Studies Of Anti-mutation Active Constituents From Inonotus Obliquus On Anti-proliferation And Inducing Apoptosis And Apoptotic Pathway On A549 Human Lung Cancer Cell Line

OBJECTIVE To observe the effects of anti-proliferation and inducing apoptosis on A549 human lung cancer cell line induced by anti-mutation active constituents from Inonotus obliquus, It will provide with evidence of experiment and theory for the development of natural medicine..METHODS MTT assay was used to dertermine the effects of anti-mutation active contituents from Inonotus obliquus on A549 human. lung cancer cell growth inhibitory. Morphological changes of the apoptosis of A549 lung cells were observed by using inverted light, fluorescent microscope and transmission electronic microscope. Apoptotic rate was calculated with terminal deoxynucleotidy transferase-mediated dUTP nick-end labeling(TUNEL). The protein expressions of ki-67 and the apoptosis-related gene such as Bcl-2, Bax, caspase-9, caspase-3, p53, Fas and FasL were detected by using immunocytochemistry. Flow cytometry was used to detecte cell cycle distribution of A549 cells and apoptotic rate.RESULTS The MTT test showed that anti-mutation active constituents fromInonotusobliquus in concentrations of 20, 40, 8Omg/Lcould inhibite the hyperplasis of A549 human lung cancer cells in a doseage and time dependent manner. The A549 human lung cancer cells treated with anti-mutation active constituents from Inonotus obliquus showed cellular volume small and round, nuclear chromatin condensation, loose between the cells and adherent ability decrease under invert microscope. The A549 human lung cancer cells in experimental group by HE staining showed morphological changes such as nuclear chromatin condensation, plmsma membrane bleb fromation and the apoptotic body formations. Human A549 lung cancer cells stained by AO/EB dounble fluorescent staining were observed under fluoresclent microscope, appearing as chromatin orange-red condensation in nuclear, orange-red or orange-yellow fluorescence disappeared and light green fluorescent apoptotic bodies around cells after induction of anti-mutation active constituents from Inonotus obliquus. Apoptotic ultrastructure of A549 human lung cancer cells were observed under transmission electronic microscope, the nuclear chromatin was condensed or aggregated under the nuclear membrane, the membrane blebs appeared and apoptotic body formations bulged bells surface. Ki-67 anti-proliferation test indicated that the anti-mutation active constituents from Inonotus obliquus of various concentration and time all inhibited A549 lung cancer cells in dose and time-dependent manner. Anti-mutation active constituents from Inonotus obliquus at final concentration of 20μg /ml could induced apoptosis on A549 human lung cancer cell in time dependent manner. FCM assay indicated that apoptotic rate increased significantly in time dependent manner in experimental group., the percentage of apoptosis rate was 18.41%,36.12%, respectively, there was statistic significance compared with different concentration(P〈0.01), A549 lung cancer cells treated with anti-mutation active constituents from Inonotus obliquus at final concentration of 20μg /ml for different time 24h, 48h, were inhibited and arrested in S phase, the cell percentage of G₀/G₁ phase and G₂/M phase decrease and that of S phase increase was observed by FCM. The expreesion of bax, caspase-9, caspase-3, p53, Fas and FasLproteins was upregulated and bcl-2 protein was downregulated after exposure to anti-mutation active constituents from Inonotus obliquus. ONCLUSIONS) A549 Human lung cancer cells was inhibited significantly by nti-mutation active constituents from Inonotus obliquus in a doseage and ime dependent manner.2) Human lung cancer A549 cells exposure to anti-mutation active constituents from Inonotus obliquus showed apoptotic changes through morphological, biochemical and FCM assay.3) Anti-mutation active constituents from Inonotus obliquus inhibited A549 human lung cancer cells growth through interfering with cell cycle, mainly arrested in S phase.4) Apoptosis might be mediated by down-regulation of apoptosis-regulated gene bcl-2 and up-regulation of apoptosis-regulated gene bax, caspase-9, p53, caspase-3, Fas, FasL. Whicth were related to the start of conducing caspase apoptosis message. This type of apoptosis' starting and performing is through death receptor pathway and mitochondrial pathway.