Photosensitized Oxidative Damage from a New Perspective: The Influence of Before-Light and After-Light Reaction Conditions.

Photooxidative damage is heavily influenced by the presence of bioactive agents. Conversely, bioactive agents influence the local environment, which in turn is perturbed by photooxidative damage. These sorts of processes give rise to a version of the "chicken-and-egg" quandary. In this Perspective, we probe this issue by referring to photooxidative damage in one direction as the light-dark (L-D) sequence and in a second direction as the dark-light (D-L) sequence with a reversed cause and effect. The L-D sequence can lead to the downstream production of reactive molecular species (RMS) in the dark, whereas the D-L sequence can be a pre-irradiation period, such as an additive to limit cellular iron levels to enhance biosynthesized amounts of a protoporphyrin sensitizer. A third direction comes from L-D or D-L sequences, or both simultaneously, which can also be useful for optimizing photodynamics. Photodynamic optimization will benefit from understanding and quantitating unidirectional L-D and D-L pathways, and bidirectional L-D/D-L pathways, for improved control over photooxidative damage. Photooxidative damage, which occurs during anticancer photodynamic therapy (PDT), will be shown to involve RMS. Such RMS include persulfoxides (R2S+OO-), NO2•, peroxynitrate (O2NOO-), OOSCN-, SO3•-, selenocyanogen [(SeCN)2], the triselenocyanate anion [(SeCN)3-], I•, I2•-, I3-, and HOOI, as well as additives to destabilize membranes (e.g., caspofungin and saponin A16), inhibit DNA synthesis (5-fluorouracil), or sequester iron (desferrioxamine). In view of the success that additive natural products and repurposed drugs have had in PDT, a Perspective of additive types is expected to reveal mechanistic details for enhanced photooxidation reactions in general. Indeed, strategies for how to potentiate photooxidations with additives remain highly underexplored.

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.

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.

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.

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.

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.

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.

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.

Pathophysiological potential of lipid hydroperoxide intermembrane translocation: Cholesterol hydroperoxide translocation as a special case.

Peroxidation of unsaturated phospholipids, glycolipids, and cholesterol in biological membranes under oxidative stress conditions can underlie a variety of pathological conditions, including atherogenesis, neurodegeneration, and carcinogenesis. Lipid hydroperoxides (LOOHs) are key intermediates in the peroxidative process. Nascent LOOHs may either undergo one-electron reduction to exacerbate membrane damage/dysfunction or two-electron reduction to attenuate this. Another possibility is LOOH translocation to an acceptor site, followed by either of these competing reductions. Cholesterol (Ch)-derived hydroperoxides (ChOOHs) have several special features that will be highlighted in this review. In addition to being susceptible to one-electron vs. two-electron reduction, ChOOHs can translocate from a membrane of origin to another membrane, where such turnover may ensue. Intracellular StAR family proteins have been shown to deliver not only Ch to mitochondria, but also ChOOHs. StAR-mediated transfer of free radical-generated 7-hydroperoxycholesterol (7-OOH) results in impairment of (a) Ch utilization in steroidogenic cells, and (b) anti-atherogenic reverse Ch transport in vascular macrophages. This is the first known example of how a peroxide derivative can be recognized by a natural lipid trafficking pathway with deleterious consequences. For each example above, we will discuss the underlying mechanism of oxidative damage/dysfunction, and how this might be mitigated by antioxidant intervention.

Nitric oxide-elicited resistance to anti-glioblastoma photodynamic therapy.

Glioblastoma multiforme is a highly aggressive primary brain malignancy that resists most conventional chemoand radiotherapeutic interventions. Nitric oxide (NO), a short lived free radical molecule produced by inducible NO synthase (iNOS) in glioblastomas and other tumors, is known to play a key role in tumor persistence, progression, and chemo/radiotherapy resistance. Site-specific and minimally invasive photodynamic therapy (PDT), based on oxidative damage resulting from non-ionizing photoactivation of a sensitizing agent, is highly effective against glioblastoma, but resistance also exists in this case. Studies in the authors' laboratory have shown that much of the latter is mediated by iNOS/NO. For example, when glioblastoma U87 or U251 cells sensitized in mitochondria with 5-aminolevulinic acid -induced protoporphyrin IX were exposed to a moderate dose of visible light, the observed apoptosis was strongly enhanced by an iNOS activity inhibitor or NO scavenger, indicating that iNOS/NO had increased cell resistance to photokilling. Moreover, cells that survived the photochallenge proliferated, migrated, and invaded more aggressively than controls, and these responses were also driven predominantly by iNOS/NO. Photostress-upregulated iNOS rather than basal enzyme was found to be responsible for all the negative effects described. Recognition of NO-mediated hyper-resistance/hyper-aggression in PDT-stressed glioblastoma has stimulated interest in how these responses can be prevented or at least minimized by pharmacologic adjuvants such as inhibitors of iNOS activity or transcription. Recent developments along these lines and their clinical potential for improving anti-glioblastoma PDT are discussed.

Nitric Oxide Inhibition of Chain Lipid Peroxidation Initiated by Photodynamic Action in Membrane Environments.

Iron-catalyzed, free radical-mediated lipid peroxidation may play a major role in tumor cell killing by photodynamic therapy (PDT), particularly when membrane-localizing photosensitizers are employed. Many cancer cells exploit endogenous iNOS-generated NO for pro-survival/expansion purposes and for hyper-resistance to therapeutic modalities, including PDT. In addition to inhibiting the pro-oxidant activity of Fe(II) via nitrosylation, NO may intercept downstream lipid oxyl and peroxyl radicals, thereby acting as a chain-breaking antioxidant. We investigated this for the first time in the context of PDT by using POPC/Ch/PpIX (100:80:0.2 by mol) liposomes (LUVs) as a model system. Cholesterol (Ch or [14C]Ch) served as an in-situ peroxidation probe and protoporphyrin IX (PpIX) as photosensitizer. PpIX-sensitized lipid peroxidation was monitored by two analytical methods that we developed: HPLC-EC(Hg) and HPTLC-PI. 5α-hydroperoxy-Ch (5α-OOH) accumulated rapidly and linearly with irradiation time, indicating singlet oxygen (1O2) intermediacy. When ascorbate (AH-) and trace lipophilic iron [Fe(HQ)3] were included, 7α/7ß-hydroperoxy-Ch (7-OOH) accumulated exponentially, indicating progressively greater membrane-damaging chain lipid peroxidation. With AH-/Fe(HQ)3 present, the NO donor SPNO had no effect on 5α-OOH formation, but dose-dependently inhibited 7-OOH formation due to NO interception of chain-carrying oxyl and peroxyl radicals. Similar results were obtained when cancer cells were PpIX/light-treated, using SPNO or activated macrophages as the NO source. These findings implicate chain lipid peroxidation in PDT-induced cytotoxicity and NO as a potent antagonist thereof by acting as a chain-breaking antioxidant. Thus, unless NO formation in aggressive tumors is suppressed, it can clearly compromise PDT efficacy.

Negative effects of tumor cell nitric oxide on anti-glioblastoma photodynamic therapy.

Glioblastomas are highly aggressive brain tumors that can persist after exposure to conventional chemotherapy or radiotherapy. Nitric oxide (NO) produced by inducible NO synthase (iNOS/NOS2) in these tumors is known to foster malignant cell proliferation, migration, and invasion as well as resistance to chemo- and radiotherapy. Minimally invasive photodynamic therapy (PDT) sensitized by 5-aminolevulinic acid (ALA)-induced protoporphyrin IX (PpIX) is a highly effective anti-glioblastoma modality, but it is also subject to NO-mediated resistance. Studies by the authors have revealed that glioblastoma U87 and U251 cells use endogenous iNOS/NO to not only resist photokilling after an ALA/light challenge, but also to promote proliferation and migration/invasion of surviving cells. Stress-upregulated iNOS/NO was found to play a major role in these negative responses to PDT-like treatment. Our studies have revealed a tight network of upstream signaling events leading to iNOS induction in photostressed cells and transition to a more aggressive phenotype. These events include activation or upregulation of pro-survival/ pro-expansion effector proteins such as NF-κB, phosphoinositide-3-kinase (PI3K), protein kinase-B (Akt), p300, Survivin, and Brd4. In addition to this upstream signaling and its regulation, pharmacologic approaches for directly suppressing iNOS at its activity vs. transcriptional level are discussed. One highly effective agent in the latter category is bromodomain and extra-terminal (BET) inhibitor, JQ1, which was found to minimize iNOS upregulation in photostressed U87 cells. By acting similarly at the clinical level, a BET inhibitor such as JQ1 should markedly improve the efficacy of anti-glioblastoma PDT.

Bystander Effects of Nitric Oxide in Cellular Models of Anti-Tumor Photodynamic Therapy.

Tumor cells exposed to stress-inducing radiotherapy or chemotherapy can send signals to non- or minimally exposed bystander cells. Bystander effects of ionizing radiation are well established, but little is known about such effects in non-ionizing photodynamic therapy (PDT). Our previous studies revealed that several cancer cell types upregulate inducible nitric oxide synthase (iNOS) and nitric oxide (NO) after a moderate 5-aminolevulinic acid (ALA)-based PDT challenge. The NO signaled for cell resistance to photokilling as well as greater growth, migration and invasion of surviving cells. Based on this work, we hypothesized that diffusible NO produced by PDT-targeted cells in a tumor might elicit pro-growth/migration responses in non-targeted bystander cells. In the present study, we tested this using a novel approach, in which ALA-PDT-targeted human cancer cells on culture dishes (prostate PC3, breast MDA-MB-231, glioma U87, or melanoma BLM) were initially segregated from non-targeted bystanders via impermeable silicone-rimmed rings. Several hours after LED irradiation, rings were removed, and both cell populations analyzed for various post-hν responses. For a moderate and uniform level of targeted cell killing by PDT (~25%), bystander proliferation and migration were both enhanced. Enhancement correlated with iNOS/NO upregulation in surviving targeted cells in the following order: PC3 > MDA-MB-231 > U87 > BLM. If occurring in an actual tumor PDT setting and not suppressed (e.g., by iNOS activity or transcription inhibitors), then such effects could compromise treatment efficacy or even stimulate disease progression if PDT's anti-tumor potency is not great enough.

Upstream signaling events leading to elevated production of pro-survival nitric oxide in photodynamically-challenged glioblastoma cells.

Nitric oxide (NO) generated endogenously by inducible nitric oxide synthase (iNOS) promotes growth and migration/invasion of glioblastoma cells and also fosters resistance to chemotherapy and ionizing radiotherapy. Our recent studies revealed that glioblastoma cell iNOS/NO also opposes the cytotoxic effects of non-ionizing photodynamic therapy (PDT), and moreover stimulates growth/migration aggressiveness of surviving cells. These negative responses, which depended on PI3K/Akt/NF-κB activation, were strongly suppressed by blocking iNOS transcription with JQ1, a BET bromodomain inhibitor. In the present study, we sought to identify additional molecular events that precede iNOS transcriptional upregulation. Akt activation, iNOS induction, and viability loss in PDT-challenged glioblastoma U87 cells were all strongly inhibited by added l-histidine, consistent with primary involvement of photogenerated singlet oxygen (1O2). Transacetylase p300 not only underwent greater Akt-dependent activation after PDT, but greater interaction with NF-κB subunit p65, which in turn exhibited greater K310 acetylation. In addition, PDT promoted intramolecular disulfide formation and inactivation of tumor suppressor PTEN, thereby favoring Akt and p300 activation leading to iNOS upregulation. Importantly, deacetylase Sirt1 was down-regulated by PDT stress, consistent with the observed increase in p65-acK310 level, which fostered iNOS transcription. This study provides new mechanistic insights into how glioblastoma tumors can exploit iNOS/NO to not only resist PDT, but to attain a more aggressive survival phenotype.

Bystander effects of nitric oxide in anti-tumor photodynamic therapy.

Ionizing radiation of specifically targeted cells in a given population is known to elicit pro-death or pro-survival responses in non-targeted bystander cells, which often make no physical contact with the targeted ones. We have recently demonstrated a similar phenomenon for non-ionizing photodynamic therapy (PDT), showing that prostate cancer cells subjected to targeted photodynamic stress stimulated growth and migration of non-stressed, non-contacting bystander cells. Diffusible nitric oxide (NO) generated by stress-upregulated inducible nitric oxide synthase (iNOS) was shown to play a dominant role in these responses. Moreover, target-derived NO stimulated iNOS/NO induction in bystanders, suggesting a NO-mediated feed-forward field effect driven by targeted cells surviving the photodynamic challenge. In this research highlight, we will review these findings and discuss their potential negative implications on clinical PDT outcomes and how these might be mitigated through pharmacologic use of select iNOS inhibitors.

Enhanced aggressiveness of bystander cells in an anti-tumor photodynamic therapy model: Role of nitric oxide produced by targeted cells.

The bystander effects of anti-cancer ionizing radiation have been widely studied, but far less is known about such effects in the case of non-ionizing photodynamic therapy (PDT). In the present study, we tested the hypothesis that photodynamically-stressed prostate cancer PC3 cells can elicit nitric oxide (NO)-mediated pro-growth/migration responses in non-stressed bystander cells. A novel approach was used whereby both cell populations existed on a culture dish, but made no physical contact with one other. Visible light irradiation of target cells sensitized with 5-aminolevulinic acid-induced protoporphyrin IX resulted in a striking upregulation of inducible nitric oxide synthase (iNOS) along with NO, the level of which increased after irradiation. Slower and less pronounced iNOS/NO upregulation was also observed in bystander cells. Activation of transcription factor NF-κB was implicated in iNOS induction in both targeted and bystander cells. Like surviving targeted cells, bystanders exhibited a significant increase in growth and migration rate, both responses being strongly attenuated by an iNOS inhibitor (1400W), a NO scavenger (cPTIO), or iNOS knockdown. Incubating bystander cells with conditioned medium from targeted cells failed to stimulate growth/migration, ruling out involvement of relatively long-lived stimulants. The following post-irradiation changes in pro-survival/pro-growth proteins were observed in bystander cells: upregulation of COX-2 and activation of protein kinases Akt and ERK1/2, NO again playing a key role. This is the first reported evidence for NO-enhanced bystander aggressiveness in the context of PDT. In the clinical setting, such effects could be averted through pharmacologic use of iNOS inhibitors as PDT adjuvants.

Multiple Means by Which Nitric Oxide can Antagonize Photodynamic Therapy.

Photodynamic therapy (PDT) is a unique site-specific treatment for eradicating a variety of solid tumors, including prostate, lung, bladder, and brain tumors. PDT is a three-component modality involving (i) administration of a photosensitizing agent (PS), (ii) PS photoexcitation by visible or near-infrared light, and (iii) molecular oxygen. Upon photoexcitation, PS gives rise to tumor-damaging reactive oxygen species, most prominently singlet oxygen (1O2). Previous studies revealed that endogenous nitric oxide (NO) in various mouse tumor models significantly reduced PDT effectiveness. Recent studies in the authors' laboratory indicated that NO produced by photostressed tumor cells per se can elicit anti-PDT effects. For example, breast cancer COH-BR1 and prostate cancer PC3 cells exhibited a rapid and prolonged upregulation of inducible nitric oxide synthase (iNOS) after sensitization with 5- aminolevulinic acid (ALA)-induced protoporphyrin-IX, followed by broad-band visible irradiation. Use of iNOS inhibitors and NO scavengers demonstrated that iNOS/NO played a key role in cell resistance to apoptotic photokilling. Moreover, cells surviving an ALA/light challenge proliferated, migrated, and invaded more rapidly than controls, again in iNOS/NOdependent fashion. Thus, NO was found to play a crucial role in various manifestations of enhanced aggressiveness exhibited by remaining live cells. Recent work has revealed that induced NO in PDT-targeted PC3 cells can also translocate and increase aggressiveness of non-targeted bystander cells. These negative and potentially tumor-promoting side effects of NO in PDT may be averted through use of iNOS inhibitors as adjuvants. Each of the above aspects of PDT antagonism by NO will be discussed in this review.

Antagonistic Effects of Endogenous Nitric Oxide in a Glioblastoma Photodynamic Therapy Model.

Gliomas are aggressive brain tumors that are resistant to conventional chemotherapy and radiotherapy. Much of this resistance is attributed to endogenous nitric oxide (NO). Recent studies revealed that 5-aminolevulinic acid (ALA)-based photodynamic therapy (PDT) has advantages over conventional treatments for glioblastoma. In this study, we used an in vitro model to assess whether NO from glioblastoma cells can interfere with ALA-PDT. Human U87 and U251 cells expressed significant basal levels of neuronal NO synthase (nNOS) and its inducible counterpart (iNOS). After an ALA/light challenge, iNOS level increased three- to fourfold over 24 h, whereas nNOS remained unchanged. Elevated iNOS resulted in a large increase in intracellular NO. Extent of ALA/light-induced apoptosis increased substantially when an iNOS inhibitor or NO scavenger was present, implying that iNOS/NO was acting cytoprotectively. Moreover, cells surviving a photochallenge exhibited a striking increase in proliferation, migration and invasion rates, iNOS/NO again playing a dominant role. Also observed was a large iNOS/NO-dependent increase in matrix metalloproteinase-9 activity, decrease in tissue inhibitor of metalloproteinase-1 expression and increase in survivin and S100A4 expression, each effect being consistent with accelerated migration/invasion as a prelude to metastasis. Our findings suggest introduction of iNOS inhibitors as pharmacologic adjuvants for glioblastoma PDT.

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.

Macrophage mitochondrial damage from StAR transport of 7-hydroperoxycholesterol: implications for oxidative stress-impaired reverse cholesterol transport.

StAR family proteins in vascular macrophages participate in reverse cholesterol transport (RCT). We hypothesize that under pathophysiological oxidative stress, StARs will transport not only cholesterol to macrophage mitochondria, but also pro-oxidant cholesterol hydroperoxides (7-OOHs), thereby impairing early-stage RCT. Upon stimulation with dibutyryl-cAMP, RAW264.7 macrophages exhibited a strong time-dependent induction of mitochondrial StarD1 and plasma membrane ABCA1, which exports cholesterol. 7α-OOH uptake by stimulated RAW cell mitochondria (like cholesterol uptake) was strongly reduced by StarD1 knockdown, consistent with StarD1 involvement. Upon uptake by mitochondria, 7α-OOH (but not redox-inactive 7α-OH) triggered lipid peroxidation and membrane depolarization while reducing ABCA1 upregulation. These findings provide strong initial support for our hypothesis.