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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 is not affected by the expression of multidrug resistant protein. The MTS assay was applied to assess the viability of MRP1 overexpressing multidrug resistant 293/MRP1 cell (open circles) and its drug sensitive parent cell line 293 (close circles) subjected to doxorubicin exposure (A) or to INA-UV treatment (B). A. The cells were exposed to doxorubicin at the indicated concentrations for 72 hours and then subjected to the MTS assay. Data are mean ± S.D. B. The cells were subjected to INA-UV treatment and then subjected to the MTS assay 24 hours post treatment. Data are mean ± S.D of seven measurements.
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Figure 5: INA-UV treatment is not affected by the expression of multidrug resistant protein. The MTS assay was applied to assess the viability of MRP1 overexpressing multidrug resistant 293/MRP1 cell (open circles) and its drug sensitive parent cell line 293 (close circles) subjected to doxorubicin exposure (A) or to INA-UV treatment (B). A. The cells were exposed to doxorubicin at the indicated concentrations for 72 hours and then subjected to the MTS assay. Data are mean ± S.D. B. The cells were subjected to INA-UV treatment and then subjected to the MTS assay 24 hours post treatment. Data are mean ± S.D of seven measurements.

Mentions: An important problem that arises during chemotherapy is the emergence of drug resistant cells [22]. A major contributor of that phenomenon is an increased efflux of the drugs facilitated by proteins such as the P-glycoprotein (Pgp) or the multidrug resistance protein (MRP) [23-25]. To test whether INA was affected by this phenomenon we used 293/MRP1 cells, a stably transfected 293 cell line that continuously expresses the MRP1 gene at high levels [26]. Indeed, those cell lines exhibited a much lower sensitivity to the commonly used chemotherapeutic agent doxorubicin (figure 5A). However, the IC50 of INA-UV cell killing was insensitive to the presence of the multidrug resistance associated gene MRP1 (figure 5B, table 1). Similarly, INA-UV treatment was not significantly affected by Pgp over-expression (table 1).


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 is not affected by the expression of multidrug resistant protein. The MTS assay was applied to assess the viability of MRP1 overexpressing multidrug resistant 293/MRP1 cell (open circles) and its drug sensitive parent cell line 293 (close circles) subjected to doxorubicin exposure (A) or to INA-UV treatment (B). A. The cells were exposed to doxorubicin at the indicated concentrations for 72 hours and then subjected to the MTS assay. Data are mean ± S.D. B. The cells were subjected to INA-UV treatment and then subjected to the MTS assay 24 hours post treatment. Data are mean ± S.D of seven measurements.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: INA-UV treatment is not affected by the expression of multidrug resistant protein. The MTS assay was applied to assess the viability of MRP1 overexpressing multidrug resistant 293/MRP1 cell (open circles) and its drug sensitive parent cell line 293 (close circles) subjected to doxorubicin exposure (A) or to INA-UV treatment (B). A. The cells were exposed to doxorubicin at the indicated concentrations for 72 hours and then subjected to the MTS assay. Data are mean ± S.D. B. The cells were subjected to INA-UV treatment and then subjected to the MTS assay 24 hours post treatment. Data are mean ± S.D of seven measurements.
Mentions: An important problem that arises during chemotherapy is the emergence of drug resistant cells [22]. A major contributor of that phenomenon is an increased efflux of the drugs facilitated by proteins such as the P-glycoprotein (Pgp) or the multidrug resistance protein (MRP) [23-25]. To test whether INA was affected by this phenomenon we used 293/MRP1 cells, a stably transfected 293 cell line that continuously expresses the MRP1 gene at high levels [26]. Indeed, those cell lines exhibited a much lower sensitivity to the commonly used chemotherapeutic agent doxorubicin (figure 5A). However, the IC50 of INA-UV cell killing was insensitive to the presence of the multidrug resistance associated gene MRP1 (figure 5B, table 1). Similarly, INA-UV treatment was not significantly affected by Pgp over-expression (table 1).

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