Evaluation Of Anticancer Function Of Anhua Black Tea Extract

Experiment purposeIn recent years,the black tea in the tea market share is becoming biger and it also got the praise of consumers.There are two main reasons,one is the unique taste of black tea and excellent quality,and on the other hand,it has very good health care function.The health effects of black tea include lipid-lowering,anti-oxidation,anti-cancer,regulation of intestinal immune function,etc.With the rising incidence of cancer in the world,anticancer effect has become a hot topic in the research of black tea.Our province is rich in resources of Anhua tea.Investigating anticancer function of Anhua tea extract can greatly improve the added value.In this study,colorectal cancer cells were used to evaluate the anticancer effect of Anhua black tea extract and the molecular mechanism of Anhua tea was also investigated.Our research will provide scientific evidence to direct people’s diet and develop functional food,health care products.Experimental methodsIn this study,we aimed to evaluate the anticancer function of black tea from two aspects:inhibiting cell proliferation of colorectal cancer and inducing apoptosis of colorectal cancer cells.The effect of Anhua black tea extract on proliferation of colorectal cancer cells was analyzed by MTS assay and carcinogenesis-related gene expressions were analyzed by real-time fluorescence quantitative PCR(RT-qPCR)to assess the effect of black tea extract on gene expressions in colorectal cancer cells.The effect of black tea extract on transcription factor AP-1 and NF-κB activities was assayed by luciferase reporter gene system.The effect of black tea extract on MAPK signaling activation was analyzed by phosphorylated Western blotting.Effect of tea extract on colorectal cancer cell apoptosis,the cell morphology,cell nucleus changes were assessed by Hoechst33258 staining.Flow cytometry was used to further confirm the effect of Anhua black tea extract on the apoptosis of colorectal cancer cells.Western blot was used to analyze the impact of Anhua black tea extract on the expressions of Bcl-2 family proteins and Caspase.The project will elucidate the molecular mechanism of Anhua black tea extract on inhibiting the proliferation and promoting apoptosis of colorectal cancer cells.Experimental results(1)Anhua black tea extract can inhibit the proliferation of colorectal cancer cells.MTS assay was used to evaluate cell survival and proliferation in this study.After treating colorectal cancer cells HCT116 with 0 g/mL,100 g/mL,200 g/mL and 400 g/mL concentrations of Anhua black tea extract for 48h,our data showed that Anhua black tea extract can significantly inhibited the proliferation of HCT116 cells.With the increase of the concentration of Anhua black tea extract,the inhibition percentage is increased.(2)Anhua black tea extract can regulate the key gene expressions of cell cycleAfter treating HCT116 cells with different concentration of Anhua black tea extract,total RNAs were extracted by Trizol Extract Kit and key target genes of cell cycle were analyzed by RT-qPCR.Anhua black tea extract can inhibit mRNA expression levels of cyclinD1 and VEGF with dosage-dependent manner.Cyclin dependent kinase inhibitor(CDKI)is closely related to the proliferation of cancer cells.The experiment found that Anhua black tea extract obviously inhibited mRNA expressions of p16,p21,p27 and p53 in a dose-dependent manner;but it has no effect on the expressions of p15,p18 and p19.It suggests that Anhua black tea extract can inhibit the activition of the CDK2/CDK4/cyclinD complex and take part in inhibiting colorectal cancer cell proliferation.(3)The effect of Anhua black tea extract on transcription activities of AP-1 and NF-κB in the colorectal cancer cellsThe transcription factor AP-1 and NF-κB play an important role in the occurrence and development of cancer.In p16,p21,p27,p53,cyclin,D1 and VEGF gene promoter region,there were DNA binding sites of AP-1 and NF-κB in the promoter of these genes.AP-1 and NF-κB are important transcription factors regulating the expression of CDKI gene;at the same time,cyclin D1 expression is also regulated by the two transcript factors.After AP-1 and NF-κB reporter genes(luciferase plasmids)were transfected by Lipofectamin 2000 into HCT116 cells,colorectal cancer cells were treated by different concentrations of Anhua black tea extract.HCT116 cell lysis was extracted and luciferase activities were assessed.The results showed that Anhua black tea extract can inhibit the ranscription activity of AP-1,and also inhibit NF-κB transription activity,suggesting that Anhua black tea extract may inhibit AP-1 and NF-κB transcription activities,which downregualte the expression of p16,p21,p27,p53,cyclin,D1 and VEGF etc..(4)Effect of Anhua black tea extract on MAPK activation in colorectal cancer cellsMAPK signaling pathway is an abnormal activation signal pathway in cancer cells,which is closely related to the proliferation and apoptosis of cancer cells.The transcription factor AP-1 and NF-κB is the target protein of MAPK downstream activation,such as JNK can directly activate c-Jun,which activates AP-1.After the treatment of colorectal cancer cells with Anhua black tea extracts,Western blot analysis showed that Anhua black tea extract could inhibit the phosphorylation of ERK1/2 and JNK in a dose-dependent manner.On the contrary,Anhua black tea extract could promote the phosphorylation of p38MAPK.The results suggest that Anhua black tea extract could inhibit the proliferation of cancer cells and activate the signal pathway related to the apoptosis of cancer cells,such as p38MAPK.(5)Anhua black tea extract promotes apoptosis of colorectal cancer cellsAfter different concentrations of Anhua black tea extract treating colorectal cancer cells,Hoechst33258 staining was used to assess the effect of Anhua black tea extract on cell apotosis.Our results showed that the low concentration of tea extract can lead to apoptosis of colorectal cancer cells HCT116,high concentration of black tea extract could significantly promote apoptosis of colorectal cancer cells.The fluorescence staining experiments showed that Anhua black tea extract results in the nuclear condensation,nuclear fragmentation and apoptotic bodies.Flow cytometry was used to further confirm that Anhua black tea extract can promote apoptosis of colorectal cancer cells.(6)Effect of Anhua black tea extract on the expression of apoptosis related genes in colorectal cancer cellsBcl-2 superfamily is very important to maintain the stability of the cells,the Bcl-2 superfamily contains both pro-apoptotic proteins,such as Bax,and also includes the inhibiting apoptosis proteins such as Bcl-2.After different concentrations of Anhua black tea extract treating colorectal cancer cells,Western blot analysis showed that Anhua black tea extract can promote the expression of Bax gene,and inhibit the expression of Bcl-2 gene,black tea extract broke the imbalance of Bax/Bcl-2 ratio,which promote colorectal cancer cell apoptosis,leading to activation of Caspase-3.Conclusion:Based on the above experimental research,the study approve Anhua black tea extract can inhibit the proliferation of colorectal cancer cells and its molecular mechanism:Anhua black tea extract→MAPK activation↓→AP-1,NF-κB activities↓→ cyclin D1 expression↓,VEGF expression ↓,CDKI expression↑→cell cycle arrest→inhibiting cancer cell proliferation.The study found that Anhua tea extract can promote colorectal cancer cell apoptosis and its possible molecular mechanism:Anhua black tea extract→Bcl-2 expression↓、Bax expression↑→Caspase-3 activation→apoptosis of colorectal cancer cells.