RESUMEN
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.
Asunto(s)
Colesterol/análogos & derivados , Isoindoles , Células Intersticiales del Testículo , Compuestos de Organoselenio , Testosterona , Ratas , Masculino , Ratones , Animales , Células Intersticiales del Testículo/metabolismo , Testosterona/farmacología , Testosterona/metabolismo , Colesterol/metabolismo , Fosfoproteínas/metabolismoRESUMEN
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.
Asunto(s)
Neoplasias , Fotoquimioterapia , Humanos , Óxido Nítrico/metabolismo , Apoptosis , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Regulación hacia ArribaRESUMEN
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.
Asunto(s)
Neoplasias , Fotoquimioterapia , Humanos , Óxido Nítrico/metabolismo , Efecto Espectador , Neoplasias/metabolismo , Ácido Aminolevulínico/farmacología , Ácido Aminolevulínico/uso terapéutico , Línea Celular Tumoral , Fármacos Fotosensibilizantes/farmacología , Fármacos Fotosensibilizantes/uso terapéuticoRESUMEN
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.
Asunto(s)
Colesterol/análogos & derivados , Células Intersticiales del Testículo/metabolismo , Peroxidación de Lípido , Mitocondrias/metabolismo , Fosfolípido Hidroperóxido Glutatión Peroxidasa/metabolismo , Esteroides/metabolismo , Animales , Carbolinas/farmacología , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Colesterol/metabolismo , Fluorescencia , Isoindoles/farmacología , Células Intersticiales del Testículo/efectos de los fármacos , Peroxidación de Lípido/efectos de los fármacos , Masculino , Ratones , Mitocondrias/efectos de los fármacos , Membranas Mitocondriales/efectos de los fármacos , Membranas Mitocondriales/metabolismo , Compuestos de Organoselenio/farmacología , Fosfatidilcolinas/metabolismo , Fosfolípido Hidroperóxido Glutatión Peroxidasa/antagonistas & inhibidores , Progesterona/biosíntesis , Sustancias Protectoras/farmacologíaRESUMEN
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.
RESUMEN
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.
RESUMEN
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.