Effects Of Hydroxysafflor Yellow A On Tumor Stem Cell Marker Aldehyde Dehydrogenase

The malignant tumor easy to relapse after the treatment of metastasis is the main cause of death in cancer patients. Increasing the sensitivity of tumor cells to various treatments, delaying tumor recurrence or metastasis would be an important means to improve the survival of cancer patients. Cancer stem cell theory think that cancer cells derived from cancer stem cells, it is the result of long-term external stimuli. Therefore, the development of specific clinical drugs has important implications for treatment.Acetaldehyde dehydrogenase 1(ALDH1, EC 1.2.1.10)is a class of zinc-directed enzymes under the aldehyde dehydrogenase (ALDH) family. Which involved in the variety of metabolism,thus plays an important role in normal development and tissue homeostasis.ALDH1+tumor cells have biological characteristics of tumor stem cells, including self-renewal, proliferation, migration, and high tumorigenicity. In vitro growth curve by MTT and colony formation assay, tumor sphere culture, transwell experiments, nude mice experiments, showing ALDH1+cells have a stronger Stem cell properties than ALDH 1-cells.Hydroxysafflor yellow A (HSYA) is a natural antioxidant found in Carthamus tinctorius L (safflower). HSYA has one phenolic hydroxyl group and it is one of the most potent natural antioxidants displaying important pharmacological activities,Based on its phenolic hydroxyl structure and antioxidant capacity, we hypothesized and deduced that HSYA can inhibit ALDH1.In this study, we investigated the mechanism of HSYA binding to ALDH1 through integrating study of kinetic analysis and computational simulations.METHOD1 Measure the ALDH1 enzyme activity affected by different concentrations of HSYA and record the values.2 Keep the same substrate concentration as a constant,the concentration of HSYA and ALDH1 as variable, and then measure the ALDH1 enzyme activity and plotted to determine the type of inhibit.3 Keep the enzyme concentration as a constant and the concentration of HSYA and substrate as variable, and mesure the value and make plot, judge the parameters of inhibition kinetics.4 Fixed the concentration of substrate and enzyme,use different high concentration of HSYA mesure the activity by different time,make the time process of enzyme’s activity change plot.5 Apply Intrinsic and ANS-binding fluorescence method to analysis the features of 3D structural.6 Use computer simulation homology modeling to predict the 3D structure of ALDH1 and the binding sites with HSYA.RESULTS1 Inhibition effects of hydroxysafflor yellow A on ALDH1:the activity and structureThe results showed that HSYA significantly inhibited ALDH1 activity in a dose-dependent manner,The concentration of HSYA that led to a 50%loss in activity (IC50) was estimated to be 0.22±0.01 mM (n= 3).The plots of the remaining activity versus [E] at various inhibitor concentrations indicating that the HSYA-induced inhibition was reversible.The results showed changes only in the value of the apparent Vmax, which indicates that HSYA induced the typical type of non-competitive inhibition,it rarely affected the substrate access to the active site,Ki= 0.4238 ±0.2168 mM(n= 3)The results indicated that the catalytic activities were not changed detectably with time from the lowest to the highest HSYA concentrations. Only the first rapid inactivation, which occurred in less than 30 sec, significantly changed the enzyme activity.We found that HSYA had a quenching effect on intrinsic fluorescence,without significant red-shift, which gradually occurred with increasing concentration, the ALDH1 activity was also gradually inhibited (see Fig.1), showing that HSYA binding to ALDH1 and loss of enzyme activity were synchronized.By applying equation (5), we calculated the binding constant, K= 45.648±0.519 M-1 (n= 3), and the binding number, n= 1.274± 0.011.Compared to the native state of ALDH1, the ANS-binding spectra showed an increase in hydrophobicity with increasing HSYA concentration.implying that hydrophobic surface exposure was connected to the regional unfolding and that this is also associated with the loss of activity.2 Computational docking and MD simulations of HSYA binding to ALDH1Because the crystal structure of human ALDH1 is not available, we generated a model of the structure by homology modeling. The nearest template structure was lbxsa (sheep liver cytosolic aldehyde dehydrogenase).We used plausible docking of NADH (1,4-dihydronicotinamide adenine dinucleotide) with ALDH1 to find plausible cofactor binding regions on the enzyme..From the analysis of the interaction patterns from the MD results, we concluded that rings R1 and R2 are inserted into the protein for ligand binding.According to these results, we propose that HSYA binds to several residues located in the cofactor binding site of ALDH1, and these interactions induce the inactivation of ALDH1 by blocking NADH production, which is non-competitive inhibition behavior.CONCLUSIONthe principal results of our study indicate the following:(1) HSYA reversibly inhibits ALDH1 catalysis in a non-competitive manner;(2) the inactivation process mediated by HSYA is a fast process with no detectable kinetic time-course;(3) HSYA binding can promote the regional unfolding of ALDH1, which enhances the hydrophobicity of the surface and is accompanied by the loss of activity;and (4) HSYA can interact with several residues in the cofactor binding sites on ALDH1, and structurally, the phenolic hydroxyl ring plays the key active role in ligand binding and inhibition.