Preliminary Studies On The Mechanisms Of Shikonin-induced Human Hepatoma Cell Apoptosis In Vitro

Hepatocellular carcinoma (HCC) is the fifth most common tumor worldwide and continues to have a poor prognosis. Despite of surveillance efforts, most tumors are diagnosed at late stages. In China, hepatoma is so high incidence that it has been the main threat of people's health in our country and has the front rank of the tumor cancers. So far, most antineoplastic drug all have strong cytotoxicity, but the toxic side effects are significant to limit extensive application and efficacy. Therefore, ti is forced on looking for natural anticancer drugs which are insignificant toxicity, safe and effective. Shikonin is extraction of Lithospermum root, which is fat-soluble naphthoquinone compounds with strong antitumor effect. Then shikonin is expected to be an effective drug for treatment of HCC. Hepatoma Cell Line SMMC-7721 as target was used in this study.Test of shikonin induced SMMC-7721 apoptosis and its related mechanisms were carried out in order to provide proof of cancer treatment of shikonin.1. Study on hepatoma carcinoma cell proliferation inhibited by shikoninThe growth inhibition of human hepatoma cells lines SMMC-7721 and human normal liver cells HL-7702 treated by various concentration of shikonin after different time were observed with MTT assay.Results showed that shikonin could inhibit the growth of SMMC-7721 cells and HL-7702 cells and it presented time-and concentration-dependent. However, the effect of shikonin on HL-7702 cell was significantly strong than that of HL-7702 cells.2.Studies on apoptosis of human hepatoma cell induced by shikoninMotphological changes of SMMC-7721 apoptosis were observed with flouorescance microscop, confocal microscope and electron microscopy. Cell cycle changes and the rates of apoptosis changes were detected by flow cytometry, and the apoptosis of hepatoma cells tunel were detected by DNA agarose gel electrophoresis. Characteristics of typical apoptosis such as nuclear shrinkage, fragmented nuclei et al, could be seen in SMMC-7721 cells treated with shikonin. By flow cytometry, it has been found that cell cycle was inhibited in G0/G1 phase, the ratio of the G0/G1 phase was gradually increased while the proportion of S phase was reduced as the dose of medicine was increased, the proportion of G2/M was not appreciably changing, and the rate of apoptosis showed a clear dose- and time- dependent."DNA ladder"was found by agarose gel electrophoresis, which was the typical characteristics of cell apoptosis.3. Preliminary studies on the mechanisms of shikonin-induced human hepatoma cell apoptosisConcentrations of intracellular calcium and mitochondrial membrane potential changes were determined by flow cytometry, the caspase-3 protein activity was detected with reader, and bcl-2, p53, bax, and caspase-3 gene mRNA levels were detected by semi-quantitative PCR. preliminary studies on the mechanisms of shikonin-induced hepatoma cell line SMMC-7721 apoptosis. The results showed that concentrations of intracellular calcium and mitochondrial membrane potential were continuously reduced, bcl-2 gene had no significant change, while bax, caspase-3, p53 gene expression increased significantly than the control group treaed by shikonin.This shows that mechanism of apoptosis may be way to induce apoptosis through mitochondrial signal pathway.