A novel approach to target hypoxic cancer cells via combining β-oxidation inhibitor etomoxir with radiation

Arpit Dheeraj,1,2 Chapla Agarwal,1 Isabel R Schlaepfer,3 David Raben,4 Rana Singh,2 Rajesh Agarwal,1 Gagan Deep5–7 1Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, Aurora, CO, USA; 2School of Life Sciences, Jawaharlal Nehru University, New Delhi, India; 3Division of Medical Oncology, University of Colorado Anschutz Medical Campus, Aurora, CO, USA; 4Department of Radiation Oncology, University of Colorado Anschutz Medical Campus, Aurora, CO, USA; 5Department of Cancer Biology, Wake Forest School of Medicine, Winston-Salem, NC, USA; 6Department of Urology, Wake Forest School of Medicine, Winston-Salem, NC, USA; 7Wake Forest Baptist Comprehensive Cancer Center, Winston-Salem, NC, USA Background: Hypoxia in tumors is associated with resistance towards various therapies including radiotherapy. In this study, we assessed if hypoxia in cancer spheres could be effectively reduced by adding etomoxir (a β-oxidation inhibitor) immediately after cell irradiation. Methods: We employed cancer cells’ sphere model to target hypoxia. Confocal imaging was used to analyze hypoxia and expression of specific biomarkers in spheres following various treatments (radiation and/or etomoxir). Results: Etomoxir (32.5 μM) treatment improved the radiation (2.5 Gy) efficacy against growth of lung adenocarcinoma H460 spheres. More importantly, radiation and etomoxir combination significantly reduced the hypoxic regions (pimonidazole+ areas) in H460 spheres compared to either treatment alone. Also, etomoxir and radiation combination treatment reduced the protein level of biomarkers for proliferation (Ki-67 and cyclin D1), stemness (CD44) and β-oxidation (CPT1A) in H460 spheres. We observed similar efficacy of etomoxir against growth of prostate cancer LNCaP cells’ spheres when combined with radiation. Further, radiation treatment strongly reduced the hypoxic regions (pimonidazole+ areas) in CPT1 knockdown LNCaP cells’ spheres. Conclusions: Together, these results offer a unique approach to target hypoxia in solid tumors via combining etomoxir with radiation, thereby improving therapeutic efficacy. Keywords: Hypoxia, radiation, β-oxidation, Etomoxir, CPT1A