| Background:Euphorbia helioscopia L, a plant from Euphorbiaceae and genus Euphorbia family, is widely distributed in most part of China. As a traditional Chinese medicine, Euphorbia helioscopia L has been used for centuries to treat different diseases. Although the antitumor activity of aquatic extract of Euphorbia helioscopia L root has been studied, the anticancer active fractions and the precise anticancer mechanism of the herb remain unclear.Objective: To identify the main antiturnor active fractions of aqueous-ethanol extracts of Euphorbia helioscopia L against cancer cell lines in vitro, and to isolate the constituents of the main antitumor active fractions in order to evaluate the possible anticancer mechanisms involved. Also explore the possible antitumor mechanisms of myricetin, a compound from Euphorbia helioscopia LMethods: The systemic research ways of phytochemistry were used for extracting Euphorbia helioscopia L, and we evaluated the growth inhibitory effects of Euphorbia helioscopia L extracts on five different human cancer cell lines for screening the main active fraction with antitumor effect. Ethyl acetate extract (EAE) of Euphorbia helioscopia L was selected for further study. The microstructure and ultrastructural change of tumor cells treated with EAE was observed; apoptosis and cell cycle were analyzed by flow cytometry. The EAE fraction of Euphorbia helioscopia L were preliminary isolated by column chromatography with silica gel, Sephadex LH-20and polyamide. The structures of chemical constituents were identified on the basis of their physicochemical and spectral analysis. The inhibitory effect of myricetin on hepatoma carcinoma cell determined by MTT; effects of myricetin on cell cycle and apoptosis were detected by FCM; the levels of mRNA and proteins involved in apoptosis were measured by RT-PCR and Western Blotting, which was used to explore the potential mechanism of apoptosis in BEL-7402cells induced by myricetin. Results:Our data showed that EAE and chloroform extract (CE) could inhibit the proliferation of all five human cancer cell lines in a dose-and time-dependent manner at the concentration range of50-200ug/mL. The five tumor cell lines showed differential sensitivities to EAE and CE with SMMC-7721cells being the most sensitive to EAE and CE treatment. The highest growth inhibition rates treatments with EAE at200ug/mL for72hr were80.91%. Of note, the antiproliferative effect of EAE was strongest among the four tested extracts at all observed time points. On the other hand, there were no obvious inhibitory effects of petroleum ether extract (PEE) and n-butanol extract (NBE) observed on these cells during the same time periods. Compared with the control group, the morphology of tumor cells changed obviously, and the decrease of cell number and cell fragments were observed. EAE mainly arrested cells in G-l phase in a dose-and time-dependent manner and reduced the percentage of cells in S-phase. Moreover, compared with controls, EAE treatment at100-200ug/mL concentration range induced a marked increase of subdiploid peak. Treatment with EAE at200ug/mL concentration for72hr resulted in the highest percentage of cells (11.18%) in Sub-Gl phase of cell cycle. After treatment with EAE at150and200ug/mL concentrations, EAE significantly induced apoptosis of SMMC-7721, and the total percent of apoptosis cells increased from13.6%to47.53%. The ultrastructural changes in EAE-treated cells included chromatin condensation, chromatin marginalization, organelle swelling, and cytoplasmic vacuolization. In addition, an abundance of autophagic vacuoles was observed in the cytoplasm and these autophagic vacuoles contained degraded organelles.5compounds were isolated from EAE of Euphorbia helioscopia L, their chemical structures were elucidated by various spectroscopic methods. They were identified as gallic acid (1), myricetin (2), quercetin (3), kaempferol (4), hyperoside (5). Cell proliferation in hepatoma carcinoma cell line BEL-7402, SMMC-7721and HepG2were inhibited effectively by myricetin in a dose-and time-dependent manner. BEL-7402cell showed the most sensitive to myrecetin. The antiproliferative rate of myricetin at100-200uM concentration for72hr was30.82%,39.64%and46.59%, respectively. Myricetin mainly increased the percentage of cells in S phase in a dose-and time-dependent manner. A remarkably high subdiploid peak was also observed in the treatment group. Myricetin at200uM concentration for72hr resulted in the highest percentage of cells (17.59%) in Sub-G1phase of cell cycle. After treatment with myricetin at150,200μM concentrations, compared with the control group, the percentage of apoptotic cells was significantly increased, and the total percent of apoptosis cells increased from4.7%to82.41%. The mRNA expression of Bax, cytochrome C, caspase-3, caspase-8, Fas and FADD were increased markedly, however, the levels of Bcl-2, P53and NF-kB in BEL-7402cell were reduced. The ratio of Bax/Bcl-2increased. Western blot analysis showed that the expression of caspase-3, caspase-8and Fas was elevated, and P53and NF-kB protein were down-regulated.Conclusion: EAE is the major active anticancer fraction, suggesting that EAE-induced antiproliferation may be related to apoptosis. Five chemical compounds were isolated from EAE of Euphorbia helioscopia L, their chemical structures were elucidated by various spectroscopic methods. They were identified as gallic acid (1), myricetin (2), quercetin (3), kaempferol (4), hyperoside (5). Myricetin can significantly inhibit the growth of hepatocellular carcinoma cell and induce apoptosis. The mechanism involved in apoptosis on BEL-7402cell treated with myricetin may mediated by mitochondrial pathway, Fas signaling pathway and apoptosis-related protein P53and NF-kB.
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