Elevated Cystatin expression in metastatic oral cancer cells confers greater resistance to TRAIL-induced apoptosis

Abstract

Background TRAIL (tumor necrosis factor–related apoptosis-inducing ligand) preferentially induces apoptosis of cancer cells without toxicity in normal cells. TRAIL plays an important role in host immunosurveillance against tumor metastasis. Cathepsin B (CB) is a mediator of apoptosis whose activity is regulated by inhibitors known as cystatins (CY). Objective To relate the TRAIL sensitivity of clonally related primary and metastatic oral cancer (OC) cells with their CB and CY levels. Study design Two pairs of primary (686Tu and 101Tu) and metastatic (686Ln and 101Ln) OC cell lines were treated with various concentrations (10-1000 ng/mL) recombinant human TRAIL protein for 14 hours. Cell viability was quantified by MTT assay. Apoptosis rate among TRAIL-treated cells was measured using the TUNEL and M30 CytoDEATH immunodetection assays. CB and CY (A, B, C, and M) levels in these cells lines were analyzed by RT-PCR and Western blots. Results Primary cells revealed greater susceptibility to TRAIL-induced killing (ED 50 of 50 and 250 ng/mL for 686Tu and 101Tu, respectively) than their metastatic clones (ED 50 of 250 and 1000 ng/mL for 686Ln and 101Ln, respectively). Primary cells in the presence of CB-specific chemical inhibitor CA074 revealed markedly increased resistance to TRAIL (ED 50 > 1000 ng/mL). Expression levels of CY were markedly higher in metastatic OC cells than in their respective primary cells whereas CB levels remain unchanged. Conclusion CB is a mediator of TRAIL-induced apoptosis in OC cells. Elevated levels of cystatins in metastatic OC cells correlate with their greater resistance to TRAIL-induced apoptosis. Our data suggest that overexpression of CY in OC cells may confer a metastatic phenotype by enhancing their resistance to TRAIL.