Inhibition And Mechanistic Study Of Matrix Metalloproteinases By Trivalent Metal Ions And Their Complexes

Matrix metalloproteinases (MMPs) are cell secreted endopeptinases whichcontain zinc dependent catalystic site. MMPs can not only degrade extracellularmatrix (ECM), but can also regulate the synthesis and secretion of cytokines, growthfactor, hormone, cell adhesion molecule acceptor, etc. MMPs involve in manyphysiological and pathological courses such as embryogenesis, organ development,wound healing, tissue remodeling, tumor, diseases of cardiovascular system, nervoussystem disease, arthritis, and respiratory diseases, etc. MMP-2, MMP-13andMMP-14in MMPs belongs to gelatinases, collagenases and membrane MMPs,respectively. With different substrate degradation specificities, they can degradedifferent types of collagens and gelatins and involve in multiple physiological orpathological processes that require ECM remodeling, and their excessive expressionsare closely associated with the migration, infiltration and transfer of cancer cells.Metal ions play important roles in many biological processes, such as cellrespiration, signal transduction, storage and metabolism. Their biological functionsinclude maintaining protein structure and function, or serving as the catalyze activitycenter of the enzyme. The disruption of homeostasis due to metal ions involves inmany diseases. And metal drugs have been clinically used in treating differentdiseases. It is found that trivalent metals and their complexes such as the lanthanideshave many biological functions, as well as resistance to bacteria, virus and tumors.In our previous work, we have studied the effective constituents in Koreanaconite roof that inhibit MMPs. The results reveals that NH4Al(SO4)212H2O, havesignificant inhibition to the MMP-2,-9. However,(NH4)2SO4and K2SO4show nosignificant inhibition to MMPs, and it can be inferred that the core of its inhibitionmight be due to trivalent aluminium. Meanwhile, transwell invasion assay showsdoes-dependent inhibition of HT-1080cell invading. And it does not affect the existence of cells at this concentration. To sum up, although we have found the aboveinhibitions, but whether they have similar action on other enzymes and what is thespecific inhibition mechanism remain unclear. In the present research, we first choosesome typical trivalent metal ions and their complexes, including trivalent lanthanides,commonly seen trivalent aluminum and ferric iron and their complexes, as well asother trivalent metal complexes, with12kinds of compounds in total. And theirinhibition to three different types of remodeled MMPs (MMP-2, MMP-13andMMP-14) are analyzed. The results show that LaCl3, TbCl3, GdCl3, YbCl3and EuCl3in the lanthanides demonstrate different inhibitions to MMP-2, MMP-13and MMP-14,and IC50<10μM. K3[Fe(CN)6], FeCl3, AlCl3and NH4Al(SO4)212H2O have excellentinhibitions to the enzyme activities of the three MMPs, and K3[Fe(CN)6] presentsparticularly prominent inhibition (IC50=0.3μM). In order to identify whether trivalentmetal ions and their complexes have specific inhibitions to remodeled proteasesMMP-2, MMP-13and MMP-14, we select the above metal ions and their compoundsand analyze the corresponding impacts on the activities of proteases B, K and L withdifferent catalytic mechanisms. K3[Fe(CN)6], FeCl3, AlCl3, NH4Al(SO4)2, LaCl3,EuCl3, TbCl3, GdCl3, YCl3, YbCl3, HAuCl4and NaAuCl4with final concentration of10μM show no significant inhibitions to both Cathepsin K and L, butinhibiteCathepsin B, while K3[Fe(CN)6] and HAuCl4show no significant inhibitionsto the above three enzymes. Therefore, we supose that K3[Fe(CN)6] and HAuCl4havespecific inhibitions to the MMPs family, especially MMP-14, and their inhibitions arenot the results of the pH values of compounds. Besides, the toxicity analysis oftrivalent metals and their complexes for HT-1080cells indicates that all of the IC50forthe inhibitions of FeCl3, AlCl3, NH4Al(SO4)212·H2O, LaCl3, EuCl3, TbCl3, GdCl3,YCl3and YbCl3are greater than350μM. And we also find that when IC50is lowerthan350μM, there is no obvious tendency of cellular poison with the increase ofcompound concentration. However, cell viability plunge at a certain concentrationabove350μM. We supose that when metal ions and their complexes attach to thesurface membranes of cells, the concentration increased to a certain degree, which leads to the sudden increase of cell permeability, so these ions and complexes entercells and cause cell death, as well as the decline of cell viability.We then analyze the inhibition mechanism and type of K3[Fe(CN)6] to MMP-14,explore the impact of K3[Fe(CN)6] on tumor cell behaviors at the cellular level, andfinally make a preliminary discussion on the mechanism of adjusting cell behaviors.Enzyme kinetic analysis results show that the inhibition of K3[Fe(CN)6] in a finalconcentration range of0-0.06μM to MMP-14is a reversible inhibition mechanism.The result of Michaelis-Menten equation, Lineweaver-Burk equation and Dixon plotsindicate that the inhibition type are concentration dependent. In the case of combiningcompetition and anti-competition inhibition, substrate and inhibitor combine withenzyme simultaneously, but the generated intermediate ternary complex can notfurther decompose, so enzyme activity begins to drop. Cytological experiment revealsthat at a concentration of0-50μM without affecting cell activity, it has significantinhibition to the migration and invasion of tumor cell HT-1080. Western blotexperiment shows that0-50μM of K3[Fe(CN)6] can inhibit the infiltration andmigration, which might be related to the down-regulated expression of MMP-14.Further experiment shows that K3[Fe(CN)6] helps down regulate the expression ofErk1/2,-catenin, P-JNK and FAK protein, which might be because that it inhibits themigration and invasion through the regulation over the above-mentioned proteins. Butthe specific regulatory mechanism needs to be further studied.Finally, we analyze the specific inhibition mechanism and inhibition type ofHAuCl4for MMP-14, and further investigate the effects of HAuCl4on tumor cellbehaviors. Our results indicated that the inhibition mechanism of HAuCl4forMMP-14is reversible. Michaelis-Menten plots, Lineweaver-Burk plots and Dixonplots results further prove that in the concentration range of0-0.6μM, HAuCl4showsnon-competitive inhibition to MMP-14, indicating that there is no competitive relationbetween primer and HAuCl4for MMP-14, and the combination of MMP-14withsubstrate or HAuCl4has on influence on its combination with the other one. However,the generated compound can not be downgraded, leading to the decrease of enzyme activity. Furthermore, the action parts are groups beyond the active site, whosestructure has nothing in common with that of substrate. As a result, the inhibition cannot be relieved. And we eliminate that the effect of compound pH. Cytologicalexperiment shows that at a concentration of0-50μM without affecting cell activity, ithas significant inhibition to the migration and invasion of tumor cell HT-1080, andindicating that HAuCl4may acts on tumor cell behaviors by inhibiting the activity ofMMP-14. Though we attempt to analyze the inhibition of the two compounds, thereason for the differences still need to be further studied.In conclution, this thesis finds and proves for the first time that trivalent metalions and their complexes show general inhibition to MMPs, and the inhibitionconcentration (IC50) is in orders of micromolar magnitude. Such inhibition mightregulate the physiological and pathological activities that MMPs participate in byregulating the functions of MMPs. Both two compounds in the form of complexK3[Fe(CN)6] and HAuCl4ion have specific inhibitions to MMP-14, and the inhibitionmechanisms are the reversible type in combination of non-competition andanti-competition and the non-competitive type, respectively. This results indicates thatthe inhibition are performed by combining the compounds with the non-active centerof MMP and changing its structure and conformation, which has not been reported sofar. We have also found that K3[Fe(CN)6] and HAuCl4can affect the behaviors oftumor cell HT-1080, and in particular, K3[Fe(CN)6] can inhibit the activity andexpression of MMP-14and several protein to inhibit migration and invasion of tumor.The research of thesis is very meaningful for the understanding of trivalent metal ionsand their complexes in regulating the activity of MMPs.