The Effect And Mechanism Of Low-polar Ginsenosides ALK As The New Chinese Patent Medicine On Anti Colorectal Cancer

BackgroundThe morbidity rate of colorectal cancer(CRC)ranks the third in the world.The conventional therapy includes surgery,chemotherapy,molecular targeted therapy and radiotherapy etc.,but these therapies cannot effectively control the progress and metastasis of tumor.The commonly used chemotherapeutic drugs and targeted therapy agents have serious side effects and toxicity,easily leads to drug resistance and so on,which are still the unsolved problems.Therefore,it is necessary to study and develop the effective,safe and new Chinese patent drugs to treat cancer.The study and development of An-liu-ke capsule(low polarity ginsenosides of ALK)as new Chinese patent medicine has been supported by key project of major new drug creation in the"11th five-year plan".Until now,we have completed the study of candidate drug and the majority work of preclinical study.The stable preparation process and reliable pilot product quality have been developed,and the analysis of its composition and content has been also completed,which mainly contains five low polar monomers of ginsenosides,incluing the F4,Rh4 and S-Rg3 etc.The acute and chronic toxicity study showed no significant toxicity,and the patent for invention has been authorized by the State Intellectual Property Office of China.Since the activity of low polarity ginsenosides ALK in anti-colorectal cancer is not clear,here it is our first time to validate the efficacy of ALK on anti-colon cancer in vivo and in vitro.Various experimental techniques have been conducted,including tumor-bearing mouse model,cytology and molecular biology experiments,and also the culture of immune dendritic cells(DC),which are core cells of the tumor microenvironment.Our study reveal the immune response efficacy of ALK in participation of anti-colorectal cancer through the induction of DC differentiation,maturation and activation function.Our study will provide the experimental evidence to expand its function and indication,and ALK is expected to be a novel new Chinese patent drug with its safety and efficacy in the treatment of leukemia and colorectal cancer.Methods1.ALK inhibits the proliferation of colon cancer cells,causes cell cycle arrest and enhances the expression of tumor suppressor genesThe dry powder of low polarity ginsenosides ALK(the capsule product named as An-liu-ke)was provided by the research and development team of new drug,Zhejiang Provincial Hospital of TCM.Human colon cancer of HT29,SW480 and SW620 cells were used as target cells.We treated cells with different concentrations of ALK and determined the inhibitary effect of ALK on cell proliferation using clony formation assay and MTT test.Flow cytometry was performed to analyse cell cycle status;confocal microscope was used to observe the expression of interferon-induced transmembrance-1(IFITM1),and DNA microarray was conducted to analyse genes expression profile induced by ALK.2.The study of ALK in anti-colon cancer using mouse xenograft modelThe HT29-GFP cells were used to construct colon cancer tumor-bearing nude mice model.Mice were divided into six groups,including the untreated control,the lower,medium and higher dosages(50,100 and 200mg/kg)of ALK treatment groups,and positive drug control groups(chemotherapeutic drugs of capecitabine,Chinese patent medicine of Shenyi capsule),respectively.At the treatment of 7 days or 14 days,the fluorescence signal value of tumor burden was examined by living small animal imaging.The volume and weight and of the tumor was measured and the histologic section of tumor tissue was used for pathologic examination.The mRNA expression levels of oncological genes were analyzed by real-time PCR,and the protein expression levels of oncological genes were detected using Western blot and immunohistochemistry.3.ALK induces the differentiation,maturation and activation of dendritic cellsBMDCs derived from mononuclear cells of mouse bone marrow were cultured in vitro,then,DCs were isolated and purified by immunomagnetic beads.Flow cytometry was performed to analyse phenotype of DCs under treatment with ALK.The colon tumor-bearing nude mice models were treated with lower,medium and higher dosages of ALK for 14 days,an immunofluorescence assay was used to determine the expression of CD83 as mature DCs surface molecule in tumor tissue from xenograft mice.Results1.Cytology and gene expression profile demonstrate the efficacy of ALK in anti-colon cancer1.1 The clony formation assay showed that proliferation of human colon cancer of HT29,SW480 and SW620 cells was significantly inhibited by ALK at the dosage of 25,50,75.and 100mg/L.The suppression rate of clony formation was from 14.47±3.74%to 100.00±0%(P<0.05,0.01).The IC50 of ALK in these three cell lines was 55.86,57.95,and 63.96mg/L,respectively.MTT test showed that inhibitory rate of ALK at the dosage of 50,75,100 and 150mg/L was from 21.45±6.16%to 96.42±1.09%(P<0.05,0.01).The IC50 of ALK in these three cell lines was 88.20,97.32 and 91.08mg/L by MTT test.It suggested that ALK effectively inhibits the proliferation of colon cancer cells in a dose-dependent manner.1.2 ALK effectively induced cell cycle arrest in G0/G1 phase the cell percentage of G0/G1 phase was significantly increased(P<0.05,0.01)in HT29 and SW480 cells when treated with ALK at 50,75 and 100mg/L,respectively.In the meeanwhile,the cell percentage of S phase was significantly decreased(P<0.05,0.01)and the proliferation index rate was obviously lower than the control(P<0.05,0.01).It indicated that ALK effectively inhibits the proliferation of colon cancer cells by blocking cell cycle in a dose-dependent manner.1.3 The IFITM1 transmembrane protein was expressed in HT29,SW480 cells,and showed strong positive reactions of green fluorescence.The green fluorescence intensity of IFITM1 protein was significantly decreased after ALK treatment,and cell morphology of HE staining also showed denaturation and degradation phenomenon,further confirming the inhibition proliferation and metastasis effect of ALK on both cancer cells.1.4 The gene expression profile analysis showed that ALK induced different genes expression associated with oncology,though upregulation of tumor-suppressor and DNA damage repair genes of p53,AKR1B10,CDKN1A,ATF3 and PER1,etc.,and downregulation of migrate related and signaling pathway genes of ANLN,DKK1 and ERCC6L,etc.It revealed the molecular mechanism of the inhibition proliferation and metastasis in colon cancer cells by ALK treatment.2.ALK inhibits tumor growth in tumor-bearing nude mice2.1 ALK effectively inhibited tumor growth in mice exnograft model.The tumor weight and volume from ALK treated groups with lower,medium and higher dosages were significantly smaller than those of untreated group(P<0.01).The body weight of mice under treatmetn with ALK was significantly weigher than those of capecitabine group and shenyi capsule group respectively(P<0.05).It suggested that ALK not only effectively inhibited tumor growth,but also provided better survival quality of mice to certain degree of advantage.2.2 The tumor-bearing nude mice were treated with ALK at lower,medium and higher dosages for 7 days.Living small animal imaging showed that the fluorescence intensity of tumor in nude mice treated with medium and higher dosage of ALK was significantly weaker than that of untreated model group(P<0.05,0.01).At the treatment of 14 days,the fluorescence signal intensity of the tumor from ALK treatment group at lower,medium and higher dosages was significantly weaker than that of control group(P<0.05,0.01).The pathological examination of tumor tissue showed denaturation and degradation phenomenon,the decreased cell density from ALK treatment groups with meidiun and higher dosages.It was proved that ALK exerts its anti-colon cancer effect in the mouse exnograft model.2.3 The tumor-bearing nude mice were treated with ALK at lower,medium and higher dosages for 7 days.Both mRNA and protein expression levels of tumor suppressor gene p53,MCC and DCC were significantly upregulated in tumor tissue from mice model treated with ALK(P<0.05,0.01).On the contrary,the mRNA and protein expression levels of oncogene c-Myc were significantly downregulated under ALK treatment(P<0.05).It indicated that ALK possesses anti-colon cancer activity in the mouse exnograft model.3.ALK induces DCs differentiation,maturation and activates their function3.1 Bone marrow-derived DCs were first prepared from mice and then treated with ALK.CD86 and CD83 positive cell percentage as DCs differentiation markers were significantly increased than those of untreated DCs(P<0.05).It suggested that ALK enhanced immune response to anti-colon cancer through promoting the differentiation and maturation of mouse BMDCs.3.2 After treated with ALK,CD83 protein expression as red fluorescence positive reaction at tumor mergins was significantly increased than that of control(P<0.05).It suggested that ALK enhances the infiltration of DCs at tumor margins,further exerting its anti-colon cancer activity..Conclusion1.For the first time,our results demonstrate the efficacy of ALK in anti-colon cancer using in vitro cell proliferation assay and microarray analysis.The molecular mechanism of ALK is associated with cell cycle arrest,upregulation of tumor suppressor genes and downregulation of oncogenes.2.ALK possesses the efficacy of anti-colon cancer in tumor-bearing nude mice.Afer treatment with ALK,the weight and volume of tumor in vivo were significantly reduced,and the fluorescence signal of tumor was significantly weakened,which was characterized by denaturation and degradation,and cell density was obviously decreased in tumor tissue.Meanwhile,the mRNA and protein expression levels of tumor suppressor genes p53,MCC and DCC were upregulated.On the contrary,the expression of oncogene c-Myc was downregulated by ALK treatment.It suggests ALK may exert its anti-colon cancer effect through the regulation of tumor related genes.3.ALK effectively induces the differentiation and maturation of mouse bone marrow-derived DCs,activates their function and enhances the infiltration of DCs at tumor margins.It suggests that ALK participates in immune response to anti-colon cancer,and it may exert multi-role and multi-target effect to anti-colon cancer.