茶多酚与抗肿瘤药物联用具有增效、 减毒以及逆转耐药性的作用, 可作为生化调节剂应用于临床。 通过荧光光谱、 紫外-可见吸收光谱、 圆二色光谱和动态光散射研究了柔红霉素 (DNR) 与人血清白蛋白 (HSA) 在模拟生理条件下的相互作用以及表没食子儿茶素没食子酸酯 (EGCG) 对DNR与HSA结合过程的影响。 通过MTT法测定了DNR单一药物、 EGCG+DNR组合药物及其与HSA的复合物对人宫颈癌HeLa细胞系的细胞毒性。 荧光猝灭结果与紫外-可见吸收差谱表明DNR与HSA之间形成静态复合物。 由荧光数据拟合得到猝灭常数、 结合常数、 结合位点数、 焓变和熵变。 正的焓变和熵变表明DNR与HSA的结合过程主要为熵驱动, 且疏水作用为结合过程的主要驱动力。 位点标记竞争实验结合同步荧光光谱表明, DNR主要结合在HSA的IIA结构域, 并且更接近色氨酸残基。 在HSA+EGCG+DNR三元体系中, 建立了三元体系的荧光数据处理模型, 并用Matlab拟合得到EGCG存在下DNR与HSA相互作用的结合位点数与结合常数均明显减小, 表明EGCG的存在降低了DNR与HSA的亲和力。 此外, 在EGCG存在下, DNR与HSA作用的结合常数随着温度升高而增加, 表明结合过程的主要作用力仍为疏水作用。 圆二色光谱和动态光散射研究表明药物与蛋白结合会影响蛋白的构象和粒径, 导致HSA的α-螺旋含量减小且粒径增加。 EGCG存在的(HSA+EGCG)+DNR三元体系中的α-螺旋含量大于相应的HSA+DNR二元体系中的α-螺旋含量, 而三元体系的水合粒径相对于二元体系的有所减小。 这均表明EGCG与DNR存在竞争结合, EGCG的存在使DNR与HSA的结合减弱, 与三元体系的荧光实验结果一致。 此外, 讨论了EGCG对DNR和HSA+DNR复合物的细胞毒性的影响, 结果表明DNR与EGCG具有协同作用且HSA可以增强DNR的细胞毒性。 所得结果可为EGCG与DNR在临床中的联合应用提供有益信息。 研究表明光谱方法可为联合药物与蛋白的相互作用研究提供有力支撑。

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.