鴻喜菇蛋白質對人類肝癌細胞 (HepG2) 生長抑制及細胞週期停滯之研究

Abstract

The mortality rate of hepatocellular carcinoma in all cancer mortality ranks second in Taiwan. There are some common treatments against this disease such as surgical resection, liver transplantation, embolization and chemotherapy. Although these treatments have been carried out for many years, they still have limitations and side effects. Therefore, exploration of effective chemotherapeutic agents from natural food is urgently needed. Hypsizigus marmoreus is a low-calorie, low-fat and high protein concent mushroom. A number of bioactive molecules including antitumor, antifungal and antioxidant substances have been identified from this mushroom. In this research, proteins in H. marmoreus were precipitated by 40~80% saturated ammonium sulfate. DEAE-Sepharose CL-6B ion exchange chromatography was used subsequently, and eluted by 0~1 M NaCl continuous or stepwise gradient. Fraction isolated by continuous gradient named HM-3, and eluted by 0.2 or 0.4 M NaCl stepwise gradient named fraction Ⅰ or Ⅱ. HM-3 at a concentration of 50 mg/ml strongly inhibited the growth of HepG2 cells by 87.41% and 88.25% for 12 and 24 hr respectively. HM-3 inhibited the growth of HepG2 cells in a concentration-dependent manner. Cells were incubated with 50 mg/ml fractionⅠ or Ⅱ for 12 and 24 hr, and results showed that fractionⅠinhibited the growth of HepG2 cells by 24.41% and 19.44%. Fraction Ⅱ had no obvious growth inhibition. Above results indicated that HM-3 had better growth inhibition than others, so we chose HM-3 to investigate the mechanism of growth inhibition. G1 phase arrest was observed by flow cytometry when cells were incubated with HM-3 for 24 hr. G1 phase percentage ranged from 54.35% to 69.08% when concentration increased to 200 mg/ml from 0 mg/ml, but no apoptosis was observed. DNA ladder was not observed by agarose gel electrophoresis. Western blotting analysis showed the expression of p53 and p21 increased in HepG2 cells with the increasing concentration of HM-3. Obvious decrease in cyclin E1 was observed and no apparent change in p27, cyclin A2, cyclin D1 and CDK2. According to these results, HM-3 might induce growth inhibition by activating p53 and p21, then inactivating cyclin E1, causing G1 phase cell cycle arrest in HepG2 cells.