P.O. Vardevanyan – D.Sc. (Biol.), Professor, Head of The Department of Biophysics, Yerevan State University
R.S. Ghazaryan – Ph.D. (Biol.), Scientific Researcher at inter-faculty scientific-research laboratory «Structural Biophysics», Department of Molecular Physics, Yerevan State University
M.A. Shahinyan – Ph.D. (Biol.), Junior Scientific Researcher, Scientific-Research Laboratory «Biophysics of Subcellular Structures», Department of Biophysics, Yerevan State University
M.V. Minasyants – Post-graduate Student, Laboratory Assistant, Scientific-Research Laboratory «Biophysics of Subcellular Structures», Department of Biophysics, Yerevan State University
S.N. Hakobyan – Ph.D. (Biol.), Associate Professor, Department of Engineering, Armenian State Engineering University
Doxorubicin is an anticancer drug which may interact both with natural and synthetic double-stranded (ds) nucleic acids mainly via intercalation binding mechanism. Nowadays, doxorubicin is successfully used in cancer treatment as chemotherapy drug, although mechanisms of influence of this compound are not fully clarified. In this work, we have investigated in vitro the interactions of doxorubicin with ds-nucleic acids, which were previously irradiated with electromagnetic waves, both with resonant (64.5 and 50.3 GHz) and non-resonant (48.3 GHz) frequencies of water structures.
We have obtained absorption spectra for doxorubicin complexes withunirradiated and irradiated (90 minutes) ds-nucleic acids in visible region of the spectrum, where nucleic acids do not have absorbance. It has been revealed that the absorption spectra for doxorubicin complexes with unirradiated and irradiated ds-nucleic acids are alike and the manner of their changes during titration is quite similar. From the absorbance curves, free (Cf) and bound (Cb) doxorubicin concentrations were calculated and binding isotherms were built.
By using determined K values, the change of Gibbs free energy (∆G), the change of entropy (∆S) and the change of enthalpy (∆H) for binding process were calculated.
Absorption isotherms were built in Scatchard coordinates (r/Cf dependence from r) from the absorption spectra of doxorubicin complexes with unirradiatedand irradiated ds-nucleic acids. As it may be seen from the gained data, doxorubicin interacts via intercalation mechanisms both with unirradiated and irradiated ds-nucleic acids. At the same time, calculations have revealed that doxorubicin have different binding constants for unirradiated and irradiated ds-nucleic acids.
From table one may assume that doxorubicin forms with B-DNA (extracted from calf thymus) stronger complex, in contrast with A-DNA (poly[rG], poly[rC]).
In case of ds-nucleic acids which were irradiated via electromagnetic waves with resonant 64.5 and 50.3 GHz frequencies of water structures, the value of binding constant, which characterize the strength of complex, is bigger almost by an order in contrast with same parameter of B-DNA and twice bigger in comparison with A-DNA. Thermodynamic parameters of doxorubicin complex-formation with ds-nucleic acids, irradiated with non-resonant rays (for instance 48.3 GHz) practically remain constant.
Based on the gained data, it was revealed that as a result of irradiation of ds-nucleic acids via electromagnetic waves with resonant 64.5 and 50.3 GHz frequencies, in more hydrated form of nucleic acid (B-DNA) takes place stronger dehydration, which allows doxorubicin molecule to penetrate deep into space between base pairs of ds-nucleic acid and as a consequent the value of binding constant grows. As it follows from obtained data, the change of entropy for doxorubicin complex-formation previously irradiated with resonant frequencies ds-nucleic acids is bigger, in contrast with unirradiated nucleic acids. During complex-formation of doxorubicin with irradiated via resonant frequencies ds-nucleic acids thermodynamic parameters of binding changes, which depends on the conformational state of ds-nucleic acid.
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