In Vitro Cell Growth Inhibition Induced By Artesunate In Myelodysplastic Syndrome And Its Underling Mechanisms

Objectives:The myelodysplastic syndrome (MDS) includes a group of incurable heterogeneous hematologic malignant diseases characterized by ineffective haematopoiesis and having the tendency to transform to acute myeloid leukaemia (AML). Patients with MDS are often the aged ones. Because the elder are unable to withstand intensive chemotherapy and bone marrow transplantation, treatment of MDS in elderly patients is difficult and the effect is limited. High-risk MDS is characterized by unregulated proliferation and apoptosis of hematopoietic cells in the bone marrow with cytopenia in peripheral blood and high tendency to progress to AML. On the other hand, MDS patients have impaired activity of bone marrow stromal cells which reduced the hematopoietic recovery ability after MDS patients accepting chemotherapy or hematopoietic stem cell transplantation.In the past decade, two DNMT inhibitors,5-aza-cytidine (Aza) and5-aza-2’-deoxycytidine (DAC), confirmed by the FDA for MDS treatment have been applied into clinical treatment, and led to complete and partial responses in10%-20%, transfusion independence in approximately50%. However, due to the non-targeted cytotoxicity, potential carcinogenicity, drug-resistant development as well as the expensive treatment costs, the usage of Aza and DAC was confined to some extent. So there is an urgent necessary in searching for safe, effective and affordable agents for improving the survival and transfution dependence for patients with High-risk MDS, who can not undergo allogeneic stem cell transplantation.Artesunate (ART), a semi-synthetic derivative of artemisinin extracted from the Chinese herb Artemisia annua, is a safe and effective antimalarial drug. Recently, several studies showed that ART could inhibit the growth of various carcinoma cell lines by inducing programmed cell death such as apoptosis and necrosis. The low dose ART is safe to normal cells, while the high dose ART show embryotoxicity and reversible kidney damage and bone abnormalities in animal experiments. However, it is not known whether artesunate can exert theraputic effect on the high-risk MDS cells. Therefore, in the present study, High-risk MDS cell line, SKM-1cells and primary blast cells from high-risk MDS patients were used to evaluate the growth inhibition effect of ART on high-risk MDS cells and to investigate the underlying mechanisms.Because some previous clinical and experimental studies have shown that epigenetic abnormalities play a key role in MDS genesis, development and progression to AML, so epigenetic abnormalities are very promising targets in MDS treatment. The expression level of DNMT1, which plays an important role in maintaining aberrant methylation transcription level, was found significantly higher in AML and MDS than that in normal control. So the expression levels of DNMT1mRNA, protein and its related genes in SKM-1cell were detected respectively before and after ART exposure in order to provide laboratory evidence for the usage of ART in MDS treatment.Methods:1After being treated with ART (1,5,10μmol/L), the viability of SKM-1cells and the primary blast cells from high-risk MDS patients were detected by CCK-8assay. The cells were divided into different group:the blank control, solvent control, ART treatment group, Trolox+ART group, and Ac-DEVD-CHO+ART group. The cytotoxicity ART (at final concentrations of1,5,10,100and200μmol/L) was also detected in normal control bone marrow mononuclear cells, bone marrow stromal cells. Positive synergies effect of DAC and ART design by factorial analysis was evaluated by CCK-8kit and then defined by the combination index (CI).2The morphological changes of MDS primary blast cells and SKM-1cells treated with10μmol/L ART for24hours was observed under microscope with Wright-Giemsa staining.3After24h ART exposure, SKM-1cells cycle distribution and sub-diploid peak was detected on flow cytometry by PI staining.4SKM-1cell MMP changes after one hour ART (at final concentration of1,5,10μmol/L) treatment, and10μmol/L ART for0.5h, lh,3h,6h,12h,24h, were detected by JC-1fluorescent dye staining.5After treated with1,5,10μmol/L ART for6hours, expression level of caspase-8, caspase-9, the activity of caspase-3, PARP, bid, AIF was detected by western blot (WB), at the same time, apoptosis regulatory proteins such as BCL-2, bax, Bad, P-Bad, survivin, XIAP were also detected. More over, as the main signal proteins in PI3K/AKT signal pathway, which is the cell survival pathway, the expression level of Pten, Akt, P-Akt (Ser473) and P-Akt (Thr308) was detected by WB in the first part of this study. In the second part, SKM-1cells, treated by1,5,10μmol/L for6h or10μmol/L ART for3h,6h,12h, were collected to detect and the expression level of DNMT1protein was also measured by WB.6After treated by10μmol/L ART for0.5,1and3hours respectively, the calcium concentration in SKM-1cells were detected by Fluo-3-Am fluorescent probe.7After treated by10μmol/L ART for0.5,3,6,12and24hours respectively, the ROS concentration in SKM-1cells were detected by DCFH-DA fluorescent probe.8After treated by10μmol/L ART for6hours, AIF nuclear translocation in SKM-1cells was detected by immuno-fluorescence staining.9DNMT1mRNA expression in SKM-1cells were detected by RT-PCR. The cultured cells were collected separately after the exposure to1,5,10μmol/L ART for6h or10μmol/L ART for3h,6h,12h. The solvent control and blank control were used as quality control.10The mRNA expression level of p15INK4B in SKM-1cells which had been treated with1,5,10μmol/L ART, was detected by real-time fluorescence quantitative PCR. The PBS group and80μmol/L DAC group were used as solvent control and positive control respectively.11The p15INK4B promoter methylation status in SKM-1cells after treated by10μmol/L ART or80μmol/L DAC for12h were evaluated by methylation-specific PCR.12Apoptosis rate in SKM-1cells treated by DAC or ART or the combination of both were detected on flow cytometry by Annexin V/PI staining.Result:1ART inhibited the growth of MDS cells in a time-dependent and dose-dependent manner. In view of ART IC50at24h,48h and72h is15.41μmol/L,3.43μmol/L and1.92μmol/L, respectively, MDS cell was sensitive to ART.48h’s IC50of MDS primary blast cells was36.05μmol/L,118μmol/L,34.72μmol/L, respectively; while the48h’s IC50of normal control bone marrow mononuclear cells was195.14μmol/L,531.88μmol/L, and1683.96μmol/L, respectively;48h’s IC50of normal control bone marrow stromal cells was227.026μmol/L,154.03μmol/L,212.49μmol/L respectively. The antioxidant trolox pretreatment decreased the growth inhibition effect of ART in SKM-1cells. Caspase3,7inhibitor Ac-DEVD-CHO partially reduced the growth inhibition effect of ART in SKM-1cells.2Wright-Giemsa staining showed ART could induce apoptosis by the appearing of apoptotic bodies, nuclear fragmentation in MDS cells.3After treated by1,5,10μmol/L ART for24hours, the SKM-1cell cycle was arrested at G0/G1phase with increase in sub-diploid peak in a dose-dependent manner (p<0.005).4After6hours treatment with1,5,10μmol/L ART, SKM-1cell lysates was examed by WB. AIF translocated into the nucleus in dose-dependent manner. The change of AIF localization has positive correlation with ART dosage (p<0.005), the Pearson correlation coefficient=0.740, P=0.003. ART at high-dose activated csapase-9via the endogenous pathway, followed by the activation of caspase3and then PARP was cut into an active form in dose-dependent manner (p<0.005). The change in caspase-9protein expression level was slightly negative correlated with ART dosage (the Pearson correlation coefficient=-0.656, P=0.010). Active caspase-3and PARP were slightly positive correlated with ART dosage. The Pearson correlation coefficient were0.553,0.614respectively (P=0.031,0.017, respectively). Caspase-8and bid in SKM-1cells after treated by1,5,10μmol/L ART did not change significantly (p>0.05). AIF nuclear translocation in SKM-1cells after6h ART(10μmol/L) exposure was clearly shown by laser scanning confocal microscopy with indirect immuno-fluorescence staining.5MMP in SKM-1cells after1hour ART (1,5,10μmol/L) exposure decreased in dose-dependent manner, F=230.12, P<0.005(ANOVA); MMP in SKM-1cells after10μmol/L ART (0.5,1,3,6,12,24hours) exposure also declined in time-dependent manner (Dunnett T3, P<0.05, the Pearson correlation analysis, r=0.702, p=0.011).6Intracellular Calcium ion increased in SKM-1cells after treated by10μmol/L ART for0.5h, and this change continued to6h when comparing with the control (ANOVA F=274.848,P<0.005). Intracellular ROS level in SKM-1cells after10μmol/L ART exposure increased at6h, and this change continued to12h when comparing with the control (ANOVA, F=255.266, P <0.005). But Intracellular ROS level in SKM-1cells were not significantly different when compared with the control group at0.5h,3h and24h (P>0.05).7After treated by1,5,10umol/L ART for6h, SKM-1cells lysis were detected by WB. Bad protein expression increased, but the difference compared with the control was not significant,(ANOVA, F=0.598, P=0.639), while the P-Bad protein expression was significantly decreased when compared with the control group(F=22.034, P<0.005). The P-Bad protein expression showed a highly negative correlation with ART dosage (pearson correlation coefficient=-0.909, P<0.005). BCl-2/Bax ratio decreased in dose-dependent manner (control Vs1μmol/L, of ART Vs5μmol/L of ART Vs10μmol/L, of ART,1.775±0.24Vs1.2033±0.01Vs1.1743±0.26Vs0.833±0.02, F=14.23, P<0.005(ANOVA). The BCl-2/Bax ratio was negatively correlated to ART dosage (pearson correlation coefficient=-0.866, P<0.005). XIAP expression decreased, but the difference was not significant (P>0.05). Survivin significantly decreased compared with the control, F 19.172, P=O.001(ANOVA); the decreasing in Survivin highly negatively correlated with ART dosage (the Pearson correlation coefficient=-0.849, P <0.005).8After the SKM-1cells treated by1,5,10μmol/L ART for6h, Pten, Akt, P-Akt (Ser473) and P-Akt (Thr308) which are belonging to PI3K/AKT pathway in the cells lysis were detected by WB. When compared with the control group, Pten increased, the single factor analysis of variance:F=10.003, p=0.004(ANOVA), in dose-dependent manner, the Pearson correlation coefficient=0.873,p<0.005; P-Akt (Ser473) showed a downward trend, F=14.836, p=0.001(ANOVA), in dose-dependent manner, the Pearson correlation coefficient=-0.854, p<0.005; p-Akt (Thr308) showed a downward trend, F=5.851, p=0.021(ANOVA), in dose-dependent manner, the Pearson correlation coefficient=-0.539, p>0.05; Akt showed a statistically significant decrease, F=8.177, p=0.008(ANOVA), but the dose dependence was not significant, the Pearson correlation coefficient=-0.210, p=0.513.9After treated by ART, SKM-1cells total RNA were detected by RT-PCR. DNMT1mRNA expression was down-regulated in dose-dependent and time-dependent manner.10After treated by ART, DNMT1expression in the SKM-1cell lysis was down-regulated in time-dependent but not dose-dependent.11As a target gene of DNMT1, p15INK4B expression increased in SKM-1cells after ART treatment in a dose-dependent manner. After ART exposure, P15INK4B promoter methylation status decreased while the promoter un-methylation status significantly increased.12SKM-1cell apoptosis ratio induced by the combination of ART with DAC was higher than that induced by any agent alone.5μmol/L ART treatment of SKM-1cells showed a significant enhanced sensitivity to apoptosis induced by DAC (1-1600μmol/L), the DAC and ART showed a positive synergistic effect (CI=0.63,0.5,0.28,0.46,0.19,P<0.005). ART (5μmol/L) enhanced apoptosis induced by the DAC (8μmol/L) than any agent alone (P<0.005), Ac-DEVD-CHO pretreatment could not suppress the enhanced apoptosis effect induced by the combination of DAC with ART in SKM-1cells (P<0.005).Conclusion:1ART could inhibit the growth of high-risk MDS cell line SKM-1cells and primary blast cells from high-risk MDS patients. The inhibitory effect of ART on the growth of bone marrow stromal cells and bone marrow mononuclear cells from patients with non blood disease was mild.2ART arrested the cell cycle of SKM-1cells at the G0/G1phase, accompanied with dose-dependent sub-diploid peak.3ART could induce apoptosis in SKM-1cells and primary blast cells from high-risk MDS patients in vitro.4The early event in SKM-1cell damage induced by ART is the increase in intracellular calcium concentration and then the decrease in MMP, followed by intracellular ROS releasing to mediate sustained and severe injury of the cells.5Apoptosis in SKM-1cells induced by ART is mainly initiated by the active endogenous pathway, and then the SKM-1cells undergo apoptosis though caspase dependent and independent pathway.6The decrease in expression level of BCl-2/bax ratio, p-bad and survivin was one of the main mechanisms of apoptosis induced by ART in the level of apoptosis related molecules.7The down regulation of Pten/PI3K/Akt signaling pathway was one of the mechanisms related with apoptosis induced by ART.8ART could inhibit the expression of DNMT1mRNA and protein to restore the expression of p15INK4b in SKM-1cells, have p15INK4b play its tumor suppressor role.9ART enhanced apoptosis in SKM-1cells induced by DAC mainly through caspase-3-independent pathway.