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

The Salmonella effector SseJ disrupts microtubule dynamics when ectopically expressed in Normal Rat Kidney cells

Salmonella effector protein SseJ is secreted by Salmonella into the host cell cytoplasm where it can then modify host cell processes. Whilst host cell small GTPase RhoA has previously been shown to activate the acyl-transferase activity of SseJ we show here an un-described effect of SseJ protein production upon microtubule dynamism. SseJ prevents microtubule collapse and this is independent of SseJ's acyl-transferase activity. We speculate that the effects of SseJ on microtubules would be mediated via its known interactions with the small GTPases of the Rho family

Nitric Oxide Antagonism to Anti-Glioblastoma Photodynamic Therapy: Mitigation by Inhibitors of Nitric Oxide Generation

Many studies have shown that low flux nitric oxide (NO) produced by inducible NO synthase (iNOS/NOS2) in various tumors, including glioblastomas, can promote angiogenesis, cell proliferation, and migration/invasion. Minimally invasive, site-specific photodynamic therapy (PDT) is a highly promising anti-glioblastoma modality. Recent research in the authors’ laboratory has revealed that iNOS-derived NO in glioblastoma cells elicits resistance to 5-aminolevulinic acid (ALA)-based PDT, and moreover endows PDT-surviving cells with greater proliferation and migration/invasion aggressiveness. In this contribution, we discuss iNOS/NO antagonism to glioblastoma PDT and how this can be overcome by judicious use of pharmacologic inhibitors of iNOS activity or transcription

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

Nitric oxide-elicited resistance to anti-glioblastoma photodynamic therapy

Glioblastoma multiforme is a highly aggressive primary brain malignancy that resists most conventional chemo- and 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