Background and Aims: Hepatocellular carcinoma (HCC) is one of the most common cancers in the world and patients with advanced HCC face a dismal prognosis due to lack of effective therapy. Survivin, a member of the family of inhibitor of apoptosis proteins, is highly upregulated in HCC as well as in experimentally induced intrahepatic tumors. Doxorubicin, the only known chemotherapeutic agent for HCC, is cardiotoxic in addition to several well known side effects.
Methods: Survivin gene deletion was established in HepG2, Hep3B, and PLC/PRF/5 human HCC cells using CRISPR/Cas9 system. All the three HCC cells in culture were treated with doxorubicin at various concentrations before and after survivin gene knockout upto 72 hr. TUNEL assay and FACS analysis were performed to demonstrate the induction of apoptosis after doxorubicin treatment. Western blotting was carried out for cleaved fragments of caspase-9 and caspase-3 as well as major apoptotic executioner molecules.
Results: Cell viability studies depicted around 20% cell death at 24 h, 50% at 48 h, and 80% at 72 h after treatment with doxorubicin at 1 µM (final concentration in the media). Among the three cell lines studied, Hep3B cells were more susceptible to doxorubicin compared to HepG2 and PLC/PRF/5 cells. After deletion of survivin gene, the dosage of doxorubicin could reduce to five fold (200 nM, final concentration in the media) with the same cytotoxic effect before the knockout of survivin gene. HCC cells treated with reduced doses of doxorubicin depicted induction of apoptosis that was proved with TUNEL assay and FACS analysis as wells as increased levels of cleaved caspases and major apoptotic executioner molecules.
Conclusions: Our studies demonstrated that blocking of survivin molecule with effective methods would be a successful approach to treat primary hepatic tumors with low and safe doses of doxorubicin and other anticancer agents.
Note: This abstract was not presented at the meeting.
Citation Format: Joseph George, Nobuhiko Hayashi, Takashi Saito, Kazuaki Ozaki, Nobuyuki Toshikuni, Mutsumi Tsuchishima, Mikihiro Tsutsumi. Deletion of survivin sensitize human hepatocellular carcinoma cells to low dose of doxorubicin and induce apoptosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2165. doi:10.1158/1538-7445.AM2017-2165