Abstract:

Objective To investigate the effects of Anhydroicaritin （AHI）， an isopentenylated flavo-noid compound， on proliferation， migration and apoptosis of human hepatocarcinoma cell line MHCC-97H. Methods Human hepatocarcinoma cell line MHCC-97H and human normal liver cell line L02 were cultured in vitro. MHCC-97H cells were treated with 0， 20， 40， 80， 120， 160， 200 μg/ml of AHI respectively and L02 cells were treated with 0， 25， 50， 100， 150， 200， 400， 500 μg/ml of AHI respectively. CCK-8 and clone formation assay were used to detect cell proliferation. Scratch test was used to explore cell migration ability. Hoechst33342 assay and flow cytometer were used to detect cell apoptosis. The expressions of apoptosis-related proteins were detected by Western blotting. Results The cell viabilities of MHCC-97H cells treated with 0， 20， 40， 80， 120， 160， 200 μg/ml of AHI for 24 h were （100.00±0.00）%， （97.41±2.10）%， （96.58±3.23）%， （87.72±4.85）%， （78.33±3.76）%， （56.97±2.61）% and （15.25±2.51）% respectively， and there was a statistically significant difference （F=429.20， P<0.001）. There were statistically significant differences between 0 μg/ml and 80， 120， 160， 200 μg/ml of AHI treatment （all P<0.001）. The cell viabilities of L02 cells treated with 0， 25， 50， 100， 150， 200， 400， 500 μg/ml of AHI for 24 h were （100.00±0.00）%， （96.82±3.79）%， （95.36±3.43）%， （90.79±5.75）%， （77.67±5.66）%， （63.98±5.22）%， （34.22±4.01）% and （33.84±4.41）% respectively， and there was a statistically significant difference （F=233.20， P<0.001）. There were statistically significant differences between 0 μg/ml and 100， 150， 200， 400， 500 μg/ml of AHI treatment （all P<0.05）. The 24 h half maximal inhibitory concentration （IC₅₀） value of AHI treated L02 cells was （300.20±17.10） μg/ml， which was significantly higher than that of MHCC-97H cells [（158.60±5.50） μg/ml]， and there was a statistically significant difference （t=13.65， P<0.001）. The cell clone numbers of MHCC-97H cells treated with 0， 120， 160 and 200 μg/ml of AHI for 24 h were 1 993.00±46.29， 1 355.00±54.84， 998.33±21.03 and 218.33±35.95 respectively， and there was a statistically significant difference （F=954.80， P<0.001）. There were statistically significant differences between 0 μg/ml and 120， 160， 200 μg/ml of AHI treatment （all P<0.001）. The healing rates of MHCC-97H cells treated with 0， 120， 160 and 200 μg/ml of AHI for 24 h were （51.68±1.93）%， （16.04±0.73）%， （8.88±0.31）% and （-6.94±0.46）% respectively， and there was a statistically significant difference （F=1 616.00， P<0.001）. There were statistically significant differences between 0 μg/ml and 120， 160， 200 μg/ml of AHI treatment （all P<0.001）. Hoechst33342 experiment showed that MHCC-97H cells treated with 0 μg/ml AHI showed uniform dark blue with a complete nuclear state under inverted microscope. Compared with 0 μg/ml AHI treated cells， cells in the 120， 160， 200 μg/ml AHI treatment groups wrinkled and broken， and nuclei were also morphologically abnormal， with some nuclei stained bright blue， and the situation became more obvious with increasing dose. The apoptosis rates of MHCC-97H cells treated with 0， 120， 160 and 200 μg/ml AHI for 24 h were （10.51±0.56）%， （42.23±0.87）%， （61.92±0.52）% and （72.05±0.74）% respectively， and there was a statistically significant difference （F=4 677.00， P<0.001）. There were statistically significant differences between 0 μg/ml and 120， 160， 200 μg/ml of AHI treatment （all P<0.001）. There were statistically significant differences among the different expression levels of Bax， Cleaved Caspase-3/Caspase-3， Cleaved Caspase-9/Caspase-9， and Bcl-2 proteins in MHCC-97H cells of 0， 120， 160， and 200 μg/ml of AHI treatment （F=30.43， P<0.001； F=212.80， P<0.001； F=475.30， P<0.001； F=10.75， P=0.004）. The Bax protein expression of 160 and 200 μg/ml was significantly increased than that of 0 μg/ml AHI （both P<0.001）. The Cleaved Caspase-3/Caspase-3， Cleaved Caspase-9/Caspase-9 protein expressions of 120， 160 and 200 μg/ml were significantly increased than those of 0 μg/ml AHI （all P<0.001）. The Bcl-2 protein expression of 120， 160， 200 μg/ml was significantly decreased compared with that of 0 μg/ml AHI （all P<0.05）. Conclusion AHI can inhibit the proliferation and migration of hepatocellular carcinoma cell line MHCC-97H， and promote its apoptosis.

Key words: Liver neoplasms, Cell proliferation, Cell movement, Apoptosis, Anhydroicaritin