| Part I:In Vitro Investigation of Mechanism of Modified CAG in Combination with Decitabine Therapy on Acute Myeloid LeukemiaObjective:To explore the anti-leukemia mechanism of modified CAG combined decitabine on leukemia cell lines U937 and Kasumi-1 by detecting cell proliferation, apoptosis, differentiation, the proportion of S phase and the expression levels of c-myc, Bcl-2, caspase-3 and caspase-9 proteins, respectively.Methods:Human acute myeloid leukemia cell lines U937 and Kasumi-1 were intervened by decitabine alone (DAC group), CAG alone (CAG group), pre-treatment decitabine in combination with CAG (D-CAG group) or post-treatment decitabine in combination with CAG (CAG-D group). We detected the proliferation of U937 and Kasumi-1 cells in different administration groups with CCK-8 assay. The cell apoptosis marker of, Annexin V/PI, the cell membrane surface antigen CDllb and the cell cycle of U937 and Kasumi-1 cells in different groups were detected by flow cytometry. The expression of proteins such as c-myc, Bcl-2, caspase-3 and caspase-9 were analyzed respectively by Western blotting.Results:1. The cell variability of DAC group, CAG group, D-CAG group and CAG-D group compared with control group in U937 were 35.3±6.6%、52.0±4.3%,16.0±1.0%、 36.8±14.6%, respectively. The cell variability rate was significantly inhibited in D-CAG group compared with other four groups (P<0.05). In Kasumi-1 cells, the cell variability of DAC group, CAG group, D-CAG group and CAG-D group compared with control group were 87.0±3.6%、37.0±1.0%、33.0±2.0%,33.0±3.6%, respectively. There were no statistical significance between CAG group and CAG combined decitabine group.2. In U937 cells, the apoptosis rates of control group, DAC group, CAG group, D-CAG group and CAG-D group were 2.7±1.6%,24.3±6.4%,16.7±11.7%,65.7±17.4%, 24.7±2.9%, respectively. The apoptosis rate was significantly increased in D-CAG group compared with other four groups (P<0.05). In Kasumi-1 cells, the apoptosis rates of control group, DAC group, CAG group, D-CAG group and CAG-D group were 6.0±0.5%, 20.3±2.2%,36.2±0.67%,45±0.9%,42.4±1.5%, respectively. The apoptosis rate were significantly increased in D-CAG group and CAG-D group (P<0.05).3. In U937 cells, the expression rate of cell-differentiation antigen CD11b in control group, DAC group, CAG group, D-CAG group and CAG-D group were 1.1±.1%,4.2±2.5%= 76.3±0.3%、2.7±2.0%、22.9±7.2%, respectively. The expression rate of CD11b was significantly increased in CAG group, then the CAG-D group, compared with other four groups (P< 0.05); however, the similar trend was not found in Kasumi-1 cells.4. In U937 cells, the cell proportion of S phase in control group, DAC group, CAG group, D-CAG group and CAG-D group were 43.0±3.6%,42.7±5.5%,61.7±6.7%,52.3±7.5%, 23.3±6.1%, respectively. The cell proportion of G2/M phase in each group were 7.0±2.4%= 10.5±5.6%,26.1±5.9%,9.0±2.2%,54.7±10.6%, respectively. The cell proportion of S phase was significantly increased in CAG group and significantly decreased in CAG-D group (P<0.05). In Kasumi-1 cells, the cell proportion of S phase in control group, DAC group, CAG group, D-CAG group, CAG-D group were 43.7±2.9%,41.7±0.6%, 32.0±2.0%.28.7±5.7%,34.7±1.5%, respectively. The cell proportion of G2/M phase in each group were 7.8±0.1%、21.8±2.9%,9.9±4.2%、11.2±5.5%、9.7±1.4%, respectively. The cell proportion of S phase was significantly decreased in CAG group, D-CAG group and CAG-D group compared with control group and DAC group (P< 0.05), but there were no statistically difference between the above three groups. There was no statistically difference in each group except DAC group in G2/M phase.5. In U937 and Kasumi-1 cells, the protein expression of c-myc and Bcl-2 were down-regulated and the apoptosis-related proteins caspase-3 and caspase-9 were up-regulated in D-CAG group and CAG-D group.Conclusion:1. Low-dose decitabine combined CAG have a synergistic effect on the suppression of cell proliferation in U937 cell line, especially in D-CAG group. The same phenomenon was not found in Kasumi-1 cell.2. CAG can induce cell differentiation obviously, then the CAG-D group in U937 cells. The same phenomenon was not found in Kasumi-1.3. Low-dose decitabine combined CAG (especially in D-CAG group) have a synergistic effect on induction of cell apoptosis in U937 and Kasumi-1 cell lines.4. Due to the high sensitivity to decitabine in U937 cells, CAG treatment increased the S phase of cell proportion significantly, then followed by decitabine administration decreased the ratio of S phase. The proportion of G2/M phase was also increased compared with D-CAG group. The similar phenomenon was not found in Kasumi-1.5. The synergistic suppressive effect on cell proliferation and induction of cell apoptosis in CAG combined decitabine group might be related to down-regulation of c-myc and Bcl-2 proteins and up-regulation of caspse-3 and caspase-9 proteins.Part II:Efficacy and safety of adjusted CAG in combination with decitabine in older acute myeloid leukemiaObjective:To evaluate the efficacy and safety of a novel induction chemotherapy using adjusted CAG regimen (low dose cytarabine and aclarubicin in combination with colony stimulating factor) for priming firstly, following by decitabine (DAC) (Abbreviated as CAG-D regimen) when administered in elderly (= 60 years) patients with acute myeloid leukemia (AML).Methods:Patients who aged>60 years with newly diagnosed or relapsed/refractory AML were treated with one or two courses of aCAG-D regimen. Adverse reactions and the recovery time of bone marrow were investigated. Complete remission rate, overall survival and disease free survivals were recorded respectively, to evaluate the efficacy of the treatment.Results:The median age of the 12 patients (seven men and five women) available for evaluation, which consisted of nine newly-diagnosed patients and three refractory/relapsed patients, was 67 years old (range of 60-75 years). A total of nine patients (75.0%) achieved complete remission (CR) after the aCAG-D treatment, in eight of whom achieved CR after only one treatment cycle. In nine newly-diagnosed patients, eight cases achieved CR (88.9%) and another got partial remission (PR). Meanwhile, three other refractory/relapsed patients achieved CR, PR, no response (NR), respectively. In four patients who were classified in M5 subtypes, three cases achieved CR and another PR. The overall response rate (ORR) and CR after two treatment cycles were 91.6% and 75.0%, respectively. In patients with CR, the median time was 24.2 days (range of 16-43 days) for granulocyte recovery and 17.9 days (range of 11-37 days) for platelet recovery. After a median follow-up of 11 months (range of 4.52 months), one patient died due to secondary infections; one patient showed a relapse; five patients died due to progression of the disease; five patients maintained CR. The median overall survival (OS) was 13 months (range of 4.5-22 months) for those who achieved CR, and was significantly longer than that of patients who did not achieved CR (5 months) (P< 0.05). The main adverse events were grade IV bone marrow suppression (12/12 patients) and grade I-IV secondary infection (11/12 patients). There was no treatment-related mortality during induction therapy period.Conclusion:The regimen combined aCAG and decitabine demonstrated superior efficacy for elderly AML patients, especially in newly-diagnosed patients. At the same time, incidence of bone marrow suppression and secondary infection increased, thus more supported care should been given. In general, aCAG-D regimen was well tolerated by the elderly patients with newly-diagnosed or relapsed/refractory AML and showed promising clinical efficacy.Conclusion of the whole paperIt is the mechanism of CAG combined decitabine that mainly inhibited cell proliferation and promoted cell apoptosis in leukemia cell lines, especially in pre-treatment decitabine in combination with CAG.CAG followed by decitabine can enhance the efficacy of targeted leukemia cells of S phase and partly remained cell-differentiation ability. Decitabine increased cytotoxicity partly through activation of caspase-9 and caspase-3 and inhibition of c-Myc and bcl-2. It is concluded that aCAG-D regimen might be effective with tolerable adverse effects for elderly patients with newly-diagnosed or relapsed/refractory AML patients and worth to further clinical study. |
PDF Full Text Request