Studies Of Ethanolic Extract From Boschniakia Rossica On Anti-proliferation And Inducing Apoptosis On Human Hepatocellular Carcinoma Cell Line SMMC-7721

OBJECTIVE To observe the effects of anti-proliferation and inducing apoptosison human hepatocellular carcinoma cell line SMMC-7721 induced by ethanolicextract from Boschniakia rossica,and provide the experimental evidence forBoschniakia rossica as a new anti-tumor drug.METHODS SMMC-7721 cells was cultured in vitro.The inhibition on theproliferation of SMMC-7721 cells by ethanolic extract from Boschniakia rossicawas viewed through the method of MTT assay.Morphological changes of theapoptosis of SMMC-7721 cells were detected by inverted microscope,lightmicroscope and fluorescent microscope,and the ultrastructure of SMMC-7721cells was observed by transmission electronic microscope.Flow cytometry wasused to detect cell cycle distribution and apoptotic rate of SMMC-7721 cells.RESULTS 1)The MTT test showed that SMMC-7721 cells exposured to ethanolicextract from Boschniakia rossica at final concentrations of 0.25-1.0g/L wereinhibited for 6h,12h and 24h, in dose and time dependent manner.The MTT testshowed the inhibitory rate was 97.88%,at final concentration of 1.0g/L for 24h.2)After treatment with extract from Boschniakia rossica,the SMMC-7721 cellsgrew slowly,turned round,vacuoles appeared in cytoplasm and the adherent abilitydecrease under invert microscope.The SMMC-7721 cells in experimental group byHE staining showed the volume of cells became small, nucleus condensed roundmasses,and acidophilia of cytoplasm was enhanced. SMMC-7721 cells stained byAO/EB double fluorescent staining showed,the late apoptotic cells appeared brightorange chromatin highly condensed,the nucleus became fragmented.Aftertreatment with 0.5g/L final concentration extract from Boschniakia rossica for12h,apoptotic ultrastructure of SMMC-7721 cells were detected under transmissionelectronic microscope,the microvilli defluviu- med,chromatin condensed to theperiphery of the nucleus and aggregated into masses,the cells shrinked,formedblebs and cytoplasmic buds.3) The apoptotic percentages of the SMMC-7721 cells after treating with the ethanolic extract from Boschniakia rossica with dose of0.5g/L, for 12h,18h and 24h were 9.64%,11.65%,32.91%,respectively, there wasstatistic significance compared with control group.SMMC-7721 cells treated withthe ethanolic extract from Boschn- iakia rossica at final concentration of 0.5g/L for12h, 18h and 24h were arrested in G₀/G₁ phase,the cell percentage of S phasedecreased, while that of G₀/G₁ phase increased by FCM.CONCLUSIONS1) Ethanolic extract from Boschniakia rossica inhibited proliferation ofSMMC-7721, cells in vitro in dose and time-dependent manners.2) SMMC-7721 cells which were treated with ethanolic extract from Boschniakiarossica showed apoptotic changes by morphological analysis and FCM.Induced apoptosis may be one of the mechanisms of ethanolic extract fromBoschniakia rossica on treating carcinoma.3) Ethanolic extract from Boschniakia rossica inhibited SMMC-7721 cellsproliferation through interfering with cell cycle mainly arrested in G₀/G₁ phase,ethanolic extract from Boschniakia rossica can inhibit the proliferation ofSMMC-7721 cells, which may be another mechanism of ethanolic extract fromBoschniakia rossica on treating carcinoma.