The additive effects of gamma secretase inhibitor and ionizing radiation in MDA-MD-231 breast cancer cell line

Abstract

14594 Background: The Notch signaling pathway is an evolutionarily conserved developmental pathway whose importance has become increasingly recognized in cancer biology. Gamma secretase inhibitors(GSI) are a novel class of agents which prevent the generation of the active domain of Notch molecules, and suppress Notch signaling downstream. GSI has been shown to have synergistic activity against breast cancer in vitro when combined with HER-2 antibodies. This study investigates whether GSI may enhance the effect of radiation on breast cancer cells. Method: The MDA-MD-231 breast cancer cell line has been shown previously to have high Notch activity. The effects of radiation, GSI, and GSI combined with radiation were studied by DNA content analysis and MTT assay after 7 days of 7.5 μM GSI treatment, cells were irradiated after day 1 of GSI treatment with 5 Gy; Cell cycle studies after 5 μM GSI treatment for 48 hrs and 9 Gy 24 hrs after GSI added; Clonogenicity, after exposure to 0, 2, 4, 6, & 8 Gy with or without 7.5 μM GSI for 6 weeks beginning 24 hrs prior to irradiation, was evaluated by soft agar growth assay. Expression of cyclins A, B (Notch targets), and NOXA (inhibited by Notch) were investigated via western blotting. After treated with GSI 7.5 μM for 48 hrs and 5 Gy 24 hrs after GSI added, cells were harvested and lysed. Results: MTT assay and DNA content analysis demonstrated significant additive effects when GSI was combined with radiation. Cell cycle analysis demonstrated increased apoptosis with a decrease in S-phase fraction with combined treatment vs. GSI or radiation alone; enhanced G1 arrest, and a trend toward enhanced G2/M arrest with combined treatment comparing to radiation alone. Soft-agar assay demonstrated decreased clonogenicity with combined 7.5 μM GSI and single-dose radiation exposure compared to radiation alone. By western blotting, GSI alone or combined with radiation was able to attenuate cyclin A expression but no additional increase in NOXA was found under these conditions. Conclusions: This study identified a potential additive effect between GSI and ionizing radiation in breast cancer in vitro. This is at least partially attributed to the inhibitory effect of GSI on cell proliferation pathways. These findings will guide further in vitro and in vivo studies. No significant financial relationships to disclose.