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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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Related in: MedlinePlus

INA-UV treatment blocks SDF1α induced CXCR4 signaling. The fluorescence emission of Fura 2 was recorded at 510 nm using simultaneous excitation at 340 and 380 nm. The ratio of the 2 emission signals obtained, 340/380, is plotted over time. SDF1α was added at time 50 sec as indicated and further on the ionophore 4-bromo A-23187 was added at time ~160 sec as indicated. The same experiment was carried on SupT1 cells (dark curve) or SupT1 cells treated with 15 μM INA-UV (grey curve).
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Figure 7: INA-UV treatment blocks SDF1α induced CXCR4 signaling. The fluorescence emission of Fura 2 was recorded at 510 nm using simultaneous excitation at 340 and 380 nm. The ratio of the 2 emission signals obtained, 340/380, is plotted over time. SDF1α was added at time 50 sec as indicated and further on the ionophore 4-bromo A-23187 was added at time ~160 sec as indicated. The same experiment was carried on SupT1 cells (dark curve) or SupT1 cells treated with 15 μM INA-UV (grey curve).

Mentions: CXCR4 is a seven transmembrane G protein-coupled chemokine receptor that is over-expressed in a variety of tumors and involved in tumor metastasis [28]. In cell membranes, blocking the signaling by INA-UV treatment of another G protein-coupled receptor, human chorionic gonadotropin (hCG) has been previously documented [18]. We wished to determine whether INA-UV treatment would alter signaling via CXCR4. Binding of the CXCR4 ligand SDF-1α to its receptor induces a calcium flux in cells, which is monitored by ratio of fluorescent signals at 340 and 380 nm excitation, respectively, and 510 nm emission using the fluorescent calcium indicator dye, Fura 2 pre-loaded into the cells [29,30]. Upon addition of SDF1α we observed calcium flux in the control SupT1 cells but not in cells pretreated with INA-UV (figure 7) suggesting an inhibition of CXCR4 signaling. By contrast, INA-UV treatment had no effect on the action of the calcium ionophore 4-bromo A-23187 that directly mediates calcium flux across membranes. When added to SupT1, in both cases the cytosolic concentration of calcium rose to a similar level as was shown by the increase of the fura 2 ratio signal (Figure 7). Hence the effect of INA-UV was at the level of CXCR4 signaling and not a general effect on intracellular calcium accumulation.


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 blocks SDF1α induced CXCR4 signaling. The fluorescence emission of Fura 2 was recorded at 510 nm using simultaneous excitation at 340 and 380 nm. The ratio of the 2 emission signals obtained, 340/380, is plotted over time. SDF1α was added at time 50 sec as indicated and further on the ionophore 4-bromo A-23187 was added at time ~160 sec as indicated. The same experiment was carried on SupT1 cells (dark curve) or SupT1 cells treated with 15 μM INA-UV (grey curve).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: INA-UV treatment blocks SDF1α induced CXCR4 signaling. The fluorescence emission of Fura 2 was recorded at 510 nm using simultaneous excitation at 340 and 380 nm. The ratio of the 2 emission signals obtained, 340/380, is plotted over time. SDF1α was added at time 50 sec as indicated and further on the ionophore 4-bromo A-23187 was added at time ~160 sec as indicated. The same experiment was carried on SupT1 cells (dark curve) or SupT1 cells treated with 15 μM INA-UV (grey curve).
Mentions: CXCR4 is a seven transmembrane G protein-coupled chemokine receptor that is over-expressed in a variety of tumors and involved in tumor metastasis [28]. In cell membranes, blocking the signaling by INA-UV treatment of another G protein-coupled receptor, human chorionic gonadotropin (hCG) has been previously documented [18]. We wished to determine whether INA-UV treatment would alter signaling via CXCR4. Binding of the CXCR4 ligand SDF-1α to its receptor induces a calcium flux in cells, which is monitored by ratio of fluorescent signals at 340 and 380 nm excitation, respectively, and 510 nm emission using the fluorescent calcium indicator dye, Fura 2 pre-loaded into the cells [29,30]. Upon addition of SDF1α we observed calcium flux in the control SupT1 cells but not in cells pretreated with INA-UV (figure 7) suggesting an inhibition of CXCR4 signaling. By contrast, INA-UV treatment had no effect on the action of the calcium ionophore 4-bromo A-23187 that directly mediates calcium flux across membranes. When added to SupT1, in both cases the cytosolic concentration of calcium rose to a similar level as was shown by the increase of the fura 2 ratio signal (Figure 7). Hence the effect of INA-UV was at the level of CXCR4 signaling and not a general effect on intracellular calcium accumulation.

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