The Effect And Related Mechanisms Of Pterostilbene Intervening EMT Process And Stemness To Inhibit The Metastasis In Triple Negative Breast Cancer

ObjectiveIn this study,the effects of Pterostilbene(PTE)on invasion and metastasis of triple-negative breast cancer were studied at the tumor cell level in vitro and in mice with spontaneous lung metastasis,and the related molecular mechanism was discussed.Methods1.In vitro:①MTT assay was used to detect the effect of PTE on the proliferation of MDA-MB-231 cells;②Transwell migration and Transwell invasion assay were used to observe the effect of PTE on migration and invasion of MDA-MB-231 cells;③The effect of PTE on the self-renewal ability of MDA-MB-231 cells was detected by serum-free suspension culture;④Flow cytometry and Western blot were used to detect the effect of PTE on stem cell marker CD44+/CD24-and stem-related protein of MDA-MB-231 cells;⑤Immunofluorescence and Western blot were used to detect the effect of PTE on EMT markers of MDA-MB-231 cells;⑥Transcriptome sequencing technology and Western blot were used to detect the effect of PTE on the expression of metastasis-related markers;⑦Western blot was used to detect the effect of PTE on the expression of related markers of AKT/GSK3β/β-catenin pathway.2.In vivo:4T1 cells were inoculated into the fourth pair of breast fat pads on the right side of BALB/c mice to establish the model of spontaneous lung metastasis breast tumor in mice.①Observe the status of mice and record their body weight during the experiment;②Observe the effect of PTE on the growth of lung metastasis nodules in tumor-bearing mice with triple negative breast cancer;③HE staining to observe the pathological changes of mouse organs(heart,liver,spleen,lung,kidney)in each group;④Immunohistochemical was used to detect the expression of E-cadherin,N-cadherin,MMP2,CD44 and β-catenin in tumor.Results1.Cellular level①PTE inhibits the proliferation and self-renewal ability of MDA-MB-231 cells:MTT experiment results show that the survival rate of MDA-MB-231 cells is significantly reduced in a time-dose-dependent manner after PTE treatment(P<0.001);MDA-MB-231 cells cultured in serum-free suspension after the mammosphere was treated with PTE,it was significantly reduced compared with the control group.PTE can inhibit the proliferation and self-renewal ability of MDA-MB-231 cells.②PTE inhibits the invasion and migration of MDA-MB-231 cells:The results of Transwell migration and Trans well invasion experiments show that compared with the control group,PTE can inhibit the migration and invasion of MDA-MB-231 cells(P<0.01,P<0.001);The results of transcriptome sequencing technology showed that PTE can inhibit the expression of metastasis-promoting genes and enhance the expression of anti-metastatic genes;Western blot results showed that compared with the control group,PTE can inhibit the protein expression of metastasis-related markers MMP2,MMP9,and Galectin-1(P<0.01,P<0.05,P<0.01).③PTE inhibits the stemness expression of MDA-MB-231 cells:Flow cytometry results show that PTE can reduce the ratio of stemness markers CD44+/CD24-;Western blot results show that compared with the control group,PTE can inhibit the protein expression of MDA-MB-231 cell stemness related markers CD44,SOX2,OCT4(P<0.001,P<0.001,P<0.01).④PTE inhibits the EMT process of MDA-MB-231 cells:The results of cellular immunofluorescence experiments show that the fluorescence intensity of E-cadherin increases after PTE treatment,while the fluorescence intensity of N-cadherin and Vimentin decreases;Western blot experiment results showed that compared with the control group,PTE can enhance the protein expression of E-cadherin in MDA-MB-231 cells(P<0.001),and reduce the protein expression of N-cadherin,Vimentin,ZEB1,SNAI1,and TWIST2(P<0.05,P<0.01,P<0.01,P<0.01,P<0.01).⑤PTE regulates AKT/GAS3β/β-catenin pathway:Western blot results show that PTE can inhibit the expression of p-AKT,p-GSK3β,β-catenin(Nucleus),c-MYC and CyclinDl protein(P<0.01,P<0.001,P<0.01).2.Overall level① Body weight:During the experiment,there was no obvious change in the body weight of mice treated with PTE.② PTE cannot significantly inhibit the tumor in situ,but it can significantly reduce the number of metastatic nodules in the lungs of tumor-bearing mice(P<0.001).③ The results of HE staining show that PTE can inhibit the number of nodules in the lung and liver of tumor-bearing mice;Cisplatin can cause a certain degree of damage to the spleen and kidney of tumor-bearing mice,but the other organs(heart,liver,lung,kidney)of the mice in the PTE treatment group have no obvious pathological changes.④ The results of Immunohistochemical show that PTE could significantly reduce the expression levels of N-cadherin,CD44 and MMP2 in tumor tissues(P<0.01,P<0.01,P<0.05).Conclusion1.PTE could regulate AKT/GSK-3β/β-catenin signal pathway to interfere with EMT progression and stemness of MDA-MB-231 cells,thus inhibit invasion and metastasis of MDA-MB-231 cells.2.PTE could inhibit lung metastasis in tumor-bearing mice.