| ObjectiveLeukemic stem cells (LSCs) possess the abilities of unlimited self-renewal, highly proliferation and multipotential differentiation. The overwhelming majority of conventional chemotherapy agents are inefficient or ineffective to LSC which are mainly located at GO phase of cell cycle, and the cells are able to survive chemotherapy and become the key of drug-resistance and relapse in leukemia. In this study, we investigate whether IFN-α is capable of activating and enforcing the quiescent LSC to enter the active cell cycle and improves the sensitivity to anticancer drug adriamycin, and the feasibility of curing leukemia with a combining strategy of IFN-a and conventional chemotherapeutic agents via efficiently eliminating the queiscent/dormant LSC is discussed.MethodsThe multidrug-resistant leukemia K562/ADM cells and its parental drug-sensitive K562cells were used as the model cells, and the LSCs were sorted separately from the2cell populations with the magnetic activated cell sorting (MACS) method. The morphological and ultra-structural changes of the cells were observed by using optical microscope and transmission electron microscopy. The survival of LSC was measured with trypan blue staining, and an MTT assay was used to detect the proliferating activity of the cells. Flow cytometry (FCM) was employed to deteminate the purity and relative proportion of the LSC, the distribution of cell cycle, the cellular apoptosis, the cellular apoptosis and expression of Ki-67protein. The expression of P53gene mRNA were analyzed by real-time quantative reverse transcription-polym erase chain reaction(RT-PCR). Results1Sorting and characteristics of LSCThe LSCs marked with CD34+CD38-CD123+phenotypes, sorted from K562cells and K562/ADM cells with MACS, were provided with the purity, viability and G0/G1phase cell proportion of above90%. Compared to the un-sorted K562and K562/ADM population cells, the LSCs displayed the morphological feature of smaller volume, larger nucleus/cytoplasm ratio, compact chromatin and heterochromatin, and like lymphocytes. The morphology of sorted LSCs with MACS was in line with the characteristics of stem cells, and the cells were in a relatively inactive status.2IFN-α enhances the proliferation-inhibiting and apoptosis-inducing effects of adriamycinCombination of IFN-a with adriamycin significantly augmented the proliferation-inhibiting and apoptotic effects of adriamycin on K562cells and K562/ADM cells, and the apoptosis-inducing effects of IFN-a combined with adriamycin on the cells by AnnexinV/PI staining are more powerful than that of adriamycin alone. The augumention of IFN-a on the effects of adriamycin to K562/ADM cells, was more effective than that to K562cells. The results suggested that IFN-a possessed the potential to improve the sensitivity of K562cells and K562/ADM cells to adriamycin.IFN-a significantly augmented the sensitivity of LSCs to adriamycin. Combination of IFN-a with adriamycin markedly enhanced the apoptosis-inducing effects of adriamycin to LSCK562cells, LSCs from K562population, and LSCK562/ADM cells, LSCs from K562/ADM population. Treated with IFN-a and adriamycin, The numbers of apoptotic LSCs(Annexin-V+LSC) in K562cell population, compared to adriamycin alone, increased by about25%, and the sensitivity of sorted LSCK562cells to adriamycin increased by about20%. In the same way, IFN-a also significantly augment the apoptosis-sensitivity of the LSCs in K562/ADM cell population and the sorted LSCK562/ADM cells to adriamycin. Thess indicated that IFN-a possesses the potential to improve the sensitivity of drug-resistant LSCK562cells and LSCK562/ADM cells to adriamycin by driving the cells to enter the cell division cycle. 3IFN-α regulates the cell-cycle distribution and the proliferating state of the leukemia stem cellsTreated with the different doses of IFN-α, there was a slight decrase of the cells in G0/G1phase, and a slight increase in S phase and Ki-67+cells in K562population. But IFN-α obviously the cell-cycle distribution and Ki-67protein expression of K562/ADM cells, the cells decreased in G0/G1phase and increased in S phase. The Ki-67positive cells in K562/ADM population rised significantly.After the treatment with1×105IU/ml of IFN-α, the cells in G0/G1phase of LSCK562cells decreased and that in S phase increased markedly, the proportion of Ki-67+cells in LSCK562cells was1.6folds higher over untreated LSCK562cells. The LSCK562/ADM cells, derived from the K562/ADM population, revealed an obvious decrease in G0/G1phase and an increase in S phase after IFN-α adminstration. The Ki-67+cell number in IFN-α treated LSCK562/ADM cells was2.2folds higher than that in untreated LSCK562/ADM cells.4IFN-α inhibits the expression of P53geneThe LSCK562cells from K562population and LSCK562/ADM cells from K562/ADM population were treated with IFN-α alone or joined adriamycin for48h, the expression of P53gene mRNA in LSCK562cells reduced to43%,33%and9%of control cells, respectively, and that in LSCK562/ADM cells dropped to21%,24%and61%of control, respectively.ConclusionIFN-α regulates the expression of P53gene the cell-cycle distribution to activate the exit of the leukemia stem cells, sorted from drug-resistant K562/ADM cell and the parental K562cell populations, out of the queiscence or dormance and entrance of an active cell cycle to improve the sensitivity to anti-proliferative chemotherapeutic agent Adriamycin, and IFN-α is more efficient to K562/ADM cells than K562cells. |
PDF Full Text Request