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OBJECTIVE:To identify the active anti-tumor constituents in the extract from Danshen(Radix Salviae Miltiorrhizae) and investigate the mechanisms underlying the actions.METHODS:First,we introduced a two-step counter-current chromatography to extract the therapeutically active diterpenoid,tanshinone from Danshen(Radix Salviae Miltiorrhizae).The cholecystokinin(CCK-8) method was used to evaluate the inhibitory effect of diterpenoid tanshinone in liver cancer QGY-7703,lung cancer PC9,lung cancer A549,gastric cancer MKN-45,gastric cancer HGC-27,colon cancer HCT116,myeloma cell U266/RPMI8226,and human breast cancer MCF-7 in vitro.Fluorescence staining was used to observe the cytotoxicity ofditerpenoid tanshinone on PC9 cells.The Western blot was used to detect apoptosis-related protein poly ADP-ribose polymerase(PARP),cysteinyl aspartate specific proteinase3/9(caspase3/9),and cleaved-cysteinyl aspartate specific proteinase3/9(cleaved-caspase3/9).The endoplasmic reticulum stress-related activating transcription factor 4(ATF4),phosphorylated eukaryotic initiation factor 2α(p-e IF2α),and phosphorylated jun amino-terminal kinase(p-JNK),and caspase-12 were also analyzed using the Western blot.RESULTS:Diterpenoid tanshinone inhibited the nine human tumor cell lines,with an IC50 of4.37-29 μg/m L,with the PC9 and MCF-7 displaying the lowest values.Fluorescence staining showed a lethal effect of diterpenoid tanshinone on PC9 cells.The Western blot showed that the expression of caspase3/9 protein and ATF-4 protein decreased gradually.However,the PARP,cleaved-caspase 3/9and the expression of p-e IF2 α,P-JNK,and caspase-12 increased gradually,in a dose-dependent fashion.CONCLUSION:We successfully introduced a two-step counter-current chromatography method to extract diterpenoid tanshinone,and demonstrated its antitumor activity.Diterpenoid tanshinone can induce apoptosis in nine human cancer cell lines.
OBJECTIVE: To identify the active anti-tumor constituents in the extract from Danshen (Radix Salviae Miltiorrhizae) and investigate the mechanisms underlying the actions. METHHODS: First, we introduced a two-step counter-current chromatography to extract the therapeutically active diterpenoid, tanshinone from Danshen (Radix Salviae Miltiorrhizae). The cholecystokinin (CCK-8) method was used to evaluate the inhibitory effect of diterpenoid tanshinone in liver cancer QGY-7703, lung cancer PC9, lung cancer A549, gastric cancer MKN-45, -27, colon cancer HCT116, myeloma cell U266 / RPMI8226, and human breast cancer MCF-7 in vitro. Fluorescence staining was used to observe the cytotoxicity of diterpenoid tanshinone on PC9 cells. Western blot was used to detect apoptosis-related protein poly ADP -ribose polymerase (PARP), cysteinyl aspartate specific proteinase 3/9 (9), and cleaved-cysteinyl aspartate specific proteinase 3/9 (9). The endoplasmic reticulum stress-related activator ating transcription factor 4 (ATF4), phosphorylated eukaryotic initiation factor 2α (pe IF2α), and phosphorylated jun amino-terminal kinase (p-JNK), and caspase-12 were also analyzed using the Western blot .RESULTS: Diterpenoid tanshinone inhibited the nine human tumor cell lines, with an IC50 of 4.37-29 μg / m L, with the PC9 and MCF-7 displaying the lowest values. Fluorescence staining showed a lethal effect of diterpenoid tanshinone on PC9 cells. The Western blot showed that the expression of caspase 3/9 protein and ATF-4 protein decreased gradually. However, the PARP, cleaved-caspase 3/9 and the expression of pe IF2 alpha, P-JNK, and caspase-12 increased gradually, in a dose-dependent fashion .CONCLUSION : We successfully introduced a two-step counter-current chromatography method to extract diterpenoid tanshinone, and demonstrated its antitumor activity. Diterpenoid tanshinone can induce apoptosis in nine human cancer cell lines.