The tumor bystander effect describes the ability of cells sustaining therapeutic treatment damage to transmit distress signals to neighboring unharmed cells and change their behavior. Photodynamic therapy (PDT), which uses light for activation of photosensitizing drugs in targeted lesions to produce cytotoxic damage, is one of the cancer treatment modalities inducing highly robust bystander effects. This is caused to a large extent by the propensity of PDT for creating extensive lipid peroxidation damage and related instigation of ferroptotic cell death. Lipid peroxides appear to be major participants in intercellular signaling generating the bystander effect and orchestrating ferroptosis that appears to have a pivotal role in propagating the lethal (therapeutically beneficial) form of bystander response. In contrast, nitric oxide (NO) released from PDT-treated cancer cells has emerged as a major signal for therapeutically detrimental bystander effects due to promoting tumor growth and metastasis. It is becoming increasingly evident that exploiting the bystander response can be an attractive strategy for improving tumoricidal depth with consequently elevated tumor cure rates following PDT treatment. A two-pronged approach is proposed for achieving this goal: (i) inactivating iNOS enzyme with a specific pharmacologic inhibitor, and (ii) amplifying the induction of cytotoxic ferroptosis in nearby cells initially unaffected by photodamage, thereby limiting their ability to promote tumor expansion.

Oxidative stress associated with atherogenesis is often accompanied by fatty acid streak and foam cell formation due to uncontrolled oxidation of cholesterol (Ch) and other unsaturated lipids. We hypothesized that under persistent oxidative stress and elevated Ch conditions, Ch hydroperoxides (ChOOHs) are the most cytotoxic Ch metabolites delivered with oxidized low-density lipoprotein (oxLDL). We further hypothesized that in addition to macrophages, LDL-bearing ChOOHs can damage endothelial cells (ECs), leading to cellular accumulation of Ch oxidation products (ChOX) as well as Ch itself. We tested our hypotheses, focusing on the lower ends of EC susceptibility to ChOX-LDL damage. We used acetylated LDL (AcLDL) which, like parental LDL, is recognized by EC scavenger receptors. Among the ChOXs studied, 7-hydroperoxy-Ch (7-OOH) in AcLDL was taken up most rapidly by ECs, followed by 7 = O and 7-OH; however, 7-OOH was the most cytotoxic. Interestingly, sub-lethal doses of AcLDL-borne 7-OOH (but not other ChOXs) significantly stimulated EC proliferation. We confirmed 7-OOH's cytotoxic role by knocking down or overexpressing seleno-peroxidase GPx4, the only enzyme capable of direct ChOOH detoxification in situ. StAR family proteins are known to mediate intracellular Ch transport: StarD1 in mitochondria and StarD4 in cytosol. In this study, we found that transport of AcLDL-borne 7-OOH can also be StAR-mediated, StarD1 knockdown causing a much greater inhibitory effect than StarD4 knockdown. Collectively, our findings provide new evidence for mitochondrial site-specific cytotoxicity of ldl-borne 7-OOH in vascular endothelial cells and demonstrate the protective role of endogenous GPx4 in mitigating this damage.

Most malignantly transformed cells are metabolically rewired to promote their survival and progression, even under conditions that would be unfavorable for normal counterparts. Arguably the most impactful metabolic transformation and recognized cancer hallmark is the reprogrammed lipid metabolism. Lipids are not only primary constituents of cell membranes but essential participants in fundamental cellular functions including cell signaling, protein regulation, energy provision, inflammation, and cell-cell interaction. Engagement of lipids in critical physiological functions in cells is additionally accentuated upon malignant transformation. Pivotal roles of lipids as influential inter- and intracellular signaling molecules, particularly under conditions of hyper oxidative stress, are delineated. Elaborated in more detail are SCAP/SREBP pathway and sphingolipid signaling cascades due to their roles of principal signaling networks determining tumor therapy responses. In the concluding section, an overview is provided of the process of lipid peroxidation and its impact in cancer cells sustaining oxidative stress with the outline of cell signaling functions of primary and secondary lipid peroxidation products. Much remains to be learned about the consequences of the fact that the lipid peroxidation process can extend beyond the site of initiation owing to (either spontaneous or transfer protein-mediated) translocation of peroxy radical species disseminating their impact to other subcellular sites.

Significance:Intralipid, a soybean oil-based lipid emulsion, is widely used in photomedicine to enhance light distribution due to its strong scattering properties. Although the optical characteristics of Intralipid are well documented, interactions with the reactive molecular species (RMS) generated during photodynamic therapy (PDT) and the impact of such interactions on therapeutic outcomes remain poorly understood. We reveal that Intralipid actively influences PDT response in vitro, beyond its role as a scattering agent. Aim:We examined how Intralipid affects the optical and photodynamic behavior of benzoporphyrin derivative (BPD), a clinical photosensitizer, in solution and across four ovarian cancer cell lines. Approach:The photodynamic properties of BPD, with and without Intralipid, were analyzed using fluorescence spectrometry and RMS probes, and PDT-induced oxidation of Intralipid components was characterized using LC-MS. The effects of Intralipid on BPD-PDT were evaluated under various conditions. Results:Intralipid reduced BPD photobleaching and RMS generation, suggesting RMS quenching. Extensive oxidation of Intralipid components was observed following PDT. Finally, Intralipid significantly modified BPD-PDT efficacy across all four cell lines, depending on photosensitizer-light interval, dose, and incubation time. Conclusions:Intralipid acts as a bioactive modulator of PDT response, highlighting the need for further investigations both in vitro and in vivo.

Resistance to apoptosis-inducing chemotherapy is a major factor contributing to treatment failure and poor survival outcomes in high-grade serous ovarian cancer (HGSOC). Ferroptosis, a regulated form of cell death driven by lipid peroxidation, has emerged as a promising effector mechanism because it remains available in HGSOC cells with impaired apoptosis signaling. While most research has focused on pharmacological ferroptosis inducers, there is growing interest in strategies that could trigger lipid autoxidation through externally delivered energy, such as photons. Photodynamic therapy (PDT), which utilizes light and light-activatable photosensitizers to generate reactive molecular species, offers a means of initiating lipid peroxidation with a high degree of precision and minimal systemic toxicities. However, the precise lipid targets of PDT, the influence of varying tumor lipidomic landscapes, and the role of ferroptosis sensitivity on PDT-lipid interactions have yet to be elucidated. In this study, we systematically compare PDT to ferroptosis induced by the inhibition of glutathione peroxidase 4, focusing on lipid redox states and composition in HGSOC cell lines. While PDT was similarly effective in both ferroptosis-sensitive and -resistant cells, its effects on cellular lipidomes differed markedly. PDT robustly induced lipid radical formation in both cell types; however, a dose-dependent accumulation of lipid hydroxides and hydroperoxides was only observed in ferroptosis-sensitive cells rich in unsaturated phospholipids. Further analysis revealed a significant overlap in lipid oxidation targets between PDT and ferroptosis. Notably, in both cell types, and in vivo, PDT upregulated ceramides, a lipid class strongly associated with mitochondrial apoptosis. In summary, PDT exhibited comparable efficacy in both ferroptosis-sensitive and -resistant cells by triggering a combination of lipid peroxidation and ceramide upregulation, suggesting the activation of both ferroptosis and apoptosis pathways. Further studies are needed to explore the role of PDT-induced lipidomic changes in the initiation of various cell death pathways and in overcoming chemoresistance in HGSOC.

Non-invasive photodynamic therapy (PDT) is an increasingly applied modality for solid tumors. Many malignant cells that can withstand PDT upregulate nitric oxide, which not only acts cytoprotectively, but stimulates growth and migration of surviving cells. These anti-PDT responses are still not well-recognized by PDT researchers and practitioners. We provide here a background on these negative effects and offer perspectives on how they can be suppressed for a therapeutic benefit, e.g. by inhibiting transcription of inducible nitric oxide synthase.

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 center dot, peroxynitrate (O2NOO-), OOSCN-, SO3 center dot-, selenocyanogen [(SeCN)(2)], the triselenocyanate anion [(SeCN)(3)(-)], I-center dot, I-2(center dot-), I-3(-), 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.

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.

Hydroperoxides of unsaturated membrane lipids (LOOHs) are the most abundant non-radical intermediates generated by photodynamic therapy (PDT) of soft tissues such as tumors and have far longer average lifetimes than singlet oxygen or oxygen radicals formed during initial photodynamic action. LOOH-initiated post-irradiation damage to remaining membrane lipids (chain peroxidation) or to membrane-associated proteins remains largely unrecognized. Such after-light processes could occur during clinical oncological PDT, but this is not well-perceived by practitioners of this therapy. In general, the pivotal influence of lipids in tumor responses to PDT needs to be better appreciated. Of related importance is the fact that most malignant tumors have dramatically different lipid metabolism compared with healthy tissues, and this too is often ignored. The response of tumors to PDT appears especially vulnerable to manipulations within the tumor lipid microenvironment. This can be exploited for therapeutic gain with PDT, as exemplified here by the combined treatment with the antitumor lipid edelfosine.

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.

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.

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.

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.

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.

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.

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.

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.

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/7β-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.

Many malignant tumors use endogenous nitric oxide (NO) to promote survival, growth, and metastatic migration. This NO, which is typically generated by inducible nitric oxide synthase (iNOS), can also antagonize various anti-cancer therapies and its source is most often assumed to be constitutive or pre-existing iNOS. In this paper, we provide evidence (i) that many different cancer cells exhibit resistance to oxidative killing by photodynamic therapy (PDT), and (ii) that cells surviving the challenge grow, migrate and invade more aggressively, as do nontargeted bystander cells. Accompanying these effects are activation or upregulation of pro-survival/progression effector proteins such as NF-kappa B, Akt, and Survivin. Observed in the author's laboratory, these responses were not attributed to basal iNOS/NO in most cases, but rather to NO from enzyme that was strongly upregulated by photodynamic stress. Each of these effects and how they can be mitigated by inhibitors of iNOS activity or transcription, or by NO scavengers will be discussed. When approved for clinical use, such pharmacologic agents could improve PDT efficacy as well as reduce potentially negative side-effects of this therapy.