Effect of Combination of Aspirin and TRAIL in HeLa Cervical Cancer Cells

Abstract

Purpose: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor (TNF) family of cytokines and induces apoptosis in most tumor cells. Although TRAIL is considered a promising drug for cancer therapies because of its tumor selectivity, many tumors are resistant to TRAIL. Thus TRAIL-resistant cancer cells must be sensitized first to become responsive to TRAIL. In this study, I studied whether pretreatment by aspirin augmented TRAIL-induced apoptotic death in HeLa cells, which derived from cervical cancer and investigated the underlying mechanism.

Methods: Cell viability and proliferation were assessed by MTT assay and cytotoxicity was analyzed by LDH assay. Annexin-V /PI staining and sub G1 analysis were used for evaluation of apoptotic cells. We measured mitochondrial membrane potential in HeLa cells undergoing apoptosis by using JC-1. Protein level changes were documented by western blot analysis.

Results: Aspirin inhibited proliferation of HeLa cells in the time- and dose-dependent manners. The combined treatment of aspirin with TRAIL strongly enhanced TRAIL-induced apoptotic cell death in HeLa cells. It activated caspase-8, -9 and -3 and caused the caspase-dependent loss of MMP and the release of cytochrome c from mitochondria to cytosol. The apoptotic characteristics enhanced by the combined treatment were inhibited by a pan-caspase inhibitor z-VAD-fmk. Interestingly, TRAIL caused the activation of ERK, whereas the ERK activation was blocked by aspirin. As a result, the activation of ERK decreased and additionally, Mcl-1 also decreased in combination treatment.

Conclusion: TRAIL in combination with aspirin significantly increase apoptosis in HeLa cells through caspase-and mitochondrial-dependent pathway. Mechanism of such a promoted apoptotic effect might be associated with that TRAIL-induced ERK activation which triggers survival signal to HeLa cells is blocked by pretreatment of aspirin. Inhibition of ERK activation enhances TRAIL-induced caspase activation, which rapidly cleavages Mcl-1. It could also trigger translocation of cleaved Bid to mitochondria to activate mitochondrial pathway, which also amplifies caspase activation. Eventually, pre-treated aspirin could block the survival signal and enhance caspase-and mitochondrial-dependent apoptotic cell death in HeLa cells treated with TRAIL.