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  • Abnormal expression and higher activity of GLO I have

    2022-05-13

    Abnormal expression and higher activity of GLO I have been detected more in various tumor MMP-2/MMP-9 Inhibitor I than in normal cell samples., , , , , , , Among cancer cell lines, lung carcinoma cells frequently showed higher GLO1 activity. Human NSCLC cell line NCI-H522 cells possess high activity and expression of GLO I., , bromobenzylglutathione cyclopentyl diester (BBGC), one of the GSH analog inhibitors of GLO I, has been reported to strongly suppress proliferation of NCI-H522 cells. Furthermore, it has been reported that RNAi knockdown of GLO I in NCI-H522 cells significantly reduces cell growth and induces apoptosis. In order to test whether piceatannol is effective in suppressing growth of NCI-H522 cells, we examined its cellular antiproliferative effect on NCI-H522 cells. NCI-H522 cells were treated with 10, 30, 50, 80 and 100μM of piceatannol for 24, 48 and 72h. The cell viabilities (% of control) were measured by using WST-8 assay. As shown in , piceatannol treatment is revealed to suppress the proliferation of NCI-H522 cells in a dose- and time-dependent manner. The EC values of piceatannol for NCI-H522 cells at 24, 48 and 72h treatments were about 53, 23 and 17μM, respectively (). Expectedly, piceatannol was found to have antiproliferative effect on GLO I highly expressed NCI-H522 cancer cells. Another human NSCLC cell line NCI-H460 cells lower express GLO I than NCI-H522 cells. BBGC has been reported to more strongly suppress proliferation of NCI-H522 cells, which has higher GLO I activity, than that of GLO I lower-expressing NCI-H460 cells, implying that NCI-H522 cells are more strongly dependent on GLO I for cell growth than NCI-H460 cells. In order to elucidate whether or not the suppression of cell growth by piceatannol is correlated with GLO I inhibition sensitivity, we compared the antiproliferative effect of piceatannol between NCI-H522 MMP-2/MMP-9 Inhibitor I and NCI-H460 cells. Both cells were treated with 50μM of piceatannol for 24, 48 and 72h. The cell viabilities (% of control) were measured by using WST-8 assay. As shown in , the decrease of the cell viability by piceatannol treatment was more significant in NCI-H522 cells than that of NCI-H460 cells. These results indicate that piceatannol can preferentially suppress the cell growth of GLO I-dependent cancer cells. To comfirm the contribution of GLO I inhibition by piceatannol to the antiproliferative effect on NCI-H522 cells, we investigated the effect of transient overexpression of GLO I on piceatannol-induced suppression of NCI-H522 cell proliferation. NCI-H522 cells were transfected with expression vector for human GLO I or mock-transfected with empty vector. After 24h post-transfection, the cells were treated with indicated concentrations of piceatannol for 72h. Cell viability (% of control) was measured by using WST-8 assay. As shown in , overexpression of GLO I partially restored cell viability in piceatannol-treated NCI-H522 cells. This result suggests that the up-regulation of GLO I expression resulted in less sensitization of cancer cells to piceatannol, and that inhibition of GLO I by piceatannol treatment contributes to the suppression of the cell proliferation in cancer cells. Our results suggest that piceatannol could be an important natural lead compound for the development of GLO I inhibitory anticancer drugs. That is, the structure of piceatannol could provide a valuable scaffold to design unique human GLO I specific inhibitors. In order to examine the binding mode of piceatannol in human GLO I activity pocket, we performed computational molecular docking study using Autodock Vina. The geometry of piceatannol was prepared by ChemBioDraw Ultra and converted to 3D structure by OpenBabel. A number of crystal structures of human GLOI with its inhibitor were deposited to the Protein Data Bank (PDB). To date, no crystal structure of human GLOI complexed with flavonoid derivative is available. Recently, the crystal structure of mouse GLOI complexed with baicalein, which is one of the flavonoids, has been published (PDB id: ). Therefore, we constructed the homology model of human GLO I based on the crystal structure of mouse GLO I-baicalein complex using SwissModel server. The docking compatible structure formats of the homology model was prepared by AutoDockTools-1.5.7. For docking with Autodock Vina, the grid size was set to (x,y,z)=(20,20,20) and the grid center was set to (x,y,z)=(−10.407,−5.554,0.829). The other parameters were using the default values. Interestingly, two binding modes of piceatannol were determined. One form, named as Binding mode A, is similar to the binding mode of baicalein, a GLO I inhibitory flavonoid (A). The other form, named as Binding mode B, is similar to the binding mode of -(-hydroxy--indophenylcarbamoyl) glutathione (HIPC-GSH), a GSH analog inhibitor of GLO I (B). The predicted binding affinity of the Binding mode A was calculated to be −6.5kcal/mol, whereas that of the Binding mode B was −7.2kcal/mol. These results imply that the Binding mode B has stronger inhibitory activity to GLO I than the Binding mode A. Importantly, these two binding modes have a common feature, i.e. the benzene ring of piceatannol having two adjacent hydroxy groups (-, -) locates near the divalent zinc in the active site, so that this -diol could be able to chelate with the active site zinc (C). The predicted interactions between the hot amino acids of human GLO I and piceatannol molecule in the Binding modes A and B are shown in D. In the Binding mode A, one hydroxy group forms a hydrogen bond to Thr182B of human GLO I. Hydrophobic bonds were formed with Phe63A, Met158B and Met180B of human GLO I. The -diol of piceatannol chelates to the active site zinc. On the other hand, in the Binding mode B, two hydroxy groups form three hydrogen bonds to Asn104A, Arg38A and Asn119B of human GLO I. Hydrophobic bonds were formed with Phe68A and Met158B of human GLO I. One of the hydroxy group of the -diol of piceatannol also chelates with the active site zinc. It is noted that only piceatannol, which show stronger inhibitory effect on human GLO I, has -diol among the -stilbene compounds (). Also, baicalein, 5,6,7-hydroxy flavone, has shown to chelate to zinc in the active site of GLO I with its 6,7-hydroxy groups in the crystal structure of mouse GLO I/baicalein complex. Thus, the chelation of the -diol to zinc in the active site is significantly important to the inhibition of human GLO I. Intriguingly, the IC value of baicalein for human GLO I inhibition was calculated to be 11.0μM, which is approximately 14-fold as compared with that of piceatannol (IC=0.76μM). Unlike piceatannol, baicalein cannot bind to GLO I as the Binding mode B, because baicalein, which has no hydroxy group on its benzene ring, cannot form hydrogen bonds to Asn104A, Arg38A and Asn119B of human GLO I. These observations suggest that the dual binding potential of piceatannol may contribute to the more potent inhibitory activity to human GLO I.