Background/Objectives Oncogene-targeted therapies against growth factor receptors are effective treatment options for driver mutation-positive non-small cell lung cancer (NSCLC). The third-generation EGFR-Tyrosine Kinase inhibitor (TKI) osimertinib, is a standard first-line therapy for patients with EGFR-mutated NSCLC cancer. Acquired resistance to osimertinib is a significant problem that limits survival. In addition to that clinical data show approximately 15% of EGFR mutant non-small cell lung cancer patients have innate resistance to Osimertinib. Tumor heterogeneity and multiple resistance mechanisms add to the complexity of EGFR-mutated NSCLC. Tumor cells that acquire resistance, independent of the mechanism, experience replication stress (RS) as proliferation resumes. Methods We have employed a series of non-small cell lung cancer models to assess the efficacy of combining osimertinib with NERx-329, targeting RS via inhibiting RPA, and the impact of these treatments on growth and damage signaling pathways. Results We demonstrate that targeting RS with RPAi treatment induces cell death in multiple EGFR-mutant cell lines alone and when combined with osimertinib. Dissection of signaling pathways revealed that RPAi treatment does not block osimertinib inhibition of EGFR signaling. Analysis of DNA damage response (DDR) signaling reveals that NERx329 potentiates the osimertinib-dependent downregulation of Chk1 activity and expression. The Chk1 loss was shown to be dependent on proteasome degradation as proteasome inhibitor MG-132 restores Chk1 activity and expression. Conclusions From these data, we infer that targeting RS via RPAi NERx-329 in combination with osimertinib is an effective strategy and represents a promising drug combination targeted therapy to enhance efficacy and limit the development of resistance. Simple Summary This research aims to assess a new mechanism to potentiate osimertinib treatment of EGFR-mutated non-small cell lung cancer. Targeting replication stress with the small molecule Replication proteins A inhibitor (RPAi), NERx-329, induces cancer cell death and enhance the effectiveness of osimertinib. The findings suggest that combining NERx-329 with osimertinib could be a promising strategy to improve treatment outcomes and reduce resistance. Importantly, we demonstrate that neither RPAi or osimertinib alter the anticancer mechanism of each single agent, providing a new approach to increase therapeutic efficacy and delay or eliminate the development of drug resistance. ### Competing Interest Statement JJT is a co-founder and CSO of NERx Biosciences. RB and SJ declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. * EGFR : Epidermal growth factor receptor RPA : Replication protein A TKI : Tyrosine kinase inhibitor RS : Replication stress DDR : DNA damage response NSCLC : Non-small cell lung cancer ssDNA : Single-strand DNA

Abstract Lung cancer is the leading cause of cancer related deaths worldwide with the majority (~80%) of patients diagnosed with non-small lung cancer (NSCLC). Targeted therapies directed against tumor initiating growth factor receptors are effective treatment options for driver mutation positive NSCLC. Osimertinib is a third-generation EGFR-Tyrosine Kinase inhibitor (TKI) that has activity against EGFR exon 19, exon 21, and T790M mutations. Unfortunately, Osimertinib treated patients acquire resistance and median progression free survival is 18 months. Moreover, Osimertinib does not cure stage IV EGFR mutant NSCLC. The acquisition of secondary mutations leading to activation of bypass pathways, MET amplification, EMT, compensatory pathway activation and histological transformation to small cell phenotype are major causes of acquiring resistance to EGFR TKIs. Proliferating cancer cells regularly experience a low level of replication stress. Additionally, receptor tyrosine kinases such as EGFR are known to interact with DNA repair proteins and impact DNA damage repair following chemotherapy, radiation therapy, and EGFR TKI treatment. However, the role of DNA repair pathways in EGFR-TKI resistance is unknown and the interaction between EGFR pathway, DNA damage, and repair pathways has not been fully elucidated. At low-level replicative stress promotes genomic instability but at a high level through mitotic catastrophe it causes cell death. Replication protein A is a critical sensor of the DNA damage response detecting replication stress. The small molecule inhibitor of RPA, NERx 329, sequesters active RPA and induces replication catastrophe and cell death. Here we demonstrate that NERx329 in combination with Osimertinib enhanced Osimertinib mediated cell death. CCk-8 assays performed after 48h of NERx329 and Osimertinib combination treatment result in a robust decrease in IC50 compared to single agent therapy. Dissection of signaling pathways leading to this death-promoting effect of NERx329 showed a combination of bypass pathway marker (AXL) and compensatory pathway marker (STAT) inhibition in AXL-high expressing EGFR mutant lung cancer cells. Whereas inhibition of STAT pathway was responsible for the death-promoting effect of NERx329 in AXL-low expressing EGFR mutant lung cancer cells. AXL and STAT inhibition are associated with induced DNA damage and impaired DNA repair. From these data, we infer that DNA damage repair pathways could be involved in TKI resistance and NERx329 could be a promising drug candidate for combination targeted therapy. Citation Format: Reshma Bhowmick, John J. Turchi, Shadia I. Jalal. Evaluation of replication protein A inhibitor, NERx329 in combination with EGFR mutant targeted therapy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3856.

Abnormal migration and proliferation of endothelial cells (EC) drive neovascular retinopathies. While anti-VEGF treatment slows progression, pathology is often supported by decrease in intraocular pigment epithelium-derived factor (PEDF), an endogenous inhibitor of angiogenesis. A surface helical 34-mer peptide of PEDF, comprising this activity, is efficacious in animal models of neovascular retina disease but remains impractically large for therapeutic use. We sought smaller fragments within this sequence that mitigate choroidal neovascularization (CNV). Expecting rapid intravitreal (IVT) clearance, we also developed a method to reversibly attach peptides to nano-carriers for extended delivery. Synthetic fragments of 34-mer yielded smaller anti-angiogenic peptides, and N-terminal capping with dicarboxylic acids did not diminish activity. Charge restoration via substitution of an internal aspartate by asparagine improved potency, achieving low nM apoptotic response in VEGF-activated EC. Two optimized peptides (PEDF-335, 8-mer-and PEDF 336, 9-mer) were tested in a mouse model of laser-induced CNV. IVT injection of either peptide, 2-5 days before laser treatment, gave significant CNV decrease at day + 14 post laser treatment. The 8-mer also decreased CNV, when administered as eye drops. Also examined was a nanoparticle-conjugate (NPC) prodrug of the 9-mer, having positive zeta potential, expected to display longer intraocular residence. This NPC showed extended efficacy, even when injected 14 days before laser treatment. Neither inflammatory cells nor other histopathologic abnormalities were seen in rabbit eyes harvested 14 days following IVT injection of PEDF 336 ( > 200 mu g). No rabbit or mouse eye irritation was observed over 12-17 days of PEDF 335 eye drops (10 mM). Viability was unaffected in 3 retinal and 2 choroidal cell types by PEDF 335 up to 100 mu M, PEDF 336 (100 mu M) gave slight growth inhibition only in choroidal EC. A small anti-angiogenic PEDF epitope (G-Y-D-L-Y-R-V) was identified, variants (adipic-Sar-Y-N-L-Y-R-V) mitigate CNV, with clinical potential in treating neovascular retinopathy. Their shared active motif, Y - - - R, is found in laminin (Ln) peptide YIGSR, which binds Ln receptor 67LR, a known high-affinity ligand of PEDF 34-mer.

Abstract Purpose: Ovarian cancer is the deadliest gynecologic malignancy with limited treatment options and novel therapies urgently needed. Immunosuppressive microenvironment is critical for tumor progression and immune checkpoint inhibitors, which enable T-cell anticancer immunity revolutionized the outcomes in multiple cancer types. However, this approach had limited success in ovarian cancer. Our small therapeutic peptides, derived from an endogenous type 2 tumor suppressor, Pigment Epithelium-Derived Factor (PEDF), act through an alternative immune mechanism, repolarization of tumor-associated macrophages (TAMs) to the tumor-suppressive phenotype.Experimental Design: Short peptides based on the PEDF's active domain were modified for improved stability and efficacy. Two peptides (PMD-427, PMD-336) were tested in preclinical ovarian cancer models using the human chemoresistant cell line, OvCar-3, and transformed mouse cell line ID8. We also performed mechanistic analysis of the peptides' anti-tumor action, including effects on macrophages cytotoxic, cytokine secretion and migratory activity in vitro and in vivo. Results: PEDF peptide PMD-427 caused > 20-fold reduction in tumor burden. PMD-427 induced selective apoptosis in ovarian cancer cells but not in normal ovarian epithelium. This selectivity was based on context-specific modulation of extrinsic death cascades, Fas and FasL. More importantly, PMD-427 peptides also stimulated macrophage polarization from M2 to M1 phenotype as was evidenced by the shift in cytokine profile (decreased IL-10 and increased IL-12 expression), altered morphology (increased number of dendrite-like-processes) and other changes in M2 markers (attenuated PD-L1 expression). M2/M1 macrophage polarization was also evident by tumor immunostaining. Critically, PMD peptides ovarian cancer cell killing by macrophages as was determined in co-culture studies; this fratricidal activity was reliant on the expression of TRAIL by the macrophages and of its cognate receptor, DR5 by ovarian cancer cells, respectively. Combined with enhanced macrophage motility as observed by time-lapse micropscopy, these changes resulted in increased macrophage recruitment to the tumors and enhance killing of the cancer cells in vivo. The key role of macrophages in the anti-cancer effects of PMD peptides was confirmed by depletion of macrophages in ovarian tumor bearing mice using clodronate liposomes. Conclusions: We have generated a first-in-class multi-targeted peptide drug, which promotes macrophage polarization that results in eradication of ovarian tumors in mice. Citation Format: Reshma Bhowmick, Elena Vinokour, Michael Paul Plebanek, Marisol Villanueva, Victor Shifrin, Jack Henkin, Ignacio Melgar-Asensio, James Petrik, Raghu Kallurie, Olga V. Volpert. Reprogramming of tumor-associated macrophages by a short synthetic peptide eradicates ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3133.

Metastatic cancers produce exosomes that condition pre-metastatic niches in remote microenvironments to favor metastasis. In contrast, here we show that exosomes from poorly metastatic melanoma cells can potently inhibit metastasis to the lung. These “non-metastatic” exosomes stimulate an innate immune response through the expansion of Ly6C low patrolling monocytes (PMo) in the bone marrow, which then cause cancer cell clearance at the pre-metastatic niche, via the recruitment of NK cells and TRAIL-dependent killing of melanoma cells by macrophages. These events require the induction of the Nr4a1 transcription factor and are dependent on pigment epithelium-derived factor (PEDF) on the outer surface of exosomes. Importantly, exosomes isolated from patients with non-metastatic primary melanomas have a similar ability to suppress lung metastasis. This study thus demonstrates that pre-metastatic tumors produce exosomes, which elicit a broad range of PMo-reliant innate immune responses via trigger(s) of immune surveillance, causing cancer cell clearance at the pre-metastatic niche.

The Ran-binding protein 2 (Ranbp2/Nup358) is a cytoplasmic and peripheral nucleoporin comprised of 4 Ran-GTP-binding domains (RBDs) that are interspersed among diverse structural domains with multifunctional activities. Our prior studies found that the RBD2 and RBD3 of Ranbp2 control mitochondrial motility independently of Ran-GTP-binding in cultured cells, whereas loss of Ran-GTP-binding to RBD2 and RBD3 are essential to support cone photoreceptor development and the survival of mature retinal pigment epithelium (RPE) in mice. Here, we uncover that loss of Ran-GTP-binding to RBD3 alone promotes the robust age-dependent increase of ubiquitylated substrates and S1 subunit (Pmsd1) of the 19S cap of the proteasome in the retina and RPE and that such loss in RBD3 also compromises the structural integrity of the outer segment compartment of cone photoreceptors only and without affecting the viability of these neurons. We also found that the E2-ligase and partner of Ranbp2, ubc9, is localized prominently in the mitochondrial-rich ellipsoid compartment of photoreceptors, where Ranbp2 is also known to localize with and modulate the activity of mitochondrial proteins. However, the natures of Ranbp2 and ubc9 isoforms to the mitochondria are heretofore elusive. Subcellular fractionation, co-immunolocalization and immunoaffinity purification of Ranbp2 complexes show that novel isoforms of Ranbp2 and ubc9 with molecular masses distinct from the large Ranbp2 and unmodified ubc9 isoforms localize specifically to the mitochondrial fraction or associate with mitochondrial components, whereas unmodified and SUMOylated Ran GTPase are excluded from the mitochondrial fraction. Further, liposome-mediated intracellular delivery of an antibody against a domain shared by the mitochondrial and nuclear pore isoforms of Ranbp2 causes the profound fragmentation of mitochondria and their delocalization from Ranbp2 and without affecting Ranbp2 localization at the nuclear pores. Collectively, the data support that Ran GTPase-dependent and independent and moonlighting roles of Ranbp2 or domains thereof and ubc9 control selectively age-dependent, neural-type and mitochondrial functions.

We discovered recently that human breast cancer cells subjected to photodynamic therapy (PDT)-like oxidative stress localized in mitochondria rapidly upregulated nitric oxide synthase-2 (NOS2) and nitric oxide (NO), which increased resistance to apoptotic photokilling. In this study, we asked whether human prostate cancer PC-3 cells would exploit NOS2/NO similarly and, if so, how proliferation of surviving cells might be affected. Irradiation of photosensitized PC-3 cells resulted in a rapid (<1 h), robust (similar to 12-fold), and prolonged (similar to 20 h) post-irradiation upregulation of NOS2. Caspase-3/7 activation and apoptosis were stimulated by NOS2 inhibitors and a NO scavenger, implying that induced NO was acting cytoprotectively. Cyclic GMP involvement was ruled out, whereas suppression of pro-apoptotic JNK and p38 MAPK activation was clearly implicated. Cells surviving photostress grew back similar to 2-times faster than controls. NOS2 inhibition prevented this and the large increase in cell cycle S-phase occupancy observed after irradiation. Thus, photostress upregulation of NOS/NO elicited both a pro-survival and pro-growth response, both of which could compromise clinical PDT efficacy unless suppressed, e.g. by pharmacological intervention with a NOS2 inhibitor. (C) 2013 Elsevier Ireland Ltd. All rights reserved.

Abstract Photodynamic therapy (PDT) employs a photosensitizing agent, molecular oxygen, and photosensitizer-exciting light to produce reactive oxygen species that lead to tumor eradication. Nitric oxide (NO) produced by tumor or tumor vasculature cells could be pro-carcinogenic by inhibiting apoptosis and/or promoting angiogenesis and tumor growth. We recently showed that photoactivation of 5-aminolevulinic acid (ALA)-generated protoporphyrin IX in mitochondria of COH-BR1 breast tumor cells strongly upregulated inducible nitric oxide synthase (iNOS) and steady state NO level in these cells. Including the iNOS inhibitor 1400W during photochallenge dramatically enhanced caspase-9 activation and Annexin-assessed apoptotic cell killing while reducing NO production assessed by the intracellular NO probe DAF-2DA. Short hairpin RNA (ShRNA)-based iNOS knockdown (kd) produced similar results, confirming iNOS involvement. An apoptosis-promoting effect of iNOS inhibition was also observed when breast MDA-MB231 and prostate PC-3 cancer cells were subjected to ALA/light stress. We have now examined the signaling events associated with iNOS-mediated hyperresistance of COH-BR1 cells using a combination of immunocytochemistry, western blotting, immunoprecipitaion, and iNOS-kd methods. A soluble guanylyl cyclase inhibitor failed to stimulate ALA/light-provoked apoptosis, ruling out cGMP involvement in stress resistance. ALA/light activated PI3-kinase-dependent signaling via phosphorylation-inactivation of the tumor suppressor PTEN, leading to phosphorylation-activation of pro-survival Akt. Inhibition of PI3K by Wortmannin prevented ALA/light-induced Akt activation as well as iNOS upregulation while enhancing apoptotic photokilling. Moreover, photostress activation of PI3K/Akt was accompanied by a cytosol-to-nucleus translocation of the iNOS transcription factor NF-κB, and an inhibitor of NF-kB activation prevented iNOS induction while stimulating apoptosis. Furthermore, iNOS-kd as well as 1400W treatment resulted in intensified and more prolonged activation of MAP kinases JNK and p38α by ALA/light stress. In addition, photostressed iNOS-kd cells exhibited p53 upregulation and Survivin inactivation/downregulation. The following general mechanism of NO-based cytoprotection is suggested from these and related findings: Photostress activation of PI3K/Akt suppresses pro-apoptotic MAP kinases and p53 while upregulating/activating Survivin via NF-κB-mediated induction of iNOS/NO. This study provides important new insights into photostress-elicited pro-survival signaling that could reduce PDT effectiveness, and suggests iNOS inhibitor-based interventions for counteracting this. (Supported by NIH Grant CA70823 and a grant from the MCW Cancer Center.)

Photodynamic therapy (PDT) employs photoexcitation of a sensitizer to generate tumor-eradicating reactive oxygen species. We recently showed that irradiating breast cancer COH-BR1 cells after treating with 5-aminolevulinic acid (ALA, a pro-sensitizer) resulted in rapid upregulation of inducible nitric oxide (NO) synthase (iNOS). Apoptotic cell killing was strongly enhanced by an iNOS inhibitor (1400 W), iNOS knockdown (kd), or a NO scavenger, suggesting that NO was acting cytoprotectively. Stress signaling associated with these effects was examined in this study. ALA/light-stressed COH-BR1 cells, and also breast adenocarcinoma MDA-MB-231 cells, mounted an iNOS/NO-dependent resistance to apoptosis that proved to be cGMP-independent. Immunocytochemistry and subcellular Western analysis of photostressed COH-BR1 cells revealed a cytosol-to-nucleus translocation of NF-κB which was negated by the NF-κB activation inhibitor Bay11. Bay11 also enhanced apoptosis and prevented iNOS induction, consistent with NF-κB involvement in the latter. JNK and p38 MAP kinase inhibitors suppressed apoptosis, implicating these kinases in death signaling. Post-irradiation extent and duration of JNK and p38 phosphorylation were dramatically elevated by 1400 W or iNOS-kd, suggesting that these activations were suppressed by NO. Regarding pro-survival stress signaling, rapid activation of Akt was unaffected by 1400 W, but prevented by Wortmannin, which also enhanced apoptosis. Thus, a link between upstream Akt activation and iNOS induction was apparent. Furthermore, p53 protein expression under photostress was elevated by iNOS-kd, whereas robust Survivin induction was abolished, consistent with p53 and Survivin being negatively and positively regulated by NO, respectively. Collectively, these findings enhance our understanding of cytoprotective signaling associated with photostress-induced NO and suggest iNOS inhibitor-based approaches for improving PDT efficacy.

Many tumor cells produce nitric oxide (NO) as an antiapoptotic/progrowth molecule which also promotes antiogenesis and tumor expansion. This study was designed to examine possible antagonistic effects of endogenous NO on tumor eradication by photodynamic therapy (PDT). Using COH-BR1 breast cancer cells sensitized in mitochondria with 5-aminolevulinic acid (ALA)-generated protoporphyrin IX as a model for ALA-based PDT, we found that caspase-9 activation and apoptotic death following irradiation were strongly enhanced by 1400W, an inhibitor of inducible nitric oxide synthase (iNOS). RT-PCR and Western analyses revealed a substantial upregulation of both iNOS mRNA and protein, beginning ca 4 h after irradiation and persisting for at least 20 h. Accompanying this was a strong 1400W-inhibitable increase in intracellular NO, as detected with the NO probe, DAF-2-DA. Short hairpin RNA-based iNOS knockdown in COH-BR1 cells dramatically reduced NO production under photostress while enhancing caspase-9 activation and apoptosis. These findings suggest that cytoprotective iNOS/NO induction in PDT-treated tumor cells could reduce treatment efficacy, and point to pharmacologic intervention with iNOS inhibitors for counteracting this.

Photodynamic therapy (PDT) employs a photosensitizing agent, molecular oxygen, and visible light to generate reactive species that kill tumor and tumor vasculature cells. Nitric oxide produced by these cells could be procarcinogenic by inhibiting apoptosis or promoting angiogenesis and tumor growth. The purpose of this study was to determine whether tumor cells upregulate NO as a cytoprotective measure during PDT. Breast tumor COH-BR1 cells sensitized in their mitochondria with 5-aminolevulinic acid (ALA)-derived protoporphyrin IX died apoptotically after irradiation, ALA- and light-only controls showing no effect. Western analysis revealed that inducible nitric oxide synthase (iNOS) was upregulated >3-fold within 4 h after ALA/light treatment, whereas other NOS isoforms were unaffected. Exposing cells to a NOS inhibitor (L-NAME or 1400W) during photochallenge enhanced caspase-3/7 activation and apoptotic killing up to 2- to 3-fold while substantially reducing chemiluminescence-assessed NO production, suggesting that this NO was cytoprotective. Consistently, the NO scavenger cPTIO enhanced ALA/light-induced caspase-3/7 activation and apoptotic kill by >2.5-fold. Of added significance, cells could be rescued from 1400W-exacerbated apoptosis by an exogenous NO donor, spermine-NONOate. This is the first reported evidence for increased tumor cell resistance due to iNOS upregulation in a PDT model. Our findings indicate that stress-elicited NO in PDT-treated tumors could compromise therapeutic efficacy and suggest NOS-based pharmacologic interventions for preventing this.

Intraflagellar transport (IFT) provides a mechanism for the transport of cilium-specific proteins, but the mechanisms for linkage of cargo and IFT proteins have not been identified. Using the sensory outer segments (OS) of photoreceptors, which are derived from sensory cilia, we have identified IFT-cargo complexes containing IFT proteins, kinesin 2 family proteins, two photoreceptor-specific membrane proteins, guanylyl cyclase 1 (GC1, Gucy2e) and rhodopsin (RHO), and the chaperones, mammalian relative of DNAJ, DnajB6 (MRJ), and HSC70 (Hspa8). Analysis of these complexes leads to a model in which MRJ through its binding to IFT88 and GC1 plays a critical role in formation or stabilization of the IFT-cargo complexes. Consistent with the function of MRJ in the activation of HSC70 ATPase activity, Mg-ATP enhances the co-IP of GC1, RHO, and MRJ with IFT proteins. Furthermore, RNAi knockdown of MRJ in IMCD3 cells expressing GC1-green fluorescent protein (GFP) reduces cilium membrane targeting of GC1-GFP without apparent effect on cilium elongation.

A novel protease was purified to homogeneity from the latex of Pedilanthus tithymaloids by a simple purification procedure involving ammonium sulfate precipitation and cation-exchange chromatography. The molecular weight of the protease was estimated to be approximately 63.1 kDa and the extinction coefficient (epsilon(1%)(280nm)) was 28.4. The enzyme hydrolyzes denatured natural substrates like casein, azoalbumin and azocasein with a high specific activity but little activity towards synthetic substrates. The pH and temperature optima were pH 8.0-9.5 and 65-70 degrees C, respectively. The proteolytic activity of the enzyme was inhibited by different protease-specific inhibitors (e.g., thiol, serine, metallo, etc.) up to a certain extent but not completely by any class of inhibitors. The enzyme was relatively stable towards pH change, temperature, denaturants and organic solvents. The enzyme consists of five disulfide bridges compared to three observed in most plant cysteine proteases. Overall, the striking features of this protease are its high molecular weight, high cysteine content and only partial inhibition of activity by different classes of protease inhibitors contrary to known proteases from other plant sources. The enzyme is named as pedilanthin as per the protease nomenclature.

Purpose Photodynamic Therapy (PDT) is an antitumor modality that employs a photosensitizing agent, molecular oxygen, and visible light to produce reactive species that kill tumor/tumor vasculature cells in a site‐specific manner. Nitric oxide (NO) at low levels can inhibit apoptosis and promote angiogenesis and tumor growth. We showed recently that NO from a chemical donor/activated macrophages induced a resistance to photokilling in protoporphyrin IX (PpIX)‐sensitized COH‐BR1 breast tumor cells. Signaling events associated with this hyperresistance was examined in the present study. Methods COH‐BR1 cells were sensitized with PpIX in mitochondria via metabolism of ALA and irradiated in the presence/absence of SPNO‐derived NO. Apoptotic/necrotic photokillling was assessed by fluorescence microscopy using Hoechst/PI staining. Redox signaling associated with MAP kinase phosphorylation‐activation and heme oxygenase‐1 (HO‐1) upregulation was examined by immunoblotting. Results NO delivered during irradiation of ALA‐treated cells protected them against apoptotic death. This was accompanied by a large induction of HO‐1 and an increase in the activation of overall p38 and ERK1/2, but not JNK. Conclusions These findings suggest that antioxidant stress protein, HO‐1 plays a major role in NO's anti‐PDT effects and is associated with activation of the anti‐apoptotic p38 and ERK1/2 signaling pathways.

Antitumor photodynamic therapy (PDT) employs a photosensitizing agent, molecular oxygen, and visible light to produce reactive oxygen species that can destroy tumor and tumor vasculature cells. NO produced by these cells could be procarcinogenic by inhibiting apoptosis and promoting angiogenesis and tumor growth. We recently showed that NO from a chemical donor or activated macrophages makes COH-BR1 breast tumor cells more resistant to photokilling sensitized by 5-aminolevulinic acid (ALA)-generated protoporphyrin IX (PpIX). Signaling events associated with this hyperresistance have now been examined. ALA-treated COH-BR1 cells containing mitochondria-localized PpIX died mainly by apoptosis after being irradiated. Underlying redox signaling associated with MAP kinase (ERK1/2, p38, JUN) phosphorylation–activation, and heme oxygenase-1 (HO-1) upregulation was studied using immunoprecipitation and Western blot methodology. ALA/light treatment resulted in activation of proapoptotic JNK and p38α, and deactivation of prosurvival p38β and ERK1/2. Involvement of both JNK and p38 in apoptosis was established by using a specific inhibitor for each. Spermine NONOate-derived NO, introduced immediately before irradiation, provided substantial protection against apoptosis. This was accompanied by greater HO-1 induction and a strong inhibition of each MAP kinase effect seen in the absence of NO. Downstream of JNK and p38α activation, a marked upregulation/activation of proapoptotic Bax and Bid was observed along with down-regulation of antiapoptotic Bcl-xL, each response being reversed by NO. These findings provide new insights into signaling activity associated with the intrinsic apoptotic pathway in ALA-PDT and how this activity can be modulated by NO.