Apoptosis In Human K562 Cells Research Articles

SRPK1‑siRNA suppresses K562 cell growth and induces apoptosis via the PARP‑caspase3 pathway

Serine-arginine protein kinase1 (SRPK1) has been used as an important signal mediator, and is associated with cancer development. However, studies have yet to determine whether SRPK1 suppresses leukemia cell growth and induces apoptosis. Studies have also yet to reveal the underlying mechanisms. In the present study, the effects of downregulating SRPK1 gene expression on chronic myeloid leukemia cell lines (K562cells) were investigated through RNA interference (RNAi) and the proliferation inhibition and apoptosis induction of SRPK1 in K562 cells were analyzed. K562 cells were transfected with two different concentrations of siRNA, and the transfection efficiency was detected via flow cytometry. The expression of SRPK1 was detected via reverse transcription‑quantitative polymerase chain reaction. K562 cell proliferation and apoptosis were analyzed using MTT and flow cytometry respectively. The roles of caspase‑3, poly(ADP‑ribose) polymerase (PARP), p53 and B-cell lymphoma (Bcl)‑2/Bcl‑2 associated X, apoptosis regulator (Bax) proteins in the apoptosis of human K562 cells were further examined through western blot analysis. The SRPK1 expression was lower in the K562cells transfected with SRPK1‑siRNA compared with untransfected cells. The inhibition rate in the transfected groups was increased compared with the untransfected groups. Compared with control groups, the number of apoptotic cells in the SRPK1‑silenced groups increased. The number of early apoptotic cells also increased. The cleaved caspase‑3, cleaved PARP and p53 expression levels were significantly increased in the RNAi groups compared with control groups. Conversely, the Bcl‑2/Bax rate was significantly lower. In conclusion, the knockdown of the SRPK1 gene by RNAi inhibited the proliferation of K562cells and induced their apoptosis. Apoptosis was induced by the activation of the PARP‑caspase3 pathway.

Gnidilatimonoein from Daphne mucronata induced differentiation and apoptosis in leukemia cell lines

Among various treatments, plants play a crucial role in cancer chemotherapy. Base on literature data, different species of the thymeleaeceae family have been found to be good sources for anticancer agents. Therefore, our laboratory has initiated a research project on evaluating the anticancer properties of the Iranian medical plant, Daphne mucronata (Thymeleaeceae). Gnidilatimonoein is a new diterpene ester compound that is purified from Daphne mucronata with antiproliferative and anti-metastatic effects in different tumor cell lines. Based on the inhibitory role of this agent on the cell cycle progression of K562 cell line, we were interested to check gnidilatimonoein for induction of differentiation and apoptosis in different human leukemia ML-1, HL-60 and K562 cells. Cell viability was determined by trypan blue exclusion method. Cell differentiation was studied in terms of morphology, NBT reduction in leukemia cells (HL-60 and ML-1) and benzidine staining in erythroleukemia (K562) cells. Apoptosis was detected by fluorescence microscopy (Acridine orange/Ethedium bromide double staining), annexineV flow cytometry and DNA electrophoresis. After 48 hours of treatment with 10 µM gnidilatimonoein, 80-70% of HL-60 and ML1 cells showed NBT reduction. Indeed gnidilatimonoein induced monocyte differentiation of myeloid leukemia HL-60 and ML-1 cells but not K562 erythroluekemia cells. In this condition, we observed the growth inhibitory effect (60%) without substantial loss of viability (

Sorbitol-induced apoptosis of human leukemia is mediated by caspase activation and cytochrome c release

It has been reported that sorbitol induces apoptosis in several cancer cell lines. However, the molecular mechanism underlying the sorbitol-induced apoptotic process is not yet clearly understood. In the present study, the intracellular signaling pathways of sorbitol-induced apoptosis in human K562 cells were investigated using both morphological analysis and DNA fragmentation technique. In this study, we demonstrated that sorbitol-induced apoptosis in human K562 cells is a concentration- and time-dependent manner. This sorbitol-induced apoptosis in human K562 cells was also accompanied by the up-regulation of Bax, and down-regulation of p-Bcl-2, but no effect on the levels of Bcl-X(L). Moreover, the sorbitol treatment resulted in a significant reduction of mitochondria membrane potential, increase in the release of mitochondrial cytochrome c (cyt c), and activation of caspase 3. Furthermore, treatment with caspase 3 inhibitor (z-DEVD-fmk) was capable of preventing the sorbitol-induced caspase 3 activity and cell death. These results clearly demonstrate that the induction of apoptosis by sorbitol involves multiple cellular/molecular pathways and strongly suggest that pro- and anti-apoptotic Bcl-2 family proteins, mitochondrial membrane potential, mitochondrial cyt c, and caspase 3, they all participate in sorbitol-induced apoptotic process in human K562 cells.

Cartilage polysaccharide induces apoptosis in human leukemia K562 cells

In this study, we extracted a polysaccharide (short-chain polysaccharide [PS]) from porcine cartilage and examined its function in chronic myeloid leukaemia by using human K562 cells and mouse L1210 cells. Results of cell proliferation assay indicated that PS inhibited cancer cell growth at different concentrations, while it had little effect on normal cells. The presence of morphological aspects of apoptosis, such as nuclear shrinkage, was shown in H&E stained sections. The occurrence of PS-induced apoptosis was confirmed by TUNEL assay and cell cycle analysis. The results of immunofluorescent staining indicated the molecular mechanism underlying. Through interfering with the cell cycle of tumor cells, PS may induce apoptosis by downregulating the expression level of cyclin D1 and upregulating the level of p21 protein. Correlation analysis of apoptosis and MAPK suggested that inactivation of ERK was crucial for PS induced apoptosis, while JNK phosphorylation had a small effect and p38 was not involved. In vivo assay showed that PS inhibited L1210 cell growth in vivo and prolonged the life span of L1210-bearing mice. We conclude that PS is a polysaccharide with anticancer effects and induced apoptosis in human K562 cells.

Alterations of Ceramide/Sphingosine 1-Phosphate Rheostat Involved in the Regulation of Resistance to Imatinib-induced Apoptosis in K562 Human Chronic Myeloid Leukemia Cells

In this study, mechanisms of resistance to imatinib-induced apoptosis in human K562 cells were examined. Continuous exposure to stepwise increasing concentrations of imatinib resulted in the selection of K562/IMA-0.2 and -1 cells, which expressed approximately 2.3- and 19-fold resistance, respectively. Measurement of endogenous ceramides by high performance liquid chromatography/mass spectroscopy showed that treatment with imatinib increased the generation of ceramide, mainly C18-ceramide, which is generated by the human longevity assurance gene 1 (hLASS1), in sensitive, but not in resistant cells. Inhibition of hLASS1 by small interfering RNA partially prevented imatinib-induced cell death in sensitive cells. In reciprocal experiments, overexpression of hLASS1, and not hLASS6, in drug-resistant cells caused a marked increase in imatinib-induced C18-ceramide generation, and enhanced apoptosis. Interestingly, there were no defects in the levels of mRNA and enzyme activity levels of hLASS1 for ceramide generation in K562/IMA-1 cells. However, expression levels of sphingosine kinase-1 (SK1) and generation of sphingosine 1-phosphate (S1P) were increased significantly in K562/IMA-1 cells, channeling sphingoid bases to the sphingosine kinase pathway. The partial inhibition of SK1 expression by small interference RNA modulated S1P levels and increased sensitivity to imatinib-induced apoptosis in resistant cells. On the other hand, forced expression of SK1 in K562 cells increased the ratio between total S1P/C18-ceramide levels approximately 6-fold and prevented apoptosis significantly in response to imatinib. Additional data indicated a role for SK1/S1P signaling in the up-regulation of the Bcr-Abl expression at the post-transcriptional level, which suggested a possible mechanism for resistance to imatinib-mediated apoptosis. In conclusion, these data suggest a role for endogenous C18-ceramide synthesis mainly via hLASS1 in imatinib-induced apoptosis in sensitive cells, whereas in resistant cells, alterations of the balance between the levels of ceramide and S1P by overexpression of SK1 result in resistance to imatinib-induced apoptosis.

Berbamine-Induced Apoptosis in Human Leukemia K562 Cells.

Purpose: To investigate apoptosis-inducing effects of Berbamine on human leukemia cells and to explore the underlying mechanism.Materials and methods: Berbamine was dissolved in 0.9% sodium chloride to an initial concentration of 1mg/ml and subsequently diluted to desired concentrations with cell culture medium. MTT was used to examine the effect of Berbamine on cell proliferation of K562 cells. Characteristic cellular morphological changes were used as indicators of apoptosis in K562 cells while the rate of apoptosis was measured by flow cytometry assay. Expression levels of apoptosis related genes bcl-2 and bax were determined by RT-PCR and the levels of bcr/abl were evaluated by nested-PCR. Levels of Caspase 3 were measured by flow cytometry assay.Results: Berbamine inhibited the cell proliferation significantly and in a dose-dependent manner in tested K562 cells. Its IC50 value was 5.23ug/ml. As determined by morphological observations and flow cytometry assay, Berbamine was able to induce apoptosis of K562 cells within 6 hours. The apoptosis rate of K562 was also dose-dependent. Steady-state transcript levels of bcr/abl decreased dramatically (half-quantity ratio from 1.284 to 0.506 within 72 hours following 8mg/ml Berbamine treatment. On the other hand, the protein levels of Caspase 3 surged from 18.36% to 38.25% (p<0.001) within 24 hours after treatment of 12mg/ml Berbamine. During the same period, no changes of bcl-2 or bax transcript levels were detected in the cells that were treated with 8mg/ml Berbamine.Conclusions: Our results suggest that Berbamine is a potent inhibitor of cell proliferation and a strong inducer of apoptosis in human K562 cells. The Berbamine-induced apoptosis pathway involves down regulation of bcr/abl and up regulation of Caspase 3 expressions. Neither bcl-2 nor bax plays substantial roles in Berbamine-induced K562 cell apoptosis.