title: An HPLC method associated with a thermodynamic analysis to compare the binding of TRAIL and its nanovectorized form to death receptors DR4 and DR5 and their relationship to cytotoxicity.
abstract: TRAIL is a member of the TNF family of cytokines which induces apoptosis of cancer cells via its binding to its cognate receptors, DR5 a high affinity site and DR4 a site of low affinity. Our working group has recently demonstrated that nanovectorization of TRAIL with single wall carbon nanotubes (abbreviated NPT) enhanced TRAIL affinity to the high affinity site DR5 and increased pro apoptotic potential in different human tumor cell lines. In this paper, the DR4 low affinity site was immobilized on a chromatographic support and the effect of temperature on a wide temperature range 1°C-50°C was studied to calculate the thermodynamic parameters of the binding of TRAIL and NPT to DR4 and DR5 receptors. For the first time the heat capacity changes for the different binding processes were determined. At a physiological pH (7.4) the heat capacity changes for the binding of NPT to DR4 and DR5 were respectively equal to -0.91kJ/molK and -0.28kJ/molK and those obtained for the binding of TRAIL to DR4 and DR5 were respectively equal to -1.54kJ/molK and -1.05kJ/molK. By the use of differential scanning calorimetry (DSC), a phase transition (∼12°C for DR5, ∼4°C for DR4) between a disordered (low temperature) and an ordered (high temperature) solid like state visualized in the receptor structure confirmed the temperature dependence of binding affinity enthalpy ΔH for soluble TRAIL and its nanovectorized form to its cognate receptors. In the low temperature domain, the positive ΔH values contribute non-favourably to the free energy of binding, TRAIL and NPT described similar affinities for DR4 and DR5. For the high temperature domain, negative ΔH values indicated that van der Waals interactions and hydrogen bonding are engaged favourably at the ligand - receptor interface. Above 30°C, their rank-ordered affinities were thus strongly different in the sequence: TRAILDR4<NPTDR4<TRAILDR5< NPTDR5. The nanovectorization of TRAIL enhanced its binding to both DR4 and DR5 receptors at 37°C and could potentially sensitized cancer cells to TRAIL induced apoptosis through simultaneous activation of DR4 and DR5 as described in this paper for the non-small lung carcinoma cell line (H1703), the two hepatocarcinoma cell lines (SK-Hep1, HUH) and the colon carcinoma cell line (HCT116WT). The excellent linear coefficient (r2=1) for the plot comparing NPT cytotoxicity to TRAIL cytotoxicity confirmed a high degree of similarity for the mechanism of cytotoxicity of TRAIL and NPT between these four cell lines expressing DR4 and DR5 receptors. The slope (0.10) and the negative intercept (-1.23) of this plot indicated that NPT was much more efficient in these four cell lines as compared to TRAIL alone. As well, it was shown that the extra cellular acidosis led to the protonation of the TRAIL residue histidine by flipping the His switch to the on position with a concomitant decrease in affinity for DR4 and DR5 receptors. The highest affinity for NPT to DR4 and DR5 receptors observed at low pHs was due to the less accessibility of the His molecular switch to be protonated when TRAIL was immobilized on CNTs.
authors.affiliation: Univ Franche - Comté, F-25000 Besançon, France; EA481 Neurosciences Intégratives et Cliniques/Pôle Chimie Analytique Bioanalytique et Physique (PCABP), F-25000 Besançon, France; CHRU Besançon, Pôle Pharmaceutique, F-25000 Besançon, France.
authors.forename: Yves Claude
authors.initials: YC
authors.lastname: Guillaume
authors.affiliation: Univ Franche - Comté, F-25000 Besançon, France; EA481 Neurosciences Intégratives et Cliniques/Pôle Chimie Analytique Bioanalytique et Physique (PCABP), F-25000 Besançon, France.
authors.forename: Lydie
authors.initials: L
authors.lastname: Lethier
authors.affiliation: Univ Franche - Comté, F-25000 Besançon, France; EA481 Neurosciences Intégratives et Cliniques/Pôle Chimie Analytique Bioanalytique et Physique (PCABP), F-25000 Besançon, France. Electronic address: claire.andre@univ-fcomte.fr.
authors.forename: Claire
authors.initials: C
authors.lastname: André
mesh_terms: D017209:Apoptosis; D045744:Cell Line, Tumor; D002470:Cell Survival; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D055767:Immobilized Proteins / Q000737:chemistry / Q000378:metabolism; D037742:Nanotubes, Carbon / Q000737:chemistry; D009369:Neoplasms / Q000378:metabolism* / Q000473:pathology; D044367:Phase Transition; D011485:Protein Binding; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand / Q000737:chemistry / Q000378:metabolism*; D053221:TNF-Related Apoptosis-Inducing Ligand / Q000737:chemistry / Q000378:metabolism*; D013816:Thermodynamics
chemical_list: D055767:Immobilized Proteins; D037742:Nanotubes, Carbon; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand; D053221:TNF-Related Apoptosis-Inducing Ligand; C505653:TNFSF10 protein, human
keywords: Binding; Cell viability; DSC; Death receptors; HPLC; Phase transition; TRAIL; Thermodynamic