INA is a highly hydrophobic molecule that partitions with high specificity in cellular membranes . This high hydrophobicity along with its photoactivable property makes it a specific probe for transmembrane anchors of membrane proteins. We have exploited this specificity towards transmembrane anchors to inactivate a variety of viral membrane proteins for design of new vaccine candidates [15, 17]. In this study we studied the effects of INA-UV treatment on whole cells. Our results indicate that treatment of cell lines with INA by itself was non toxic; however, in the presence of UV light the treatment mediated loss in viability of numerous cancer cells.
The photoactivation of INA is mediated by its azido group that is sensitive to UV irradiation. Upon UV irradiation, a highly reactive nitrene radical is formed that covalently reacts with transmembrane proteins in its vicinity . Such covalent modifications of proteins has been shown to result in the complete loss of infectivity of several enveloped viruses [14–17] and correlates with the loss of membrane fusion function of the viral fusion proteins [16, 17]. The transmembrane segments of the fusion proteins of retroviruses  and influenza virus  are indeed critical for the full fusion process to take place. Although replacement of the transmembrane anchor of influenza virus hemagglutinin with a lipid-anchor obliterated its full fusion capacity, the lipid-anchored hemagglutinin still promoted the hemifusion phenotype leading to the mixing of the outer layers of the viral and target cell membranes . Consistent with this, INA-UV treatment of influenza preserved the hemifusion phenotype confirming the requirement of the transmembrane anchor for full fusion activity and the specific effect of INA on the transmembrane anchor without effecting the function of extracellular domains .
Treatment of cells with INA and UV irradiation resulted in complete loss of viability in a variety of cancer cell lines (table 1). This effect was shown to be dependent on the concentration of INA used and strictly required activation by UV light (figure 1). We show here that INA activation induces cell death via characteristics of apoptosis as determined by mitochondrial membrane depolarization, phosphatidyl serine presentation, PARP cleavage and DNA fragmentation in treated cells. At lower doses of INA, apoptosis could be reversed by ZVADfmk, a potent pan caspases inhibitor. Caspase activation was also detected by FITC-VAD-FMK 24 h post INA-UV treatment showing the involvement of caspases in this process and confirming the apoptotic pathway. However, at higher concentrations of INA, caspase activation is decreased and accordingly ZVADfmk becomes ineffective in preventing mitochondrial depolarization and PS presentation suggesting a caspase independent pathway of apoptosis as seen with other treatments like hexaminolevulinate PDT of lymphoma cells . A caspase independent cell death referred to as sub apoptosis has been previously documented . The death inducing stimulus might be such that apoptosis can be achieved through release of apoptosis inducing factors (AIF) from mitochondria without the participation of caspases .
As the dosage of INA is increased, more targets within the transmembrane region and possibly other membrane compartments are likely to be reached by the treatment and this can alter the mechanisms inducing the death of the treated cells. If activated INA will covalently react with any protein that is deeply anchored in the lipid bilayer, the consequence of this covalent modification will depend on the function of this portion of the particular protein. Understanding the effect of INA on cellular proteins is important for determining the mechanism of apoptosis induction by INA and its development as an anti cancer agent.
It has been previously reported that INA-UV treatment of cell membranes induces inactivation of gonadotropin receptor by uncoupling of the response of adenylate cyclase to gonadotropins . While binding of chorionic gonadotropin and the luteinizing hormone to gonadotropin receptor were preserved, the binding failed to induce stimulation of the adenylate cyclase pathway. On the other hand, this pathway was shown to be still functional when stimulated directly with NaF. The luteineizing hormone receptor is a member of the G protein-coupled receptor, a family of receptors displaying seven predicted transmembrane helices. CXCR4, another member of this family, has recently been shown to be overexpressed in many cancer cell lines [39, 40]. This overexpression is largely due to the hypoxic conditions in the tumor environment  and can favor the metastasis of cancer cells through its ligand SDF1α [42–44]. We show here that INA-UV treatment blocks CXCR4 mediated calcium signaling generated by SDF1α stimulation. At the same time the calcium gradient is preserved in the INA-UV treated cells as it is still sensitive to the effect of calcium ionophores indicating that the integrity of the treated cell membranes is not compromised. These data indicate a direct inactivation of CXCR4 receptor signaling by INA-UV treatment.
Similarly, the activity of MRP1 a member of the ABC transporters superfamily that along with Pgp is involved in the drug resistance phenotype in various cancers  was also affected by INA-UV treatment. Although no crystal structure is yet available for this protein, ABC transporters are thought to be composed of clusters of predicted transmembrane helices . Overexpression of drug resistance genes makes cells several orders of magnitudes less sensitive to conventional chemotherapeutic agents. The mechanism of action is not clearly understood but the working hypothesis of "hydrophobic vacuum cleaner" has been proposed [7, 45] whereby the hydrophobic chemotherapeutic agents while partitioning into the membrane will interact with the transporter within the lipid domain of the bilayer and be pumped outwards. The ability of INA to covalently react with members of ABC transporters has been previously demonstrated . We show here that this interaction leads to the inhibition of MRP1 function and drug efflux. Our data indicate that cell killing induced by irradiation at given concentrations of INA is not affected by the presence of MRP1 or Pgp. Although INA labeling of MRP1 has previously been demonstrated , the lack of difference in IC50 for INA killing between MRP1+ and MRP1- cells suggests that either INA is not a substrate for MRP1 or that the Kd for INA binding to MRP1 is much higher than the IC50 for INA to kill cells by irradiation. Therefore irradiation in the presence of INA, and possibly other hydrophobic alkylating agents, appears to be an effective modality of killing of multi-drug resistant cells.
Growth receptor signaling via IGF1R and EGFR has been known to induce cell survival and proliferation in cancer cells via activation of the PI3Kinase and Akt pathway. The tyrosine kinase IGF1R mediated Akt phosphorylation was not affected by INA treatment in cells incubated with IGF1. Interestingly, increased levels of Akt phosphorylation were observed in INA treated cells in the absence of IGF1 stimulation, which was further enhanced upon incubation with IGF1. This suggests that INA treatment may activate IGF1R consistent with reports that a mutation in the transmembrane anchor of IGF1R can result in activation of the receptor . The effects of INA can be interpreted through intramolecular effects via the chemical modification introduced by the covalent addition of a hydrophobic moiety in the transmembrane segment of a protein. However protein-protein and protein-lipid interactions are also likely to be affected by this modification. Proper signaling within cells relies on a dynamic reorganization upon stimuli of the signaling receptors within different domains of the membrane that are thought to assemble and disassemble for the signaling to proceed . We show here by FRAP analysis that INA-UV treatment considerably reduces the mobile fraction of proteins within plasma membranes. Whether this is due to a partial aggregation of membrane proteins and/or a reorganization of domains to accommodate the enhanced hydrophobicity needs to be further studied. The enhanced basal activation of Akt following INA and light treatment might also be a result of receptor immobilization/redistribution. Furthermore, the ability of IGF1 to stimulate the tyrosine kinase IGF1R receptor is considerably amplified by INA treatment. IGF1R has been shown to relocalize in membrane "raft" microdomains in MCF7 cells upon stimulation with IGF1  and activation of Akt also appears to rely on its membrane redistribution  in membrane "raft" microdomains . Nevertheless Akt activation induced by INA treatment does not prevent the cells to undergo apoptosis suggesting the mechanism of cell death induced by INA is independent of Akt signaling pathway.
In this report we show the potency of INA as a novel and efficient photoactivable chemotherapeutic agent. The activity of this treatment is strictly dependent on activation by UV light and is mediated by the covalent reaction of INA with membrane embedded domains of proteins. Unlike conventional photodynamic sensitizer that are dependent on reactive oxygen species for activity, reaction of INA with membrane proteins has been shown to be increased under hypoxic conditions . Such hypoxic conditions are common in tumors micro environment and present a major challenge for other photosensitizers. Furthermore, while INA can be directly activated by UV, an equivalent activation can be obtained through energy transfer processes called photosensitization using a variety of chromophores as photosensitizers . The result presented here show that INA is a very potent light activatable therapeutic agent whose targets and mechanism of action are very different from existing PDT agents. Those properties of INA make it a unique candidate for use in photoactivated cancer chemotherapy.