Norcantharidin-induced Apoptosis of AGS Human Gastric Cancer Cells Through Reactive Oxygen Species Production, and Caspase- and Mitochondria-dependent Signaling Pathways

Abstract

Norcantharidin (NCTD) was purified from mylabris, the dried body of the Chinese blister beetle. NCTD has been shown to exhibit anticancer activities in many human cancer cell lines, but there are no reports to show whether it induces apoptosis of human gastric cancer cells. Therefore, in the present study, we investigated NCTD-induced cell death and associated protein expression in human gastric cancer AGS cells in vitro. Cell morphological changes, viability and cell-cycle distribution were examined and analyzed by phase-contrast microscopy and flow cytometric assays. Flow cytometry was also used to measure the levels of reactive oxygen species (ROS), Ca²⁺, mitochondrial membrane potential (Ψ_m) and activity of caspases. The results indicated that NCTD induced cell morphological changes, reduced total viable cell number and induced G₀/G₁ phase arrest. NCTD also increased ROS production and reduced the Ψ_m and increased caspase-9 activity in AGS cells. Western blotting also found that NCTD increased the pro-apoptotic proteins such as BCL2-associated X protein (BAX) and BH3 interacting-domain death agonist (BID) and increased the release of cytochrome c, apoptosis inducing factor (AIF) and endonuclease G (Endo G) release from mitochondria in AGS cells. NCTD also significantly increased the expression of active forms of caspase-3 and -8 and -9 and reduced the expression of caspase-4 and -12 in AGS cells. Based on these observations, we suggest that NCTD-induced apoptotic cell death may be through mitochondria- and caspase-dependent pathways.

Keywords: AGS human gastric cancer cells; Norcantharidin; ROS; apoptosis; mitochondria; reactive oxygen species.