Enhancement of Apo2L/TRAIL (tumor necrosis factor–related apoptosis-inducing ligand)–induced apoptosis in non–small cell lung cancer cell lines by chemotherapeutic agents without correlation to the expression level of cellular protease caspase-8 inhibitory protein

Abstract

Objective:Apo2L/tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) is a potential anticancer drug that promotes apoptosis specifically in tumor cells. Because not all cancer cells are susceptible to Apo2L/TRAIL, the aim of our study was to determine whether non–small cell lung cancer cells can be sensitized by chemotherapeutic agents for Apo2L/TRAIL-induced apoptosis. In addition, endogenous expression levels of the caspase-inhibiting cellular protease caspase-8 inhibitory protein (C-FLIP) were measured to investigate partial resistance to Apo2L/TRAIL.Methods:Six human lung cancer cell lines (A549, NCI-H358, Calu1, Calu6, SkMes1, and SkLu1) were incubated with soluble Apo2L/TRAIL and two different concentrations each of cisplatin, paclitaxel, doxorubicin, 5-fluorouracil, and camptothecin. After 24 hours the rate of apoptosis was measured by annexin V/propidium iodide staining followed by FACScan analysis. Expression levels of C-FLIP in cell lines and lung cancer biopsy specimens were determined by Western blotting.Results:Treatment of lung cancer cells with Apo2L/TRAIL alone resulted in apoptotic cell death in four cell lines (P < .001). Combining Apo2L/TRAIL and chemotherapeutic agents enhanced the rate of apoptosis significantly. Statistical analysis revealed a synergistic effect of Apo2L/TRAIL in combination with 1.8 mmol/L camptothecin and 100 μmol/L cisplatin, each in four of the six cell lines (P < .002). Western blot analysis showed that sensitization to Apo2L/TRAIL did not correlate with the expression of cellular protease caspase-8 inhibitory protein. Furthermore, no increased cellular protease caspase-8 inhibitory protein levels relative to those in normal lung tissue could be found in non–small cell lung cancer specimens from 12 patients. Conclusion: Apo2L/TRAIL-induced apoptosis in non–small cell lung cancer cell lines is significantly enhanced by chemotherapeutic agents. Resistance and sensitization to Apo2L/TRAIL are not correlated with the endogenous expression level of cellular protease caspase-8 inhibitory protein, implying that in non–small cell lung cancer other mechanisms are responsible for inhibition of the Apo2L/TRAIL pathway. Even though the molecular mechanism remains unclear, the combination of Apo2L/TRAIL with chemotherapy may be a promising treatment modality for non–small cell lung cancer.