The Mechanism Of Rhein Hybrid 7F Targeting EGFR And Rac1 Inhibiting Invasion And Metastasis Of TNBC

BackgroundBreast cancer has become the malignant tumor with the highest diagnosis rate and mortality among women in the world,and the triple negative breast cancer（TNBC）is a breast cancer subtype characterized by the deletion of estrogen receptor（ER）,progesterone receptor（PR）and human epidermal growth factor（Her-2）.TNBC has the characteristics of easy metastasis,easy recurrence and early age of onset and there is no clear treatment target and targeted drugs.A new quinazoline Rhein hybrid 7F（7F for short）was designed and synthesized using molecular docking computer-aided drug design technology with EGFR and Rac1 proteins as targets.ObjectiveIn this paper,metastatic TNBC MDA-MB-231 cells,low metastatic breast cancer MCF-7 and normal breast epithelial MCF-10A were used to study the effects of 7F on the morphological changes,cell movement,invasion,cell cycle,apoptosis and other functions.By verifying the regulation of EGFR and Rac1targets,the mechanism of 7F on breast cancer cell death and its effect on related proteins were revealed.Finally,the mechanism involved in the inhibition of MDA-MB-231 invasion and metastasis of triple negative breast cancer cells by7F was determined,and a new compound inhibiting triple negative breast cancer was obtained.Methods1.Molecular docking software MOE.2008 was used to evaluate the interaction between gefitinib,Rhein,7F docking with Rac1 and EGFR proteins.MTT assay was used to assess the tumor activity of gefitinib,Rhein and 7F;Western Blot was used to detect the regulation of 7F on EGFR and Rac1 in MDA-MB-231 and MCF-7 cells.2.The toxicity of 7F on various tumor cells was determined by MTT and CCK-8 methods.The effect of 7F on cell proliferation was observed by colony formation assay.The morphological and ultrastructural changes of organelles were observed by HE staining and electron microscope,respectively.Apoptosis and cell cycle distribution were detected by flow cytometry.Western Blot was used to detect the expression of apoptosis-related proteins Caspase-3,Caspase-7,Caspase-9,Bcl-2,Bax,Survivin and PARP.Finally,the mode of cell death induced by 7F was determined..3.Scratch and invasion assays were used to study the ability of 7F to inhibit the invasion and metastasis of MDA-MB-231 cells.The angiogenesis assay was used to explore the effect of 7F on the angiogenesis ability of cells.Western Blot was used to detect EMT-related proteins includingβ-catenin,Vimentin,Snail,E-cadherin,and matrix metalloproteinases MMP-2,MMP-7,MMP-9.Finally,the ability and potential mechanism of 7F inhibiting cell invasion and metastasis were clarified.Results1.The binding energy scores of rhein,gefitinib and 7F docking with EGFR protein were-16.330 Kcal/mol,-27.211 Kcal/mol and-35.325 Kcal/mol,respectively.The binding energy scores of 7F,rhein,gefitinib docking with Rac1 were-31.215 Kcal/mol,-12.759 Kcal/mol and-25.953 Kcal/mol,respectively.The lower the score,the stronger the protein binding ability.IC₅₀values on ovarian cancer,liver cancer,osteosarcoma and breast cancer cells were lowerthan that of Rhein and gefitinib.Meanwhile,the IC₅₀of normal breast MCF-10A cells was higher than that of MDA-MB-231 cells,showing a cell selectivity of 7F.Western Blot results showed that with the increase of 7F concentration,the protein expressions of EGFR and P-EGFR decreased in MDA-MB-231 cells.In MCF-7 cells,EGFR expression was decreased at 8μM7F,and with the increase of 7F concentration,P-EGFR protein expression gradually decreased.2.The IC₅₀ values of breast cancer MDA-MB-231 and MCF-7 cells treated with 7F for 48h were（2.53±0.83）μM and（7.54±1.25）μM,respectively,and the colony formation assay showed that 7F could significantly inhibit the proliferation of breast cancer cells.7F had a significant effect on the cell cycle distribution and apoptosis rate in MDA-MB-231 cells.With the increase of 7F concentration,it gradually blocks the MDA-MB-231 cell cycle at the G₂/M phase.The apoptosis rate of MDA-MB-231 was（5.23±1.44）%,（33.99±1.56）%,（77.37±3.55）%and（89.37±5.19）%,when the concentration of 7F was 0,1,2,4μM,respectively.Compared with the control group,the difference was statistically significant（P<0.001）.7F had no effect on the cell cycle in MCF-7 cells.When the concentration of 7F was 0,4,8,16μM,the apoptosis rates of MCF-7 was（4.43±0.71）%,（30.91±4.01）%,（39.37±3.07）%and（51.10±3.84）%,respectively.Compared with the control group,the difference was statistically significant（P<0.001）.In the two kinds of breast cancer cells treated with 7F,the nucleus were shrank,the cytoplasmic content decreased,the cell volume decreased and the junction disappeared.Under the electron microscopy,it can be seen that the nuclei were clustered at the edge,the nucleolus were fragmented,the mitochondria were swelled,the crest disappeared,and obvious vacuoles appeared in mitochondria.Apoptotic bodies can be found in the cells,showing typical morphological changes of apoptosis.With the increase of the 7F concentration,the expression of anti-apoptotic proteins such as survivin and Bcl-2 decreased,while the expression of pro-apoptotic proteins such as Bax increased.Moreover,Caspase-3,caspase-7,caspase-9 and other key apoptotic proteases were activated.Cleaved-caspase-9,Cleaved-caspase-3,Cleaved-PARP and other cleaved proteins appeared.It is speculated that 7F may inhibit the proliferation of breast cancer cells by inducing apoptosis.3.7F significantly inhibited cell migration and invasion,and inhibited angiogenesis or mimicry in MDA-MB-231 cells and human umbilical vein endothelial（HUVECS）cells.Western Blot showed that matrix metalloproteinases MMP-2,MMP-7,MMP-9,Snail protein with the 7F concentration increasing,epithelial-mesenchymal transition（EMT）marker proteinβ-catenin,Vimentin protein were decreased,while E-cadherin protein was increased.The results indicated that 7F could reverse the EMT of MDA-MB-231 cells.Conclusion1.7F synthesized by combining the anthraquinone skeleton of natural products with a drug active group quinazoline,is a new anthraquinone hybrid with better anti-tumor activity superior to lead compound Rhein.7F has a good inhibitory effect on many kinds of tumor cells.It can inhibit the expression of EGFR and Rac1 of TNBC at the same time,and may be a dual-target small molecule inhibitor of EGFR and Rac1 protein.2.7F inhibits the proliferation of breast cancer MDA-MB-231 and MCF-7cells,and its effect is superior to cisplatin.Its mechanism is related to regulating the expression of Bax,Bcl-2,caspase-3,PARP and other proteins and inducing cell apoptosis.3.7F can significantly inhibit the invasion and metastasis of TNBC by inhibiting tumor angiogenesis and epithelial mesenchymal transformation.