Treatment of small cell lung cancer with TRA-8 in combination with cisplatin and radiation.

Abstract

Limited stage small cell lung cancer (SCLC) represents a minority of SCLC. Despite extensive clinical trials, standard treatment remains cisplatin-based chemotherapy and thoracic irradiation (TI). This study focused on the interaction of cisplatin/radiation with the anti-human DR5 monoclonal antibody TRA-8 in SCLC cells. TRA-8 binds specifically to DR5 and has been shown to activate apoptosis. Four human SCLC cell lines were utilized for experimentation (SCLC-41, SCLC-58, SCLC-68, and SCLC-74). Immunoblot analysis was used to determine relative protein levels of DR5, DR4 and pro-caspase 8 for each cell line. Using a tetrazolium-based assay (XTT), the IC(50) values for cisplatin with or without TRA-8 were determined for the SCLC cell lines. Four SCLC lines were assayed with a combination of TRA-8 (10 μg/ml), 2 Gy radiation and various concentrations of cisplatin. Apoptosis was evaluated using Annexin V-FITC and cleaved caspase immunoblotting. Using a SCLC-58 subcutaneous xenograft model, treatment began 21 d after tumor cell injection. Treatment included weekly cisplatin (4 mg/kg) and radiation of 1 Gy (24 h after cisplatin) and TRA-8 (200 μg) was administered i.p. twice weekly for three weeks. Immunoblot analysis showed similar levels of DR5 for all cell lines with variable levels of DR4. Various concentrations of TRA-8 antibody (≤ 10 μg/ml) induced no significant cytotoxicity in the SCLC cell lines. The in vitro combination treatment with TRA-8 (10 μg/ml), 1.25 μg/ml cisplatin and 2 Gy radiation showed increased cytotoxicity when compared to combinations without TRA-8. Furthermore, the triple combination demonstrated the greatest amount of apoptosis as measured by Annexin V staining. The in vivo studies showed the combination of 1G y, cisplatin and TRA-8 extended the tumor doubling time to 44 d as compared to any doublet treatment groups that ranged from 12 to 20 d. Analysis of survival data showed 100% of the combination group (RT+cisplatin+TRA-8) were alive 65 d after treatment began whereas all doublet treatment groups showed 50% or less survival. These studies showed increased cytotoxicity when TRA-8 was added to radiation/cisplatin in SCLC. This effect was demonstrated in vitro and in vivo. TRA-8 represents a promising new agent in the treatment of SCLC.