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Photo-activation of the hydrophobic probe iodonaphthylazide in cells alters membrane protein function leading to cell death.

Viard M, Garg H, Blumenthal R, Raviv Y - BMC Cell Biol. (2009)

Bottom Line: 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.It reacts with membrane proteins to alter the normal physiological function resulting in apoptosis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Nanobiology Program, Center of Cancer Research, National Cancer Institute, Frederick, Maryland, USA. viardm@mail.ncifcrf.gov

ABSTRACT

Background: Photo-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.

Results: INA 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.

Conclusion: INA 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.

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INA-UV treatment does not inhibit IGF1 signaling. Akt phosphorylation was assessed by Western detection and quantified. The different samples correspond to MCF7 cells untreated, exposed or not to 1% DMSO, INA at the indicated concentrations and/or IGF1. All the samples exposed to DMSO or INA were irradiated with UV. The Akt phosphorylation signal was normalized to the signal obtained with MCF7 exposed only to IGF1. The Western signal obtained with GAPDH on the same samples is shown for loading control.
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Figure 8: INA-UV treatment does not inhibit IGF1 signaling. Akt phosphorylation was assessed by Western detection and quantified. The different samples correspond to MCF7 cells untreated, exposed or not to 1% DMSO, INA at the indicated concentrations and/or IGF1. All the samples exposed to DMSO or INA were irradiated with UV. The Akt phosphorylation signal was normalized to the signal obtained with MCF7 exposed only to IGF1. The Western signal obtained with GAPDH on the same samples is shown for loading control.

Mentions: Growth factor receptors including EGFR and IGF1R are often overexpressed in tumor cells. Overexpression of IGF1R is associated with increased survival and proliferation of tumor cells. Hence we wished to determine whether INA-UV had an effect on IGF1R signaling and whether it was related to the apoptosis seen in previous experiments. The binding of IGF1 to its receptor activates the PI3kinase pathway leading to Akt phosphorylation at serine 473 [31], which has been reported to be involved in apoptosis inhibition. As can be seen on figure 8, the pretreatment of MCF7 cells with INA-UV leads to an amplification of Akt phosphorylation induced by IGF1 compared to untreated cells. While DMSO partially contributed to this effect, further amplification was observed upon irradiation in the presence of INA. Interestingly we observe that INA-UV treatment alone, in the absence of IGF1, can partially induce 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 in fact activate signaling via IGF1R contrary to the results seen with other receptors. These results underscore the complex physiological outcome of INA interaction with cellular receptors and the diversity in the signal transduction mechanism by different receptors.


Photo-activation of the hydrophobic probe iodonaphthylazide in cells alters membrane protein function leading to cell death.

Viard M, Garg H, Blumenthal R, Raviv Y - BMC Cell Biol. (2009)

INA-UV treatment does not inhibit IGF1 signaling. Akt phosphorylation was assessed by Western detection and quantified. The different samples correspond to MCF7 cells untreated, exposed or not to 1% DMSO, INA at the indicated concentrations and/or IGF1. All the samples exposed to DMSO or INA were irradiated with UV. The Akt phosphorylation signal was normalized to the signal obtained with MCF7 exposed only to IGF1. The Western signal obtained with GAPDH on the same samples is shown for loading control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2666636&req=5

Figure 8: INA-UV treatment does not inhibit IGF1 signaling. Akt phosphorylation was assessed by Western detection and quantified. The different samples correspond to MCF7 cells untreated, exposed or not to 1% DMSO, INA at the indicated concentrations and/or IGF1. All the samples exposed to DMSO or INA were irradiated with UV. The Akt phosphorylation signal was normalized to the signal obtained with MCF7 exposed only to IGF1. The Western signal obtained with GAPDH on the same samples is shown for loading control.
Mentions: Growth factor receptors including EGFR and IGF1R are often overexpressed in tumor cells. Overexpression of IGF1R is associated with increased survival and proliferation of tumor cells. Hence we wished to determine whether INA-UV had an effect on IGF1R signaling and whether it was related to the apoptosis seen in previous experiments. The binding of IGF1 to its receptor activates the PI3kinase pathway leading to Akt phosphorylation at serine 473 [31], which has been reported to be involved in apoptosis inhibition. As can be seen on figure 8, the pretreatment of MCF7 cells with INA-UV leads to an amplification of Akt phosphorylation induced by IGF1 compared to untreated cells. While DMSO partially contributed to this effect, further amplification was observed upon irradiation in the presence of INA. Interestingly we observe that INA-UV treatment alone, in the absence of IGF1, can partially induce 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 in fact activate signaling via IGF1R contrary to the results seen with other receptors. These results underscore the complex physiological outcome of INA interaction with cellular receptors and the diversity in the signal transduction mechanism by different receptors.

Bottom Line: 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.It reacts with membrane proteins to alter the normal physiological function resulting in apoptosis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Nanobiology Program, Center of Cancer Research, National Cancer Institute, Frederick, Maryland, USA. viardm@mail.ncifcrf.gov

ABSTRACT

Background: Photo-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.

Results: INA 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.

Conclusion: INA 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.

Show MeSH
Related in: MedlinePlus