Nitric oxide-mediated resistance to photodynamic therapy in a human breast tumor xenograft model: Improved outcome with NOS2 inhibitors

Many malignant tumors employ iNOS-derived NO to resist eradication by chemotherapeutic agents or ionizing radiation. In this study, we determined whether human breast carcinoma MDA-MB-231 cells in vitro and in vivo as tumor xenografts would exploit endogenous iNOS/NO to resist the cytotoxic effects of 5-aminolevulinic acid (ALA)-based photodynamic therapy (PDT). Broad band visible irradiation of ALA-treated cells resulted in a marked after-light upregulation of iNOS protein which persisted for at least 24 h. Apoptotic killing of ALA/light-challenged cells was significantly enhanced by iNOS inhibitors (1400W, GW274150) and a NO trap (cPTIO), implying that stress-induced iNOS/NO was acting cyto-protectively. We found that cells surviving the photostress proliferated and migrated more rapidly than controls in 1400W- and cPTIO-inhibitable fashion, indicating iNOS/NO involvement. Female SCID mice bearing MDA-MB-231 tumors were used for animal model experiments. ALA-PDT with a 633 nm light source caused a significant reduction in post-irradiation tumor growth relative to light-only controls, which was further reduced by administration of 1400W or GW274150, whereas 1400W had little or no effect on controls. Immunoblot analyses of tumor samples revealed a progressive post-PDT upregulation of iNOS, which reached >5-times the control level after six days. Correspondingly, the nitrite/nitrate level in post-PDT tumor samples was substantially higher than that in controls. In addition, a 1400W-inhibitable upregulation of pro-survival/progression effector proteins such as Bcl-xL, Survivin, and S100A4 was observed after in vitro and in vivo ALA-PDT. This is the first known study to demonstrate iNOS/NO-induced resistance to PDT in an in vivo human tumor model. (C) 2016 Elsevier Inc. All rights reserved.