Studies Of Low-dose Ethanol Suppress Hepatocellular Carcinoma Cell Proliferation

Objective:To investigate the anti-cancer effect and mechanism of low-dose ethanol suppresshepatocellular carcinoma proliferation.Methods:l)The human hepatocellular carcinoma cells line SMMC-7721 were cultured in RPMI640 medium with 10% FBS, 37℃, 5% CO₂. Hepatocellular carcinoma cells were divided into three groups: the control group, the group treated by ethanol and the group treated by ethanol combined with catalase. The cells morphologic changes were evaluated by microscopy.2) SMMC-7721 cells were treated with 2%, 4% and 6% ethanol one or two hours respectively, then stained with DCFH-DA, the intracellular hydrogen peroxide (H₂O₂) levels were analyzed on a flow cytometer using excitation at 488 nm and emission at 600 nm.3) SMMC-7721 cells were incubated for 12h, 24h and 48 h with 4% concentrations of ethonal or 4% concentrations of ethonal combined with catalase, the growth inhibition rate was detected by trypan blue staining.4) SMMC-7721 cells were treated with 4% ethanol after 3, 6 and 12 hours respectively, Mitochondrial membrane potential (â–³ Ψ m) was measured by flow cytometry using the DIOC₆, a fluorescent dye being shown to be selectively accumulated in the mitochondria of living cells by a mechanism which depends on â–³ Ψ m, DIOC₆ was excited at 488nm and detected at 525nm.5) For measurement of cellular sub-DNA content, flow cytometric analysis was used. Three groups cells were strained with propidium iodide (PI) after 24h and 48h treatment. PI wasexcited at 488nm, and fluorescene was analyzed at 620nm. Results:1) Morphological alterations have taken place between the control group and the group treated by ethanol after 24h or 48h treament under microscopy. Cells treated with ethanol were shrinked, separated and stained by trypan blue, which happened on cells incubated with ethanol and catalase too.2) When SMMC-7721 cells were treated with 2%, 4%, and 6% ethanol, the intracellular H2O2 level ascended after an hour, more after two hours. They were 13.47+1.07%, 54.03 ±3.02%, 64.981 ±3.19%, and 38.97±3.65%, 95.59±1.35%, 97.02±1.50% respectively. Compared with the control group, whose H2O2 level was 0.87 ±0.085 and 1.01 ±0.02%, they have statistic significance (PO.05). But to 2 hours groups, 4% ethonal compared with 6% ethanol, the results showed they have no statistic significance (P>0.05).3) Ethanol can suppress SMMC-7721 cells growth. When SMMC-7721 cells was treated with 4% ethanol on 12h, 24h and 48h, the growth inhibition rate was 26.21 ±1.60%, 65.80± 2.17%, 86.91 ±2.60%, CAT can reduce the growth inhibition rate, which was 14. 88±2.35%, 49.09±3.46% and 72.95 ±2.61% when cells were treated with ethanol and CAT.4 ) When SMMC-7721 cells wrer treated with 4% ethanol after 3h, 6h and 12h, Mitochondrial membrane potential was 96. 02 ±1.68%, 59.68 ±1.47% and 16.84 ±2.06% respectively. While treated with 4% ethanol compared with CAT, Mitochondrial membrane potential was 98.92±0.60%, 73.33±2.99% and 45.76± 1.55% respectively.5) When SMMC-7721 cells were treated with 4% ethanol or 4% ethanoi compared with CAT for 24h or 48h, apoptotic sub-Gl peak was happened, the result was 25.59±3.87%, 14.59± 2.83%, 21.61 ±1.86% and 36.00±3.51%, which had not happened on the control groups.Conclusion:1) Ethanol can suppress the hepatocellular carcinoma cells line smmc-7721 proliferation.2) Ethanol induced smmc-7721 cells apoptosis was one of the reasons of ethanol suppress thehepatocellular carcinoma cells smmc-7721 proliferation.3) Ethanol induced the hepatocellular carcinoma cells apoptosis was caused by adding H2O2 damaged mitochondrial membrane potential.