Regulation of ferroptotic cancer cell death by GPX4.

Ferroptosis is a form of nonapoptotic cell death for which key regulators remain unknown. We sought a common mediator for the lethality of 12 ferroptosis-inducing small molecules. We used targeted metabolomic profiling to discover that depletion of glutathione causes inactivation of glutathione peroxidases (GPXs) in response to one class of compounds and a chemoproteomics strategy to discover that GPX4 is directly inhibited by a second class of compounds. GPX4 overexpression and knockdown modulated the lethality of 12 ferroptosis inducers, but not of 11 compounds with other lethal mechanisms. In addition, two representative ferroptosis inducers prevented tumor growth in xenograft mouse tumor models. Sensitivity profiling in 177 cancer cell lines revealed that diffuse large B cell lymphomas and renal cell carcinomas are particularly susceptible to GPX4-regulated ferroptosis. Thus, GPX4 is an essential regulator of ferroptotic cancer cell death.

Upregulation of iNOS/NO in Cancer Cells That Survive a Photodynamic Challenge: Role of No in Accelerated Cell Migration and Invasion.

Anti-tumor photodynamic therapy (PDT) is a unique modality that employs a photosensitizer (PS), PS-exciting light, and O2 to generate cytotoxic oxidants. For various reasons, not all malignant cells in any given tumor will succumb to a PDT challenge. Previous studies by the authors revealed that nitric oxide (NO) from inducible NO synthase (iNOS/NOS2) plays a key role in tumor cell resistance and also stimulation of migratory/invasive aggressiveness of surviving cells. iNOS was the only NOS isoform implicated in these effects. Significantly, NO from stress-upregulated iNOS was much more important in this regard than NO from preexisting enzymes. Greater NO-dependent resistance, migration, and invasion was observed with at least three different cancer cell lines, and this was attenuated by iNOS activity inhibitors, NO scavengers, or an iNOS transcriptional inhibitor. NO diffusing from PDT-targeted cells also stimulated migration/invasion potency of non-targeted bystander cells. Unless counteracted by appropriate measures, all these effects could seriously compromise clinical PDT efficacy. Here, we will review specific examples of these negative side effects of PDT and how they might be suppressed by adjuvants such as NO scavengers or inhibitors of iNOS activity or expression.

Pro-Tumor Activity of Endogenous Nitric Oxide in Anti-Tumor Photodynamic Therapy: Recently Recognized Bystander Effects.

Various studies have revealed that several cancer cell types can upregulate inducible nitric oxide synthase (iNOS) and iNOS-derived nitric oxide (NO) after moderate photodynamic treatment (PDT) sensitized by 5-aminolevulinic acid (ALA)-induced protoporphyrin-IX. As will be discussed, the NO signaled cell resistance to photokilling as well as greater growth and migratory aggressiveness of surviving cells. On this basis, it was predicted that diffusible NO from PDT-targeted cells in a tumor might enhance the growth, migration, and invasiveness of non- or poorly PDT-targeted bystander cells. This was tested using a novel approach in which ALA-PDT-targeted cancer cells on a culture dish were initially segregated from non-targeted bystander cells of the same type via impermeable silicone-rimmed rings. Several hours after LED irradiation, the rings were removed, and both cell populations were analyzed in the dark for various responses. After a moderate extent of targeted cell killing (~25%), bystander proliferation and migration were evaluated, and both were found to be significantly enhanced. Enhancement correlated with iNOS/NO upregulation in surviving PDT-targeted cancer cells in the following cell type order: PC3 > MDA-MB-231 > U87 > BLM. If occurring in an actual PDT-challenged tumor, such bystander effects might compromise treatment efficacy by stimulating tumor growth and/or metastatic dissemination. Mitigation of these and other negative NO effects using pharmacologic adjuvants that either inhibit iNOS transcription or enzymatic activity will be discussed.

Anti-steroidogenic effects of cholesterol hydroperoxide trafficking in MA-10 Leydig cells: Role of mitochondrial lipid peroxidation and inhibition thereof by selenoperoxidase GPx4.

Steroid hormone synthesis in steroidogenic cells requires cholesterol (Ch) delivery to/into mitochondria via StAR family trafficking proteins. In previous work, we discovered that 7-OOH, an oxidative stress-induced cholesterol hydroperoxide, can be co-trafficked with Ch, thereby causing mitochondrial redox damage/dysfunction. We now report that exposing MA-10 Leydig cells to Ch/7-OOH-containing liposomes (SUVs) results in (i) a progressive increase in fluorescence probe-detected lipid peroxidation in mitochondrial membranes, (ii) a reciprocal decrease in immunoassay-detected progesterone generation, and ultimately (iii) loss of cell viability with increasing 7-OOH concentration. No significant effects were observed with a phospholipid hydroperoxide over the same concentration range. Glutathione peroxidase GPx4, which can catalyze lipid hydroperoxide detoxification, was detected in mitochondria of MA-10 cells. Mitochondrial lipid peroxidation and progesterone shortfall were exacerbated when MA-10 cells were treated with Ch/7-OOH in the presence of RSL3, a GPx4 inhibitor. However, Ebselen, a selenoperoxidase mimetic, substantially reduced RSL3's negative effects, thereby partially rescuing the cells from peroxidative damage. These findings demonstrate that co-trafficking of oxidative stress-induced 7-OOH can disable steroidogenesis, and that GPx4 can significantly protect against this.

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

Endogenous nitric oxide (NO) generated by inducible NO synthase (iNOS) promotes glioblastoma cell proliferation and invasion and also plays a key role in glioblastoma resistance to chemotherapy and radiotherapy. Non-ionizing photodynamic therapy (PDT) has anti-tumor advantages over conventional glioblastoma therapies. Our previous studies revealed that glioblastoma U87 cells up-regulate iNOS after a photodynamic challenge and that the resulting NO not only increases resistance to apoptosis but renders surviving cells more proliferative and invasive. These findings were largely based on the effects of inhibiting iNOS activity and scavenging NO. Demonstrating now that iNOS expression in photostressed U87 cells is mediated by NF-κB, we hypothesized that (i) recognition of acetylated lysine (acK) on NF-κB p65/RelA by bromodomain and extra-terminal (BET) protein Brd4 is crucial; and (ii) by suppressing iNOS expression, a BET inhibitor (JQ1) would attenuate the negative effects of photostress. The following evidence was obtained. (i) Like iNOS, Brd4 protein and p65-acK levels increased severalfold in photostressed cells. (ii) JQ1 at minimally toxic concentrations had no effect on Brd4 or p65-acK up-regulation after PDT but strongly suppressed iNOS, survivin, and Bcl-xL up-regulation, along with the growth and invasion spurt of PDT-surviving cells. (iii) JQ1 inhibition of NO production in photostressed cells closely paralleled that of growth/invasion inhibition. (iv) Finally, at 1% the concentration of iNOS inhibitor 1400W, JQ1 reduced post-PDT cell aggressiveness to a far greater extent. This is the first evidence for BET inhibitor targeting of iNOS expression in cancer cells and how such targeting can markedly improve therapeutic efficacy.

Photodynamic therapy of cancer: an update.

Photodynamic therapy (PDT) is a clinically approved, minimally invasive therapeutic procedure that can exert a selective cytotoxic activity toward malignant cells. The procedure involves administration of a photosensitizing agent followed by irradiation at a wavelength corresponding to an absorbance band of the sensitizer. In the presence of oxygen, a series of events lead to direct tumor cell death, damage to the microvasculature, and induction of a local inflammatory reaction. Clinical studies revealed that PDT can be curative, particularly in early stage tumors. It can prolong survival in patients with inoperable cancers and significantly improve quality of life. Minimal normal tissue toxicity, negligible systemic effects, greatly reduced long-term morbidity, lack of intrinsic or acquired resistance mechanisms, and excellent cosmetic as well as organ function-sparing effects of this treatment make it a valuable therapeutic option for combination treatments. With a number of recent technological improvements, PDT has the potential to become integrated into the mainstream of cancer treatment.

Upregulation of nitric oxide in tumor cells as a negative adaptation to photodynamic therapy.

One of the advantages of PDT is that it can often circumvent tumor resistance to chemotherapeutic agents such as cisplatin and doxorubicin. However, pre-existing and acquired resistance to PDT has also been demonstrated. One type of resistance, which involves nitric oxide (NO) generated by inducible nitric oxide synthase (iNOS/NOS2) in tumor cells, was discovered in the author's laboratory. When subjected to a 5-aminolevulinic acid (ALA)-based photodynamic challenge, several cancer lines, including breast, prostate, and glioma, underwent intrinsic apoptosis that could be substantially enhanced by iNOS enzymatic inhibitors or a NO scavenger, implying iNOS/NO-mediated resistance. In most cases, iNOS was significantly upregulated by the challenge and this appeared to be more important in the hyper-resistance than pre-existing enzyme. Of added importance was our observation that cells surviving ALA/light treatment typically exhibited a more aggressive phenotype, proliferating and migrating/invading more rapidly than controls in iNOS/NO-dependent fashion. Most of these in vitro PDT findings have recently been confirmed at the in vivo level, using a human breast tumor xenograft model. We have also shown that upregulated iNOS in PDT-targeted cells can elicit a pro-growth/migration response in non-targeted bystander cells, NO again playing a key role. Post-PDT resistance and potentially dangerous hyper-aggressiveness can be attenuated by inhibitors of iNOS enzymatic activity, some of which have seen pharmacologic use in non-cancer or PDT settings. These various aspects of PDT antagonism by tumor iNOS/NO and how they might be overcome will be discussed in this review. Lasers Surg. Med. 50:590-598, 2018.© 2018 Wiley Periodicals, Inc.

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 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.

Impairment of Macrophage Cholesterol Efflux by Cholesterol Hydroperoxide Trafficking: Implications for Atherogenesis Under Oxidative Stress.

OBJECTIVE: Oxidative stress associated with cardiovascular disease can produce various oxidized lipids, including cholesterol oxides, such as 7-hydroperoxide (7-OOH), 7-hydroxide (7-OH), and 7-ketone (7=O). Unlike 7=O and 7-OH, 7-OOH is redox active, giving rise to the others via potentially toxic-free radical reactions. We tested the novel hypothesis that under oxidative stress conditions, steroidogenic acute regulatory (StAR) family proteins not only deliver cholesterol to/into mitochondria of vascular macrophages, but also 7-OOH, which induces peroxidative damage that impairs early stage reverse cholesterol transport. APPROACH AND RESULTS: Stimulation of human monocyte-derived THP-1 macrophages with dibutyryl-cAMP resulted in substantial upregulation of StarD1 and ATP-binding cassette (ABC) transporter, ABCA1. Small interfering RNA-induced StarD1 knockdown before stimulation had no effect on StarD4, but reduced ABCA1 upregulation, linking the latter to StarD1 functionality. Mitochondria in stimulated StarD1-knockdown cells internalized 7-OOH slower than nonstimulated controls and underwent less 7-OOH-induced lipid peroxidation and membrane depolarization, as probed with C11-BODIPY (4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-inda-cene-3-undecanoic acid) and JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benzimidazolylcarbocyanine iodide), respectively. Major functional consequences of 7-OOH exposure were (1) loss of mitochondrial CYP27A1 activity, (2) reduced 27-hydroxycholesterol (27-OH) output, and (3) downregulation of cholesterol-exporting ABCA1 and ABCG1. Consistently, 7-OOH-challenged macrophages exported less cholesterol to apoA-I or high-density lipoprotein than did nonchallenged controls. StarD1-mediated 7-OOH transport was also found to be highly cytotoxic, whereas 7=O and 7-OH were minimally toxic. CONCLUSIONS: This study describes a previously unrecognized mechanism by which macrophage cholesterol efflux can be incapacitated under oxidative stress-linked disorders, such as chronic obesity and hypertension. Our findings provide new insights into the role of macrophage redox damage/dysfunction in atherogenesis.

Accelerated migration and invasion of prostate cancer cells after a photodynamic therapy-like challenge: Role of nitric oxide.

Employing an in vitro model for 5-aminolevulinic acid (ALA)-based photodynamic therapy (PDT), we recently reported that human prostate cancer PC3 cells rapidly and persistently overexpressed inducible nitric oxide synthase (iNOS) and nitric oxide (NO) after a moderate ALA/light challenge. The upregulated iNOS/NO was shown to play a key role in cell resistance to apoptotic photokilling and also in the dramatic growth spurt observed in surviving cells. In the present study, we found that PC3 cells surviving an ALA/light insult not only proliferated faster than non-stressed controls, but migrated and invaded faster as well, these effects being abrogated by an iNOS inhibitor or NO scavenger. Photostressed prostate DU145 cells exhibited similar behavior. Using in-gel zymography, we showed that PC3 extracellular matrix metalloproteinase-9 (MMP-9) was strongly activated 24 h after ALA/light treatment and that MMP-9 inhibitor TIMP-1 was downregulated, consistent with MMP-9 involvement in enhanced invasiveness. We also observed a photostress-induced upregulation of α6 and ß1 integrins, implying their involvement as well. The MMP-9, TIMP-1, and integrin effects were strongly attenuated by iNOS inhibition, confirming NO's role in photostress-enhanced migration/invasion. This study reveals novel, potentially tumor-promoting, side-effects of prostate cancer PDT which may be averted through use of iNOS inhibitors as PDT adjuvants.

Tumor-generated nitric oxide as an antagonist of photodynamic therapy.

Nitric oxide (NO) is a multifunctional free radical molecule produced naturally by nitric oxide synthase (NOS) enzymes. Many tumors exploit NO for survival and growth signaling, and also to thwart the effects of therapeutic treatments, including PDT. The anti-PDT effects of NO were discovered using animal tumor models, but the mechanisms involved are still not fully understood. Recent in vitro studies on breast and prostate cancer cells have shown that inducible NOS (iNOS) along with NO is dramatically upregulated after an ALA-PDT-like challenge. Cells were more resistant to apoptosis after a photochallenge and survivors grew, migrated, and invaded more rapidly, iNOS/NO playing a key role in all these effects. This perspective briefly reviews what is currently known about NO's negative effects on PDT and some of the signaling mechanisms involved. It also provides insights into how these effects may be attenuated by pharmacologic use of iNOS inhibitors.

Deleterious cholesterol hydroperoxide trafficking in steroidogenic acute regulatory (StAR) protein-expressing MA-10 Leydig cells: implications for oxidative stress-impaired steroidogenesis.

Steroidogenic acute regulatory (StAR) proteins in steroidogenic cells are implicated in the delivery of cholesterol (Ch) from internal or external sources to mitochondria (Mito) for initiation of steroid hormone synthesis. In this study, we tested the hypothesis that under oxidative stress, StAR-mediated trafficking of redox-active cholesterol hydroperoxides (ChOOHs) can result in site-specific Mito damage and dysfunction. Steroidogenic stimulation of mouse MA-10 Leydig cells with dibutyryl-cAMP (Bt2cAMP) resulted in strong expression of StarD1 and StarD4 proteins over insignificant levels in nonstimulated controls. During incubation with the ChOOH 3ß-hydroxycholest-5-ene-7α-hydroperoxide (7α-OOH) in liposomes, stimulated cells took up substantially more hydroperoxide in Mito than controls, with a resulting loss of membrane potential (ΔΨm) and ability to drive progesterone synthesis. 7α-OOH uptake and ΔΨm loss were greatly reduced by StarD1 knockdown, thus establishing the role of this protein in 7α-OOH delivery. Moreover, 7α-OOH was substantially more toxic to stimulated than nonstimulated cells, the former dying mainly by apoptosis and the latter dying by necrosis. Importantly, tert-butyl hydroperoxide, which is not a StAR protein ligand, was equally toxic to stimulated and nonstimulated cells. These findings support the notion that like Ch itself, 7α-OOH can be transported to/into Mito of steroidogenic cells by StAR proteins and therein induce free radical damage, which compromises steroid hormone synthesis.

Cholesterol Hydroperoxide Co-trafficking in Testosterone-generating Leydig Cells: GPx4 Inhibition of Cytotoxic and Anti-steroidogenic Effects.

Trafficking of intracellular cholesterol (Ch) to and into mitochondria of steroidogenic cells is required for steroid hormone biosynthesis. This trafficking is typically mediated by one or more proteins of the steroidogenic acute regulatory (StAR) family. Our previous studies revealed that 7-OOH, a redox-active cholesterol hydroperoxide, could be co-trafficked with Ch to/into mitochondria of MA-10 Leydig cells, thereby inducing membrane lipid peroxidation (LPO) which impaired progesterone biosynthesis. These negative effects of 7-OOH were inhibited by endogenous selenoperoxidase GPx4, indicating that this enzyme could protect against 7-OOH-induced oxidative damage/dysfunction. In the present study, we advanced our Leydig focus to cultured murine TM3 cells and then to primary cells from rat testis, both of which produce testosterone. Using a fluorescent probe, we found that extensive free radical-mediated LPO occurred in mitochondria of stimulated primary Leydig cells during treatment with liposomal Ch+7-OOH, resulting in a significant decline in testosterone output relative to that with Ch alone. Strong enhancement of LPO and testosterone shortfall by RSL3 (a GPx4 inhibitor) and reversal thereof by Ebselen (a GPx4 mimetic), suggested that endogenous GPx4 was playing a key antioxidant role. 7-OOH in increasing doses was also cytotoxic to these cells, RSL3 exacerbating this in Ebselen-reversable fashion. Moreover, GPx4 knockdown increased cell sensitivity to LPO with reduced testosterone output. These findings, particularly with primary Leydigs (which best represent cells in intact testis) suggest that GPx4 plays a key protective role against peroxidative damage/dysfunction induced by 7-OOH co-trafficking with Ch.

Trafficking of oxidative stress-generated lipid hydroperoxides: pathophysiological implications.

Lipid hydroperoxides (LOOHs) are reactive intermediates that arise during peroxidation of unsaturated phospholipids, glycolipids and cholesterol in biological membranes and lipoproteins. Non-physiological lipid peroxidation (LPO) typically occurs under oxidative stress conditions associated with pathologies such as atherogenesis, neurodegeneration, and carcinogenesis. As key intermediates in the LPO process, LOOHs are susceptible to one-electron versus two-electron reductive turnover, the former exacerbating membrane or lipoprotein damage/dysfunction and the latter diminishing it. A third possible LOOH fate is translocation to an acceptor membrane/lipoprotein, where one- or two-electron reduction may then ensue. In the case of cholesterol (Ch)-derived hydroperoxides (ChOOHs), translocation can be specifically stimulated by StAR family trafficking proteins, which are normally involved in Ch homeostasis and Ch-mediated steroidogenesis. In this review, we discuss how these processes can be impaired by StAR-mediated ChOOH and Ch co-trafficking to mitochondria of vascular macrophages and steroidogenic cells, respectively. The protective effects of endogenous selenoperoxidase, GPx4, are also discussed. This is the first known example of detrimental ChOOH transfer via a natural Ch trafficking pathway and inhibition thereof by GPx4.

Hyper-Aggressiveness of Bystander Cells in an Anti-Tumor Photodynamic Therapy Model: Role of Nitric Oxide Produced by Targeted Cells.

When selected tumor cells in a large in vitro population are exposed to ionizing radiation, they can send pro-survival signals to non-exposed counterparts (bystander cells). If there is no physical contact between irradiated and bystander cells, the latter respond to mediators from targeted cells that diffuse through the medium. One such mediator is known to be nitric oxide (NO). It was recently discovered that non-ionizing anti-tumor photodynamic therapy (PDT) can also elicit pro-survival/expansion bystander effects in a variety of human cancer cells. A novel silicone ring-based approach was used for distinguishing photodynamically-targeted cells from non-targeted bystanders. A key finding was that NO from upregulated iNOS in surviving targeted cells diffused to the bystanders and caused iNOS/NO upregulation there, which in turn stimulated cell proliferation and migration. The intensity of these responses depended on the extent of iNOS/NO induction in targeted cells of different cancer lines. Moreover, the responses could be replicated using NO from the chemical donor DETA/NO. This review will focus on these and related findings, their negative implications for clinical PDT, and how these might be averted by using pharmacologic inhibitors of iNOS activity or transcription.

Permeabilization of the mitochondrial outer membrane by Bax/truncated Bid (tBid) proteins as sensitized by cardiolipin hydroperoxide translocation: mechanistic implications for the intrinsic pathway of oxidative apoptosis.

Cytochrome c (cyt c) release upon oxidation of cardiolipin (CL) in the mitochondrial inner membrane (IM) under oxidative stress occurs early in the intrinsic apoptotic pathway. We postulated that CL oxidation mobilizes not only cyt c but also CL itself in the form of hydroperoxide (CLOOH) species. Relatively hydrophilic CLOOHs could assist in apoptotic signaling by translocating to the outer membrane (OM), thus promoting recruitment of the pro-apoptotic proteins truncated Bid (tBid) and Bax for generation of cyt c-traversable pores. Initial testing of these possibilities showed that CLOOH-containing liposomes were permeabilized more readily by tBid plus Ca(2+) than CL-containing counterparts. Moreover, CLOOH translocated more rapidly from IM-mimetic to OM-mimetic liposomes than CL and permitted more extensive OM permeabilization. We found that tBid bound more avidly to CLOOH-containing membranes than to CL counterparts, and binding increased with increasing CLOOH content. Permeabilization of CLOOH-containing liposomes in the presence of tBid could be triggered by monomeric Bax, consistent with tBid/Bax cooperation in pore formation. Using CL-null mitochondria from a yeast mutant, we found that tBid binding and cyt c release were dramatically enhanced by transfer acquisition of CLOOH. Additionally, we observed a pre-apoptotic IM-to-OM transfer of oxidized CL in cardiomyocytes treated with the Complex III blocker, antimycin A. These findings provide new mechanistic insights into the role of CL oxidation in the intrinsic pathway of oxidative apoptosis.

Intermembrane Translocation of Photodynamically Generated Lipid Hydroperoxides: Broadcasting of Redox Damage.

Lipid hydroperoxides (LOOHs), including cholesterol- and phospholipid-derived species, are reactive intermediates that arise during photosensitized peroxidation of unsaturated lipids in biological membranes. These intermediates may appear in cancer cell membranes during anti-tumor photodynamic therapy (PDT). Photodynamically generated LOOHs have several different fates, including (a) iron-catalyzed one-electron reduction to free radical species which trigger damaging chain peroxidation reactions, (b) selenoperoxidase-catalyzed two-electron reduction to redox-inert alcohols (LOHs), and (c) spontaneous or protein-mediated translocation to other lipid membrane compartments where (a) or (b) may take place. These different LOOH fates will be described in this review, but with special attention to category (c), which the authors were the first to describe and characterize. Seminal early findings on cholesterol hydroperoxide (ChOOH) translocation and its potential negative consequences will be discussed. In reviewing this work, we wish to congratulate Jean Cadet, for his many outstanding accomplishments as a photobiologist and P&P editor.

The Negative Impact of Cancer Cell Nitric Oxide on Photodynamic Therapy.

Numerous studies have shown that low-flux nitric oxide (NO) in tumors produced mainly by inducible nitric oxide synthase (iNOS/NOS2) can signal for angiogenesis, inhibition of apoptosis, and promotion of cell growth, migration, and invasion. Studies in the authors' laboratory have revealed that iNOS-derived NO in various cancer cell types elicits resistance to cytotoxic photodynamic therapy (PDT) and moreover endows PDT-surviving cells with more aggressive proliferation and migration/invasion. In this chapter, we describe how cancer cell iNOS/NO in vitro can be monitored in different PDT model systems (e.g., a targeted cell-bystander cell model) and how pharmacologic interference with basal and PDT-upregulated iNOS/NO can significantly improve PDT outcomes.

Role of nitric oxide in hyper-aggressiveness of tumor cells that survive various anti-cancer therapies.

Low level nitric oxide (NO) produced by inducible NO synthase (iNOS) in many malignant tumors is known to play a key role in the survival and proliferation of tumor cells. NO can also induce or augment resistance to anti-tumor treatments such as platinum-based chemotherapy (CT), ionizing radiotherapy (RT), and non-ionizing photodynamic therapy (PDT). In each of these treatments, tumor cells that survive the challenge may exhibit a striking increase in NO-dependent proliferative, migratory, and invasive aggressiveness compared with non-challenged controls. Moreover, NO from cells directly targeted by PDT can often stimulate aggressiveness in non- or poorly targeted bystander cells. Although NO-mediated resistance to many of these therapies is fairly-well recognized by now, the hyper-aggressiveness of surviving cells and bystander counterparts is not. We will focus on these negative aspects in this review, citing examples from the PDT, CT, and RT publications. Increased aggressiveness of cells that escape therapeutic elimination is a concern because it could enhance tumor progression and metastatic dissemination. Pharmacologic approaches for suppressing these negative responses will also be discussed, e.g., administering inhibitors of iNOS activity or iNOS expression as therapeutic adjuvants.

Relationship between oxidizable fatty acid content and level of antioxidant glutathione peroxidases in marine fish.

Biological membranes can be protected from lipid peroxidation by antioxidant enzymes including catalase (CAT) and selenium-dependent glutathione peroxidases 1 and 4 (GPx1 and GPx4). Unlike GPx1, GPx4 can directly detoxify lipid hydroperoxides in membranes without prior action of phospholipase A(2). We hypothesized that (1) GPx4 is enhanced in species that contain elevated levels of highly oxidizable polyunsaturated fatty acids (PUFA) and (2) activities of antioxidant enzymes are prioritized to meet species-specific oxidative stresses. In this study we examined (i) activities of the oxidative enzyme citrate synthase (CS) and antioxidant (CAT, GPx1 and GPx4) enzymes, (ii) GPx4 protein expression, and (iii) phospholipid composition in livers of five species of marine fish (Myxine glutinosa, Petromyzon marinus, Squalus acanthias, Fundulus heteroclitus and Myoxocephalus octodecemspinosus) that contain a range of PUFA. GPx4 activity was, on average, 5.8 times higher in F. heteroclitus and S. acanthias than in the other three marine fish species sampled. Similarly, activities of CAT and GPx1 were highest in S. acanthias and F. heteroclitus, respectively. GPx4 activity for all species correlates with membrane unsaturation, as well as oxidative activity as indicated by CS. These data support our hypothesis that GPx4 level in marine fish is a function, at least in part, of high PUFA content in these animals. GPx1 activity was also correlated with membrane unsaturation, indicating that marine species partition resources among glutathione-dependent defenses for protection from the initial oxidative insult (e.g. H(2)O(2)) and to repair damaged lipids within biological membranes.