The Study Of Hedgehog And STAT3 Signaling Pathways In T-cell Lymphoma

T-cell lymphomas are lymphoid malignancies with aggressive clinical course and poor prognosis. There is great heterogeneity in the pathology characteristics, clinical manifestation, and biological behavior between different subtypes. The disease can be involved in the lymph nodes, liver, spleen, bone marrow, skin, etc. T-cell lymphomas are usually classified as Clinical stage Ⅲ/Ⅳ, and accompanied by a variety of clinical syndrome (e.g. hemophagocytic syndrome). T-cell lymphomas accounts for 10%-15% of NHL in Europe and the United States, while 25%-35% in our country, exhibiting obvious regional and ethnic characteristics.The progress of the treatment for T-cell lymphomas is dismal due to the lower incidence, ill-defined pathogenisis, and the lack of a multicenter clinical trial. No uniformed therapeutic strategies have been achieved. CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone) or CHOP-like regimen is still considered the "standard therapy" despite consistent results showing that is largely ineffective compared to diffuse large-B-cell lymphoma (DLBCL). Although the clinical outcome of the patients has improved with enhanced combination chemotherapy and hematopoietic stem cell transplantation (HSCT), refractory relapse remains challenging in the treatment of PTCL and the 5-year survival rate is less than 30%. Novel effective agents are needed to be developed.Up to now, the etiology of T-cell lymphoma remains to be elucidated. Various pathogenic factors may contribute to the pathogenesis of T-cell lymphoma, such as virus infection (e.g. EBV, HTLV), physicochemical stimulus, immunodeficiency, genetic variation. As is known, normal signal transduction is the foundation of life activities in human body. The dysregulation of many signaling pathways participate in the malignant transform, angiogenesis, invasion, migration of tumors. Previous literatures have reported that many signaling pathways (e.g. Notch, Wnt, PI3K/AKT, and P53 pathway) were aberrantly activated in T-cell lymphomas, and some component molecules in these pathways were associated with the relapse and chemo-resistance of disease. To investigation the signaling pathway in T-cell lymphomas will provide rationale for exploring novel treatment targets and agents to improve the survival of patients.The hedgehog (Hh) signaling pathway, a highly conserved pathway, has been described to be over-expressed in a wide range of neoplasm. Especially the critical transcription factor GLI1 (glioma-associated oncogene-1) was activated in various tumor tissues, suggesting the important role of Hh pathway in oncogenesis. Signal transducer and activator of transcription 3 (STAT3) was known as an important effector of multiple growth factor receptors and cytokine signals. The constitutive activation of STAT3 will result in the up-regulation of many downstream targets, which facilitate tumourigenesis.Increasing evidence has demonstrated that dysregulation of Hh and STAT3 pathways could participate in oncogenesis and facilitate tumor suivival by promoting cell proliferation and preventing cell apoptosis, inducing angiogenesis and chemo-resistance, etc. However the regulations of these two pathways in the pathogenesis of T-cell lymphoma were poorly understood and deserve more extensive study. This study investigated the effects of GANT61 (GLI1 inhibitor) and WP1066 (STAT3 inhibitor) in T-cell lymphoma cells, and explored the potential molecular mechanisms of the antitumor effects which provide new insight into the diagnosis and targeted treatment of T-cell lymphoma.Part I. Antitumor effect and mechanisms of GANT61 in T-cell lymphoma cellsObjective:T-cell lymphomas are lymphoid malignancies with aggressive clinical course and poor prognosis. The etiology remains to be elucidated, and no uniformed therapeutic strategies have been achieved. The hedgehog (Hh) pathway, aberrantly activated in a number of tumors, is extensively studied with respect to its interaction with other oncogenic pathways. Increasing evidence has shown that aberrant activation of signal transducer and activator of transcription-3 (STAT3) and suppressor of cytokine signaling 3 (SOCS3) may be associated with the development of various malignancies, including hematologic tumors. It has also been established that inhibition of Hh pathway has antitumor efficacy in T-cell lymphoma cells. Glil inhibitor GANT61 is a promising Hh pathway inhibitor in anticancer therapy. This study attempts to investigate the effect of GANT61 on T cell lymphoma cells and its role in the involvement of STAT3 pathway.Material and Methods:1. Specimen collection;2. Immunohistochemistry (IHC);3. Isolation of peripheral T lymphocyte cells;4. Cell culture;5. Silencing of GLI1 gene using lentiviral vector-mediated RNA interference;6. Cytotoxicity assays using the Cell Counting Kit-8 (CCK-8) method;7. Assessment of cell apoptosis by annexinV/7-aminoactinomycin D (7-AAD) assay;8. Protein extraction and western-blot analysis;9. Co-Immunoprecipitation (Co-IP) analysis;10. Statistical analysis.Results:1. Immunohistochemistry analysis exhibited that the expressions of GLI1, p-STAT3, STAT3 and SOCS3 were detected in 28/35(80.0%),26/35(74.2%),32/35(91.4%), and 24/35(68.6%) of T-cell lymphoma cases respectively, while the staining of these protein was too weak to be detected in the 15 cases of reactive hyperplasia of lymph node (RHL) tissues. Furthermore, Analysis results showed that both p-STAT3 and SOCS3 protein expression were positively correlated with GLI1 in T-cell lymphomas (P<0.05).2. Western blot analysis also showed higher expressions of GLI1, p-STAT3, STAT3, and SOCS3 in three T-cell lymphoma cells (Jurkat, Karpass299 and Myla3676 cells) compared to peripheral blood T lymphocytes freshly isolated from healthy donors. Moreover, the interaction of GLI1 and p-STAT3 in these cells was observed in the Western blot analysis of Co-Immunoprecipitation (Co-IP).3. The cell counting kit-8 (CCK-8) assay showed that viability of the three cell lines (Jurkat, Karpass299 and Myla3676 cells) decreased in a dose-dependent manner after a 48-hour GANT61 treatment at different concentrations. The half-maximal inhibitory concentration (IC50) values of GANT61 at 48h were calculated as following:Jurkat cells,13.761±0.81μM; Karpass299 cells,6.81±0.91μM; and Myla3676 cells, 10.23±0.94 μM.4. GANT61 induced apoptosis and decreased the protein expression of GLI1, p-STAT3 and SOCS3 in the three cells (Jurkat, Karpass299 and Myla3676 cells) after a 24-hour treatment of GANT61 in a dose-dependent manner.5. Knockdown of GLI1 by lentivirus-mediated RNA interference inhibited the proliferation and induced apoptosis, as well as down-regulated the protein expression of GLI1, p-STAT3 and SOCS3 in these T-cell lymphoma cells.Conclusions:These results showed that protein expression of GLI1, p-STAT3, STAT3 and SOCS3 was up-regulated in T-cell lymphoma tissues and cell lines. Inhibition of Glil by GANT61 or RNA interference could attenuate proliferation and induce apoptosis of Jurkat, Karpass299 and Myla3676 cells. Moreover, the p-STAT3 and SOCS3 were decreased accompanied with the inhibition of GLI1. Our findings indicated a potential mechanism for the antitumor activity of GANT61 which might inhibit viability of T-cell lymphoma cells at least partially by down-regulating p-STAT3 and SOCS3. The crosstalk between Hh pathway and STAT3 pathway in T cell lymphomas deserves more exploration. Targeting the Hh pathway with GANT61 may be a novel therapeutic strategy for T-cell lymphomas.Part Ⅱ. The inhibitory effect of WP1066 in nasal-type NK/T-cell Iymphoma cellsObjective:Nasal-type NK/T-cell lymphoma (nasal NKTCL) is an aggressive hematological neoplasm with poor prognosis, and its incidence is higher in Asia than it is in Western countries. Even though two-thirds of nasal NKTCL patients present with localized lesions, the prognosis of nasal NKTCL remains dismal because of its aggressive course and resistance to conventional chemotherapy. Increasing evidence suggests that aberrant activation of signal transducer and activator of transcription-3 (STAT3) is related to numerous malignancies. WP1066, a novel promising STAT3 inhibitor, appears to be highly effective against human cancer cells in vitro and in xenograft animal models, including renal cell carcinoma and malignant glioma cells. In this present study, we investigated the inhibitory effect and the underlying mechanisms of WP1066 (a novel selective STAT3 inhibitor) in the nasal NKTCL cells.Material and Methods:1. Specimen collection;2. Immunohistochemistry (IHC);3. Cell culture;4. Immunofluorescence (IF) analysis;5. Cytotoxicity assays using the Cell Counting Kit-8 (CCK-8) method;6. Analysis of cell apoptosis by flow cytometry with annexin V/7-aminoactinomycin D (7-AAD) assay;7. Protein extraction and western-blot analysis;8. RNA extraction and real-time quantitative PCR;9. Statistical analysis.Results:1. The Immunochemistry (IHC) analysis showed 21/28 (75.0%) nasal NKTCL tissues harbored constitutively expressions of phospho-STAT3 (p-STAT3) which were positively correlated with the Ki-67 levels (P< 0.05).2. The cell counting Kit-8 and flow cytometric analysis showed that WP1066 could inhibit proliferation and induce apoptosis of SNK6 cells in dose-dependent manner at 24h (P< 0.05).3. Western-blot and Immunofluorescence (IF) detection exhibited that WP1066 could inhibit the phosphorylation of STAT3 in SNK6 cells.4. Western-blot and RT-PCR demonstrated that WP1066 could down-regulated the protein and mRNA expressions of the pro-survival molecules (including c-Myc, cyclinD1, and Bcl-2) in SNK6 cells.Conclusions:Our results showed that both the nasal NKTCL tissues and cell line (SNK6) harbored constitutively activation of STAT3. WP1066 could inhibit proliferation and induce apoptosis of SNK6 cells. Furthermore, the down-regulation of c-Myc, cyclinD1, and Bcl-2 may partly contribute to the potential mechanisms for the antitumor effect of WP1066 in nasal NKTCL cells. These findings suggested that STAT3 activation represented a potential target in nasal NKTCL. WP1066 may be a promising agent to improve the treatment of nasal NKTCL.