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Background: Hepatocellular carcinoma (HCC) is the most common malignant primary liver tumor worldwide. Systemic treatment in advanced disease has been limited to sorafenib, a broad spectrum tyrosine kinase inhibitor, with many adverse side effects and suboptimal outcomes. Our lab has investigated triptolide, a diterpene triepoxide, as a potential chemotherapeutic option. This study evaluates the response of HuH-7 and Hep3B HCC cells to triptolide, with or without combination therapy with sorafenib.

Methods: HuH-7 and Hep3B HCC cell lines were treated in vitro with triptolide and/or sorafenib at varying concentrations. Cell viability (MTT assay), caspase activation (Promega), and Annexin V positivity (Guava Nexin) were then assessed. Real-time PCR was utilized to determine the changes in mRNA levels, and Western blots were used for evaluation of protein expression.

Results: Triptolide and sorafenib were equally effective at reducing cell viability in Hep3B cells, at low concentrations (FIGURE 1). Within 72 hours of 25 nM triptolide treatment, 80% cell death was noted, and similar a reduction in cell viability was seen with 2.5 uM sorafenib. Increased concentrations of either drug achieved minimal increases in cell death. In distinction to the Hep3B cell line, the HuH-7 cells were more resistant to single agent treatment. Sorafenib treatment (2.5 uM) resulted in 70% cell death at 72 hours, whereas triptolide (100 nM) resulted in 40% cell death (FIGURE 1). Combination therapy was attempted in this cell line. Notably, a significant reduction in cell viability was found using lower concentrations of each drug, in comparison to either drug concentration alone, with less than 20% cell viability at 72 hours (FIGURE 2).

Cell death with both treatments resulted in increased caspase-3 activation and Annexin V positivity in both cell lines, confirming apoptosis. Evaluation of mRNA and protein levels in response to triptolide showed significant downregulation of the heat shock protein cascade, with levels of HSF-1 decreased in both cell lines. Downstream expression of HSP70 and HSP27, known upregulated proteins in metastatic HCC disease, were also significantly decreased.

Conclusions: Treatment of advanced HCC is currently limited to sorafenib therapy, with many adverse side effects and suboptimal outcomes. We have shown that triptolide treatment in vitro induces HCC cell death by apoptosis, with decreased expression of proteins found to be normally upregulated in metastatic disease. While triptolide therapy alone results in significant cell death in Hep3B cells, combination therapy with sorafenib, both at low concentrations, results in notably superior cell death to either treatment alone in the more resistant HuH-7 cells. Our study suggests triptolide may serve as a therapeutic option for advanced HCC. Orthotopic mouse model studies are underway.