Andrographolide Derivative ADA On Apoptosis And Its Mechanism Of Breast And Lung Cancer

Malignant tumor is a huge threat to our life and development.With the aggravation of hazerecently,lung cancer has been the disease with the highest morbidity among malignant tumors.The new morbidity of lung cancer is about seven hundred and thirty-three thousand peryear[1].And breast cancer has been the most dangerous malignant tumor for women,the incidence of which is keeping rising in these years.So researchingpathogenesis of tumor has gradually become a new hotspot.The occurrence of tumor is closely related to cell mutation,that is to say,normal cells are transformed and overgrowth beacuse of the activation of oncogenes and the inactivation of anti-oncogenese.Theresearchers focused on cell differentiation and division,but they did not pay attention to the relation between cell death andtumorigenesis before.Now the study shows that the reason of the overgrowth of cell proliferation is the lack of dead cell.That means the apoptpsis did not really happen.[2].There are more and more researches about apoptosis recently.What's more,therelationbetween apoptosis andrumorigenesis has been gradually recognized by researchers.The theory of apoptosis provides a new theoretical basis for anti-tumor.Andrographis paniculata?Burm.f.?Nees,commonly known as‘king of bitter'.It has been widelyused for centuries in Asian for the treatment of sore throat,flu and upper respiratory tract infectins.Recently,it's newly pharmacological antitumor activitywas found.ADA,an andrographolide derivatives,was synthesized by semi-synthetic method.he anti-proliferationeffects of ADA were assessed by MTT assay in A549 and MCF-7 cells.Apoptotic morphological changes of A549 andMCF-7cellsafter ADA treatment wereobservedbyhigh content screening?HCS?after AO/EB doublestaining,DAPIandRh123/PI,Apoptotic number was by using flow cytometry.The expression of Bcl-2,Bax,caspase3,NF-?B p65,p-I?B?,I?B?and p-IKK?were measured using western bloting.MTTassaydemonstratedthatADAexertedpotentanti-tumoreffectinasignificantconcentration-dependent manner.The IC₅₀ values ofA were 25.49±4.02?M and 19.5±2.82?M;The IC₅₀ values ofADA were9.65±1.50?M and 6.4±3.29?M after 24h treatmentrespectively.And the anti-tumor effects were more powerful than andrographolide both in A549and MCF-7 cells.The stain result of DAPI and AO/EB shows that cell of dosing treatment group changed obviously such as the apoptosis characters of absent of nucleus chromatin condensation compared with the blank control group,margination and so on.According to the picture of double staining,ADA in the dosing treatment group nucleus gave out fluorescent orange compared with the blank control group.The fluorescence intensity?P<0.01?enhanced significantly as increasing the concentration of dosing.It shows that cell apoptosis in morphology appears.The result of flow cytometry shows that apoptosis rates of the dosing treatment group enhancedsignificantly compares with the blank control group.The apoptosis rates of A549 are 6.41%±2.51,9.28%±1.86,21.85%±7.75,32.46%±7.61.And the apoptosis rates of MCF-7 are 4.34%±1.12,21.22%±2.09,32.46%±7.61,19.06%±1.23.The apoptosis rates of ADA after twenty-four hours all enhanced significantly?P<0.01?,which shows that ADA can induce breast cancer cell MCF-7 and lung cancer cell A549 apoptosis.The result of Rh123 shows that mitochondrial membrance potential repressed after acting onADA for twenty-four hours.mitochondrial membrance potential repression is regarded as the sign of apoptosis.Rhodamine fluorescence decreased significantly as increasing the concentration of ADA?P<0.01?,which shows that cells lost the ability to combine with Rhodamine123.The experiment of Western blot shows:MCF-7 caspase 3 were activated and expressionincreased?P<0.01?.The expression of pro-apoptotic protein Bax increased?P<0.01?.The expression of anti-apoptosis Bcl-2 decreased?P<0.05?,which suggests that ADA played a role in the regulation among Bax,Bcl-2?and caspase 3.The expression of phosphoralation I?B?,IKK?and NF-?B p65 in nucleus decreased?P<0.01?,but the expression of p65 in nucleus increased?P<0.01?.It shows that ADA also took part in the regulation with signal pathway NF-?BAccording to the results above,Andrographolide derivative ADA can induce breast cancerMCF-7 and lung cancer A549 apoptosis.The mechanism is explained by controling endogenous mitochondria of apoptosis passway and takes part in the regulation with signal pathway.