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

Abstract

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 cytoprotectively. 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.

Nitric oxide antagonism to glioblastoma photodynamic therapy and mitigation thereof by BET bromodomain inhibitor JQ1

“…10B). A similar response was observed previously for a breast cancer cell line (52). JQ1 strongly reduced Bcl-xL expression in U87 control cells, consistent with others' results (51), and also attenuated its post-PDT upregulation; e.g.…”

“…However, an indirect effect due to iNOS down-regulation was also possible, since NO is known to signal for survivin induction (49,50). Similar direct and indirect effects of JQ1 may have occurred in the case of Bcl-xL, given our recent evidence that Bcl-xL upregulation in PDT-stressed breast cancer cells was suppressed by iNOS enzyme inhibitors (52). Whether low level iNOS-derived NO has any influence on p21 expression is not known.…”

“…Some of these inhibitors (L-NIL, GW274150) have already been safely tested in clinical trials, although these had no relationship to cancer or PDT (28,71). As an intermediary proof-ofconcept, we recently showed that ALA-PDT suppression of mouse-borne human breast tumor xenografts was substantially augmented by administration of 1400W or GW274150, whereas no significant effect was observed on control tumor growth (52). Consistently, iNOS protein in tumor samples was strongly upregulated after ALA-PDT, and NO-derived NO 2 -levels were also elevated relative to control levels -and in 1400W-inhibitable fashion (53).…”

Modulation of the Anti-Tumor Efficacy of Photodynamic Therapy by Nitric Oxide

“…Immune-deficient female mice bearing human breast carcinoma MDA-MB-231 tumor xenografts were subjected to ALA-PDT using a 633 nm light source. Treated animals exhibited a significant reduction in tumor growth relative to light-only controls over a 12-day post-irradiation period, and 1400 W reduced growth even further [55]. Analysis of post-PDT tumor samples revealed a strong elevation in iNOS and NO-derived nitrite levels.…”

“…DU145 cells also upregulated iNOS under photostress, but less extensively than PC3 counterparts [57]. It is important to note that all experiments described up to this point [50,51,52,53,54,55,56,57] started with cells at confluence no greater than ~60%. At significantly higher confluences, e.g., >90%, the stress from inter-cell contact was sufficient to elevate iNOS [58] and this could have resulted in an under-estimation of the elevation caused by photostress.…”