Study On The Inhibitory Effects And Mechanisms Of The Chinese Medicine Yifei Tongluo Formula And Its Improved Prescription On The Growth And Metastasis Of Lung Cancer

Lung cancer has become the most malignant tumor with the highest morbidity and mortality due to the characteristics of high metastasis,drug resistance and easy recurrence,threatening the human health and quality of life.Because of the latency during the early detection of lung cancer,about 70%of patients are in advanced stage at the time of diagnosis missing the opportunity for surgery.Traditional treatments such as chemotherapy and radiotherapy are the main means to treat lung cancer.However,there are many disadvantages of traditional treatments for instance multi-drug resistance,serious side effects,and poor therapeutic effect.In recent years,although targeted therapy and immunotherapy for lung cancer have made great progress,the 5year survival rate of lung carcinoma is only 17.4%.Therefore,it is an urgent problem to develop drugs with better efficacy,less toxicity and multi-target for the treatment of lung cancer.As the progress of lung cancer is accompanied by the immune disorder of the body,Chinese medicine formula plays an increasingly important role in the treatment of lung cancer in recent years,which can comprehensively adjust the immune state of the body,inhibit the tumor growth and metastasis,prolong the survival time and significantly improve the quality of life of patients.Chinese medicine compound has unique advantages of multi-component,multi-target and less toxicity.Therefore,it is of great significant to seek effective Chinese medicine compound to inhibit the growth and metastasis of lung cancer.Yifei Tongluo(YFTL),a Chinese herbal formula,exerts anti-tuberculosis effects through immune regulation,anti-multidrug resistance and detoxification,which is clinically used for the treatment of multidrug resistance and extensively multidrugresistant tuberculosis.Several studies demonstrated that YFTL could regulate immune function and balance Th1/Th2 immune response.Modern pharmacological studies have shown that kinds of herbs and main active ingredients of YFTL have anti-tumor and anti-metastatic activities,and play important roles in inhibiting cell proliferation,inducing apoptosis,inhibiting angiogenesis,reducing inflammation,and reversing multi drug resistance.In addition,the prescription of YFTL is in line with the Chinese medicine principle of treatment for lung cancer.Therefore,the activity and mechanism of YFTL on the growth and metastasis of lung cancer were firstly studied in vivo.Secondly,in order to seek a more simplified and effective Chinese medicine compound,we improved the YFTL consisting of 11 kinds of herbal medicine according to the compatibility principle of YFTL,the mechanism of anti-lung cancer metastasis,the theory of Chinese medicine and the research results of modern pharmacology.The improved prescription of YFTL(CMFE)were composed of Ganoderma lucidum,Salvia miltiorrhiza,Radix stemonae and Herba ardisia japonica.The inhibitory effects of CMFE on the growth and metastasis of lung cancer were studied on the whole animal,cell and molecular levels,and the possible molecular mechanism of CMFE was discussed to provide experimental and theoretical basis for the development of antilung cancer drugs.The main research contents and results of this subject have the following aspects.(1)The inhibitory effect and mechanism of YFTL on the tumor growth and metastasis in Lewis lung carcinoma miceWe determined the effect of YFTL in the inhibition of tumor growth and metastasis to the lung in Lewis lung carcinoma mice and explored the underlying mechanism.The results showed that YFTL significantly inhibited the tumor growth and prolonged the overall survival in Lewis lung cancer mice without exhibiting distinct toxicity.At doses of 200 mg/kg and 400 mg/kg,YFTL suppressed the tumor growth by 48.4%and 62.0%(p<0.001),respectively.Western Blot results showed that YFTL could induce tumor cell apoptosis by up-regulating the p53 and Bax expression,and down-regulating Bcl2 expression.Additionally,YFTL treatment resulted in a markedly decreased number of surface lung metastatic lesions compared with the model control group.The inhibitory rate of lung metastasis with low and high doses of YFTL was 47.1%(p<0.01)and 61.5%(p<0.001),respectively.YFTL also inhibited the microvessel density(MVD)in tumor tissues,down-regulated the expression of VEGF,MMP-2,MMP-9,Vimentin in tumor tissues and lung tissues,and up-regulated the expression of Ecadherin,suggesting that YFTL could suppress the angiogenesis and the epithelialmesenchymal transition(EMT)of the tumor.Besides,YFTL could inhibit the development of tumor by regulating the immune fuction including up-regulating the expression of IL-2 and IFN-?,down-regulating the expression of inhibitory cytokines IL-10 and TGF-? in tumor,promoting T lymphocytes proliferation,increasing NK cell cytotoxicity,up-regulating the ratio of CD4+/CD8+and NK cells,and down-regulating the proportion of Treg cells.Mechanistic studies indicated that PI3K/Akt and MAPK pathways may be involved in YFTL-induced apoptosis,and YFTL could suppress the angiogenesis and EMT through inhibiting Akt,ERX1/2 and TGF?1/Smad2 pathways to inhibit the tumor metastasis.(2)The influence of CMFE on the growth and metastasis of A549 cellsWe evaluated the effect of CMFE on proliferation,apoptosis,invasion and metastasis of human adenocarcinoma A549 cells,and explored its possible mechanism.The results of MTT assay showed that CMFE significantly inhibited the proliferation of A549 cells in a time and concentration-dependent manner.The IC50 values of CMFE for 24 h,48 h,and 72 h were 330.2±9.5 ?g/mL,125.3±7.2 ?g/mL and 56.0±3.2 ?g/mL,respectively.The cell cycle and apoptosis of A549 cells were detected by Flow cytometry.CMFE could arrest A549 cells in G2/M phase,which involved up-regulation of p53,p21 expression and down-regulation of Cyclin B1 expression in A549 cells.CMFE could induce A549 cells apoptosis by up-regulating the expression of p53 and Bax,down-regulating the expression of Bcl-2,promoting cytochrome C release,and activating caspase 9 and PARP.The results of Transwell migration and invasion assays showed that CMFE observably inhibited the abilities of migration and invasion of A549 cells.CMFE increased the expression of the epithelial phenotype marker E-cadherin and suppressed the expression of the esenchymal phenotype marker Vimentin.CMFE also inhibited the MMP-2 and MMP-9 expression.Mechanistic studies indicated that CMFE activated JNK and p38 MAPK pathways and inhibited ERK,PI3K/Akt and STAT3 pathways involved in the effect of anti-proliferation,apoptosis induction,antiinvasion and anti-migration in A549 cells.(3)The inhibitory effect and mechanism of CMFE on the tumor growth and metastasis in Lewis lung carcinoma miceA mouse model of Lewis lung cancer was established to investigate the effect of CMFE on the tumor growth and lung metastasis formation,and to evaluate the underlying molecular mechanism.The results showed that CMFE inhibited the tumor growth in lewis lung carcinoma mouse model with no obvious side effects and prolonged the overall survival of tumor-bearing mice.At doses of 200 mg/kg and 400 mg/kg,CMFE suppressed the tumor growth by 51.2%and 67.4%(p<0.001),respectively.CMFE could induce tumor cell apoptosis by up-regulating the expression of p53 and Bax,down-regulatiing the expression of Bcl-2,promoting cytochrome C release,and activating PARP.In addition,CMFE obviously inhibited the lung metastasis of Lewis lung cancer.The inhibitory rate of lung metastasis with low and high doses of CMFE was 59.2%(p<0.01)and 78.6%(p<0.001),respectively.CMFE inhibited the expression of CD34 and VEGF in tumor tissues.CMFE also downregulated the expression of MMP-2 and MMP-9,and up-regulated the expression of Ecadherin in tumor tissues and lung tissues.Moreover,the results of immune regulation showed that CMFE markedly increased the expression of IL-2 and IFN-y,downregulated the inhibitory factors such as IL-10,TGF-? and VEGF,and increased the infiltration of CD8+T cells and the expression of CD86 in tumor tissues.CMFE also promoted T lymphocytes of splenocytes proliferation,increased NK cell cytotoxicity,up-regulated CD4+/CD8+ ratio and the proportion of NK cells,and down-regulated the proportion of Treg cells.Our findings suggest that CMFE could inhibit the tumor growth and metastasis by antagonizing the immunosuppression of tumor microenvironment and enhancing body's immune function.Mechanistic studies indicated that the apoptosis induced by CMFE was mainly related to the regulation of PI3K/Akt and MAPK pathways.CMFE inhibited tumor metastasis by the inhibition of ERK1/2,PI3K/Akt/mTOR and STAT3 pathways,suppressing the angiogenesis and weakening the ability of invasion and metastasis.CMFE could antagonize the immunosuppressive microenvironment of tumor which may be related to the inhibition of PI3K/Akt/mTOR and STAT3 pathway.(4)The protective effect of bergenin on cyclophosphamide-induced immunosuppressionBergenin is the main active ingredient of YFTL and CMFE.The protective effect of bergenin on cyclophosphamide(CTX)-induced immunosuppression was investigated to fill the deficiency in this aspect.The results showed that bergenin increased the index of immune organs,the level of IgM and IgG,peritoneal macrophage function,the proliferation of T and B lymphocytes,NK and CTL cell activities,and CD4+/CD8+ ratio.Besides,bergenin also had the ability to modulate the Th1/Th2 balance.Bergenin also prevented the decrease in numbers of peripheral RBC,WBC and platelets induced by CTX,providing supportive evidence for their anti-leukopenia activities.More over,bergenin reversed the decrease in the total antioxidant capacity including activities of superoxide dismutase(SOD),catalase(CAT)and glutathione peroxidase(GSH-Px)induced by CTX.In conclusion,bergenin protected against CTXinduced adverse reactions by enhancing immune functions and augmenting antioxidative activity and could be considered as a potential immunomodulatory agent.In summary,we for the first time provide evidence that YFTL can effectively inhibit tumor growth and metastasis in Lewis lung cancer mice.Then,we improved the prescription of YFTL,and the anti-lung cancer effect and mechanism of the improved prescription(CMFE)in vitro and in vivo were studied.Our findings suggest that YFTL and CMFE could prevent the growth and metastasis of lung cancer through modulation of multiple signaling pathways,involving the inhibition of proliferation,induction of apoptosis,suppression of invasion and metastasis ability,attenuation of angiogenesis,and regulation of the immune function of tumor-bearing mice,which provides experimental and theoretical basis for the application and development of Chinese medicine compound in the treatment of lung cancer.Compared with YFTL,CMFE has higher anti-tumor metastasis activity,simpler components and lower cost,suggesting that CMFE can be considered as a potential prescription with the advantages of low toxicity and multi-target for the treatment of lung cancer.In addition,our data showed that bergenin could be considered as a potential immunomodulatory agent,which provides experimental basis for its combination with chemotherapy drugs and application in the treatment of immunosuppressive diseases.