Synergistic Anticancer Effects Of Combinations Of Tanshinone ?A With Andrographolide And Apigenin

Tanshinone ?A(Tan ?A)is one of the main components of Salvia miltiorrhiza,which has a variety of biological activities and antitumor effects with low toxicity.However,due to its poor water solubility and anti-tumor effects it is difficult to be used in clinic.In this study,taking Tan ?A as the target compound and proliferation inhibition of tumor cells as screening criteria,a large number of screening experiments were done at the cellular level to select natural compounds that had synergistic antitumor effects with Tan ?A from the natural compound library.Two natural compounds Andrographolide(Andro)and Apigenin(Api)were screened out to have synergistic antitumor effects with Tan ?A.This study focused on the synergistic antitumor mechanisms of the two pairs of compounds,Tan ?A with Andro,and Tan ?A with Api,and the synergistic antitumor effects in vivo were evaluated.The main methods,results and conclusions are as follows:(1)Screening natural compounds which have synergistic antitumor effects with Tan ?ATan ?A and Andro were found out to have synergistic effects on the proliferation inhibition of MCF7,BGC823 and SMMC7721 tumor cell lines by MTT methods.The combination index(CI)was 0.26±0.02.The IC50 of Andro to MCF7 cells is 183.23 ?M,and the IC50 of Tan ?A was 51.46 ?M.When the two compounds were combined used,the IC50 of Andro was 38.45 ?m to MCF7 cells and of Tan ?A was 9.61 ?M.The combination of Tan ?A and Api has synergistic inhibitory effects on the proliferation of BGC823,MCF7 and SMMC7721 cell lines.The combination index Cl was 0.28±0.01.The IC50 of Api to BGC823 cells was 131.28 ?M,and the IC50 of Tan ?A was 61.46 ?M.When the two compounds were combined used,the IC50 of Api to BGC823 cells was 22.30 ?M and of Tan ?A was 11.15 ?M.(2)Synergistic antitumor effects of Tan ?A and Andro on MCF7 human breast cancer cell line.The synergic effects of Tan ?A combined with Andro on MCF7 cell apoptosis were verified by AV-PI double staining.The antioxidant N-acetylcysteine(NAC)could reverse the toxicity of Andro on MCF7 cells and the synergistic effects of Andro with Tan ?A,but it could not reverse the toxicity of Tan ?A on MCF7 cells,indicating that reactive oxygen species(ROS)is one of the key factors of the synergistic mechanisms.Two oxidants L-buthionine-sulfoximine(BSO)and diethyl dithio carbamic acid silver salt(DETC)had synergistic antitumor effects with Tan ?A,and the effects could also be antagonized by antioxidants NAC,which further proved that ROS was the key factor of the synergistic effects of Tan ?A with Andro on MCF7 cells.On the other hand,Andro was proven to form covalent coupling with the Glutathione(GSH)analog NAC by using liquid chromatography and mass spectrometry methods.It was further confirmed that the reaction blocked the-SH on GSH and reduced the reduction of GSH in cells by 5,5'-Dithiobis-(2-nitrobenzoic acid)(DTNB)oxidation methods.The intracellular level of H2O2and superoxide anion were detected by two dye probes:2',7'-Dichlorodihydro-fluorescein diacetate(DCFH-DA)and Dihydroethidine(DHE).The results showed that Tan?A combined with Andro significantly increased the intracellular level of ROS compared with the single use.Western blot assays were used to investigate the effects of Tan ?A combined with Andro on the expression of p53,Bax and PUMA in MCF7 cells.The results showed that when Andro combined with Tan HA,the expression of p53,Bax and PUMA were significantly upregulated.When MCF7 cells were pretreated with p53 inhibitor Pifithrin-?(Pft-?),the toxicity of Tan ?A combined with Andro reduced by half,and the CI value of Tan ?A with Andro increased from 0.26±0.02 to 1.18±.15.This suggested that p53 was another key mechanism of Andro and Tan?A synergistic antitumor activities.When MCF7 cells were treated with Tan ?A and Andro combination,both p53 signals and ROS were significantly enhanced.Tan ?A combined with DNA activated the expression of p53,and Andro blocked the sulfhydryl group of GSH,increased the intracellular H2O2 level.The overexpression of p53 would further aggravate the production of ROS,and oxidative stress could further activate the overexpression of p53 through direct p53 upregulations or DNA damage repair pathways.Therefore,the crosstalk between ROS and p53 signal pathways resulted in synergistic effects of Tan ?A with Andro on MCF7 cells.(3)Synergistic antitumor effects of Tan ?A and Api on BGC823 human gastric cancer cell lineThe results of AV-PI double staining and western blot assay showed that Tan ?A combined with Api significantly upregulated the expression of p53,Bax/Bcl-2 ratio and enhanced the cell apoptosis.The antioxidant NAC could neither reverse the toxicity of Api and Tan ?A when treated alone on BGC823 cells,nor the synergistic effects of Api and Tan ?A combination,which showed that ROS was not the key factor of Api and Tan ?A synergistic effects on BGC823 cells.PI staining and western blot were used to investigate the effects of Tan ?A and Api on cell cycle and cyclin B1,D1 level of BGC823 cells.The results showed that the cell cycle was blocked in G2/M phase(24 h),G1 and G2/M phase(48 h)and S phase(72 h)in turn with the passage of time.The cell level of cyclin B1,D1 also changed with cell cycle.The interactions of Tan ?A and Api with DNA were analyzed by circular dichroism(CD)and DNA thermal denaturation curve methods The results showed that when the two compounds mixed with CT-DNA together,Api interacted with DNA in a groove binding and external stacking manner;and Tan ?A changed the base stacking structure of DNA skeleton;DNA thermal denaturation experiment showed that:when Tan ?A and Api combined with DNA,the thermal denaturation curve changed a lot,indicating that the combination of Tan ?A and Api with DNA had a significant impact on the stability of DNA.The above results confirmed that Tan ?A combined with DNA in the way of cha nging the base stacking structure of DNA skeleton,and API combined with DNA in a groove binding and external stacking manner.Both of them act on DNA together,ac celerate cell cycle arrest and promote cell apoptosis,which were the core mechanisms of their synergistic effects.The antitumor experiments in vivo(S180 tumor bearing mice model)showed that Api(60 mg/kg)and Tan ?A(30 mg/kg)had no significant antitumor effects by oral administration alone.The antitumor effects of the combination of Api and Tan ?A were significant.The inhibition rate of mice tumor is 37.6%,which was similar to that of cyclophosphamide(30 mg/kg),and the toxicity was lower than that of cyclophosphamide(30 mg/kg).In conclusion,in this study Tan ?A and Andro were found for the first time to have synergistic antitumor effects.Tan ?A and Andro acted on p53 and ROS signal pathways respectively,and the mutual activations between p53 and ROS signal pathways are the critical mechanisms of the synergistic effects.Tan ?A and Api were found for the first time to have synergistic antitumor effects.Tan ?A and Api combined with DNA by changing DNA skeleton base accumulation and external groove binding mode respectively,which aggravated cell cycle arrest and promoted cell apoptosis.