Study Of Effect Of Trichostatin A On Biological Process Of Burkitt's Iymphoma Cells And Its Mechanism

Burkitt's lymphoma(BL)is one of the most aggressive forms of B-cell NHL,which is also considered as one of the fastest growing amongst malignancies.The current treatments of BL including chemotherapy,radiotherapy,or combined with monoclonal antibodies,often lead to relapse and resistance.Thus,targeted therapy is still needed for patients with poor prognosis or relapse.Epidermal growth factor receptor substrate 8(EPS8)is a docking protein that may be phosphorylated by multiple receptor tyrosine kinases[1],participating in a variety of signal transduction,promoting tumorigenesis and metastasis[2-4].In addition,EPS8 is gradually considered to be a marker of poor prognosis[5-7],which affects the survival of patients.However,the study of EPS8 on Burkitt's lymphoma has never been reported yet.The balance of acetylation and deacetylation of histone can maintain normal function of the cells[8].The disorder of histone acetylation can cause changes in chromatin structure,leading to transcriptional imbalance related to cell cycle,cell differentiation and apoptosis.Histone deacetylase inhibitor(HDACi)is a new and effective antitumor compound,which mainly promotes histone acetylation and regulates chromatin structure,thereby affecting gene transcription and increasing the expression of tumor suppressor genes.HDACi can lead to cell cycle arrest,cell differentiation and apoptosis[9,10,11].Up to now,Trichostatin A(TSA)is considered to be one of the most effective HD AC inhibitor and few studies have been reported on Burkitt's lymphoma.What's more,the relationship between EPS8 and TSA on BL remains obscure.This study was to explore the effect of TSA on cell proliferation of Burkitt's lymphoma cells and the relationship between EPS8 expression and TSA in vitro and in vivo.Then we further explored the mechanism of TSA and the effect of TSA when combined with chemotherapeutic drugs epirubicin in order to provide a new direction for the treatment of Burkitt's lymphoma.Objective:1.To investigate the effect of TSA on Ramos cells and Namalwa cells proliferation and its molecular mechanism.2.In Ramos cells and Namalwa cells,stably overexpressing EPS8 and EPS8 knockdown were constructed by lentivirus transfection.3.To explore the effect of the combination of TSA and chemotherapeutic drugs epirubicin on Ramos cells and Namalwa cells.4.To investigate the effect of TSA and EPS8 in animal model of Burkitt's lymphoma.Methods:1.The sensitivity of Ramos and Namalwa cells to TSA were detected by MTT.The effect of TSA on the cell cycle of Ramos and Namalwa cells were detected by flow cytometry PI staining.The changes of apoptosis in Ramos and Namalwa cells induced by TSA were detected by flow cytometry.2.The expression of proteins in Ramos and Namalwa cells were detected by Western blot.3.Vectors of shRNA-EPS8 and overexpression of EPS8 were constructed and transducted into Ramos and Namalwa cells to pack up lentivirus interfering expression of EPS8.The proliferation of Ramos and Namalwa cells after silencing EPS8 and overexpression of EPS8 were detected by Trypan blue exclusion.The sensitivity of cells to TSA after silencing EPS8 and overexpression of EPS8 were detected by MTT.Western Blot was used to detect the effect of TSA on the expression of Phospho-Erkl/2 pathway proteins before and after silencing EPS8 or overexpression of EPS8.4.The effect of TSA and Epirubicin alone or in combination on Ramos and Namalwa cells activity were detected by MTT.Western blot was used to detect the expression of EPS8 and its downstream Phospho-Erkl/2 pathway after the treatment of TSA and Epirubicin alone or in combination.5.Mice were subcutaneously implanted with Namalwa-scramble or Namalwa-shRNA-EPS8 cells.Results:1.Ramos cells were more sensitive to TSA as compared to Namalwa cells.And the effect of TSA on cell viability of Ramos and Namalwa cells were dose-and time-dependent.There were significant differences of the percentage of G0/G1,S and G2/M phase in Ramos cells and Namalwa cells after treating with different concentration of TSA.With the increase of the concentration,the rate of apoptosis increased in the two cells.2.Western blot suggested that the expression of EPS8,Phospho-Erkl/2 pathway proteins,anti-apoptotic protein Bcl2 and cycle protein cyclinDl in Ramos and Namalwa cells decreased after TSA treatment.3.EPS8 knockdown suppressed cell proliferation of Ramos and Namalwa cells.Conversely,overexpression of EPS8 could increase cell proliferation.Silencing of EPS8 could decrease the activity of Phospho-Erkl/2 pathway,and EPS8 knockdown and TSA had a synergistic effect.Overexpression of EPS8 could activate the Phospho-Erkl/2 pathway,and could weaken the effect of TSA on Burkitt's lymphoma in some degree.4.Compared with TSA or Epirubicin alone,the activity of EPS8 and its downstream Phospho-Erk1/2 pathway in Ramos and Namalwa cells were significantly inhibited in a combination.5.Compared with Namalwa-scramble group,the tumor volume in Namalwa-shRNA-EPS8 group and TSA group was smaller,and the tumor volume was the smallest in the combination group.There were no significant differences of the weight of mice between the groups.Conclusion:1.TSA could inhibit cell proliferation of Ramos and Namalwa cells and promote cell cycle arrrest and cell apoptosis.2.Silencing of EPS8 could inhibit cell proliferation of Burkitt's lymphoma cells,reduced the activity of the Phospho-Erkl/2 pathway and had a synergistic effect with TSA.However,overexpression of EPS 8 had an opposite effect and could reduce the effect of TSA.3.TSA enhanced chemosensitivity of Burkitt's lymphoma cells.4.Knockdown of EPS8 enhanced the effect of TSA in inhibiting tumor growth in the Balb/c-nu mice models of Burkitt's lymphoma.