The Effect And Molecular Mechanism For Artesunate On The CD34～+ Cell Separated From K562 Cell Line

ObjectiveChronic myeloid leukemia(CML)originated from the proliferation of a magligant hematopoietic stem cell colon. Until now,tradional chemotherapy and radiotherapy are still the main therapies in leukemic treatment. But these remedies have many side effects, and the ratio of relapse is also very high. What's more , it greatly damages the immunity and hematopoiesis. Finding natural effective ingredients extracted from Chinese tradional medicine , which has the effect inducing apoptosis of tumor cells and little side effects, has become the new hope of leukemic therapy.Artemisinin extracted from Chinese tradional medicine has specific sesquiterpene lactone endoperoxide. Being one of the derivatives, artesunate has been used for a long time as effective antimalarial drug. Recent materials show that artesunate has effect on several kinds of cancer cell line including K562 cell line.But these studies still remain four main questions as follows. Firstly, they only focus on the levels of cancer cell lines rather than cancer stem cells(CSC). Secondly, the effort of artesunate for the leukemic model mouse has not been reported. Thirdly, there are very few materials about the toxicity of the drug. Last, the anti-tumor of molecular mechanism for artesunate is not very clear.Thus, in the study, we investigated the effect of artesunate on the CD34+leukemic stem cell-like cell separated from K562 cell line and the CD34+HSC/HPC from umbilical cord blood, the effect on the leukemic model mouse, the effect on the bcr-abl mRNA, Bcl-XL mRNA. Our purpose is to clarify the effect and the toxicity of artesunate, the anti-tumor of molecular mechanism and to provide the theoretical basis and the experimental evidence of its clinic application.Methods1. The separation of CD34+cells from K562cell line was established by magnetic activated cell corting(MACS) according to the strategy of positive selection. These technologies as identification were used including morphology, the flow cytomery, immunocytochemistry, CFCs, RT-PCR. Using the technique of single-cell clone, the CD34+cells from K562cell line were cultured in IMDM culture medium and growth factors.2. The leukemic animal model (SCID mouse) was successfully copied by transplanting the CD34+cells from K562cell line. The effect of the drug was studied by comparing the sickness, the period of survival, the inhibition rate of the tumor, the peripheral blood ,the bone marrow, and the expression level of bcr-abl gene of tumor cells.3. The CD34+cells from K562cell line and the CD34+ hematopoietic stem/progenitor cell (HSC/HPC)from umbilical cord blood were cultured together with series concentrations of AST.The effect on the capabilities of proliferation and multilineage differentiation was studied by methylcellulose assay systems. The apoptosis was examined with morphology, TUNNEL, the flow cytomery stained by anti Annexin V-FITC and PI. The differentiation was studied by immunocytochemistry.4.The apoptosic molecular mechanism of CD34+cells from K562cell line induced by artesunate was analyzed through the expression of bcr-abl gene and Bcl-XL by RT-PCR. Results1. Separation, purification and identification of CD34+cells from K562cell lineThe separated CD34+cells from K562cell line had biological characteristics of lukemic stem cell-like cell such as keeping the same percentage of CD34, the capabilities of proliferation and multilineage differentiation, more G0/G1cells, strong tumorgenicity and possessed more P–gp.2. Establishment and identification of a transplantable lukemic SCID mouse modelThe mouse model bearing leukemia could be established by inoculating CD34+cells from K562cell line into SCID mouse. Compared with the common K562cell, CD34+cells from K562cell line owned stronger tumorgenicity.3. The effect of the CD34+cells from K562cell line induced by artesunate in vitro3.1The numbers of CFC were markly decreased by artesunate within dose range of 12.5μg /ml～50μg/ml .The ratio of inhibitory of CFC was 100% induced by 50μg /ml artesunate.3.2Artesunate had a significant apoptosis inducing effects on CD34+cells from K562cell line in both time-and dose dependent manner.3.3 Artesunate might induce CD34+cells from K562cell line to differeniate toward granulocyte.4.The effect of artesunate on transpianting leukemic animal model (SCID mouse)4.1After injection of AST to leukemic animal model, the common condition of model SCID mouse was more improved than that of the control group, the weight was higher than that of the control group, surviving time significantly prolonged.4.2 The number peripheral blood cells was significantly decreased after treatment for 2 weeks.4.3The tumor weight of treated group was also lower than that of untreated group, and the inhibition rate was 37.85% and 48.65%.5. The effect of the CD34+ hematopoietic stem/progenitor cell from umbilical cord blood induced by artesunate5.1 Artesunate, however, within dose range of 50μg /ml,had little inhibitory effects on the CD 34+HSC/HPC.The numbers of CFC were not also affected.5.2 Artesunate within dose range of 50μg /ml,had not apoptosis inducing effects on hematopoietic stem/progenitor cells.6. The primary study of apoptosic molecular mechanism of CD34+cells from K562cell line induced by artesunate The expression of bcr-abl mRNA and Bcl-XL mRNA could be down-regulated in a dose-response manner induced by AST.Conclusion1.The experiments in vitro and vivo show that CD34+cells from K562 cell line have biological characteristics of lukemic stem cell-like cell.2. Artesunate has the effect on the inhibition of the cellular growth, on the apoptosis inducing and induce CD34+cells from K562cell line to differeniate toward granulocyte.3. Artesunate has the effect on the leukemic animal model (SCID mouse) which was established by transplanting CD34+cells from K562cell line.4. Artesunate less than the dose range of 50ug/ml,had little inhibitory effects and apoptosis inducing effects on the CD34+ HSC/HPC from umbilical cord blood .The numbers of CFC were not also affected. 5. The mechanism of the anti-leukemia effect of AST was might be related to downregulated bcr-abl mRNA ,BCL-XLmRNA and thus induced apoptosis.