Lapatinib In Combination With Bortezomib Inhibits Breast Cancer Cells Growth And The Activity Of Bortezomib To Lapatinib-resistant Breast Cancer Cell

Objective:(1) To explore the effect oflapatinib and bortezomib,alone or in combination,to the growth,cell cycles and signalling transduction pathway of breast cancer cell lines.(2) To determine the activity of bortezomib to lapatinib-resistant breast cancer cell.Methods:(1) The effect of lapatinib and bortezomib to growth of SK-BR3 breast cancer cell was assayed by CCK-8 kit.The combined effect of the two agents was tested by combination index(CI) of Chou and Talary.The effect of lapatinib and bortezomib to SK-BR3 cell cycles distribution was assessed by flow cytometry, and an annexin V/propidium iodide assay was used to identify induction of apoptosis.The expression of Her2 signal transduction pathway proteins,Her2,p-Her2,p-Akt,p-Erkl/2,Akt,Erkl/2,influenced by lapatinib and bortezomib was assayed by western-blot.In the mean time,the level of P27 protein expression was assayed.(2) Lapatinib-resistant breast cancer cell was cultured.The effect of bortezomib to lapatinib-resistant breast cancer cell proliferation was assayed by CCK-8 kit.The effect of bortezomib to lapatinib-resistant cell cycles distribution was assayed by FCM.The expression of Her2 signal transduction pathway proteins, Her2,p-Her2,p-Akt,p-Erkl/2,Akt,Erkl/2,p-4E-BP1,influenced by bortezomib was assayed by western-blot.The level of P27 protein expression was also assayed.Results:(1) The growth inhibition of lapatinib and bortezomib to SK-BR3 breast cancer cell was dose-dependent.The combined effect of the two agents was additive,but not synergistic,because combination index(CI) of them was equal to 1. As the concentration of lapatinib increased,the percentage of the G0/G1 phase of SK-BR3 breast cancer cell also increased(P＜0.05);the percentage of the S phase of SK-BR3 breast cancer cell also decreased(P＜0.05).The effect of bortezomib to SK-BR3 breast cancer cell cycle distribution was similar to that of lapatinib.The effect of lapatinib in combination with bortezomib was more significant than either of the two agents.Apoptosis of SK-BR3 breast cancer cell induced by lapatinib and bortezomib was related to the concentration of the two agents(P＜0.05,P＜0.05), and the percentage of apoptosis of SK-BR3 breast cancer cell treated by combination therapy significantly increased.Western-blot assay showed that lapatinib can downregulate the expression of p-Her2,p-Akt,p-Erk1/2 proteins,the effect was doseand time-dependent.While lapatinib had no influence on the expression of Her2,Akt,Erk1/2 proteins.Contrary to lapatinib,p-Erk1/2 protein of SK-BR3 breast cancer cell treated by bortezomib was uprgulated.When SK-BR3 breast cancer cell treated by lapatinib and bortezomib,p-Her2,p-Akt proteins was downregulated more significantly.Lapatinib with higher concentration(1μM) can abrogate the role of bortezomib to Erk pathway.Lapatinib and bortezomib can upregulate the expression of P27 protein,and the effect of combined therapy was more significant.The effect of lapatinib and bortezomib to Her2 signal transduction pathway of BT474 breast cancer cell was relatively not obvious.(2) The growth inhibition of bortezomib to lapatinib-resistant SK-BR3 breast cancer cell was time- and dose-dependent.As the concentration of bortezomib increased,the percentage of the G2/M phase of lapatinib-resistant cell also increased(P＜0.05);the percentage of the S phase of lapatinib-resistant cell also decreased(P＜0.05).bortezomib can downregulate the expression of p-Her2,p-Akt proteins,the effect was dose- dependent.While bortezomib had no significant influence on the expression of Her2,Akt,Erk1/2,p-Erk1/2 proteins.Bortezomib can upregulate the expression of P27 protein,as the concentration increased.Conclusions:(1) Lapatinib and bortezomib can inhibit the growth of SK-BR3 breast cancer cell line,the effect of the two agents was additive.Lapatinib in combination with bortezomib can upregulate the expression of P27 protein,and strongly inhibit activity of PI3K-Akt pathway.Lapatinib can abrogate the upregulation role of bortezomib to Ras-MAPK pathway.(2) Bortezomib can inhibit proliferation of lapatinib-resistant SK-BR3 breast cancer cell line.Bortezomib can inhibit activity of PI3K-Akt pathway,can strongly upregulate the expression of P27 protein,but had no significant influence on Ras-MAPK pathway.Bortezomib could prevent or delay the resistance of SK-BR3 breast cancer cell to lapatinib.