Experimental Study On The Effects Of Tanshinone â…¡A On The Inhibition Of Growth And Induction Of Apoptosis In The Human Tumor Cell Lines

Objective:At present malignant tumor has become a major cause which influences people's quality of life and threatens people's health severely. So the urgent affairs are to study the pathogenesis of tumor and develop effective anti-tumor drugs which can help the sufferers racked with cancer improve their quality of life and prolong their life span. In the past researchers summed up the pathogenesis of tumor in two aspects. One was the activation of oncogene which made cells proliferate limitlessly. The other was the block of the cellular differentiation which made immature cells multiply in large amount. Recently researchers find the maladjustment of apoptosis is also one of the important mechanisms of cancerization. Even someone thinks that cancer cells originate from cells which should but can't happen apoptosis normally. With the development of study, researchers recognize that apoptosis is a cellular voluntary death controlled by gene and different from necrosis in morphological and biochemical characters. Killing tumor cells by inducing apoptosis can protect the normal tissues of organism from intensive inflammatory reaction. Therefore researchers pay more attention to the task of developing drugs which can induce apoptosis of tumor cells. Tan ⅡA (tanshinoneⅡA) is an effective ingredient isolated from salvia miltiorrhiza BUNGE. Recent studies show that Tan ⅡA can inhibit proliferation and induce differentiation or apoptosis of many kinds of carcinoma cells. Those characters make Tan ⅡA possess good applying prospects as a new anti-tumor drug. Gastric carcinoma is a very familiar malignant tumor in our country whose mortality rate stands first on the list. So more and more studies appear which aim at the prevention and cure of gastric carcinoma. Up to the present the study about the effect of apoptosis-inducing on gastric cancer cell by Tan ⅡA hasn't been reported. So we selected BGC-823 cell line which has been cultured and certified by Chinese researchers as inducing object. We hoped the obtained results would provide more valuable information to the treatment of gastric cancer. The research about the effect of apoptosis-inducing on K562 cells by TanⅡA of low concertration has been reported interiorly. But this kind of inducing manner must take upwards of 5 days to achieve the effect of apoptosis-inducing. Therefore we attempted an inducing manner of short-cycle with high concentration in order to explore its effect. So in our study we examined the effects of apoptosis-inducing and growth-inhibition on BGC-823 and K562 cells by Tan ⅡA through the analyses of morphologic and biochemical characters and apoptosis ratio etc. At the same time we made a preliminary study of its molecular mechanism. We hoped the results would enrich the basic studyon the anti-tumor effects of Tan ⅡA from the point of apoptosis,enlarge its applying ranges and promote its clinical application. Methods:1.The preparation of drug's solution: Tan ⅡA was dissolved in absolute alcohol and reserved at 4℃away from the sunshine. Its reserving concentration was 0.5mg/ml. The experimental concentrations of Tan ⅡA were adjusted by RPMI1640 medium. 2.The suppressive effects of Tan ⅡA on the proliferation of BGC-823,K562 cancer cells were evaluated in vitro by using MTT colorimetric assay. 3.BGC-823 cells were treated with 2,5,10μg/ml Tan ⅡA for 72h respectively. K562 cells were also treated with TanⅡA at various concentrations(0.5,1,2,5,10μg/ml) for 72h. The apoptosis-related alterations in morphology were ascertained by fluorescence upside-down microscope. 4.The apoptosis-related morphological changes of BGC-823 cells induced by Tan ⅡA at various concentration(2,5,10μg/ml) for 72h and 48h were observed by HE staining. 5.DNA agarose gel electrophoresis: After being treated with 0.5～10μg/ml TanⅡA for 72h, the DNA samples of BGC-823,K562 and control cells were extracted and collected. Each of DNA samples was loaded on 1.8% agarose gel using 0.5×TBE buffer and 60 voltage,dyeing with 0.5μg/ml of ethidium bromide solution in the dark. BIO-PROFIL analysis system was used to observe and collect image.6. TanⅡA-treated cancer cells and control cells were harvested and washed once with cold PBS. Then the pellets of BGC-823 cells were suspended in 70% cold ethanol at 4℃for 24h. Precipitates were digested with 0.5% pepsin after centrifugation. And then cells were suspended in the solution of propidium iodide and incubated at 4℃in the dark. Apoptotic rates were analyzed by using flow cytometer and correlative software. Flow cytometry of TanⅡA-treated K562 cells was accomplished by using DNA-Prep COULTER Reagent Kit. 7.The expressions of Bcl-2 and Bax proteins in control cells and BGC-823 cells which treated with 10μg/ml of TanⅡA for 72h were investigated by using flow cytometer. Results: 1.The effects of TanⅡA on the proliferation and apoptosis of K562 cell line: TanⅡA inhibited markedly the proliferation of K562 cell line compared with the control group(p<0.01) in dose-dependent and time-dependent manners. The IC50 value for the treatment of 72 hours was 3.28μg/ml with 9.24μg/ml for the treatment of 48 hours. The typical apoptosis-related alterations weren't observed under the fluorescence microscope using acridine orange staining. But a large amount of necrotic cells with swoln bodies and faint fluorescence were found in the groups of high concentration(2,5,10μg/ml). The successional membrane-liked strips were presented in DNA electrophoresis of samples of Tan ⅡA-treating groups(2,5,10μg/ml). Flow cytometry of TanⅡA-treated K562 cells at various concentrations didn't show themarked increase of hypodiploid apoptotic cells with the increasement of drug's concentrations compared with the control group. 2.The effects of the growth-inhibition and apoptosis-inducing of TanⅡA on BGC-823 cell line: TanⅡA can inhibit the growth of BGC-823 cell line at several experimental concentrations upwards of 0.5μg/ml in a dose-dependent manner(r=0.947, p<0.05). The IC50 for the treatment of 48 hours was 10.59μg/ml whereas the value for 72 hours reduced to 4.58μg/ml. The typical morphological features indicative of apoptotic state were observed under the light or fluorescence microscope. The DNA-ladder was generated after the treatment of TanⅡA at the concentrations of 5 and 10μg/ml. Apoptotic rates were (20.60±1.84)%,(31.03±1.47)% in cells treated with 5,10μg/ml of TanⅡA respectively. There was marked difference compared with the control group whose apoptotic rate was (5.23 ±0.39)%. The expression of Bcl-2(apoptosis-related protein) reduced markedly but Bax increased in the cells in 10μg/ml of TanⅡA-treating group. Conclusions:1.The higher concentrations of TanⅡA(2,5,10μg/ml) didn't show the apoptosis-inducing activity to human erythroblast cell line K562. They maybe exerted anti-tumor effect by inducing necrosis. 2. TanⅡA could inhibit the proliferation and induce apoptosis of human gastric cancer cell line BGC-823. The mechanisms of apoptosis-inducing maybe associated with the...