Effects Of Bortezomib Combined With Adriamycin On The Cell Line Of The Lymphoma

The chemotherapy, the radiotherapy, and the hematopoietic stem cell transplantation were the predominant strategies for clinical therapy of lymphoma in recent years. Most recently, insights into the molecular patheogenesis of lymphoma have led to development of new targeted agents for lymphoma. Such agents include the monoclonal antibody, radioimmunotherapy, enzyme-specific inhibitors, tumor vaccine. PS-341 was the first of proteosome inhibitors on the clinical research , which has been used for multiple myelpma and some non-hodgkin lymphoma. To investigate the effect of PS-341 on the anti-proliferation of lymphoma, the cell lines of Hodgkin lymphoma and T cell lymphoma(L428, Jurkat) were used in our experiment. We explored the mechanism of PS-341 inducing apoptotic effect on cell line of the lymphoma and the synergistic efficacy of ps-341 in combination with adriamycin. which provides an experimental evidence of PS-341 for clinical treatment of lymphoma.First of all, we used MTT assay to examine the effects of PS-341 orAdriamycin on the growth of L428 cells and Jurkat cells, The results indicated that cell viability in the presence of PS-341 or Adriamycin decreased in a dose-dependent manner. The inhibitory rates of cell growth were positively correlated with PS-341 or Adriamycin concentrations (P< 0.01). The growth-inhibitory IC₅₀ of L428 values were 391.82 ng/ml; 173. 86ng/ml; 49.9ng/ml after the treatment of PS-341 for 24, 48, 72 hours, and the growth-inhibitory IC50 of L428 values were 1955.56ng/ml,464.93 ng/ml, 74. 77 ng/ml after the treatment of Adriamycin for 24, 48, 72 hours respectively. The growth-inhibitory IC₅₀ of Jurkat values were 137.64 ng/ml; 69. 50ng/ml; 31.23ng/ml after the treatment of PS-341 for 24, 48, 72 hours, and the growth-inhibitory IC₅₀ of Jurkat values were 471. 38ng/ml, 226. 57 ng/ml,72. 58 ng/ml after the treatment of Adriamycin for 24, 48, 72 hours respectively.Then, in order to investigate whether apoptosis is associated with the antitumor activity of PS-341 in L428 and Jurkat ,we observed the cell morphology using Wright-Giemsa staining, evaluated the exposure of phosphatidylserine (PS) on L428 cells and Jurkat cells after double staining with fluorescein isothiocyanate (FITC)-labeled annexin V and propidium iodide (PI). We found typical apoptotic morphology such as pyknosis and apoptotic body after L428 cells or Jurkat cells were exposed to PS-341 for 24h.. Apoptosis induced by PS-341 was also confirmed using Annexin V and PI staining to detect externalization of PS on the cell membrane. The apoptosis of cells induced by PS-341 was in a dose-dependent manner.To further investigate the mechanism possibly involved in PS-341-induced cell line of the lymphoma, we used western-blot to assay the protein expression of the caspase single and Bcl-2 family members. The raw tape of caspase-3, 8,PARP,Bcl-2 were down-regulated and the cleaved tape of caspase-3, 8,PARP,Bax protein level was up-regulatedwith the dose of PS-341 increasing. when Jurkat cells was treated by PS-341 for 24h, there were no changes in the level of Bcl-2 and Bax , other changes were the same as the L428 cell treated by PS-341 for 24h.We further examined whether Adriamycin could enhance the growth inhibitory effect of PS-341. L428 cells and Jurkat cells were treated for 24 hours by a certain dose of Adriamycin, which was more lower than the IC50, combined with a certain dose of PS-341 ,which aslo was more lower than the IC50. We could find that Adriamycin inhanced the effects of PS-341 as analyzed by MTT assay.Also the double staining with fluorescein isothiocyanate (FITC) -labeled annexin V and propidium iodide (PI) could show that Adriamycin inhance the effects of PS-341.In conclusion, PS-341 can inhibit the viability of L428 and Jurkat cells in a dose-dependent manner. And this effect is induced by apoptosis. PS-341 triggers the apoptosis of L428 cell line through the mitochondrial pathway and exogenous pathway. But PS-341 triggers the apoptosis of Jurkat cell line just through exogenous pathway .Adriamycin can enhance the cytotoxicity of PS-341.