Mechanistic Study Of The Arsenic Methylation And The Effects Of Arsenic And Selenium On The Treatment Of Leukemia

Inorganic arsenic (iAs) is one of the most significant hazards in the environment, affecting millions of people around the world. Exposure to arsenic is associated with cancers of the skin, lung, urinary bladder, kidney and liver as well as non-cancer diseases, such as diabetes mellitus, hypertension, and cerebrovascular and cardiovascular diseases. However, as an ancient drug used in traditional Chinese medicine, iAs shows anticancer activity. Since arsenic trioxide (As3+) has been successfully used in the treatment of acute promyelocytic leukemia (APL), the anticancer efficacy of arsenic attracted increasing attentions. Evidence suggests that the biotransformation of arsenic is related to its biological functions. Thus, we investigated the biotransformation of arsenic and the double-edged effects of arsenic on APL treatment in three aspects:the mechanism of arsenic methylation; the detoxification mechanism of DMPS in arsenic posing; and the mechanism for the effects of arsenic and selenium on the apoptosis and differentiation of APL cells. In the experiments, we used multiply methods, such as enzyme kinetics, spectroscopy and mass spectrometry to investigate the mechanism of arsenic methylation. We also studied the inhibitory effects of dimercaptopropanesulfonic acid (DMPS) on arsenic methylation in HepG2 cells, and explained the detoxification mechanism of DMPS in arsenic posing from metabolism. Subsequently, we used RT-PCR, western-blot and other methods of molecular biology to investigate the effects of arsenic and selenite on the apoptosis and differentiation of APL cells. Major works show as follow:1. We constructed recombinant human arsenic (Ⅲ) methyltransferase (hAS3MT) and analyzed the reaction sequence of arsenic methylation by rapid equilibrium kinetic model. The results suggest that hAS3MT-catalyzed As3+ methylation is a completely ordered reaction. S-Adenosyl-L-methionine (AdoMet) is the first-order reactant, glutathione (GSH) is the second-order reactant which binds to the hAS3MT-AdoMet compound, As3+ is the third-order reactant which binds to the hAS3MT-AdoMet-GSH compound and starts the enzymatic methylation. Similarly, AdoMet, GSH and MMA3+ subsequently bind to hAS3MT before the enzymatic methylation of monomethylarsonous acid (MMA3+). Moreover, we used UV-Vis spectroscopy, CD spectrometry and fluorescence spectrometry to investigate the interactions between substrates and hAS3MT. The results suggest that AdoMet modulates the conformation of hAS3MT to a best matched state by hydrophobic interaction. GSH increases the β-pleated sheet content of hAS3MT and exposes the active site cysteine residues for As3+ binding. Before the enzymatic methylation, As3+ binds to Cys61, Cys156 and Cys206. Subsequently, we analyzed the functions of cysteine residues by matrix-assisted laser desorption ionization-time of flight-mass spectrometry (MALDI-TOF-MS) and other methods. Two disulfide bonds of Cys368-Cys369 and Cys250-Cys32 are detected in hAS3MT. The Cys250-Cys32 disulfide bond is reduced by GSH or other disulfide bond reductants before the enzymatic methylation of As3+. In addition to exposing residues around the active sites, cleavage of the Cys250-Cys32 disulfide bond modulates the conformation of hAS3MT. We also observed the intermediate of Cys250-S-adenosylhomocysteine (AdoHcy), suggesting that Cys250 is involved in the transmethylation. GSH recoveres the catalytic activity of Cys250 by reducing the Cys250-AdoHcy disulfide bound. Cys156 and Cys206 release the methylated arsenicals from the enzyme through forming an intramolecular disulfide bond. GSH reduces the formed disulfide bond of Cys156-Cys206 to recover the activities of these cysteine residues for next cycle.2. We used HepG2 cells to investigate the detoxification mechanism of DMPS in arsenic posing. In the experiment, we analyzed the arsenic species by high performance liquid chromatography-inductively coupled plasma-mass spectrometry (HPLC-ICP-MS). Meanwhile, we used RT-PCR and western-blot to investigate the effect of DMPS on the expression of hAS3MT in HepG2 cells. Additionally, flow cytometry was used to analyze the effects of DMPS on As3+ -induced reactive oxygen species (ROS) accumulation and apoptosis in HepG2 cells. Subsequently, we investigated the inhibitory mechanism of DMPS in the As3+/MMA3+ methylation catalyzed by hAS3MT. Three aspects, the influx and efflux of arsenic, the accumulation of cellular ROS, and the enzymatic methylation of arsenic, were considered to explain the reasons for detoxification of DMPS in arsenic posing. (1). DMPS inhibits HepG2 cells influx As3+ and promotes the efflux of As3+. (2). DMPS eliminates arsenic-induced accumulation of ROS, which may decrease As3+ -induced oxidative damage. (3). DMPS inhibits the cellular arsenic methylation. On the one hand, DMPS down-regulates the expression of hAS3MT in HepG2 cells. On the other hand, DMPS competitively coordinates with As3+ to inhibit As3+ binding to hAS3MT and involving in the enzymatic methylation.3. The mechanisms of As3+, As4S4 and selenite (Se4+) on the apoptosis and differentiation of APL cells were analyzed by RT-PCR, western-blot and other molecular biological methods. As4S4 and Se4+, which are similar to As3+, induce the apoptosis and differentiation of APL cells in dose-dependent manner. The mechanism of As4S4 induced apoptosis and differentiation of APL cells is similar to As3+:at low concentrations (0.1-0.4 μM) As4S4 induces partial differentiation of APL cells through inducing the degradation of PML-RARa oncoprotein; at high concentrations (0.5-3.0 μM) it triggers apoptosis through ROS-related multiply apoptosis pathways. However, the mechanism of low concentrations of Se4+-induced apoptosis is different from As3+ and As4S4.1.0-2.0 μM Se4+ show no effects on the apoptosis and differentiation of APL cells,2.0-4.0 μM Se4+ induce the apoptosis and differentiation of APL cells. Se4+, which is similar to As3+, induces the differentiation of APL cells through inducing the degradation of PML-RARa oncoprotein. However,2.0-4.0 μM Se4+ do not induced the accumulation of ROS and promote the apoptosis of APL cells through inhibiting the nuclear factor-κB (NFκB) signal pathway. The combination of As3+ and Se4+ (2.0-4.0μM) not only enhances the apoptosis and differentiation of APL cells, but also decreases the accumulation of ROS. Thus, the combination of As3+ and Se4+ is a better therapy for APL treatment.