Molecular basis of 'hypoxic' breast cancer cell radio-sensitization: phytochemicals converge on radiation induced Rel signaling

Background: Heterogeneously distributed hypoxic areas are a characteristic property of locally advanced breast cancers (BCa) and generally associated with therapeutic resistance, metastases, and poor patient survival. About 50% of locally advanced BCa, where radiotherapy is less effective are suggested to be due to hypoxic regions. In this study, we investigated the potential of bioactive phytochemicals in radio-sensitizing hypoxic BCa cells. Methods: Hypoxic (O2-2.5%; N2-92.5%; CO2-5%) MCF-7 cells were exposed to 4 Gy radiation (IR) alone or after pretreatment with Curcumin (CUR), curcumin analog EF24, neem leaf extract (NLE), Genistein (GEN), Resveratrol (RES) or raspberry extract (RSE). The cells were examined for inhibition of NF kappa B activity, transcriptional modulation of 88 NF kappa B signaling pathway genes, activation and cellular localization of radio-responsive NF kappa B related mediators, eNos, Erk1/2, SOD2, Akt1/2/3, p50, p65, pI kappa B alpha, TNF alpha, Birc-1, -2, -5 and associated induction of cell death. Results: EMSA revealed that cells exposed to phytochemicals showed complete suppression of IR-induced NF kappa B. Relatively, cells exposed EF24 revealed a robust inhibition of IR-induced NF kappa B. QPCR profiling showed induced expression of 53 NF kappa B signaling pathway genes after IR. Conversely, 53, 50, 53, 53, 53 and 53 of IR-induced genes were inhibited with EF24, NLE, CUR, GEN, RES and RSE respectively. In addition, 25, 29, 24, 16, 11 and 21 of 35 IR-suppressed genes were further inhibited with EF24, NLE, CUR, GEN, RES and RSE respectively. Immunoblotting revealed a significant attenuating effect of IR-modulated radio-responsive eNos, Erk1/2, SOD2, Akt1/2/3, p50, p65, pI kappa B alpha, TNF alpha, Birc-1, -2 and -5 with EF24, NLE, CUR, GEN, RES or RSE. Annexin V-FITC staining showed a consistent and significant induction of IR-induced cell death with these phytochemicals. Notably, EF24 robustly conferred IR-induced cell death. Conclusions: Together, these data identifies the potential hypoxic cell radio-sensitizers and further implies that the induced radio-sensitization may be exerted by selectively targeting IR-induced NF kappa B signaling.