To Study Of The Anti-leukemia Effect Of Homoharringtonine With Idarubicin

Background:Acute myeloid leukemia(AML) is a clonal disease caused by hematopoietic stem cell abnormalities. A large number of leukemia cells are blocked in differentiation and apoptosis and stagnated at different stages of cell development. As an age-related diseases, due to the high-risk cytogenetics, poor performance and multi-drug resistance, elderly patients have difficult to tolerate intensive chemotherapy. In recent years, researchers have studied the pathogenesis of AML deeply, various drugs continue to emerge, so the clinical treatment achieves a certain effect, but the complete remission rate(CR) and overall survival(OS) is not seen much improvement, new chemotherapy regimens is also needed to improve CR and OS.Objective:To investigate the combined effect of homoharringtonine(HHT) and idarubicin(IDA) on the proliferation and apoptosis of K562 cells,and the effect on proliferation of HUVEC cells in vitro. To observe the efficacy and safety on elderly patients with acute myeloid leukemia(AML) treated with HHT and IDA.Method:1. MTT method was used to determination the effect of different concentration of HHT, IDA and HHT combined with IDA on the proliferation of K562 cells and HUVEC cells,and then the IC50 concentrations were calculated.2.After treating K562 cells with drugs for 6 hours, 12 hours and 24 hours,the flow cytometry were used to test the effect of different concentration of HHT, IDA and HHT combined with IDA on the apoptosis of K562 cells.3.After treating K562 cells for 6 hours, the RT-PCR method was used to detect the effects of HHT, IDA and HHT combined with IDA on the P53 gene expression of K562 cells.4.After treating K562 cells for 6 hours,the methylcellulose colony forming assay was used to study the effects of HHT, IDA and HHT combined with IDA on the colony formation of K562 cells.5. We retrospectively analyzed the difference of the clinical efficacy and safety of elderly AML between CHID regimen(HHT combine with IDA) group and the control group(with other regimens).Result:1.The effects of different concentrations of HHT, IDA and HHT+IDA combined effects on the proliferation of K562 cells0.005~5.12μmol/L HHT could inhibit the proliferation of K562 cells,which was in a concentration dependent manner(P < 0.05). The HHT IC50 concentration of K562 cells is 0.09μmol/L. 0.002~31.25μg/ml of IDA could inhibit the proliferation of K562 cells,which was in a concentration dependent manner(P<0.05).The K562 cells IC50 concentration of IDA is 0.14 μg/ml. HHT combined with IDA could inhibit the proliferation of K562 cells,which was in a concentration dependent manner.(P<0.05).The K562 cells IC50 concentration of HHT combined with IDA is HHT×0.80+IDA×0.80.2. The effects of different concentrations of HHT, IDA and HHT combined with IDA on the proliferation of HUVEC cells0.005~5.12μmol/L HHT could inhibit the proliferation of HUVEC cells,which was in a concentration dependent manner(P<0.05). The HHT IC50 concentration of HUVEC cells is 0.42μmol/L. 0.002~31.25μg/ml of IDA could inhibit the proliferation of HUVEC cells,which was in a concentration dependent manner(P<0.05). The IDA IC50 concentration of the HUVEC cells is 0.14 μg/ml. HHT combined with IDA could inhibit the proliferation of HUVEC cells,which was concentration-dependent(P<0.05),and the IC50 concentration of HUVEC cells is HHT×1.41 + IDA×1.41.3. The effect on apoptosis about different concentrations of HHT, IDA and HHT combined with IDA treating on K562 cells HHT could induce the K562 cells apoptosis,which was not in a concentration dependent manner(P > 0.05). After treating K562 cells with HHT × 1(IC50concentration)with the prolongation of time, the apoptosis of K562 cells increased.After treated with HHT for 24 hours,the apoptosis of K562 cells are 13.9%(P<0.05).High concentration of IDA could lead to the K562 cells necrosis largely(account for 87.5%).Low concentration of IDA could induce the K562 cells apoptosis,which was in a time-dependent manner(P < 0.05), but not in a concentration-dependent manner.HHT and IDA could induce the K562 cells apoptosis,which wasin dose-dependent and time-dependent manner(P<0.05).Treating K562 cells with IDA×0.001(0.001×IC50 concentration),the early apoptosis of K562 cells increased in a time-dependent manner,and the early apoptosis at 24 hours increased to 15.33%.Compared to the low concentration of IDA, the early apoptosis of K562 cells could up to 26.14% after treating with HHT×1 for 24 hours.4.The effect of different concentrations of HHT, IDA and HHT combined with IDA on the colony formation of K562 cells Compared with the negative group, the different concentrations of HHT, IDA and HHT combined with IDA could reduce the colony formation of K562 cells. With the increasing of HHT,IDA and HHT combined with IDA concentration, the number of colony of K562 cells decreases.At the same concentration,the difference between HHT,IDA and HHT combined with IDA groups showed no significant(P > 0.05).5. The effect of different concentrations of HHT, IDA and HHT combined with IDA on the m RNA gene expression in K562 cells The K562 cells in negative group does not express the P53 gene. The K562 cells in HHT×1(IC50 concentration) group does not express the P53 gene m RNA too.The P53 m RNA expression level of IDA×1(IC50 concentration) group is lightly higher than that of the negative group, but the P53 m RNA expression level of HHT combined with IDA group significantly increased(P<0.05).6.Clinical data14 cases of the all 16 patients treated with CHID regimen achieved complete remission, 2 cases achieved non-remission, the CR rate was 87.50%. Nine cases of 21 patients treated with other regimen achieved complete remission, 2 cases achieved partly remission, and the rest 10 cases were non- remission,the CR rate in controlgroup was 42.86%. The overall survival in CHID regimen was also longer than the control group significantly.The adverse reactions in patients of both CHID group and control group during the chemotherapy time were mainly pancytopenia. All patients of two groups were thrombocytopenia. Except for 1 case in CHID group and 2 cases in control group are not observed agranulocytosis(non-remission and direct discharge), the remain patients were neutropenia. The duration time of neutropenia in CHID group and control group were 8.60 d±4.08 d and 6.45 d±4.65 d repectively(P > 0.05), and the duration time of thrombocytopenia in both group were 6.56 d±4.43 d and 8.05 d±6.70 d repectively(P > 0.05).Other adverse reactions were bleeding, infection, liver damage, cardiac arrhythmias, pericarditis and mouth ulcers.In the CHID group, 7 cases were bleeding with grade Ⅰ,seven cases were with infection in gradeⅠ,one case of gastrointestinal reaction, 1 case of grade Ⅱ liver toxicity and three cases of grade Ⅰ,1 patients with grade Ⅳarrhythmia, 1 case of pericarditis and two cases of gradeⅡoral ulcers.In the control group,5 cases were bleeding with grade Ⅰ,and 2 patients with grade Ⅳbleeding, infection in 5 cases of grade I and 1 cases of grade II, one case of gradeⅠand 1 case of grade Ⅳ arrhythmia,one case of gradeⅠpericarditis.The two groups are not observed with kidneys and nervous system damage.Conclusion:1. HHT, IDA and HHT combined with IDA can reduce the proliferation of K562 cells and HUVEC cells, which are in a concentration-dependent manner.2. HHT can induce the K562 cells apoptosis, which is in a time dependent manner, but not in a concentration dependent, and HHT significantly promote the low concentration of IDA to induce the K562 cells apoptosis.3. HHT, IDA and HHT combined with IDA can be reduced K562 cell colony formationability in a concentration dependent manner.4. The combined effect of HHT and IDA can promote the expression of P53 m RNA in K562 cells.5.Compared to other programs group,the complete remission rate of newlydiagnosed and recurrent patients in CHID regimen group is significantly higher. In addition, the toxicity of the CHID regimen can be tolerated,and the CHID regimen can effectively prolong survival in elderly patients with AML.