表没食子儿茶素没食子酸酯对柔红霉素与人血清白蛋白相互作用影响的光谱学及细胞毒性研究

doi:10.3964/j.issn.1000-0593(2020)06-1821-07

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Abstract Tea polyphenols play roles in the augmentation of antitumor effects, the reversal of multidrug resistance, and the reduction of side effects of antitumor drugs. Tea polyphenols could be used as biochemical modulators in cancer therapy. In this study, the interaction of daunorubicin (DNR) with human serum albumin (HSA) and the effect of epigallocatechin gallate (EGCG) on the binding process under physiological condition were studied by fluorescence spectroscopy, UV-Vis absorption spectroscopy, circular dichroism (CD) spectroscopy, and dynamic light scattering (DLS). The cytotoxicity of DNR single drug, EGCG+DNR combined drug, and their complexes with HSA against human cervical cancer HeLa cell line was determined by MTT assay. Fluorescence quenching result and difference spectra of UV-Vis absorption revealed the formation of a static complex between DNR and HSA. Quenching constants, binding constants, the numbers of binding sites, enthalpy and entropy changes were obtained from fluorescence data. Positive enthalpy and entropy changes indicated that the binding of DNR to HSA was mainly driven by entropy. The hydrophobic interaction was the main driving force. Site marker competitive experiments combined with synchronous fluorescence spectra showed that DNR mainly bound to subdomain IIA of HSA and was closer to tryptophan residues. In HSA+EGCG+DNR ternary system, the fluorescence data processing model of the ternary system was established. The number of binding sites and binding constants for DNR-HSA interaction in the presence of EGCG obtained by Matlab fitting were obviously decreased in the presence of EGCG. This indicated that the presence of EGCG might decrease the binding affinities of DNR to HSA. In addition, in the presence of EGCG, the binding constant of DNR to HSA increased with the elevation of temperature. This implied that the main driving force for the binding process was still the hydrophobic interactions. CD spectroscopy and DLS studies showed that drug-protein binding could affect the conformation and particle size of the protein, resulting in a decrease in the α-helical content of HSA and an increase in particle size. The α-helical content in (HSA+EGCG)+DNR ternary system was greater than that in the corresponding HSA+DNR binary system, while the hydraulic diameter of the ternary system was smaller than that of the binary system. This indicated the presence of EGCG weakened the binding of DNR to HSA due to the competitive binding between EGCG and DNR. This conclusion was consistent with the fluorescence experiment result of the ternary system. Furthermore, the cytotoxicity of DNR and HSA+DNR complex in the presence or absence of EGCG was discussed, and the results indicated that the combination of DNR with EGCG had the synergistic effect and HSA can enhance the cytotoxicity of DNR. Obtained results would provide beneficial information on the combination of EGCG and DNR in the clinic. This study showed that the spectroscopic method could provide strong support for the study of the interaction between combined drugs and proteins.

Key words：Human serum albumin; Daunorubicin; (-)-Epigallocatechin-3-gallate; Spectroscopic technique; Cytotoxicity

Received: 2019-05-15 Accepted: 2019-10-09

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O642

Corresponding Authors: LIU Min E-mail: panpanliumin@163.com

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https://www.gpxygpfx.com/EN/10.3964/j.issn.1000-0593(2020)06-1821-07 OR https://www.gpxygpfx.com/EN/Y2020/V40/I06/1821