The Mechanism Of ALOX5 In HER-2 Positive Breast Cancer And The Application Of Inhibitor Zileuton

Objective:Explores the mechanism of ALOX5 in HER-2 positive breast cancer and the effect of the ALOX5 inhibitor,Zileuton,on the growth of HER-2 positive breast cancer.Methods:1.Immunohistochemical staining（IHC）and ELISA were used to detect the expression levels of ALOX5 and 5-HETE protein in 30 cases of breast cancer tissue samples and 10 cases of normal breast tissue samples,respectively.Three samples were taken from each patient.2.Western blot was used to detect the expression as well a phosphorylation levels of HER-2,ALOX5,ALOX5AP,paxillin,FAK,E-cadherin,vimentin,PI3K,Akt,and mTOR in the breast cancer cells.3.G-LISA was used to detect the activity of RhoA.4.HER-2 gene in SKBR3/BT-474 breast cancer cells and ALOX5 gene in MCF-7/SKBR3 breast cancer cells were knockdown by siRNA transfection.Herceptin was used to inhibit the phosphorylation of HER-2 in SKBR3 and BT-474 cells,and the activity of ALOX5 in MCF-7 and SKBR3 cells was inhibited by the ALOX5 inhibitor Zileuton.5.The pcmv3-c-his vector contained the ALOX5 gene was transfected into MCF-7 and SKBR3 cells.6.Cell proliferation was detected using the Brd U Cell Proliferation Assay Kit.Cell apoptosis was determined by labeling and analyzing the apoptotic cells using annexin V-FITC and propidium iodide（PI）apoptosis kit and flow cytometry.Cell migration was determined using Boyden chamber assay.7.HER-2 tumor-bearing animal models（SKBR3）were constructed and the effect of Zileuton on tumor growth was observed.BALB/c female nude mice were selected for tumor-burdened models.SKBR3 cells suspension（10⁷cells/ml,0.1ml）was injected into the flank of each nude mouse.Seven days after tumor transplant（size>100 mm³）,the tumor-burdened mice were randomly divided into Control group（100μl/d 20%DMSO saline,n=4）and Zileuton group（100μl/d Zileuton（100mg/kg/d）,n=4）,through oral administration,respectively.Tumor size（long diameter a×short diameter b²/2）was measured every 5 days.Twenty days later,The nude mice were euthanized by CO₂when the tumor size>150mm³.Transplanted tumors were dissected,weighed,and then stored in10%formaldehyde solution.The expression level of PCNA was used to evaluate the proliferation of the transplanted tumor cells by IHC.The apoptosis was detected by TUNEL.Results:1.The immunohistochemistry results suggested that the expres-sion level of ALOX5 in the breast cancer tissues was significantly higher than that in the normal breast tissues（P<0.05）.The ELISA results suggested that the expression level of 5-HETE was significantly increased in breast cancer tissues than in the normal breast tissues as well（P<0.05）.The expression level of ALOX5 had a 1.47–7.07 times increase in 83.33%patients（25/30）.HER-2was increased>5 times in all the patients.In 20 cases（66.67%）,the expression level of 5-HETE in the breast cancer tissue was also increased.Spearman rank correlation analysis showed that the expression of ALOX5 was significantly correlated with HER-2 expression and lymph node metastasis（P<0.05）,but not with ER and PR expression（P>0.05）.2.The western blot results suggested that the expression levels of HER-2 and ALOX5 in SKBR3 and BT-474 cells were increased significantly than that in MCF-7 cells（P<0.05）.The expression levels of 5-HETE was also increased significantly（ELISA）.3.Western blot results suggested that the expression level of HER-2 in SKBR3and BT-474 cells was decreased by knocking down the HER-2 gene（P<0.05）.The phosphorylation of HER-2 in SKBR3 and BT-474 cells was significantly inhibited by Herceptin treatment（P<0.05）,without changing the total protein expression level（P>0.05）.The expression of ALOX5 was significantly inhibited（P<0.05）without changing the expression of ALOX5AP（P>0.05）.In addition,the expression of 5-HETE was also significantly inhibited（ELISA）.4.Western blot results suggested that the expression of ALOX5 was inhibited by knocking down the ALOX5 gene（P<0.05）.4.ALOX5 gene knockdown using two independent siRNAs consistently inhibited proliferation and migration as well as inducing apoptosis in MCF-7 and SKBR3 cells（P<0.05）.Our data also suggested that the ALOX5 inhibition was more sensitive in SKBR3 cells rather than in MCF-7.The decreased growth,survival,and migration in MCF-7 and SKBR3 cells are not limited to ALOX5 gene knockdown;the treatment of a selective ALOX5 inhibitor,Zileuton,resulted in a similar phenotype in MCF-7 and SKBR3 cells with a dose-dependent manner（P<0.05）.5.Overexpression of ALOX5 significantly promoted MCF-7 and SKBR3 cells proliferation and migration（P<0.05）,rather than apoptosis（P>0.05）.6.The activity of RhoA and phosphorylation of paxillin,FAK and PI3K,Akt and mTOR in SKBR3 cells was inhibited significantly by knocking down ALOX5 gene or application of Zileuton（P<0.05）without changing the expression of paxillin,FAK and PI3K,Akt,mTOR,E-cadherin and vimentin（P>0.05）.7.Compared to the Control group,the tumor size of the tumor-bearing mouse in Zileuton group was significantly smaller than that in Control group（P<0.05）,as well as the tumor weight（2.80g±0.60 vs6.00g±2.50g,P<0.05）.The proliferation of transplanted tumor cells in the Zileuton group was significantly decreased（P<0.05）,while the apoptosis was significantly increased（P<0.05）,compared to the Control group.Conclusion:1.The expression and activity of ALOX5 in the breast cancer tissues,especially in HER-2 positive tissues is increased.The expression and activity of ALOX5 are affected by HER-2,and both have synergistic effects.2.The overexpression and activation of ALOX5 promote proliferation and migration in the breast cancer cells and inhibits apoptosis.The activation of PI3K/Akt/mTOR pathway plays an important role in this process.The promotion of cell migration is also related to ALOX5’s ability to increase RhoA activity and promote adhesion spot formation.3.The selective ALOX5inhibitor Zileuton significantly inhibits the growth of HER-2 positive breast cancer.ALOX5 inhibitor Zileuton has an inhibitory effect on HER-2 positive breast cancer,which will benefit further clinical research.