| Objective:Mantle cell lymphoma（MCL）is a type of indolent B-cell lymphoma that is highly heterogeneous,aggressive and difficult to cure.The prognosis of MCL is pretty poor,so it is an urgent need to find more effective therapies.The B cell receptor（BCR）signaling pathways are abnormally active in MCL.Bruton tyrosine kinase（BTK）is a key regulator protein in the BCR signaling pathway and can promote survival and proliferation of MCL cells by affecting signaling transduction in BCR pathways.Ibrutinib,a BTK inhibitor,was approved by FDA in November 2013for the treatment of MCL and later approved globally in 2018 due to its good clinical efficacy.However,some patients develop acquired resistance due to drug toxicity or activation of proliferation signaling pathways during treatment,which reduces the overall survival（OS）of patients and worsens the prognosis.Studies have shown that increased expression and stability of Cyclin D1 promoted the resistance of MCL cell lines to Ibrutinib.Based on previous studies of our group and literatures,heat shock protein 90（HSP90）inhibitors can reduce the expression of Cyclin D1 and Cyclin-dependent kinases 4（CDK4）,and inhibit proliferation of MCL cells.This study aims to study whether the HSP90 inhibitor Ganetespib can increase the the inhibitory effects of Ibrutinib on proliferation of MCL cells in vivo and in vitro and the relevant mechanisms.Methods:First,MTT assays were used to detect the inhibitory effects of Ibrutinib and Ganetespib on proliferation of MCL cell lines Jeko-1 and Granta-519.Subsequently,Jeko-1 and Granta-519 cells were pre-treated with Ganetespib for 12hours and then treated with Ibrutinib,and MTT and Ed U assays were used to detect whether the treatment with Ganetespib can enhance the inhibitory effects of Ibrutinib on cell proliferation in two MCL cell lines.After that,we used flow cytometry and Western Blot to evaluate the effects of combined administration on cell cycle and cell cycle biomarker proteins in Jeko-1 and Granta-519 cells.Flow cytometry and Tunel methods were used to detect the effects of combined administration on apoptosis intwo MCL cell lines,and Western Blot was used to detect changes in apoptosis-related proteins.Finally,we tested the effects of drug combination on tumor growth in nude mice xenograft tumor models,and used the immunohistochemical（IHC）staining techniques to detect proteins changes in tumor tissues related to cell proliferation,cell cycle,and apoptosis.Results:Through MTT assays,we found that both Ibrutinib and Gantespib can significantly inhibit cell proliferation of MCL cell lines Jeko-1 and Granta-519,and the half-inhibitory concentrations(IC50)of Ibrutinib in Jeko-1 and Granta-519 cell lines were 6.51±2.20μM and 5.57±1.37μM,respectively;Ganetespib’s IC50 in Jeko-1 and Granta-519 cell lines were 12.84±1.57 n M and 45.73±4.18 n M,respectively.MCL cell lines were pre-treated with Ganetespib for 12 hours and then treated with Ibrutinib,it was found that Ganetespib significantly increased the sensitivity of Jeko-1 and Granta-519 cell lines to Ibrutinib.The IC50 of Ibrutinib in Jeko-1 cell line changed from 6.51±2.20μM to 1.30±0.32μM,a decrease of about 5folds;the IC50 in Granta-519 cell line changed from 5.57±1.37μM to 2.71±0.25μM,a reduction of about 2 times.Results from Ed U experiments showed that the Ed U-positive rates of Jeko-1 cells decreased by about 33.68%after drug combination treatment;while the rates reduced by about 20.22%in the Granta-519 cells after drug combination treatment.The flow cytometry and Western Blot experiments have demonstrated that cell cycles of the two MCL cell lines was blocked in the G0/G1phase after drugs combination treatment.At the same time,the expression of Cyclin D1,CDK2,and CDK4 was down-regulated.Using flow cytometry,it was found that compared with the control group,the percentage of apoptosis cells was increased by14.78%in Jeko-1 cells and it was increased by 41.64%in Granta-519 cells when compared to the control group.The Tunel assays found that after the drug combination,the percentage of positive Tunel cells was increased by about 23.45%in Jeko-1 cells,and it was increased by about 41.64%in Granta-519 cells when compared to the control group.Western Blot was used to detect the effects of drug combination on apoptotic biomarker proteins in two MCL cell lines,and it was found that the expression of cleaved caspase-9 was increased while that of the anti-apoptotic protein BCL-2 was decreased when compared to the control and single-drug treatment groups.Through xenograft tumor models in nude mice,we found that the combined administration inhibited the tumor growth by about 74.49%when compared to the control group,and the immunohistochemical results showed that compared with the control group.And the expression of Ki-67 in tumor tissues of the combined administration group was reduced by about 81.95%;the expression of Cyclin D1protein was reduced by about 66.67%;and the expression of BCL-2 protein was reduced by about 69.44%.Conclusion:This current study found that the combination of HSP90 inhibitor Ganetespib with BTK inhibitor Ibrutinib increased the sensitivity of MCL cell lines to Ibrutinib.After the combined use of drugs,the IC50 value of Ibrutinib was decreased by a reduction of about five folds in Jeko-1 cells and the IC50 value was decreased by a reduction of about 2 times in Granta-519 cells.And the drug combination reduces the expression of Cyclin D1,CDK2,and CDK4 in MCL cell line and blocks the cell cycle process in the G0/G1 phase.At the same time,the expression of apoptotic proteins cleaved caspase-9 increased,and the expression of anti-apoptotic protein BCL-2 decreased,which promoted cell apoptosis.In addition,through xenograft tumor models in nude mice,we found that the combined administration of Ganetespib and Ibrutinib reduced the expression of Ki-67,Cyclin D1 and BCL-2 proteins in tumor tissues,and inhibited tumor growth without significant toxicity.This study will provide new treatment strategies for the treatment of MCL and hopefully improve the prognosis of MCL patients. |
