Study On Effect Of Mercuric Chloride To Leukemia And Its Mechanism

Leukemia is a common malignancy in the hematopoietic system withepidemiological survey results showing that it accounts for5%of totalmalignancies. Its mechanism: hematopoietic stem cells and progenitor cellsproduce a malignant change which renders the differentiation and maturationineffective, resulting in a cellular block in the different hematopoietic stages,and ultimately the formation of a heterogeneous group of hematopoieticmalignancies. Therefore, the treatment of leukemia today is recognized as adifficult problem in research, and exploring its effective therapy has always beenthe focus of attention of scientists.Since1969when cisplatin was found to have a significant anti-tumoractivity, metal anticancer drugs in clinical practice have attracted widespreadconcern. Much research has led to a discovery of other antineoplastic agentssuch as arsenic, mercury, ruthenium, titanium and gallium. The confirmation ofthe clinical efficacy of arsenic in treatment of leukemia is an importantbreakthrough in the research of metal treatment of leukemia. Since ancient times,arsenic sulfide ore has been used as a pigment, insecticide, and rodenticide.Arsenic can be used for malignant sores. Slight similarity in efficacy existsbetween mercury and arsenic. In the record of Chinese medicine, mercury ismainly used to detoxify and treat scabies, syphilis, malignant sore, as well as hemorrhoid and fistula. Therefore, researchers bravely predict that mercury canalso serve as anti-leukemia drug. Mercury is divided into organic and inorganicmercury. For example, methylmercury belongs to organic mercury and mercuricchloride to inorganic mercury. Methylmercury was reported to inhibit theproliferation of leukemia K562and NB4leukemia cells in research. In Japaneseliterature, mercuric chloride (MC) can cause apoptosis of leukemia HL-60cellsafter6h, and a DNA fragment and caspase-3substrate PARP were detected inapoptotic cells, so confirming activation of Caspase-3in the apoptotic process;this report also said that the initiating factor of the Caspase cascade reaction wasa release of cytochrome C (CytC) from the mitochondria into the plasma, andthe above effect began after the mitochondrial permeability transition pore (PTP)opened. The above studies give us a new idea that the mechanism of multi-drugtargets MC may have for promoting apoptosis, acting on the cell cycle andmitochondrial can be applied in leukemia treatment which is characterized byhigh morbidity, high recurrence rate and poor cure rate in order to identify amore effective treatment program for leukemia.Objective: To study the MC in vitro inhibition of APL NB4and CML K562,analyze the effect of the MC on the two types of leukemia cells and deplore themechanism of inhibition by the MC to leukemia cell proliferation; provide thetheoretical and experimental basis for the treatment of malignant tumors by theMC as a novel drug; open up the new drug way for the treatment of malignanttumors with mercury compounds. Methods: MTT was used to obtain the inhibition rate of the MC on APLNB4and CML K562cell lines; flow cytometry was used to detect the effect ofthe MC on the cell cycle and induction of apoptosis; Western blot assay wasused to detect the effect of the MC on the Bcl-2, Bax, CtyC, Caspase-3proteinexpression of the two cell lines.Results: MTT results showed that, for NB4cells, after6h of exposure to5M MC, the survival rate declined, after24h of exposure to40M MC, thesurvival rate of20.53%, and a comparison was made with the negative controlgroup (p<0.01); for K562cells, after12h of exposure to20M MC, the survivalrate declined, after24h of exposure to40M MC, the survival rate dropped to33.54%, and there was a significant difference when compared with the negativecontrol group (p<0.01). The above result indicated that the MC could inhibit theproliferation of K562and NB4cell lines in a time-and concentration-dependentmanner; MC could promote apoptosis of K562and NB4leukemia cell lines in aconcentration-dependent manner (p<0.05). The flow cytometry was used todetect the cell cycle of NB4and K562cells after24h of exposure to MC,respectively, with the following results: with the MC dose continuouslyincreasing, G2-M phase and G0-G1phase proportions increased to varyingdegrees, the S-phase ratio gradually decreased, and a significant dose-responsedependence was indicated. After24h of exposure to5μmol.L-1MC, NB4cellshad a G2-M phase ratio of25.42±0.39%, which was more significantlydifferent (p<0.05) when compared with the negative control group (12.64± 0.92%)(p <0.05). After24h of exposure to10M MC, NB4cells had a G0-G1phase ration of52.0±62.08%, which was more significantly different whencompared with the negative control group (36.43±1.51%)(p <0.05). After24h of exposure to5M MC, NB4cells had a S-phase ratio of30.35±1.98%,which was more significantly different when compared with the negative controlgroup (50.92±2.2%)(p<0.05). Detection on the effect of MC on K562cellcycles indicated that the G2/M phase percentage increased (p<0.05), S-phasepercentage gradually reduced (p<0.05), and there was no significant change inthe G0/G1phase percentage. The above result showed that the MC inhibition ofK562cell cycles was weaker than MC inhibition of NB4cell cycles. In24h ofexposure to MMC, with the dose increasing, NB4and K562cells were found tohave a gradual decrease in the Bcl-2expression, but have a gradually increasetrend in the Bax expression; caspase-3expression gradually weakened, butcaspase-3and Cyt-C expressions gradually increased, suggesting that the MCcould make the pro-apoptotic protein content to increase and promote apoptosisof NB4and K562leukemia cells.Conclusion: MC can induce apoptosis of NB4and K562leukemic cells,inhibit proliferation and arrest cellular cycles of the two leukemic cells,significantly decrease S-phase cells, and change the expression of proteins Bcl-2,Bax caspase and Cty-C relating to apoptosis of NB4Cell which are cultured for24h. The in-depth study of the mechanism of MC acting on leukemia cells canlead to the consideration of MC used in adjuvant chemotherapy for leukemia.