Abstract
BackgroundPhoto-activation of the hydrophobic membrane probe 1, 5 iodonaphthylazide (INA) by irradiation with UV light (310–380 nm) results in the covalent modification of transmembrane anchors of membrane proteins. This unique selectivity of INA towards the transmembrane anchor has been exploited to specifically label proteins inserted in membranes. Previously, we have demonstrated that photo-activation of INA in enveloped viruses resulted in the inhibition of viral membrane protein-induced membrane fusion and viral entry into cells. In this study we show that photo-activation of INA in various cell lines, including those over-expressing the multi-drug resistance transporters MRP1 or Pgp, leads to cell death. We analyzed mechanisms of cell killing by INA-UV treatment. The effects of INA-UV treatment on signaling via various cell surface receptors, on the activity of the multi-drug resistance transporter MRP1 and on membrane protein lateral mobility were also investigated.ResultsINA treatment of various cell lines followed by irradiation with UV light (310–380 nm) resulted in loss of cell viability in a dose dependent manner. The mechanism of cell death appeared to be apoptosis as indicated by phosphatidylserine exposure, mitochondrial depolarization and DNA fragmentation. Inhibition by pan-caspase inhibitors and cleavage of caspase specific substrates indicated that at low concentrations of INA apoptosis was caspase dependent. The INA-UV treatment showed similar cell killing efficacy in cells over-expressing MRP1 function as control cells. Efflux of an MRP1 substrate was blocked by INA-UV treatment of the MRP1-overexpressing cells. Although INA-UV treatment resulted in inhibition of calcium mobilization triggered by chemokine receptor signaling, Akt phosphorylation triggered by IGF1 receptor signaling was enhanced. Furthermore, fluorescence recovery after photobleaching experiments indicated that INA-UV treatment resulted in reduced lateral mobility of a seven transmembrane G protein-coupled receptor.ConclusionINA is a photo-activable agent that induces apoptosis in various cancer cell lines. It reacts with membrane proteins to alter the normal physiological function resulting in apoptosis. This activity of INA maybe exploited for use as an anti-cancer agent.
Highlights
Apoptosis mediated by INA-Ultraviolet rays (UV) is caspase dependent Caspases are cysteine proteases that are key mediators of apoptosis [19]
To determine whether apoptosis mediated via INA-UV treatment was caspase dependent, we treated SupT1 cells with ZVADfmk (40 μM) prior to INA-UV treatment
We observe that INA-UV treatment alone, in the absence of insulin-like growth factor 1 (IGF1), can partially induce Akt or protein kinase B (Akt) phosphorylation in MCF7 cells. This effect requires the reaction to transmembrane proteins with INA, since it is not observed with UV and DMSO treatment alone. These results suggest that INA-UV may activate signaling via Insulin-like growth factor 1 receptor (IGF1R) contrary to the results seen with other receptors
Summary
Cells have developed a complex architecture that relies on the compartmentalization of cellular functions within organelles that are bounded by lipid membranes. The plasma membrane constitutes a unique interface between the cytoplasm and extra cellular milieu. While ensuring the physical separation of two very different environments, a constant communication between the cell and its extracellular milieu is established by means of cell surface proteins. Signaling via membrane proteins regulate various cellular functions including cell survival, cell propagation, cell differentiation and cell migration. Membrane proteins are considered prime targets for drugs designed to combat cancer and other diseases. While inhibition of a given cell surface receptor often leads to predictable changes in cells based on the signaling cascade initiated by the receptor, it is unclear how altering cell signaling via a number of receptors would change the cell physiology
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