The Effects Of Methylprednisolone On Wnt Signaling Pathway In Chronic Lymphocytic Leukemia

BACKGROUND:Chronic lymphocytic leukemia (CLL) is a disease characterized by proliferation and abnormal progressive accumulation of aberrant monoclonal B lymphocytes with defective cell death mechanisms in peripheral blood and bone marrow, lymph nodes and spleen, which is the most prevalent leukemic disease of adults in Western countries, accounting for about1/3of adult leukemias. CLL is relatively rare in China, but as the population aging, the CLL incidence rate has increased year by year. CLL is a heterogeneous disease with significant variation in disease progression, response to therapy, and survival outcome. Recently, progress has been made in the identification of the genetic deficiencies that may underlie CLL. Cytogenetic abnormalities in CLL have been considered related to prognosis. Deletions of17p and11q, or expressions of P53and ATM have been shown to be a marker of poor prognosis. Patients with unmutated IGVH develop progressive CLL with shorter median survival than those with mutated IGVH.In recent years, as the widely application of purine analogs and monoclonal antibodies, fludarabine and cyclophosphamide combined with rituximab (FR or FCR) as the first-line chemoimmunotherapy regimens, objective response rate (ORR), complete remission (CR) and long term progression-free survival (PFS) of patients improved significantly. Despite remarkable progress in the treatment of CLL, represented in particular by introduction of chemo-immunotherapy combining purine nucleosid analogs and monoclonal antibodies, it remains an incurable disease with significant relapse rates or resistance to conventional therapy, especially for the patients with aggressive molecular characteristics such as deletion of17p or mutation of P53which do not respond to conventional treatments and have a shorter survival duration. Although allogeneic hematopoietic stem cell transplantation (allo-HSCT) has proven efficacy in high-risk CLL for younger patients with17p-abnormalities and dismal responses or relapse to conventional chemotherapy regimens, being considered as the only therapeutic modality with curative potential. But since CLL are mostly elderly, allo-HSCT can be performed in a very small subgroup of highly selected, younger with HLA matched and fit patients only. There is an urgent need to find a novel and more effective combination regimens which should be possible to overcome the chemoresistance associated with P53defects by using agents that do not depend on P53for their action.Recent insights into the pathogenesis of the disease are providing new starting points for more potent treatment options, as well as drugs designed to interfere with central pathways regulating the cell cycle (BTK inhibitors, PI3K inhibitors), the leukemic microenvironment (Bcl-2inhibitors), or novel monoclonal antibodies (Obinutuzumab, alemtuzumab) have lead to improved rates of complete remission and significant improvements in progression-free survival. Furthermore, high dose methylprednisolone (HDMP) either alone or combined with chemotherapy and/or monoclonal antibodies has been an effective salvage therapy for patients with relapsed CLL including those with unfavorable cytogenetic features. HDMP has rightly regained its place alongside more modern treatments as a important therapeutic weapon against relapsed/refractory and P53-defective CLL.HDMP appear to play an important role in the management of highly pretreated relapsed/refractory CLL. As demonstrated, glucocorticoids exert their effects through both genomic and non-genomic signaling. Publications suggested different specific aspects of glucocorticoid mediated effects:suppression of P53, bcl-2, c-myc, cyclin-D3, activation of caspase mediated apoptosis, downregulation of Lyn and SYK, or induction of autophagy. Importantly, HDMP may act independently of the P53signaling pathway which is frequently damaged in refractory CLL. Despite the frequent use of HDMP in CLL patients, little is known about the exact mechanisms implicated in glucocorticoid-induced cell death of CLL cells.It has been observed that Wnt signaling pathway has been involved in many types of cancer, and the aberrant Wnt signaling also has been implicated in the pathogenesis of CLL. Several advances have been made in targeted treatment to therapeutically inhibit Wnt signaling. Lymphoid enhancer factor-1(LEF-1) is overexpressed in primary CLL cells when compared with normal B cells and has been identified as an important regulator of pathophysiologically relevant genes in CLL, which can serve an excellent target for therapeutic intervention in CLL. OBJECTIVE:In this study, we investigate the influence of various concentrations of methylprednisolone on the proliferation and apoptosis of human CLL cell line MEC-1, and Wnt signaling pathway in CLL, attempting to explore the mechanisms implicated in glucocorticoid-induced cell death of CLL cells in search of a further theoretical basis for HDMP therapy in CLL.METHODS:Human CLL cell line MEC-1was cultured in Iscove’s Modified Dulbecco’s Medium (IMDM) with10%fetal bovine serum (FBS) maintained at37℃in5%carbon dioxide. MEC-1was incubated with methylprednisolone (Pfizer, New York, NY) at various concentrations of1uM,10μM,50μM,100μM and500μM. Cell proliferation activity was detected by CCK8assay at a particular time point, and the apoptotic effect was evaluated by TUNEL assay. Western blot was used to detect Active-caspase3, and the key proteins in Wnt signaling pathway (LEF-1, β-catenin). The quantitative real time RT-PCR was performed to assess the relative mRNA levels of β-catenin, LEF-1, c-myc and cyclin D1.RESULTS:1. Methylprednisolone suppresses the proliferation activity of MEC-1cells.The MEC-1cells were incubated with different concentrations of methylprednisolone (1uM,10uM,50uM,100uM and500uM) for different time periods (12h,24h,48h,72h and96h). CCK8assay revealed that treatment with1uM and10uM methylprednisolone could not suppresses the proliferation activity of MEC-1cells compared with the untreated control. While treatment with concentrations above50uM (50uM,100uM,500uM) significantly reduced the proliferation activity by23.34%,30.73%,30.57%after24h, and28.48%,42.35%,44.56%after48h respectively. In addition, with the extension of culture time, the inhibitory effect on proliferation activity of MEC-1cells increased gradually.2. Methylprednisolone induces apoptotic cell death of MEC-1cells.The MEC-1cells were incubated with10uM,50uM and100uM methylprednisolone respectively for12h,24h and48h. Western blot analysis revealed that treatment with concentrations above50uM promoted the relative expression of Active-caspase3after24h, suggesting high concentration of methylprednisolone can induce apoptosis. The following TUNEL experiment also confirmed the presence of cell apoptosis after24h.3. Methylprednisolone inhibit LEF-1protein expression and down-regulates mRNA levels of c-myc and cyclin D1in MEC-1cells.To further assess whether methylprednisolone-induced apoptosis was associated with the suppression of Wnt signaling pathway in MEC-1cells, LEF-1and β-catenin protein expressions were detected by Western blot, the mRNA levels of P-catenin, LEF-1, c-myc and cyclin D1were measured by quantitative RT-PCR. Following treatment with100uM methylprednisolone for24h and48h, we observed P-catenin had no obvious change in the methylprednisolone treatment process, either protein expression or the mRNA level. LEF-1protein expression was reduced by31.94%after24h and38.89%after48h in MEC-1cells, which consequently down-regulated mRNA levels of downstream target genes as c-myc and cyclinDl, while no significant effect on LEF-1mRNA level. We speculated the inhibition of LEF-1expression may be related with the influence of methylprednisolone on the regulation of protein translation or post translation.CONCLUSIONS:1. High concentration of methylprednisolone can suppresse the proliferation activity of MEC-1cells, promote the relative expression of Active-caspase3, and induce apoptotic cell death.2. Methylprednisolone can inhibit LEF-1protein expression in MEC-1cells, consequently down-regulate mRNA levels of downstream target gene as c-myc and cyclinD1, but can not affect the transcription level of β-catenin and LEF-1mRNA.3. Methylprednisolone suppresses Wnt signaling pathway by down-regulating LEF-1protein expression, indicating a novel mechanism for HDMP therapy in CLL.