01).Comparing with heat clearing and detoxicating prescription, we found sharply increased IL-6and IFN-γ in Quxie capsules(P<0.01). On laser confocal microscope we can observed LC3, KRAS, PTEN expression decreased in Quxie Capsule alternative group and Quxie Capsule (low toxicity) group. Quxie Capsule (low toxicity) group was obseved PI3K increasing.The immunological difference of Heat clearing and detoxicating and Wen Yang may be exist in a increasing of INF-γ and IL-6in the later.Conclusion:This study found Quxie capsules containing serum have inhibitive effect in vitro on HCT-116colon cancer cells,the inhibive effect is better than the other three cell tissue source cancer cells.And we found Quxie Capsule have high efficiency and low toxicity (PBMC). We found Quxie capsules containing serum can induce tumor cell death by apoptosis pathway in vitro. If removed the key two toxic ingredients of Quxie capsules(Croton and fructus gleditsiae), we found a decline of inhibition rate of tumor.Quxie Capsule shortered survival time and weight of tumor-bearing mice, but Quxie capsules combined with Sijunzitang prolonged survival.Compared with Sijunzitang combination, Quxie capsules and Sijunzitang alternative taking achieved better survival rate and inhibition rate.The study also found that Quxie Capsule and Si junzi Tang Combined with had antagonism in vitro. LC3n PI3K and PTEN expression were observed increased expression of different degree under laser scanning confocal microscope on Quxie Capsule group, Quxie Capsule without Croton group,Quxie Capsule without croton honeylocust group. We found Quxie Capsule and Sijunzitang Combined with can regulate the function of T helper cells in mice, improving Thl/Th2immune balance in a reasonable range. We found Quxie capsules (Low toxicity) has higher survival rate than Quxie capsules,the mechanism of this may exist in increasing IFN-γ and decreasing IL-6.Comparing with heat clearing and detoxicating prescription, we found sharply increased IL-6and IFN-Y in Quxie capsules.On laser confocal microscope we can observed LC3, KRAS, PTEN expression decreased in Quxie Capsule alternative group and Quxie Capsule (low toxicity) group.Quxie Capsule (low toxicity) group was obseved PI3K increasing.Base on multiple levels of past clinical and in vitro and in vivo experiments, we put forward the hypothesisthe traditional Chinese medicine Wenyang Jianpi therapy based on the immune balance.