| Summary: | 博士 === 國立臺灣大學 === 毒理學研究所 === 97 === Hepatocellular carcinoma (HCC) is the fifth most common cancer and the third leading cause of cancer death worldwide. The incidence of HCC is estimated to range from ranging 500,000 to 1,000,000 new cases annually, causing 600,000 deaths worldwide per year. Surgery with curative intent is achievable for only 15 to 25% of patients, and most patients die from locally advanced or metastatic disease in a relatively short period of time. To date, cytotoxic chemotherapy has not been a standard treatment for HCC.
With intensive research on the molecular biology of HCC, several important intracellular signaling pathways such as the Ras/Raf/MEK/ERK pathway and the PI3K/Akt/mTOR pathway have been identified as involved in the carcinogenesis and tumor progression of HCC. Recently, molecular targeted therapy, which acts on these dysregulated signal transduction pathways, has shown promise as a treatment for advanced HCC. Development of novel agents to enhance the effectiveness of treatment is mandatory.
OSU-03012 is a derivative of celecoxib, a cyclooxygenase (COX)-2 inhibitor which has been shown to induce cell death in various types of cancer cells, including prostate cancer, pancreatic cancer, and breast cancer. The mechanism of action is presumably through inhibition of the 3-phosphoinositide-dependent kinase-1 (PDK-1)/Akt signaling pathway. In addition to PDK-1/Akt signaling inhibition, OSU-03012 might also have effects on other important signaling pathways. For example, OSU-03012 has been reported to cause a PDK1/Akt-independent cell death in glioma cells. These findings suggest that OSU-03012 might be a multi-targeted inhibitor which exerts its functions in a cell type-dependent manner.
Autophagy has been recognized as a cellular catabolic degradation response to starvation or stress where cellular proteins and organelles are engulfed, digested and recycled to maintain cellular metabolism. The process of autophagy starts by sequestering a portion of the cytoplasm and intracellular organelles in a double-membrane-bound structure known as the autophagosome. These autophagosomes subsequently fuse with lysosomes to form autolysosomes, in which the sequestered contents are degraded by lysosomal hydrolases. Recent studies demonstrated that autophagy also has an active role in cell death. Autophagy or autophagic cell death, also known as type II programmed cell death, has been shown to be a response to various anticancer therapies in many kinds of cancer cells.
In this study, we showed that OSU-03012 inhibits growth of Huh7, Hep3B, and HepG2 cells within a low micromolor range. TUNEL assay and flow cytometry analysis indicated that no apoptotic cell death was induced by OSU-03012 treatment. Active caspase-3 and cleaved PARP, two biochemical markers of apoptosis, were undetectable by Western blot analysis in OSU-03012-treated Huh7 cells. OSU-03012 induced a significantly increased S-phase population in Huh7 cells. Interestingly, OSU-03012 induced autophagy in Huh7 cells, evidenced by MDC staining, electron microscopy image and Western blot analysis of MAP1-LC3, an important marker of autophagy. OSU-03012-induced autophagy as well as cytotoxicity was partially reversed by silencing ATG5, a gene involved in autophagy, or 3-MA, a widely used autophagy chemical inhibitor. The xenograft tumor model demonstrated that OSU-03012 suppressed Huh7 tumor growth. These findings suggest that autophagy is a mechanism which contributes to the in vivo cytotoxic effect of OSU-03012.
We next demonstrated that OSU-03012 induced reactive oxygen species (ROS) generation by using H2DCFDA-based flow cytometry and florescence microscopy detection. While high levels of ROS often induce apoptotic cell death through caspase activation, ROS cause autophagic cell death in different cancer cells under certain physiological conditions. The ROS scavengers N-acetylcysteine (NAC) and tiron abrogated OSU-03012-induced autophagy and subsequent cytotoxicity. We found that OSU-03012 increased ROS accumulation which in turn induced ER stress and ERK1/2 activation. Knockdown of Bip, an ER stress marker, enhanced OSU-03012-induced autophagy, while overexpression of Bip decreased OSU-03012-induced autophagy and subsequent cytotoxicity, suggesting that ER stress is involved in OSU-03012-induced autophagic cell death and Bip protects the cells from OSU-03012-induced cell death.
In parallel, we found that inhibition of ERK1/2 activated by ROS accumulation reversed OSU-03012-induced cytotoxicity in Huh7 cells. We showed that activated ERK1/2 triggered a decrease in the p27 kip1 protein level, which may result in arrested or prolonged S-phase cells. We further demonstrated that the expression of cyclin A and CDK2, two G1/S-related proteins, were increased by OSU-03012.
In conclusion, our results show that the orally bioavailable drug OSU-03012 induces autophagic but not apoptotic cell death in HCC, and that this autophagy-inducing activity is in part related to ROS accumulation. This study demonstrates a novel biological effect of OSU-03012 which supports its clinical potential as a component of therapeutic strategies for HCC.
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