P38mitogen-activated Protein Kinase Mediates Apoptosis Induced By Dexamethasone And Its Mechanism In Human Leukemia Cells

ObjectiveGlucocorticoids have occupied a central role in the treatment of hematologic malignancies, due to their ability to induce apoptosis in neoplastic lymphoid cells. Glucocorticoid resistance is present among 20% initial acute lymphoblas-tic leukemia, even 80% refractory acute lymphoblastic leukemia. Glucocorticoid resistance has been an important determinant of clinical outcome.The precise mechanism of glucocorticoid resistance has not yet been elucidated. Survivin, a member of the inhibitor of apoptosis protein family, is over-expressed in many cancers and considered to play an important role in inhibition of apoptosis. Expression of survivin correlates with proliferation, angiogenesis and multiple drugs resistance. Its expression is an unfavourable prognostic factor in several cancers. Caspase - 3, downstream effector of apoptosis, is mediated by survivin.In primary prostate PC - 3 M cells, calcitonin potently stimulated survivin synthesis and induced chemoresistance to dexamethasone. Rapamycin sensitized both MM cell lines and primary MM cells to dexamethasone - induced apoptosis. This effect was associated with a decreased expression of survivin. In HL - 60 cells, the downregulation of survivin expression and activation of caspase - 3 may be an important mechanism in leukemia cell apoptosis induced by realgar. Sodium arsenite induced apoptosis by down - regulating survivin expression in ATL cell lines. Expression of survivin and caspase genes induced by dexamethasone has not be illuminated.The mitogen - activated protein kinases ( MAPK ) superfamily of serine/ threonine kinases has emerged as an important component of cellular signal transduction. Four MAP kinase families, extracellular signal - regulated kinases (ERK) , p38 MAP kinase, c - Jun NH2 -terminal kinases (JNK) , and extracellular signal regulated kinase - 5 [ ERK5;also called Big MAP kinase - 1 ( BMK1) ], have been well characterized. The MAP kinases family members have been implicated in events necessary for proliferation, differentiation, apop-tosis, and certain kinds of stress responses . These MAP kinases are activated by specific cascades responsible for certain stimuli and eventually induce a variety of cell responses.p38 MAPK usually plays a role in regulating apoptosis, cell cycle arrest and cytokines production, et al. In the human lung adenocarcinoma cell line Arsenic and NO could downregulate survivin via activations of p38 and lead to cy-totoxicity and apoptosis. In MCF - 7 breast adenocarcinoma cells, VD3 compound - induced apoptosis was at least partially dependent on survivin downregu-lation via the activation of p38 MAPK pathway. At present, it is no report that the change of survivin and caspase - 3 induced by dexamethasone via p38 MAPK pathway in CEM cells.Glucocorticoids depend on glucocorticoid receptor (GR) to induce apoptosis. After glucocorticoids are thought to diffuse freely across the cell membrane, they interact with the glucocorticoid receptor. The ligand - free receptor is largely present in the cytoplasm as a multi - protein complex. Upon ligand activation, the receptor translocates into the nucleus and binds to glucocorticoid response elements ( GREs) where it either enhances or represses transcription of target genes. Glucocorticoid receptor, a number of nuclear hormone receptor superfamily, is mediated by many signal transduction systems. In steroid resistance patients, IL - 2 combined with IL - 4 can decrease glucocorticoid receptor ligand - binding affinity via p38MAPK. This difference in ligand - binding affinity could be blocked by the p38 mitogen - activated kinase ( MAPK) inhibitor SB203580. In human alveolar epithelial A549 cells, dexamethasone could inhibit the activation of p38MAPK. It is unclear that the effect of p38MAPK on glucocorticoid receptor function induced by dexamethasone in CEM cells.In this paper, we want to explore gene expression of survivin and caspase -3 in dexamethasone - induced apoptosis in CEM cells, to investigate the effect of p38 mitogen - activated protein kinase on the expression of survivin and caspase - 3 in dexamethasone - induced apoptosis in CEM cells, to research the effect of p38 mitogen - activated protein kinase on glucocorticoid receptors.MethodsCell viability was determined by trypan blue dye exclusion. Apoptosis was detecteded by morphology and flow cytometry. Expression of surviving caspase -3Np - p38MAPK^GRa^GRpwere analyzed by Western blot, and expression of survivin mRNA by RT - PCR.ResultsProliferation of CEM cells was inhibited by dexamethasone in dose — and time - dependent manner. The IC50 at 24h, 48h and 72 h were 9.8,0.9 and 0. 4(xM,respectively. Apoptosis was induced when the cells were treated with dexamethasone at concentration of 5jxM for 12 h. The apoptosis percentage was 14. 9% ,16. 9% ,26. 2% and 46. 2% , respectively when treatment with dexamethasone at 5fxM for 12h,24h,36h and 48h. Compared with control,treatment with dexamethasone at 5fxM for 12h,24h, 48h and 72 h resulted in decrease of sur-vivinmRNA expression to 67. 3% ,55. 0% , 38. 3% andl8. 3% , and survivin protein to 54. 6% ,45. 5% , 15. 8% ,9. 1%. The cleaved active subunits of caspase - 3 were observed following dexamethasone treatment from 24h to 72h.When treatment with SB203580 and dexamethasone for 24h to 72h, the survival percentage was increased from 62. 3% ,35. 5% and 11. 6% to 82. 8% , 54.7% and 48. 1% , respectively. Co -treatment with SB203580 and dexamethasone resulted in the decrease of apoptotic percentage from 26. 2% to 7.1% for 36h. p38 MAPK activation was apparent at 15 min, peaked at 1 h after dexamethasone treatment, and was sustained for 6 h. When treatment with SB203580 and then dexamethasone for 48 h, survivin protein was increased from15. 8% to 55. 8% and the cleaved active subunits of caspase -3 were not observed.Treatment with dexamethasone at 5jxM for 12,24, 36 and 48 h resulted in increase of GRaprotein to 117% ,121% ,122% and 125%. Unbinding to dexamethasone, GRais in the cytoplasm. Nuclear -to - cytoplasmic ratio of GRais 0. 27. Treatment with dexamethasone at the same concentration and time resulted in the nuclear - to - cytoplasmic ratio increase to 0.48,0. 59,0. 95, 2.16 and 4.08. There was no significance of GRpprotein expression. Combined treatment with SB203580 and dexamethasone resulted in the nuclear - to - cytoplasmic ratio decrease from 4. 08 to 0. 43 for 48h. The total GRaand GRp protein were unaffected.DiscussionGlucocorticoids have extensive use and efficacy in the treatment of corresponding leukemia, lymphoma and multiple myeloma. Treatment with dexamethasone at 5/xM for 12h resulted in typical apoptosis on morphology. At the same time, the apoptosis percentage was increased from 14.9% (12h)to 46. 2% (48h). Our data implicate that glucocorticoid can treat leukemia through induction apoptosis in CEM cells. But the mechanism remains largely unclear. The IAP family is a relatively new group of apoptosis regulating proteins, which consists of a number of proteins that can bind to and inhibit caspases. The accumulated data from survivin studies on human cancers suggest that survivin expression in cancer is associated with cancer progression, poor prognosis, drug resistance, and shorter patient survival. We have shown, in this paper, that the basal level of survivin expression was greater in CEM cells than that in normal cells. Higher survivin expression is associated with antiapoptosis, multiple drug resistance and proliferation.Our previous studies demonstrated that survivin was downregulated induced by bufalin in HL -60 cells. We also showed, in this report, that survivinmRNA and protein were downregulated from 67. 3% tol8. 3% , 54. 6% to 9. 7% , respectively when treatment with dexamethasone for 12h to 72h. We demonstra-ted, for the first time, that survivin correlates with dexamethasone - mediated apoptosis in CEM cells.Recent sights have indicated caspase - 3, downstream effector of apoptosis, is mediated by survivin. Caspase - 3 is activated by dexamethasone - mediated apoptosis in WEH17.2 lymphoma cells and pre - B leukemic 697 cells. Our results indicated cleaved active subunits of caspase - 3 were observed following dexamethasone treatment from 24h to 72h.In summary, this study is the first to report that downregulation survivin and activation caspase - 3 may be the mechanism of dexamethasone - mediated apoptosis.p38MAPK, a main number of MAPK family, can be activated and induce apoptosis by stress, osmotic pressure alteration and chemotherapeutics. Hallah-an has already demonstrated ATRA caused medulloblastoma cells apoptosis through p38MAPK phosphorylation. But SB203580 suppressed this action. In Hela cells, p38 MAPK was necessary for taxol - induced cell death and mitosis arrest . In this paper, we first treated with SB203580 for lh, then with dexamethasone for 24 h to 72 h, co - treatment increased cell survival and decreased the apoptotic percentage, comparison with dexamethasone alone. Our results indicate that p38 MAPK acts as an important mediator of dexamethasone - mediated apoptosis.Cheng reported p38mitogen - activated protein kinases mediated arsenic -induced down - regulation of survivin in human lung adenocarcinoma HI355 cells. Chaos results indicated that NO inhibited the expressionof survivin, which was down - regulated by the p38 MAP kinase pathway. In Park's report, p38MAPK was phosphoiylated in 5min and sustained for 3 or 6h during treatment with phytosphingosine - induced apoptosis in Jurkat and NCI - H460 cells. SB203580, a p38 MAPK specific inhibitor, suppressed phytosphingosine - induced translocation of the proapoptotic protein, Bax, from the cytosol to mitochondria , cytochrome c release, and subsequent caspase - 9 activation, indicating that activation of p38 MAPK is essential for Bax translocation to the mitochondria and subsequent mitochondria - mediated cell death pathway during phytosphingosine - induced apoptotic cell death. To determine the role of p38MAPK in dexamethasone - induced apoptosis, we first analyzed the activation status of p38 MAPK by Western blot analysis with antibodies specific to the phosphorylated form of p38MAPK. Treatment with dexamethasone resulted in a dramatic increase of the phosphorylated form of p38 MAPK, suggesting its activation in CEM cells. p38 MAPK activation was apparent at 15 min, peaked at 1 h after dexamethasone treatment, and was sustained for 6 h. These results suggest that dexamethasone can selectively induce activation of p38 MAPK during the apoptotic process of human leukemia CEM cells. On the other hand, pre-treatment with SB203580 for lh and treatment with dexamethasone for48h, expression of survivin protein was increased from 15. 8% to 55. 8% . The cleaved active subunits of caspase - 3 were not observed, indicating that SB203580 effectively suppresses the downregulation of survivin protein and activation of caspase - 3 induced by dexamethasone.In conclusion, these results suggest that activation of the p38 MAPK signaling pathway is critical in downregulating survivin protein and activating caspase - 3 during dexamethasone - induced apoptosis.Glucocorticoid - induced apoptosis mediated by the intrinsic apoptotic pathway is dependent on glucocorticoid receptor. Translocation of the receptor ligand complex to the nucleus is a critical step in this pathway. Our results demonstrate the whole GRawas increased about 117% tol25% from 12h to 48h, when treatment with DEX. GRawas transloceted to nucleus from 6h at the same concentration of DEX. Cytoplasmic - to - nuclear ratio of GRawas upregulated about 1.78 to 15. 1 times, comparison with untreatment, indicating GRawas upregulated and translocated to nucleus.Alternative splicing of the ninth and final ,GR exon gives rise to GRaand GRd proteins divergent at only the extreme carboxy termini. In this paper, western blots shown that protein expression level of GRp was no difference between treatment with DEX and untreatment. It is coincident with Matthew R. Y' s.Glucocorticoid receptor, a ligand - dependent transcription factor, is mediated by many signal transduction pathways. JNK phosphorylates GR and enhances nuclear export and reduces GR transcriptional activation. Pretreatment with SB203580 and then treatment with DEX resulted in the nuclear - to - cytoplas-mic ratio decrease of GRa from 4.08 to 0.43. The total GRaand GRp proteins remain unchangeable, indicating p38MAPK enhances GRatranslocation from cytoplasm to nucleus so as to intensify apoptosis induced by dexamethasone.Taken together, the total GRaprotein is increasd and translocates from cytoplasm to nucleus. GRpprotein remains unchangeable. p38MAPK enhances GRa translocation from cytoplasm to nucleus so as to intensify apoptosis induced by dexamethasone. The total GRaand GRpproteins are unaffected.Conclusion1. Apoptosis induced by dexamethasone in CEM cells is associated with downregulation of expression of survivin and activation of caspase - 3.2. Dexamethasone downregulates survivin and activates caspase -3 via activations of p38MAPK so as to lead to apoptosis in CEM cells.3. Expression of GRa protein is upregulated and translocated into nucleus. p38MAPK enhances GRQprotein translocation into nucleus, but GRa and GRp proteins are unaffected.