The Synergistic Effect Of Nrf2 Inhibitor In Combination With Trastuzumab In HER2-positive Tumors And Its Mechanism Of Action

BackgroundTrastuzumab is a Food and Drug Administration(FDA)-approved antibody for the treatment of HER2-positive breast cancer and gastric cancer.Despite the clinical efficacy,a majority of patients treated with trastuzumab developed resistance within 1 year of treatment initiation.Recent studies showed that Nrf2 is a key transcription factor that promotes tumor progression and drug resistance through interaction with other cell proliferation signaling pathways.Brusatol can be isolated from Brucea,and it acts as an unparalleled inhibitor of the Nrf2 protein expression.Brusatol has exhibited effective anti-tumor effects against liver cancer and lung cancer.However,few studies disclosed the anti-tumor effects of brusatol on HER2-positive tumors.ObjectiveOur present research aimed to study the synergistic effect and the mechanism of action of brusatol in combination with trastuzumab in HER2-positive tumors and provide a new insight on exploring Nrf2 as a potential new therapeutic target in overcoming trastuzumabresistant breast cancer.Methods1.Firstly,to observe the effect of brusatol on the growth and proliferation of HER2-positive BT-474,SK-BR-3 and SK-OV-3 cells by the CCK-8 assay.What′s more,to examine the inhibitory effect of brusatol on the Nrf2/HO-1 and HER2-AKT/ERK1/2signaling pathway by Western Blot assay.2.Secondly,to utilize CCK-8 assay to evaluate the activity of brusatol on cell viability and growth,and calculate combination index(CI)of brusatol plus trastuzumab by Compu Syn Software.On the other hand,to observe the in vivo inhibitory effect of brusatol plus trastuzumab in SK-OV-3 and BT-474 tumor-bearing nude mice.3.Finally,to detect the inhibitory effect of brusatol plus trastuzumab on Nrf2/HO-1 and HER2-AKT/ERK1/2 signaling pathways in BT-474 and SK-OV-3 cells and tumor tissue by Western Blot assay.Besides,to detect the effect of trastuzumab on cell proliferation and the changes upon the HER2-AKT/ERK1/2 signaling pathway after knockdown of Nrf2 by transfection of si RNA.Furthermore,to detect the level of ROS accumulation and apoptosis percentage by flow cytometry upon the treatment of trastuzumab plus brusatol.Results1.Brusatol exhibited potent anti-tumor effects on the growth and proliferation of HER2-positive BT-474,SK-BR-3 and SK-OV-3 cells.2.Brusatol effectively inhibits the Nrf2/HO-1 anti-oxidant pathway and the HER2-AKT/ERK1/2 signaling pathway in HER2-positive tumor cells.3.Trastuzumab in combination with brusatol exerts a synergistic anti-tumor effect on the growth and proliferation of BT-474 and SK-OV-3 cells in vitro.4.Compared to trastuzumab or brusatol alone,trastuzumab in combination with brusatol is more effective in inhibiting the growth of tumor in vivo.5.Trastuzumab and brusatol synergistically inhibited the Nrf2/HO-1 and the HER2-AKT/ERK1/2 signaling pathway in vivo and in vitro.6.Nrf2 knockdown sensitized SK-OV-3 cells to trastuzumab and regressed the HER2-AKT/ERK1/2 signaling pathway.7.Brusatol treatment promoted ROS accumulation and resulted in apoptosis in BT-474 and SK-OV-3 cells induced by trastuzumab.ConclusionBrusatol enhanced the sensitivity of HER2-positive cancer cells to trastuzumab by inhibiting Nrf2/HO-1 and HER2-AKT/ERK1/2 signaling pathways in vivo and in vitro.It indicated that the combination brusatol with trastuzumab has great potential to treat HER2-positive tumors in clinics.