Study On Effect Of Methyl Mercury Chloride To Leukemia And Its Mechanism

Malignant cancers seriously threaten human health and life, 5% of malignant tumors is leukemia. At present, the incidence of leukemia has been on an upward trend, so looking for the more efficient, lower toxicity anti-leukemia drug with low recurrence rate in treatments of leukemia is very important. With gradually raising awareness of leukemia, treatment of leukemia has been changed from traditional chemotherapy to the treatment of differentiation, immunotherapy, stem cell transplantation and genic therapy. The targeted therapy that the Chinese scholars advocated against leukemia genes protein, like the all-trans-retinoic acid (ATRA) and arsenic trioxide (ATO) in the treatment of APL has become a representative of targeted therapy of leukemia. The fact that Gleevec successfully treat the chronic myeloid leukemia also proves that targeted therapy can be used as a powerful tool to conquer human tumors based on the pathogenesis of the tumor .Since Rosenberg had found that cisplatin(cis-platin)had anticancer activity in 1969, platinum and platinum-group metal with anticancer different ligand structure and role of different mechanisms and target caused people's widespread interesting . Studying on Chinese medicine and arsenic's targeting role in the mechanism of antitumor made us understand the toxic substances in new ways. The researches for long time indicate that methyl mercury chloride (MMC) could induce free radicals and lipid per oxidation, non-selective increase plasma membrane permeability which led to polarization; increase intracellular Ca2+ to affect the normal cell functions; lower intracellular glutathione content; induce apoptosis ultimately. On the basis of investigation of the MMC neurotoxicity mechanism , we think it has multi-drug targets and induces apoptosis of tumor cell, We raise the new idea of using MMC to cure leukemia. In this study , we explore MMC's effect and possible mechanism to leukemia cells in vitro and vivo, using ATO as positive antineoplastic .In vitro testCultured K562 and NB4 cells with different concentrations of MMC for different time, Assay proliferation of K562 and NB4 cells; the cell's apoptosis was observed by Hoechst33258 staining with fluorescence upside-down microscope. Use FCM to determine apoptosis rate of two kinds of leukemia cells in different concentrations of MMC for 24 hours and cells cycle distribution in different MMC concentrations for 72 hours; assay the expression of two apoptosis-related protein(Bcl-2, Bax) in NB4 cells cultured with different concentrations of MMC for 24 hours by Western blotting .1. Effect of MMC inhibiting proliferation of K562 and NB4 cells.After NB4 cells cultured with 5μmol/L MMC for 6h, the survival rate decreased to 78.6%, and after cultured with 20μmol/L MMC for 24 hours, the survival rate dropped to 13.7%. After K562 cells cultured with 10μmol/L MMC for 24h, the survival rate decreased to 70.0%,and after cultured with 20μmol/L MMC for 24 hours, the survival rate dropped to 35.6%. The results showed that MMC could inhibit the proliferation of NB4 and K562 cells and have a dose-response relationship. 2. Effect of MMC inhibiting cell cycle of K562 and NB4 leukemia cells.After cultured with MMC for 72 hours, the percent of NB4 and K562 cells in G2-M phase and G0- G1 phase rose up, the percent of NB4 and K562 cells in S phase declined and the all the values showed a dose-dependent relationship, which measured by FCW.After cultured with 2.50μmol/L MMC , the percent of NB4 cells in G2-M phase (22.70±9. 79%) had significant difference(p <0.05) compared with the control group (8.40±4.09%); after cultured with 10μmol/L MMC ,the percent of NB4 cells in G0-G1 phase (51.86±2.7 3%) also had significant difference (p <0.05) compared with the control group (36.11±6.18%); after cultured with 2.50μmol/L MMC , the percent of NB4 cells in the S-phase (34.80±10.1 7%) had significant difference (p <0.05) compared with the control group (55.49±2.35%).After cultured with 5.00μmol/L MMC , the percent of K562 cells in G2-M phase(13.36±5.86%)had significant difference(p <0.05) compared with the control group(5.18±4.25%); after cultured with MMC ,although the percent of K562 cells in G0-G1 phase had the trend of rising up and the percent of K562 cells in S-phase had the trend of declining, they all had not significant difference(p>0.05)compared with the control group,The effect of MMC inhibiting cell cycle of K562 leukemia cells was weaker than that of NB4 cells.3. Effect of MMC promoting the apoptosis of K562 and NB4 leukemia cellsObserved NB4 and K562 cells of control group before being stained under the microscope, we can see cells with clear contours, smooth membrane, dense cytoplasm. Observed by upside-down fluorescence microscope after being stained by Hoechst 33258, the cells were in uniform light blue; After cultured with 10μmol/L MMC for 24 hours, the number of the two kinds of leukemia cells had significantly been reduced, and most cells had smaller volume, irregular sizes and shapes; the membrane of the cells were integrity, the cells'edge crimpled. Observed by upside-down fluorescence microscope after being stained by Hoechst 33258, we can see many intracellular concentration, shiny blue fluorescence of the nuclear and debris, which may be the predecessor of apoptotic bodies.After cultured with MMC for 24 hours, the apoptotic rates of NB4 cell and K562 cell increased with the dose increased, which determined by FCW after FITC- AnnexinⅤ/PI double staining. After NB4 cells cultured with 2.5μmol/L MMC, there was a significant difference (p <0.05) between the apoptotic rate of MMC group (20.87±3.70%)and that of the control group (11.86±3.77%); after K562 cells cultured with 5.00μmol/L MMC, there was a significant difference (p <0.05)between the apoptotic rate of MMC group (31.54±6.35%) and that of control group (10.36±3.23%).4. Effect of MMC to expression of apoptosis-related protein in NB4 cells.After cultured with MMC for 24 hours, with the dose increased, the expression of Bcl-2 in NB4 cells reduced gradually , but expression of Bax increased gradually, and the band of Bcl-2 was gradually unclear, the band of Bax gradually turned bright which detected by Western blot. All above showed that MMC could increase the pro-apoptotic protein content , decrease the antiapoptotic protein content and promote the apoptosis of NB4 cells.in vivo testSelected 4-5 weeks old BALB/C female nude mice, Separated them into three groups: normal group inoculating nothing, K562 group inoculating K562 cells with tail vein injection directly and NB4 group inoculating NB4 cells with tail vein injection after 3Gy60Co irradiation for 10min .Determined number of the tail's blood WBC diluted 20 times by using XT1800i hematology analyzer in manual way. When the number of leukocytes of the groups inoculated K562 and NB4 cells significantly increased compared with the normal group, we confirmed that we established both acute and chronic leukemia xenograft mice model. Then we divided each kinds of Leukemia mice into three groups in randomization way. (one group gave 0.1mg/10gBW MMC ,one group gave 0.1mg/10gBW ATO during each 2 days, and one for leukemia control group), the normal and the leukemia control groups gave the same amount of saline at the same time. Weighted all mice and determined some mice's WBC in tail blood each 2 days. After the acute and chronic leukemia nude mice were given three and four times MMC and ATO ,we saw the significant declining trend of WBC of MMC group , and stopped the test. During the test , we observed changes of peripheral blood smears, bone marrow smears and liver pathology, and compared the number and classification of WBC ,the viscera / body ratio, survival time of nude mice inoculating NB4 cells . 1. Effect of MMC to WBC number and cell classification of nude mice inoculating NB4 and K562 leukemia cells .After the mice were inoculated K562 and NB4 cells for half a month, the number of WBC of them became higher than that of normal group and there was a certain proportion of leukocytes in their blood. There was significant difference between values of the non-inoculated mice and inoculated mice. That meant the leukemia animal models had been established.! And the lymphocyte percentage and neutrophils percentage had different changes. All the changes of the acute and chronic leukemia mice returned to local level after being given MMC ( 0.1mg/10g BW). The number of WBC of the mice inoculated NB4 cells and given 3 times MMC reduced to 17.3×109/L from 22.8×109/L; the number of WBC of the mice inoculated K562 cells and given 4 times MMC reduced to 20.3×109/L ,and two kinds of leukocytes of MMC group also significantly reduced compared with the leukemia control group (p <0.05). Note MMC can ease abnormal symptoms of the mice inoculated NB4 and K562 cells. But withdrawal 15 days and continued feeding and observing for 15 days, number of WBC of the mice inoculated K562 cells and given 4 times MMC gradually rebound .Note Although MMC can reduce the number of WBC to some extent, it can't cure absolutely .2. Observation of MMC's affection on nude mice inoculated K562 and NB4 cells in peripheral blood smear, bone marrow smears and liver biopsyThe normal mice peripheral blood smear showed WBC fewer than RBC, and most of WBC were neutrophils and small mature lymphocytes with regular shape . Compared with that , the number of WBC of mice inoculated NB4 cells increased, there were more WBC called promyelocytic leukocytes which had irregular shape, larger than normal lymphocytes, they had more cytoplasm and more vacuoles in the loose cytoplasm than normal WBC. We found Auer body in the bone marrow smear . Peripheral blood smear of mice inoculated K562 cells showed promyelocytic cells at various stages there were more eosinophils and basophil than that of normal mice. the number of leukocytes and platelet were more than normal mice .The change of bone marrow smears of nude mice inoculated K562 and NB4 cells were not too obvious, But myeloproliferative can be seen. We also saw proliferation of later neutrophils and rod-shaped nuclear cells ,Eosinophilic and basophil also increased, and megakaryocyte cells often hyperplasiakedly increased.liver biopsy of nude mice inoculated NB4 cells showed the leukemia cells infiltration in liver vascular and liver. Electron microscope showed leukocytes with Auer bodies in liver.3. MMC's affection on ratio of viscera and weight of nude mice inoculated NB4 and K562 cells and survival time of nude mice inoculated NB4 cells.After given MMC 3 times, the weight of nude mice inoculated NB4 cells had significant difference compared with the normal control mice, and had no more significant compared with the leukemia control mice. Acute leukemia had an impact on body weight of nude mice inoculated NB4 cells , but MMC had not impact on weight yet at that this time to leukemia mice; After given MMC 4 times, the weight of nude mice inoculated K562 cells in the end of the experiment was significantly lower than that of normal control mice and leukemia control mice, the difference was significant at that time. That showed a lot of MMC may inhibit appetite and thyroid's function, caused the weight of leukemia nude mice reduce at the end of test.After given nude mice inoculating K562 and NB4 cells MMC (oral administration 0.1mg/10gBW), spleen / body ratio that compared with the respective leukemia control group decreased , and the spleen / body ratio of nude mice inoculated K562 cells had significantly difference, Note MMC can inhibit vicious increasing of spleen / body ratio caused by inoculating K562 and NB4 cells .In addition, MMC significantly prolonged survival time of mice inoculating NB4 cells.In summary, in vitro test , MMC can induce apoptosis of K562 and NB4 cells, inhabit the proliferation of them, block cell cycle of them, decrease the S-phase cells significantly, change expression of apoptosis-related protein Bcl-2 and Bax in NB4 cells cultured with MMC for 24 h . In vivo test , MMC can reduce number of WBC and leukocytes of nude mice inoculated K562 and NB4 cells, change smears of peripheral blood and bone marrow, reduce body spleen ratio, prolong survival time of mice inoculated NB4 cells. In the future, we will study on the mechanism of MMC to leukocytes further, consider adjuvant treatment for leukemia., find different formulations mercury suitable for the development of different human leukemia. Change the situation of current treatment of leukemia which is not ideal, and develop personalized treatment.