Triptolide Sensitizes Liver Cancer Cell Lines To Chemotherapy In Vitro And In Vivo

Hepatocellular carcinoma (HCC) is the fourth most common cancer and the third leading cause of cancer-related death in the world. Surgical resection is the best method to reduce the growth of HCC and improve life expectancy of HCC patients. However, more than 80% of HCC patients are diagnosed with an advanced stage or unresectable diseases. For those patients who do undergo resection, the prognosis is very poor with as high as 50% recurrence rates at 2 years. It is important for the majority of unresectable patients to seek other treatments. Current chemotherapy is not very effective, and the systemic toxicity and development of chemoresistance are the major obstacles. Thus, development of low- or non-toxic chemosensitisers to enhance the antitumor effects of conventional chemotherapeutic agents is urgently needed for treatment of this deadly disease. Triptolide is a diterpenoid isolated from the herb Tripterygium wilfordii, which has been used as an immunosuppressant in China for many years. In recent years, triptolide has shown strong anti-tumor and proapoptotic activities in many different tumor cell lines in vitro and in vivo. However, the effect of triptolide on liver cancer cell lines to chemotherapy has not yet been evaluated. In this study, we investigated whether triptolide could enhance the effect of chemotherapeutics such as cisplatin and 5-FU in vitro and in vivo on liver cancer cells.Objective: To study potential effects of triptolide on the sensitivity of human liver cancer cells to chemotherapeutics and the possible mechanisms in vitro and in vivo.Methods: HepG2 and SMMC7721 cells were treated with triptolide, 5-FU and cisplatin alone, or triptolide plus 5-FU or cisplatin for different times in vitro and in vivo. Cell viability was determined by MTT assay. Western blotting was used to evaluate protein expression. Flow cytometry was used to detect cellular apoptosis and reactive oxygen species (ROS). Caspase-3 activity assay kit was used to analyze the mechanism of cellular apoptosis. The antitumor effects of triptolide alone or in combination with chemotherapeutics were also measured in transplantation tumor models.Results:1. Triptolide enhanced cytotoxicity of cisplatin and 5-FU to liver cancer cell lines in vitroIt revealed that treatment with triptolide for 48h caused cell inhibition in a dose-dependent manner in both cell lines. The cytotoxic effect of 5-FU and cisplatin were also determined by MTT assay alone. In combination with triptolide(20ng/ml), the cytotoxicity of 5-FU and cisplatin were significantly enhanced compared to signal chemotherapy(P <0.05)。2. Triptolide enhances chemotherapy-induced apoptosis in vitroFCM analysis showed that the apoptosis population of cells were increased after treatment liver cancer cells with triptolide, CDDP and 5-FU alone for 48h (P<0.05). The apoptotic population was significantly induced by co-treatment of liver cancer lines with triptolide and chemotherapeutics, compared with signal treatment (P<0.05).3. Triptolide induces Bcl-2 and p53, but inhibits Bax and p21 expressionTriptolide markedly inhibited Bcl-2 and p53 protein expression in both liver cancer cell lines. The expression of Bcl-2 and p53 protein was further inhibited by treating the cells with triptolide and chemotherapeutics in combination, compared with triptolide or chemotherapeutics treated only. In contrast, the expression of Bax and p21 protein was significantly induced by triptolide, and further increased by triptolide and chemotherapeutics in both cell lines4. Triptolide enhances chemotherapy-induced ROS and caspase-3 activity in vitroAfter treatment with triptolide and chemotherapies for 24h, the results suggested that triptolide not only increased ROS and caspase-3 activity alone but also increased ROS and caspase-3 activity induced by antitumor drugs in liver cancer cell lines, compared to control or chemotherapeutic-only treated cells (P<0.05).5. Triptolide increases tumor susceptibility to chemotherapy in VivoMice treatment with triptolide showed significantly to reduce tumor growth in comparison to untreated controls. Triptolide also boosted the anticancerous effects of cisplatin and 5-FU in nude mice compared with cisplatin- or 5-FU-only treated tumors.Conclusion: In summary, triptolide was associated with increased cellular sensitivity of liver cancer cellls to cisplatin and 5-FU in vivo and in vitro, and may provide new liver cancer treatment strategies.