Study On The Inhibitory Effect And Mechanism Of Chelerythrine On Triple-negative Breast Cancer

Objective: Breast cancer is a disease that threatens human life and health worldwide,and is also the second leading cause of cancer deaths.Triple-negative breast cancer(TNBC)is a type of breast cancer that is more aggressive than other types of breast cancer.Traditional Chinese medicine believes that breast tumors belong to the categories of "ruyan" and "ruishi carbuncle",which are mainly caused by the deficiency of righteous qi and the imbalance of yin and yang of the viscera.At present,there are many reports on traditional Chinese medicine celandine analgesia and anti-tumor.Celandine erythrine(CHE)is one of the main active components of celandine,which has biological activities such as antibacterial and antitumor.However,there are few reports on the role of CHE in the proliferation,invasion,metastasis and tumor microenvironment of triple-negative breast cancer.We selected two triple-negative breast cancer cells,the mouse breast cancer cell line4T1 and the human breast cancer cell line MDA-MB-231.Firstly,the effects of celandine extract(CM)and its constituent CHE on the growth and viability of triple-negative breast cancer cells were analyzed.The anti-breast cancer effect of CHE was confirmed,and the apoptosis of triple-negative breast cells was induced,and the mechanism of CHE against triple-negative breast cancer tumor and metastasis was further explored.The efficacy of CHE in inhibiting the proliferation and metastasis of 4T1 cell tumor was verified in animals.In this study,the mechanism of action of CHE against triple-negative breast cancer tumors was deeply discussed,which laid the foundation for the development and clinical application of CHE new drugs.Method:Part 1 In vitro demonstration that CHE inhibits triple-negative breast cancer cell proliferation and induces apoptosis1.CM and CHE inhibit the proliferation of triple-negative breast cancer cellsThe inhibitory effect of CM and CHE on triple-negative breast cancer 4T1 and MDA-MB-231 cells was studied by MTT experiment,the inhibition rate was calculated,and the anti-triple-negative breast cancer tumor effects were compared.2.CHE induces apoptosis in triple-negative breast cancer cellsAfter 24 h of CHE treatment,(1)the cell morphology of triple-negative breast cancer cells 4T1 and MDA-MB-231 was observed under a microscope.(2)Carry out cell clone formation experiment on triple negative breast cancer cells 4T1 and MDA-MB-231 to observe clone formation.(3)Apoptosis of 4T1 and MDA-MB-231 triple-negative breast cancer cells were observed by fluorescence microscope.(4)BAX,BCL-2,Procaspase-3 and Procaspase-9 apoptosis-related proteins in 4T1 and MDA-MB-231 triple-negative breast cancer cells were detected by Western blotting after 24 hours of CHE treatment.Part II In vitro demonstration of CHE inhibiting the invasion and metastasis of triple-negative breast cancer1.After CM and CHE acted for 24 hours,the horizontal migration ability of CHE on two breast cancer cells was observed by scratch test.2.To observe the vertical migration ability of CHE on two breast cancer cells by Transwell assay.3.Western blotting was used to detect epithelial-mesenchymal transition-related proteins E-cadherin,Vimentin,transcription factors SLUG,SNAIL,MMP-2 and MMP-9proteins.Part 3: In vivo effect of CHE on triple-negative breast cancer1.CHE inhibits breast cancer in situAn orthotopic mouse model was established by injecting mouse 4T1-LUC breast cancer cells into the mouse mammary fat pad to study the inhibitory effect of CHE on triple-negative breast cancer tumors.The size of the tumor mass was measured,the in situ breast tissue tumor formation was observed by in vivo imaging system,the necrosis of the tumor mass was observed by HE staining,and the expression levels of Ki67,CD31,CD163 and Gr1+CD11b were detected by immunohistochemistry.2.CHE inhibits breast cancer metastasisThe mouse 4T1-LUC breast cancer cells were injected into the tail vein of mice to establish a metastasis model,and the effect of CHE on inhibiting the metastasis of triple-negative breast cancer cells was studied.Lung tissue tumors were observed by in vivo imaging system,and changes in lung were observed by HE staining.Part IV: The mechanism of CHE inhibiting triple-negative breast cancer1.Western blotting detection of 4T1 and MDA-MB-231 triple negative breast cancer cells agent verification.(2)The protein expression of TGF-β-related proteins TGF-βR2,p-SMAD2/3,SMAD2/3 and SMAD4.2.Western blotting detection(1)The protein expression of MAPK-related proteins p-ERK,ERK,p-JNK,JNK,p-P38 and P38 in tumor tissue of orthotopic tumor model mice,and added inhibitors to verify.The expression of apoptosis-related proteins of BAX,BCL-2,Procaspase-3 and Procaspase-9 was detected by Western blotting in the tumor mass(2)TGF-β-related proteins TGF-βR2,p-SMAD2/ 3.The protein expression of SMAD2/3 and SMAD4.The expression levels of epithelial-mesenchymal transition-related proteins E-cadherin,Vimentin,transcription factors SLUG,SNAIL,MMP-2 and MMP-9 were detected by Western blotting in lung tissue.Results: 1.The results of MTT assay showed that CM and CHE had obvious inhibitory effects on the viability of 4T1 and MDA-MB-231 triple-negative breast cancer cells.2.The results of colony formation assay and Hoechst 33258 staining assay showed that the increase of CHE concentration reduced the proliferation of 4T1 and MDA-MB-231triple-negative breast cancer cells and increased the number of apoptotic cells.3.Western blotting results showed that CHE could up-regulate BAX protein levels and down-regulate BCL-2,Procaspase-3 and Procaspase-9 protein expressions in 4T1 and MDA-MB-231 cells.4.The results of scratch experiment and Transwell experiment showed that CM and CHE inhibited the healing of scratches in 4T1 and MDA-MB-231 cells,and CHE could reduce the number of transmembrane cells in Transwell migration experiment,suggesting that CHE could affect both 4T1 and MDA-MB-231 cells.It has a certain inhibitory effect on the migration and invasion ability of triple-negative breast cancer cells.5.Western blotting results showed that CHE up-regulated the protein level of E-cadherin and down-regulated the protein expressions of Vimentin,SLUG,SNAIL,MMP-2 and MMP-9 in 4T1 and MDA-MB-231 cells.6.CHE has obvious inhibitory effect on breast cancer in situ tumor,and the tumor volume is reduced.HE staining results show that tumor necrosis increases.Immunohistochemical results show that CHE down-regulates the expressions of Ki67,CD31,CD163 and Gr1+CD11b.7.The results of Western blotting experiments showed that CHE up-regulated the BAX protein level and down-regulated the protein expressions of BCL-2,Procaspase-3 and Procaspase-9 in the tumor tissue of the orthotopic model mice,which were basically consistent with the in vitro experimental results.8.CHE has obvious inhibitory effect on breast cancer tail vein metastasis,the fluorescence area in the lung is reduced,and the fluorescence intensity is weakened;the nodules in the lung tissue are significantly reduced.9.Western blotting results showed that CHE up-regulated E-cadherin protein level and down-regulated the protein expressions of Vimentin,SLUG,SNAIL,MMP-2 and MMP-9in lung tissue of metastasis model mice,which were basically consistent with the in vitro results.10.In vitro experiments showed that CHE up-regulated the protein levels of p-JNK in two triple-negative breast cancer cells,but had no effect on the protein expressions of p-ERK,ERK,JNK,p-P38 and P38.CHE could down-regulate the protein expressions of TGF-βR2,p-SMAD2/3 and SMAD4 in 4T1 and MDA-MB-231 triple-negative breast cancer cells,but had no effect on SMAD2/3 protein.11.In vivo experiments proved that CHE up-regulated the level of p-JNK protein in tumor tissue of orthotopic model mice,but had no effect on total JNK protein,which was basically consistent with the results of in vitro experiments.CHE down-regulated the protein levels of TGF-βR2,p-SMAD2/3 and SMAD4 in the lung tissue of metastasis model mice,but had no effect on the total protein of SMAD2/3,which was basically consistent with the in vitro results.Conclusion: In this study,two triple-negative breast cancer cells,4T1 and MDA-MB-231,and 4T1 animal tumor orthotopic and metastatic models were used in this study.It was found that CHE may induce tumor apoptosis by regulating JNK signaling pathway and inhibit EMT by regulating TGF-β signaling pathway.,thereby inhibiting tumor metastasis.CHE is one of the main components of celandine alkaloids.Celandine is a traditional Chinese medicine in my country.It is widely distributed and abundant.CHE is easy to obtain.It will be expected to be developed as a drug for the treatment of aggressive and metastatic triple-negative breast cancer.The research provides experimental basis for the clinical application of traditional Chinese medicine celandine.