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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 induced apoptosis in Hela cells is also caspase dependent. A. Hela cells, pre-incubated or not with 40 μM ZVADfmk were treated with indicated amounts of INA, irradiated with UV and analyzed 24 h post treatment. Cells were stained with Annexin V and analyzed by FACS. The percentage of the population presenting high staining (PS exposure) is presented. B. Hela cells were treated with indicated amounts of INA. 24 hours upon treatment, the cells were lysed and the presence of PARP and/or cleaved PARP was assessed by Western analysis. The ratio of the two as determined by Western analysis is represented.
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Figure 4: INA-UV induced apoptosis in Hela cells is also caspase dependent. A. Hela cells, pre-incubated or not with 40 μM ZVADfmk were treated with indicated amounts of INA, irradiated with UV and analyzed 24 h post treatment. Cells were stained with Annexin V and analyzed by FACS. The percentage of the population presenting high staining (PS exposure) is presented. B. Hela cells were treated with indicated amounts of INA. 24 hours upon treatment, the cells were lysed and the presence of PARP and/or cleaved PARP was assessed by Western analysis. The ratio of the two as determined by Western analysis is represented.

Mentions: Caspases are cysteine proteases that are key mediators of apoptosis [19]. The involvement of caspases in the apoptotic pathway can be studied by the inhibition of apoptosis via the pan-caspase inhibitor Z-Val-Ala-Asp-fluoromethylketone (ZVADfmk) and the cleavage of caspase specific substrates like PARP [20,21]. 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. As seen in figure 3A and 3B INA-UV mediated apoptosis at low concentrations was inhibited by ZVADfmk confirming the role of caspases. Interestingly higher concentrations were not inhibited by ZVADfmk suggesting a different mechanism at very high concentrations. In order to monitor caspase activation in SupT1 cells 24 h post treatment with INA-UV, the cells were labeled FITC-VAD-FMK which binds irreversibly with active caspases and analyzed by flow cytometry. As seen in figure 3C, high activation of caspases is observed at 5 μM which correspond to the IC50 of INA-UV treatment of SupT1 cells. The activation of caspases was lower at higher doses as determined by FITC-VAD-FMK staining. These findings are supported by the high levels of PARP cleavage seen at low INA concentrations whereas there was little PARP cleavage at higher INA concentrations (figure 3D). PARP cleavage was prevented at all INA concentrations by ZVADfmk indicating its caspase specificity (data not shown). To generalize our conclusion about mechanisms of cell killing, we performed similar experiments with Hela cells. As shown in figure 4, the major caspase involvement in Hela cells takes place at 10 μM INA. At higher INA concentrations ZVADfmk becomes inefficient in preventing apoptosis (figure 4A) and PARP cleavage is decreased (figure 4B). The enhanced caspase activity was observed at 5 μM INA for SupT1 cells and 10 μM INA for Hela cells, which corresponds in each case to the IC50 at which INA causes cell death following photo-activation. These studies therefore suggest that in the IC50 range of concentrations of INA the apoptosis is mediated by caspases. However, at higher INA concentrations a caspase-independent apoptotic process may be involved.


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 induced apoptosis in Hela cells is also caspase dependent. A. Hela cells, pre-incubated or not with 40 μM ZVADfmk were treated with indicated amounts of INA, irradiated with UV and analyzed 24 h post treatment. Cells were stained with Annexin V and analyzed by FACS. The percentage of the population presenting high staining (PS exposure) is presented. B. Hela cells were treated with indicated amounts of INA. 24 hours upon treatment, the cells were lysed and the presence of PARP and/or cleaved PARP was assessed by Western analysis. The ratio of the two as determined by Western analysis is represented.
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Related In: Results  -  Collection

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Figure 4: INA-UV induced apoptosis in Hela cells is also caspase dependent. A. Hela cells, pre-incubated or not with 40 μM ZVADfmk were treated with indicated amounts of INA, irradiated with UV and analyzed 24 h post treatment. Cells were stained with Annexin V and analyzed by FACS. The percentage of the population presenting high staining (PS exposure) is presented. B. Hela cells were treated with indicated amounts of INA. 24 hours upon treatment, the cells were lysed and the presence of PARP and/or cleaved PARP was assessed by Western analysis. The ratio of the two as determined by Western analysis is represented.
Mentions: Caspases are cysteine proteases that are key mediators of apoptosis [19]. The involvement of caspases in the apoptotic pathway can be studied by the inhibition of apoptosis via the pan-caspase inhibitor Z-Val-Ala-Asp-fluoromethylketone (ZVADfmk) and the cleavage of caspase specific substrates like PARP [20,21]. 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. As seen in figure 3A and 3B INA-UV mediated apoptosis at low concentrations was inhibited by ZVADfmk confirming the role of caspases. Interestingly higher concentrations were not inhibited by ZVADfmk suggesting a different mechanism at very high concentrations. In order to monitor caspase activation in SupT1 cells 24 h post treatment with INA-UV, the cells were labeled FITC-VAD-FMK which binds irreversibly with active caspases and analyzed by flow cytometry. As seen in figure 3C, high activation of caspases is observed at 5 μM which correspond to the IC50 of INA-UV treatment of SupT1 cells. The activation of caspases was lower at higher doses as determined by FITC-VAD-FMK staining. These findings are supported by the high levels of PARP cleavage seen at low INA concentrations whereas there was little PARP cleavage at higher INA concentrations (figure 3D). PARP cleavage was prevented at all INA concentrations by ZVADfmk indicating its caspase specificity (data not shown). To generalize our conclusion about mechanisms of cell killing, we performed similar experiments with Hela cells. As shown in figure 4, the major caspase involvement in Hela cells takes place at 10 μM INA. At higher INA concentrations ZVADfmk becomes inefficient in preventing apoptosis (figure 4A) and PARP cleavage is decreased (figure 4B). The enhanced caspase activity was observed at 5 μM INA for SupT1 cells and 10 μM INA for Hela cells, which corresponds in each case to the IC50 at which INA causes cell death following photo-activation. These studies therefore suggest that in the IC50 range of concentrations of INA the apoptosis is mediated by caspases. However, at higher INA concentrations a caspase-independent apoptotic process may be involved.

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