Studies Of Radix Saposhnikoviae Extract Combined With ATO On Acute And Chronic Myelogenous Leukemia In Vivo And In Vitro

ObjectivesHuman acute myelogenous leukemia (AML) cell line HL-60 cells and human chronic myelogenous leukemia (CML) cell line K562 were selected in the study. To evaluate the effect of the Radix Saposhnikoviae extract alone or in combination of arsenic trioxide (ATO) in human acute myelogenous leukemia HL-60 cells and chronic myelogenous leukemia K562 cells and investigated the possible mechanism. Cellular viability was measured by MTT assay, cell cycle distribution and apoptosis were monitored by flow cytometry (FCM). As well to verify its increasing efficacy and decreasing toxicity of ATO in vivo, we established the NOD/SCID mice model suffering from the human AML.Methods1. Effects of Radix Saposhnikoviae extract on proliferation in HL-60 and K562 cells.After exposed to Radix Saposhnikoviae extract in different concentrations (1500、1250、1000、750、500、250μg/ml), the proliferation of Radix Saposhnikoviae extract was detected by MTT experiment and the 50 percent of inhibition concentration was calculated.2. Effects of Radix Saposhnikoviae extract in combination with ATO on proliferation and apoptosis in HL-60 and K562 cells.The cells were divided to four groups:control group, Radix Saposhnikoviae extract treated group, ATO treated group and Radix Saposhnikoviae extract+ATO group. Cellular proliferation was measured by MTT assay, cell cycle distribution and apoptosis were monitored by flow cytometry.3. Establish the NOD/SCID mice model suffering from human AML.NOD/SCID mice of 3～4 weeks old were raised for one week and then they were divided to 7 groups randomly which were numbered from A to G. HL-60 cells were injected into NOD/SCID mice via a tail vein (A, D:1×107; B, E:5×106; C, F:3 ×106), the mice of group A B C were irradiated with 300 cGy (60Co source) 24h earlier. The group of F is normal control group. General condition and the growth of tumor in shallow parts of mice were observed daily. The HL-60 cells which expressed CD33 of blood, liver, spleen tissues and bone marrow cells detected by flow cytometry analysis and the infiltrated organization observed by HE staining were used to identify whether the model is successfully established.4. Investigate Radix Saposhnikoviae extract could potentiate ATO activity against leukemia and decrease the toxicity of ATO by establishing the NOD/SCID mice model suffering from human AML.HL-60 cells (1×107) were injected into 4-5 weeks old NOD/SCID mice via a tail vein. Peripheral blood cell morphology was examined every week, the HL-60 cells of peripheral blood were detected by flow cytometry analysis. The NOD/SCID mice models were randomly divided into four groups:control group, Radix Saposhnikoviae extract treated group (500 mg/kg, i.g.×15d), ATO treated group (3 mg/kg, i.p. Qd X 15d), Radix Saposhnikoviae extract+ATO treated group (500 mg/kg Radix Saposhnikoviae extract i.g.+3 mg/kg ATO i.p. Qd×15d) to receive treatment for 15d after 4 weeks of leukemia cells injection. The changes of common condition and the average survival time were observed. At the end of the experiment,3 mice of every group were killed by the institutionally approved method. The damages of heart, liver, spleen, lung and kidney were examined by HE staining and microscopy.Results1. Radix Saposhnikoviae extract had inhibitory effect on K562 and HL-60 cells750,1000,1250,1500μg/ml Radix Saposhnikoviae extract had a significantly inhibitory effect to K562 cells in dose-dependent manner. OD values of 48 hours had significant difference compared with control group (P<0.05). Radix Saposhnikoviae extract of six concentrations had a significantly inhibitory effect to HL-60 cells in dose-dependent manner. OD values of 48 hours had significant difference compared with control group (P<0.05).2. Radix Saposhnikoviae extract potentiated ATO inhibiting proliferation abilityCombination treatment of Radix Saposhnikoviae extract with ATO had synergistic effects on the proliferative inhibition rate K562 and HL-60 cells. This synergistic effects showed a dose-dependent manner. The apoptosis rates of K562 cells were increased gradually after treatment of 500,250μg/ml Radix Saposhnikoviae extract with 2,1μg/ml ATO respectively, which was higher than control group or those treated by ATO alone (P<0.05). The apoptosis rates of HL-60 cells were increased gradually after treatment of 500,250μg/ml Radix Saposhnikoviae extract with 1,0.5μg/ml ATO respectively, which was higher than control group or those treated by ATO alone (P<0.05). The number of cells in G2-M phase of K562 and HL-60 increased, cells in G0-G1 phase of K562 and S phase of HL-60 decreased and cells in G0-G1 phase of HL-60 increased compared with control group after treatment of 2,1,0.5μg/ml ATO for 48h. The number of cells in S phase of K562 and the apoptosis ratio of HL-60 increased after treatment of Radix Saposhnikoviae extract combination with ATO compared with control group or those treated by ATO alone (P<0.05) and the effects is related to the dosage. The cell cycle of HL-60 after treatment of Radix Saposhnikoviae extract combination with ATO showed no regular change.3. The NOD/SCID-AML mice model were well established by the mentioned ways.About 10 days after inoculation, HL-60 cells can be detected in peripheral blood of NOD/SCID mice model. About 28 days after inoculation, hindlimbs paralysis were observed and solid tumors were found in shallow parts a week later. A large number of leukemia cells appeared in peripheral blood, bone marrow and tissues of liver, spleen and transplanted tumor demonstrated that NOD/SCID mice model suffering from human AML had already established. The mice began to die about 40 days after inoculation. Mice irradiated by 60Co were dead in 52d and average survival time was (39.33 ± 12.66) d. The rest of mice model were dead in 64d and average survival time was (55.67±10.41) d. The morbidity was 100%.4. The combined group prolonged the survival time of NOD/SCID-AML mice.There were significant differences between control group and the combination group (P<0.05). The Radix Saposhnikoviae extract group and the ATO group had no significant differences with the control group. The prolonged rates of survival time were 36% in ATO group,37.3% in Radix Saposhnikoviae extract group and 51.8% in combined treated group respectively. Pathological section showed that the damages of main organs relieved on different levels especially for the kidney.Conclusion1. Radix Saposhnikoviae extract has inhibitory effect on K.562 and HL-60 cells in a dose-dependent manner.2. Radix Saposhnikoviae extract potentiates ATO inhibiting proliferation ability in K562 cells probably by synergistically inducing cell apoptosis of ATO or through S phase arrest. Radix Saposhnikoviae extract potentiates ATO inhibiting proliferation ability in HL-60 cells probably by synergistically inducing cell apoptosis of ATO.3. Injecting HL-60 cells (3×106～1×107) into 4-5 weeks old NOD/SCID mice can successfully establish NOD/SCID mice-human AML model which has similar symptoms and infiltration of human being. NOD/SCID mouse-human leukemia model is an important and useful tool for studies on proliferation, differentiation and modulation of leukemic cells, as well as studies on screening and evaluation of anti-leukemia drugs.4. Radix Saposhnikoviae extract can potentiate ATO anticancer ability in NOD/SCID mice suffering from AML, relieve the damage in main organs.