| Chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) are both B-cell malignancies with defects in apoptosis and abnormal proliferation. With the introduction of novel therapeutic agents, the long-term survival of CLL and MCL patients has been improved. However, drug resistance remains a formidable obstacle to treatment.CLL, characterized by a gradual accumulation of leukemic cells, is an incurable disease with an extremely variable course; survival after diagnosis can range from months to decades. Genomic features such as mutational status of immunoglobulin heavy chain variable region genes (IGHV), ataxia telangiectasia mutated genes (ATM) and TP53 tumor suppressor genes,β2-microglobulin, zeta-chain-associated protein kinase 70 (ZAP70) expression, interphase cytogenetics and complex karyotype on metaphase cytogenetics, provide further differentiation of disease prognosis. MCL, a morphologically distinct subtype of aggressive B-cell non-Hodgkin lymphoma (NHL), is characterized by the cytogenetic abnormality t (11; 14) (q13; q32) and overexpression of cyclin D1. MCL comprises approximately 3-10% of NHL and is frequently resistant to conventional chemotherapy. The median survival for MCL patients is between 3-5 years. Thus, novel approaches to CLL and MCL therapy are urgently needed.Histone deacetylases (HDACs) are enzymes that regulate the structural conformation of chro matin and the posttranslational modification of numerous key proteins. HDACs play a key role in transcriptional regulation and are involved in the pathogenesis of malignancies. HDAC inhibitors represent an emerging class of therapeutic drugs that induce tumor apoptosis through damage to the mitochondria and modulating the acetylation status of a wide range of protein targets. Though several studies indicated pan-HDAC inhibitors vorinostat (suberoylanilide hydroxamic acid, SAHA) and panobinostat (LBH589) exerting potent cytotoxic effects on CLL and MCL cells in vitro, these inhibitors have not been demonstrated to have meaningful single agent activity in CLL and MCL patients. The lack of selectivity of SAHA and LBH589 also led to undesired toxic effects, including hematological, hepatic and gastrointestinal toxicity.Different factors contribute to the resistance of CLL and MCL cells to therapeutic drugs, such as intrinsic defects in apoptosis signaling pathways and interactions of malignant cells with stromal cells and immune cells in the microenvironment of bone marrow and other tissues. Bone marrow stromal cells (BMSCs) and immune cells secrete cytokines, chemokines, and other molecules that are associated with tumor survival, proliferation, and metastasis. Cytokines and chemokines secreted by BMSCs, such as Interleukin-1β (IL-1P), IL-6, IL-8, basic fibroblast growth factor (bFGF), and stromal cell-derived factor-1 (SDF-1, CXCL12), could activate signal transducer and activator of transcription 3 (STAT3) signaling pathway either directly or indirectly. Early reports implicated IL-6 and STAT3 as pro-tumorigenic factors in various human tumors, including CLL and MCL.WP1066, a small-molecule inhibitor of signal transducer and activator of transcription 3 (STAT3), has a marked antitumor effect on acute myelogenous leukemia, polycythemia vera, melanoma, malignant glioma cells and renal cell carcinoma. It suppresses the Janus kinases (JAKs)/STAT3 signaling pathway by inhibiting activation of STAT3 and downregulating the expression of JAKs upstream from STAT3. The combinations of WP1066 and HDAC inhibitors have not been evaluated in treating CLL and MCL. In this study, we will further investigate the antiproliferative and proapoptotic activity of WP1066 combined with HDAC inhibitors in CLL and MCL cells by using series of cell and molecular biology techniques. We will also explore the potential mechanisms involved to illuminate the role of the STAT3 signaling pathway in the resistance of CLL and MCL cells to HDAC inhibitors.Part I Study of STAT3 signaling pathway in the resistance of chronic lymphocytic leukemia cells to histone deacetylase inhibitorsObjective:This study was designed to illuminate the role of the STAT3 pathway on the resistance to SAHA and LBH589 in CLL cells, and to evaluate the proapoptotic activity of the combination of SAHA and LBH589 with a STAT3-selective inhibitor, WP1066.Material and Methods:1. Specimen collection and peripheral blood mononuclear cells (PBMCs) isolation;2. Cell culture and establishment of bone marrow stromal layer;3. Cytotoxicity assays using the Cell Counting Kit-8 (CCK-8) method;4. Analyses of cell apoptosis by annexin V/7-aminoactinomycin D (7-AAD) assay;5. Protein extraction and western blot analyses;6. Flow cytometric analysis of efflux of Rhodamine 123;7. RNA extraction and real-time quantitative PCR;8. Statistical analysis.Results:1. HDAC inhibitors regulated the tyrosine phosphorylation of STAT3 induced by IL-6 and acetylated STAT3 at Lys685 in CLL cellsConstitutive tyrosine phosphorylation of STAT3 (pSTAT3) was undetectable in 28 of 33 (85%) CLL samples. IL-6-induced pSTAT3 was completely abolished by 4-hour incubation with 20μM of SAHA or 100nM of LBH589 before IL-6 was added. But when CLL cells were incubated with lower concentration of SAHA or LBH589 (e.g., SAHA, 1μM; LBH589 lOnM), the level of pSTAT3 induced by IL-6 was further enhanced. Both 2.5 μM and 10 μM of SAHA markedly induced STAT3 acetylation.2. SAHA potentiated IL-6 induced upregulation of Mcl-1 and Bcl-xLTreatment of CLL cells with 10 ng/mL IL-6 for 24 hours resulted in approximately 2-fold upregulation of the antiapoptotic proteins, Mcl-1 and Bcl-xL, whereas the level of Bcl-2 protein remained constant. The upregulation of Mcl-1 and Bcl-xL induced by IL-6 was further enhanced in the presence of 2.5 μM of SAHA, whereas the level of Bcl-2 had no significant changes.3. IL-6-induced resistance of CLL cells to HDAC inhibitors was reversed by WP1066IL-6 decreased the sensitivity of CLL cells to each of the HDAC inhibitors tested, as evidenced by increases in the mean IC50 values (P< 0.01). Coincubation with 5 μM of WP1066 reversed the cytoprotection of IL-6 (P< 0.01). Analysis of annexin V-PE/7-AAD dual staining to CLL cells showed that apoptosis induction by SAHA and LBH589 was decreased by IL-6, and that this decrease could be blocked by WP1066. Moreover, western blot analysis showed the cleavage of poly(ADP ribose) polymerase (PARP) induced by SAHA or LBH589 was completely eliminated by IL-6 treatment. WP1066 in combination with HDAC inhibitors augmented the PARP cleavage induction and blocked the decrease of PARP cleavage mediated by IL-6.4. WP1066 blocked IL-6-induced STAT3 phosphorylation, but lacked sensitivity for apoptosis induction and blockade of P-glycoprotein function in resistant CLL cellsThe IC50 values of WP1066 for primary CLL cells were ranging from 3.28 μM to excess of 20uM (n=10), with a median value of 5.93 μM. WP1066 showed weak efficacy on the apoptosis induction in human p53 deleted/muteted CLL cell line MEC-1, as quantified by analysis of caspase activation. The study of Rho123 efflux assay revealed that there was only 16.87±5.69% increase of the Rho123 retention in MEC-1 cells treated with 5 μM WP1066 and 36.57 ± 7.29% increase in MEC-1 cells treated with 10 μM WP1066. In addition, there was no significant difference in mRNA levels of P-glycoprotein 1 (P-gp, MDR1) in MEC-1 cells with or without WP1066 treatment.5. Combination of SARA and WP1066 modulated STAT3 signaling pathwayWestern blot analysis showed that WP1066 dramatically reduced the level of IL-6-induced p-STAT3 in the presence or absence of SAHA. WP1066 single treatment or combined with 2.5 μM SAHA slightly decreased levels of Mcl-1 and Bcl-xL protein in primary CLL cells, whereas the level of Bcl-2 protein remained constant. Moreover, WP1066 markedly reversed the increases of Mcl-1 and Bcl-xL protein induced by IL-6 in the presence or absence of SAHA.6. WP1066 reversed the HDAC inhibitor resistance of CLL cells induced by co-culture with BMSCsCo-culture of CLL cells with a BMSC layer protected CLL cells from apoptosis induction by HDAC inhibitors. Simultaneously, co-culture of CLL cells with BMSCs resulted in activation of STAT3 in CLL cells. Pretreatment with low concentrations of SAHA or LBH589 resulted in significant enhancement of STAT3 phosphorylation, while pretreatment with 10 μM of SAHA or 100 nM LBH589 abolished phosphorylation of STAT3 induced by co-culture with BMSCs. The addition of WP1066 to the CLL cell/BMSC co-cultures not only significantly inhibited the increases of pSTAT3, but also resulted in subsequent PARP cleavage in the presence of SAHA or LBH589. Data obtained from the annexin V/7-AAD assay also showed that WP1066 reversed the resistance of CLL cells to HDAC inhibitors induced by CLL cell/BMSC co-cultures.Conclusions:The present study indicated that CLL cells were protected from HDAC inhibitor-induced apoptosis by IL-6 or co-culture with BMSCs. This protection may be mediated by activation of STAT3 signaling pathway. Furthermore, WP1066 reversed the resistances of CLL cells to SAHA and LBH589 induced by IL-6 or co-culture with BMSCs. Our findings suggest that targeting the STAT3 pathway may be a novel way to improve the efficacy of HDAC inhibitor in CLL patients by overcoming antiapoptotic signaling of the microenvironment.Part II WP1066 synergizes with vorinostat to induce apoptosis of mantle cell lymphoma cellsObjective:To investigate the antiproliferative and proapoptotic activity of WP1066 combined with SAHA in a panel of MCL cell lines, as well as the potential mechanisms involved.Material and Methods:1. Cell lines and cell culture;2. Cytotoxicity assays using the Cell Counting Kit-8 (CCK-8) method;3. Analyses of cell apoptosis by annexin V/7-aminoactinomycin D (7-AAD) assay;4. RNA extraction and real-time quantitative PCR;5. Protein extraction and western blot analyses;6. Statistical analysis.Results:1. Characterization of sensitivity of MCL cell lines to WP1066 and SAHAThe 50% inhibitory concentration (IC50) value for WP1066 single treatment was 4.71 μM in Mino cells,6.31 μM in SP53 cells,11.86 μM in Granta 519 cells and higher than 20 μM in Jeko-1 cells. Respectively, the IC50 value for SAHA single treatment was 2.64 μM,7.73 μM,10.49μM and 2.25 μM. Western blot analysis revealed high levels of p-STAT3 in untreated Granta 519 and SP53 cells, significantly lower levels in Mino cells and almost no detectable expression in Jeko-1 cells. Both Granta 519 and SP53 cells showed dose-dependent inhibition of p-STAT3 after 24 hours incubation with WP1066.2. WP1066 synergistically interacted with SAHA to inhibit growth of MCL cell linesSignificant decreases in the cell growth of Granta 519 and SP53 cells were observed in response to treatment with combined 5 μM WP1066 and 5 μM SAHA than with either agent alone, in a time-dependent manner up to 72 hours. Factional effect-Combination index (CI) curves revealed that WP1066/SAHA combination provided highly synergistic anti-MCL activity with CIs≤0.5 in all four representative MCL cell lines.3. Combination of WP1066 with SAHA inhibited STAT3 activation and induced caspase-dependent apoptosisAnnexin V/7-AAD assay showed that both WP1066 and SAHA single treatment induced cell apoptosis, and combination of the two drugs resulted in a significant increase (P< 0.01) in percentage of apoptotic cells in Granta 519 and SP53 cell lines. Moreover, western blot analysis showed that WP1066 in combination with SAHA significantly inhibited STAT3 activation and induced downregulation of pro-caspase-3 expression. Concomitantly, cleavage of PARP was further increased, whereas SAHA single treatment had only a moderate effect on its expression.4. WP1066/SAHA combination modulated multiple mRNA expressions of apoptosis regulation genesFollowing treatment with 5 μM SAHA alone or in combination with 5 μM WP1066 for 24 hours, mRNA expressions of p21Cip1 and Bax were significantly upregulated in Granta 519 cells, while mRNA expressions of c-Myc and cyclin D1 were reduced. However, no significant difference in the expression of Bcl-2 mRNA was observed in the WP1066/SAHA combination-treated and untreated Granta519 cells. The similar results were also observed in SP53 cell line.Conclusions:our study indicated that combining STAT3 inhibitor WP1066 with HDAC inhibitor SAHA resulted in synergistic antitumor activity in MCL cell lines in vitro. The mechanism involved in this drug synergism was associated with downregulation of constitutive STAT3 activation, modulation of anti- and pro-apoptotic genes expression and caspase-dependent apotosis induction. Our results suggest that agents such as WP1066 and other STAT3-selective inhibitors may be candidate drugs for future use in the treatment of MCL as adjuvants, which could overcome the drug resistance induced by the aberrant activation of STAT3 signaling pathway. |
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