The Role Of WNT/β-catenin Signaling In The Anti-leukemic Effect Of HDACis And The Mechanisms

Background:Acute T lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy, accounting for about15%and25%of acute lymphoblastic leukemia in pediatric and adult patients respectively. Despite recent progress in the treatment, the long-time survival rate is still low, especially in adult patients, underscoring the need for improved therapy. The dysregulation of T cell differentiation, apoptosis and proliferation are proved to be involved in the pathogenesis of T-ALL. Recent studies show that the altered balance of acetylation status of histone and some non-histone proteins and the dysregulation of WNT/β-catenin signaling play important role in T-ALL proliferation/apoptosis and differentiation.Acetylation and deacetylation of histone and some non-histone proteins have an important role in regulating gene transcription. The imbalance of the acetylation status is an important feature of tumor cells. HDACs, which can remove the acetyl groups from ε-N-acetyl lysines of histone and non-histone proteins, play a pivotal role in developmental processes and tumorigenesis. Histone deacetylases inhibitors (HDACis), which can inhibit the activity of HDAC and thus regulate gene transcription, are emerging as a promising new class of anticancer agents. It has been proved that HDACis induce growth arrest, differentiation, and apoptosis in leukemia cells and the molecular mechanism needs further elucidated in T-ALL cells. According to previous reports, HDACis are responsible for regulating WNT/β-catenin signaling in colon and pancreas cancer.WNT/β-catenin signaling pathway is a highly evolutionarily conserved pathway with β-catenin as the main molecule.. Although most researchers believe that the constitutive activation of WNT/β-catenin is involved in the oncogenesis of several types of cancer, however, it has been proved that the role of WNT/β-catenin signaling is multifaced in human cancer cells. WNT/β-catenin signaling is constitutive activated in colon and pancreatic cancer, interestingly, the hyper-activated WNT/β-catenin signaling by HDACis mediate the anti-cancer effect of HDACis in part. The constitutive activation of WNT/β-catenin signaling in T-ALL has been proved to be involved in the leukemia-genesis. However, to date, there has been no study of the effects of HDACis on WNT/β-catenin signaling in T-ALL.Objective:The purpose of the current study was to determine:(a) the in vitro anti-leukemia effects of two chosen HDACis, valproic acid (VPA) and suberoyl bishydroxamic acid (SBHA), on T-ALL cells,(b) the effect of VPA and SBHA on WNT/β-catenin signaling in T-ALL cells, and (c) the role of WNT/β-catenin signaling in the anti-leukemia effects of HDACis.Materials and methods:1. To study the effect of HDACis on the Biological behavior of T-ALL cells1.1Cell growth inhibition by MTT assay:Jurkat and Molt-4T-ALL cells were treated with incremental doses of VPA or SBHA for24h,48h,72h. At each time point, MTT assay were performed to detect the cell viability after the treatment with HDACis.1.2Cell cycle analysis by flow cytometry:Jurkat and Molt-4cells were cultured on12-well plates and treated with VPA (1mM) or SBHA (10μM). After HDACis treatment for24h, cells cycle distribution was analyzed by flow cytometry using propidium iodide (PI) staining.1.3Western blot analysis:Total protein was extrated from HDACis/DMSO treated Jurkat cells using RIPA lysis buffer and total protein was separated by SDS-polyacrylamide gel electrophoresis. Western blot method was used to detect the protein levels of apoptosis and cell cycle-related proteins, including cleaved Caspase-3,9, cleaved PARP and P21WAF12. To study the effect of HDACis on WNT/β-catenin signaling in T-ALL cells2.1Western blot:T-ALL Jurkat and Molt-4cells were treated with VPA (0.5,1mM) or SBHA (5,10μM) for two days, then total proteins and nuclear protein were extracted. The expression of (3-catenin was detected by western blot.2.2Immunocytochemistry: after treatment with VPA (1mM) or SBHA (10μM) for two days, Jurkat and Molt-4cells were collected and resuspended with PBS. A drop of cells was dded on coated slides, and then detected the expression and subcellular distribution of β-catenin using immunocytochemistry. The mean density of immunocytochemistry straining was analyzed by IPP software.2.3Real time RT-PCR:after treated with VPA (0.5,1mM) or SBHA (5,10μM) for two days, Jurkat and Molt-4cells were collected and total RNA were extracted. Real time RT-PCR was used to detect the mRNA level of β-catenin after the treatment of VPA or SBHA2.4Dual luciferase reporter assay:After transfected with TOPflash/MOPflash or Renilla luciferase pRL-TK, Jurkat cells were treated with VPA (1mM) or SBHA (10μM) for24h. Dual luciferase reporter assay were performed to determine the activity of beta-catenin.3. To study the relationship between the anti-leukemic effect of HDACis to T-ALL cells and unregulated WNT/β-cateninsignaling3.1Stable transfection:shRNA plasmid vectors against β-catenin and control plasmids were transfected into T-ALL Jurkat cells.48hours after transfection, cells were selected in G418containing medium for at least2weeks and the stable transfectants were named JK-β-catenin-shRNA and JK-control-shRNA respectively. The expression of β-catenin was detected by real time RT-PCR and Western blot.3.2Sensitivity of transfected cells to HDACis by MTT assay:JK-β-catenin-shRNA and JK-control-shRNA were seeded into96-well culture plates and treated with serial dilutions of VPA or SBHA for up to48hours. MTT method was used to detect live cells. IC50values, which were defined as the concentration inducing50%loss of cell viability, were calculated.3.3Apoptosis assay by flow cytometry:JK-β-catenin-shRNA and JK-control-shRNA cells were seeded into12-well culture plates and treated with VPA (1mM) or SBHA (10μM). After drug treatment for24hours, the apoptotic cells were detected by flow cytometry using Annexin V and propidium iodide (PI). The results were analyzed by WINMDR2.9soft ware.3.4Western blot for cleaved-PARP:After treated with VPA (1mM) or SBHA (10μM) for24hours, JK-β-catenin-shRNA and JK-control-shRNA cells were solubilized in RIPA lysis buffer. Total proteins were separated by10%SDS-polyacrylamide gel electrophoresis. Western blot method was used to detect cleaved PARP protein level.3.5Up regulation of β-catenin expression by BIO (6-bromoindirubin-3'-oxime):In order to chose a proper concentration of BIO for the following experiments, Jurkat cells were incubated with incremental doses of BIO, then total and nuclear proteins were extracted and the protein levels of p-catenin were detected by western blot; MTT assay were performed to detect the effect of BIO on Jurkat cells.3.6Sensitivity of Jurkat cells treated with BIO to HDACis by MTT assay:Jurkat cells were seeded into96-well culture plates and treated with serial dilutions of VPA or SBHA with or without BIO (0.5μM) for up to48hours. MTT method was used to detect live cells and IC50values for HDACis with or without BIO were calculated.Results:1. VPA and SBHA exert anti-tumor effects in T-ALL cells1.1VPA and SBHA suppressed proliferation of T-ALL cells:Jurkat and Molt-4were treated with incremental doses of VPA or SBHA, and cell viability was determined by MTT assay. We found that VPA and SBHA could significantly inhibit the proliferation of T-ALL cells even at low doses. Moreover, a dose-dependent and time-dependent growth-inhibitory effect was observed.1.2VPA and SBHA induced caspase-dependent apoptosis in T-ALL cells:Results howed that after two days of treatment with VPA or SBHA, increased cleavage of caspase-9, caspase-3and PARP in both Jurkat and Molt-4cells was noted, indicating that VPA and SBHA were capable of inducing apoptosis through the intrinsic pathway.1.3VPA and SBHA arrested cell cycle at G2/M phase in T-ALL cells:Jurkat and Molt-4cells were treated with VPA or SBHA for48h, and cell cycle profiles were monitored by flow cytometric analysis of DNA content. Results showed that percentages of cells at G2/M after treatment with VPA and SBHA were significantly higher than those treated with DMSO (Jurkat:29.09±0.91,42.42±9.66versus9.52±1.03; Molt-4:51.44±11.09,12.29±0.96versus6.70±0.39). Furthermore, western blot results showed that VPA and SBHA increased the level of p21WAFI. These results suggested that the growth inhibitory VPA and SBHA could induce G2/M phase cell cycle arrest in T-ALL cells, and increased expression of p21WAFI may be associated with the G2/M phase arrest induced by VPA and SBHA.1. VPA and SBHA activated WNT/β-catenin signaling in T-ALL cell lines1.1VPA and SBHA up-regulated β-catenin protein and promoted it nuclear location: Results form western blot showed that after Jurkat and Molt-4cells were treated with VPA or SBHA, the levels of P-catenin protein were obviously elevated both in total proteins and nuclear proteins; Results from immunocytochemistry showed that the VPA or SBHA treated cells exhibited strong staining, and the nucleus were strongly stained. In contrast, the DMSO treated cells showed weak staining mainly in the cytoplasma. Furthermore, the mean density of β-catenin staining of HDACis treated cells apparently increased compared with DMSO control in both Jurkat and Molt-4.1.2VPA and SBHA increased the activity of β-catenin in Jurkat cells:compared with DMSO treated cells, VPA and SBHA treated cells showed higher TCF reporter activity, suggesting that the TCF-dependent transcriptional activity could be activated by VPA and SBHA.1.3β-catenin mRNA level did not change in T-ALL cells upon treatment with HDACis:After treatment with VPA and SBHA for48hours, relative β-catenin mRNA expression level of T-ALL cells did not change significantly, indicating that the accumulation of active form of p-catenin resulted from post-translation modification.3. p-catenin ShRNA weakened the effects of VPA and SBHA in T-ALL cells3.1The pRNAi-CTNNB1and pRNAi-control plasmid were transfected into T-ALL Jurkat cell line, and the stable transfectants were named JK-β-catenin-shRNA and JK-control-shRNA respectively. Real time RT-PCR showed that the β-catenin mRNA level in JK-β-catenin-shRNA cells only accounts for25.35%of that in JK-control-shRNA cells. Western blot showed β-catenin protein levels were also significantly down-regulated both in whole cell and nuclear lysates, which was consistent with the results of quantitative real time RT-PCR.3.2Down-regulation of β-catenin decreased sensitivity of T-ALL cells to HDACis: MTT results showed the IC50values of VPA and SBHA for JK-p-catenin-shRNA cells were significantly higher than those for control JK-control-shRNA cells (JK-β-catenin-shRNA:VPA3.50±0.28mM, SBHA:30.32±1.82μM, JK-control-shRNA:VPA2.42±0.14mM, SBHA17.54±2.66μM, respectively).3.3Down-regulation of β-catenin compromised the ability of HDACis to induce apoptosis:JK-β-catenin-shRNA and JK-control-shRNA were exposed to1mM of VPA or10μM of SBHA respectively for18h. Quantitation of apoptotic cells was assessed by Annexin V/PI staining and flow cytometric analysis. The results suggested that the proportion of Annexin V positive cells in JK-control-shRNA increased from3.66%(DMSO control) to29.56%(1mM of VPA) or26.89%(10μM of SBHA). In contrast, the proportion of Annexin V positive cells in JK-β-catenin-shRNA increased from1.30%(DMSO control) to15.75%(1mM of VPA) or11.83%(10μM of SBHA). Being consistent, the western blot results showed that knockdown of β-catenin via shRNA decreased PARP cleavage after treatment with VPA or SBHA. 4. BIO strengthened the proliferation inhibition effect of HDACis4.1BIO increased β-catenin protein of Jurkat cells:Jurkat cells were treated with BIO for48hours, cell viability were measured with MTT assay and expression of P-catenin protein was detected by western blot. Results suggested that after treatment with BIO, the expression of β-catenin protein was up-regulated in a dose dependent manner. Even0.5μM of BIO (the proliferation inhibition rate was5.27%) could apparently up-regulate the expression of β-catenin in Jurkat cells.4.2Overexpression of β-catenin increased sensitivity of T-ALL cells to HDACis: Jurkat cells were treated with0.5μM of BIO and incremental doses of VPA or SBHA. Cells treated with VPA or SBHA alone served as controls. MTT results proved that treatment with0.5μM of BIO significantly decreased the IC50values of VPA and SBHA (HDACi+BIO:VPA1.04μM±0.01mM, SBHA7.40μM±0.81μM; HDACi alone:VPA1.38±0.13mM, SBHA22.03μM±1.07μM).Conclusion:1. VPA and SBHA activate WNT/(3-catenin signaling in T-ALL cell lines.2. VPA and SBHA have anti-leukemic effect in T-ALL cells, including inhibition of proliferation, induction of G2/M phase cell cycle arrest and apoptosis.3. HDACis exert anti-leukemic effects partly by WNT/β-catenin signaling.Using HDACis to modulate WNT/β-catenin signaling could be an attractive new therapy strategy for the treatment of T-ALL...