Cédric Volanti

Restoration of SHIP-1 activity in human leukemic cells modifies NF-kappaB activation pathway and cellular survival upon oxidative stress.

Nuclear factor-kappa B (NF-κB) is an important prosurvival transcription factor activated in response to a large array of external stimuli, including reactive oxygen species (ROS). Previous works have shown that NF-κB activation by ROS involved tyrosine phosphorylation of the inhibitor IκBα through an IκB kinase (IKK)-independent mechanism. In the present work, we investigated with more details NF-κB redox regulation in human leukemic cells. By using different cell lines (CEM, Jurkat and the subclone Jurkat JR), we clearly showed that NF-κB activation by hydrogen peroxide (H2O2) is cell-type dependent: it activates NF-κB through tyrosine phosphorylation of IκBα in Jurkat cells, whereas it induces an IKK-mediated IκBα phosphorylation on S32 and 36 in CEM and Jurkat JR cells. We showed that this H2O2-induced IKK activation in CEM and Jurkat JR cells is mediated by SH2-containing inositol 5′-phosphatase 1 (SHIP-1), a lipid phosphatase that is absent in Jurkat cells. Indeed, the complementation of SHIP-1 in Jurkat cells made them shift to an IKK-dependent mechanism upon oxidative stress stimulation. We also showed that Jurkat cells expressing SHIP-1 are more resistant to H2O2-induced apoptosis than the parental cells, suggesting that SHIP-1 has an important role in leukemic cell responses to ROS in terms of signal transduction pathways and apoptosis resistance, which can be of interest in improving ROS-mediated chemotherapies.

Downregulation of ICAM-1 and VCAM-1 expression in endothelial cells treated by photodynamic therapy

Photodynamic therapy (PDT) is a treatment for cancer and several noncancerous proliferating cell diseases that depends on the uptake of a photosensitizing compound followed by selective irradiation with visible light. In the presence of oxygen, irradiation leads to the production of reactive oxygen species (ROS). A large production of ROS induces the death of cancer cells by apoptosis or necrosis. A small ROS production can activate various cellular pathways. Here, we show that PDT by pyropheophorbide-a methyl ester (PPME) induces the activation of nuclear factor kappa B (NF-κB) in HMEC-1 cells. NF-κB is active since it binds to the NF-κB sites of both ICAM-1 and vascular cell adhesion molecule-1 (VCAM-1) promoters and induces the transcription of several NF-κB target genes such as those of IL-6, ICAM-1, VCAM-1. In contrast, expression of ICAM-1 and VCAM-1 at the protein level was not observed, although we measured an IL-6 secretion. Using specific chemical inhibitors, we showed that the lack of ICAM-1 and VCAM-1 expression is the consequence of their degradation by lysosomal proteases. The proteasome and calpain pathways were not involved. All these observations were consistent with the fact that no adhesion of granulocytes was observed in these conditions.

Involvement of Oxidative Stress in NF-κB Activation in Endothelial Cells Treated by Photodynamic Therapy¶

In human endothelial cells ECV 304 and HMEC‐1 photosensitized by pyropheophorbide‐a methylester (PPME) in sublethal conditions transcription factor Nuclear Factor kappa B (NF‐κB) activation takes place for several hours. Activated NF‐κB was functional because it stimulated the transcriptional activation of either a transfected reporter gene or the endogenous gene encoding interleukin (IL)‐8. Concomitant with NF‐κB activation, inhibitor of NF‐κBα (IκBα) was degraded during photosensitization and IκBβ, p100, p105 and IκBε were slightly modified. Reactive oxygen species (ROS) were shown to be crucial intermediates in the activation because antioxidants strongly decreased NF‐κB activation. Using both a fluorescent probe and isotope substitution, it was shown that ROS, and especially singlet oxygen (1O2), were important in the activation process. Because NF‐κB activation in the presence of ROS was suspected to proceed through a pathway independent of the IκB kinases (IKK), we demonstrated that the IKK were indeed not activated by photosensitization but required an intact tyrosine residue at position 42 on IκBα, suggesting the involvement of a tyrosine kinase in the activation process. This was further reinforced by the demonstration that herbimycin A, a tyrosine kinase inhibitor, prevented NF‐κB activation by photosensitization but not by TNFα, a cytokine known to activate NF‐κB through an IKK‐dependent mechanism.

A proinflammatory role for the cyclopentenone prostaglandins at low micromolar concentrations: Oxidative stress-induced extracellular signal-regulated kinase activation without NF-kappa B inhibition

An anti-inflammatory role and therapeutic potential for cyclopentenone PGs (cyPGs) has been suggested, based on observations that levels of cyPGs in exudates increase during the resolution phase of inflammation, and that exogenous cyPGs may attenuate the inflammatory response in vivo and in vitro mainly through inhibition of NF-κB, a critical activator of inflammatory gene expression. However, exogenous cyPGs inhibit NF-κB only at concentrations substantially higher than those of endogenous cyPGs present in inflammatory fluids, thus challenging the hypothesis that cyPGs are naturally occurring inhibitors of inflammation and suggesting that cyPGs at low concentrations might have previously unappreciated effects. In this study, using various cell types, we report that cyPGs, when used at concentrations substantially lower than required for NF-κB inhibition (viz, low micromolar concentrations), significantly potentiate the inflammatory response to TNF-α. At these concentrations, cyPGs induce production of reactive oxygen species, thereby synergizing with TNF-α to activate the extracellular signal-regulated kinase 1/2, an activation which in turn potentiates proinflammatory cytokine expression at both transcriptional and posttranscriptional levels. Our study establishes a proinflammatory role for cyPGs at low micromolar concentrations, raises the possibility that cyPGs do not act as physiologic anti-inflammatory mediators, and questions the therapeutic potential of these compounds.

Distinct transduction mechanisms of cyclooxygenase 2 gene activation in tumour cells after photodynamic therapy

Photodynamic therapy (PDT) is a minimally invasive treatment for cancer and several noncancerous proliferating cell diseases. PDT relies on the uptake of a photosensitizing compound by the pathologic tissue followed by a selective irradiation with visible light, which leads to oxidative stress-mediated cell death. However, some studies showed that PDT induces the release of proangiogenic factors, such as vascular endothelial growth factor, and/or cyclooxygenase-2 (COX-2), thereby promoting cancer cell regrowth following PDT. In this work, we focused on the molecular mechanisms regulating COX-2 expression after low-dose PDT in two cancer cell lines, namely HeLa and T24. We report that PDT induces COX-2 expression in these cells and this expression is mainly due to nuclear factor kappa B (NF-κB)-dependent transcription of cox-2 gene without any post-transcriptional regulation. However, the transduction mechanism leading to NF-κB activation and subsequent cox-2 gene transcription differs in both cell types. In T24, NF-κB activation occurs through a protein kinase C (PKC)α- and phosphoinositide-3-kinase (PI3K)-dependent I kappa B kinase (IKK) complex activation, whereas in HeLa cells, NF-κB activation is mediated by PKC- and PI3K-independent IKK complex activation.

NF-κB in Photodynamic Therapy: Discrepancies of a Master Regulator

Abstract Tumor eradication by photodynamic therapy (PDT) results from the onset of distinct killing processes. In addition to the well-known necrotic and apoptotic mechanisms, PDT initiates an inflammatory response that will indirectly contribute to tumor clearance. The NF-κB transcription factor is a major regulator of inflammation modulating the expression of cytokines, chemokines, and adhesion molecules in various cell types in response to a large number of stimuli. Besides, NF-κB regulates the expression of antiapoptotic genes, cyclooxygenases (COXs) and metalloproteinases (MMPs) as well, thereby favoring tumor cell proliferation and dissemination. In the present review, we aim to summarize the current knowledge on NF-κB status following photosensitization of cancer cells and endothelial cells. In order to unravel the NF-κB impact in PDT tumorigenicity and recurrences, we will stress the discrepancies of this major transcription factor relative to the signaling cascades underlying its activation and the cellular effects triggered by its translocation into the nucleus and its binding to its target genes.