Study On The Mechanism Of Lupeol Inhibits Triple-negative Breast Cancer Via Regulate Autophagy

ObjectiveTriple negative breast cancer is a particularly tenacious and recurrent form of breast cancer and is characterized by a lack of expression of estrogen receptor（ER/ESR-1）,progesterone receptor（PR/PGR）and human epidermal growth factor receptor（HER-2/ERBB2）.This results in a decreased effectiveness of the existing targeted treatment,making surgery and chemotherapy the predominant treatments.Unfortunately,this leads to higher tumor recurrence rate and metastasis rate,and a reduced overall survival rate in patients with triple negative breast cancer post-surgery.In light of the characteristics of triple negative breast cancer,the search for biomarkers and the development of effective treatment methods are of great importance.Traditional Chinese Medicine has demonstrated its potential in suppressing the recurrence and metastasis of triple negative breast cancer.Lupeol,a recurrent compound of this kind of traditional Chinese Medicine,has been reported to considerably curtail the activity of many types of breast cell lines.however,its effect on triple negative breast cancer has been inadequately studied.Thus,this research attempts to elucidate the part and mechanism of Lupeol in triple negative breast cancer.Methods1.Assessing the Antitumor Activity of Lupeol on MDA-MB-231 and 4T1 Human Breast Cancer Cell Lines.A CCK8 experiment and clone formation experiment were conducted to assess the impact of Lupeol on the proliferation of MDA-MB-231 and 4T1 cells;cell scratch test was used to analyze the effect of Lupeol on the migration ability of the two cell lines;flow cytometry was employed to measure the apoptotic level prior to and following administration;laser confocal microscopy evaluation was done to examine the damage of mitochondrial membrane potential before and after application,and electron microscopy was utilized to evaluate the formation of autophagic bodies before and after administration,thereby providing preliminary insights into the effects of Lupeol on type I and type II programmed death.2.Evaluating the Influence of Lupin Alcohol on Autophagy in MDA-MB-231 Cells with Triple Negative Breast Cancer.In order to ascertain the impact of Lupeol on autophagy,we first utilized m Cherry-GFP-LC3B adenovirus for transfection of MDA-MB-231 cells and then monitored the changes in autophagic flux prior to and post Lupeol administration.Subsequently,western blotting and RT-PCR were employed to establish the autophagy-related proteins and genes,and the influence of lupin on the AKT-m TOR upstream pathway of autophagy was further examined.3.Exploring the Influence of Lupeol on EMT of MDA-MB-231 Cells.The expression of E-cadherin and N-cadherin,proteins associated with EMT,was analyzed by Western blot before and after Lupeol administration.In view of the close correlation between EMT and autophagy,transcriptomics will be employed to identify genes which have been altered in MDA-MB-231 cells after Lupeol administration,followed by an enrichment screening and functional analysis of the difference genes in order to investigate how Lupeol controls autophagy and cell EMT,and thereby to explore the relationship between them.4.Exploration of the Impact of Lupin Alcohol on Triple Negative Breast Cancer in vivoThe subcutaneous transplanted tumor model and lung metastasis model were constructed to evaluate the effects of Lupeol on MDA-MB-231 cell proliferation and metastasis in vivo,corroborate the consistency of its action mechanism in vitro and in vivo,evaluate the toxicity of Lupeol on various organs of animals as well:（1）Subcutaneous transplantation tumor model:15 4-week-old BALB/c female nude mice were injected subcutaneously with MDA-MB-231 cells（1-2×10⁶ cells）.Mice were randomly divided into 3 groups（5 animals in each group）:control group（corn oil）,low-dose group（20mg/kg/2 days）,and high-dose group（40mg/kg/2 days）.On the day of the end of the experiment,the whole blood of mice was collected by blood from the orbit,and then the mice were sacrificed by cervical dislocation.The mice organs（liver,heart,spleen,lung,kidney）and tumors were removed,weighed and imaged,some tumor tissues were stored in liquid nitrogen for western blot analysis,and each organ and part of the tumor tissues were fixed with formaldehyde for further histopathological analysis.（2）Lung metastasis model:15 4-week-old female BALB/c nude mice were injected with MDA-MB-231 cells（1×10⁶ cells）through the tail vein.Mice were randomly divided into 3 groups（5 in each group）:control group（corn oil）,low-dose group（20mg/kg/2d）,high-dose group（40mg/kg/2d）.After 21 days,the lungs were separated and the number of metastatic nodules was counted.Then,part of the lung tissue is preserved in liquid nitrogen for western blot analysis,and part of the lung tissue is fixed with formaldehyde for further histopathological analysis.Results1.The CCK8 experiment results demonstrated that Lupeol significantly inhibited two triple negative breast cancer cell lines（MDA-MB-231 and 4T1）,with IC₅₀ values of45.67μM and 40.27μM.Meanwhile,it did not cause obvious toxicity to normal breast epithelial cell MCF-10A.The clonal formation experiment suggested that the number of colony formation decreased significantly with increasing Lupeol concentration（P<0.05）.Moreover,flow cytometry and laser confocal microscopy were employed to investigate the effects of Lupeol on mitochondrial membrane potential damage and apoptosis of MDA-MB-231 cells and 4T1 cells,revealing that Lupeol could induce mitochondrial damage and advance apoptosis rate of triple negative breast cancer cells,and the difference between the drug group and the blank group was significant（P<0.05）.Subsequently,under the electron microscope,it was also observed that the autophagic bodies of the two tumor cells were significantly increased（P<0.05）after Lupeol administration.2.The expression levels of LC3,P62,Beclin-1,Atg5,and Atg7 autophagic proteins were evaluated through immunofluorescence and Western blotting.Lupin was found to significantly increase the expression of these proteins in MDA-MB-231 cells（P<0.05）.Autophagic flow results further suggested that lupin might promote the early formation of autophagic bodies.Additionally,Lupeol administration was observed to significantly decrease the expression of p-Akt and p-m TOR proteins（P<0.05）.Moreover,treatment of Lupeol in combination with the Akt agonist SC79 significantly reduced Lupeol-induced autophagy promotion（P<0.05）.3.This study was conducted to assess the impact of Lupeol on epithelial mesenchymal transition（EMT）-associated proteins in triple negative breast cancer cells.The results demonstrated that Lupeol could remarkably reduce the expression of both N-cadherin and E-cadherin proteins（P<0.05）.To further explore the potential of Lupeol to stimulate autophagy and suppress EMT,transcriptome analysis was carried out to appraise the alteration of MDA-MB-231 cells prior and after its administration.The results demonstrated that Lupeol could activate multiple cellular pathways,including autophagy.Subsequently,a comprehensive screening and differential expression analysis of related genes was conducted,and ultimately six genes with the most remarkable difference in expression were established,with Twist1 atomizing out as the principal regulator of cell EMT.Verification and supplementary experiments were then implemented on Twist gene,and it was observed that the expression of Twist1 protein was significantly decreased following Lupeol treatment（P<0.05）,while the expression of Twist1 protein was boosted with the addition of the autophagy inhibitor 3-MA（P<0.05）.4.In vivo experiments were conducted using Balb/c nude mice to establish subcutaneous tumor transplantation and lung metastasis models in order to evaluate the efficacy of lupin alcohol on triple negative breast cancer.Results revealed a dose-dependent inhibition of tumor size and weight of MDA-MB-231 tumor-bearing mice treated with 20and 40 mg/kg of Lupeol（P<0.05）,while no significant differences in the weight of mice among the groups were observed.Immunohistochemical analysis showed a decrease in the expression of Ki67,a marker of cell proliferation,in the tumors of mice treated with lupin.Western-blot results showed an increase in the expression of autophagy-related proteins LC3,Beclin-1,Atg5,Atg7 and m TOR in the tumor tissue（P<0.05）,and a decrease in the expression of P62（P<0.05）following lupin administration,which was in accordance with the results from the in vitro experiments.The toxicity of Lupeol to mice showed no significant difference in the activities of ALT,AST,Cre,Bun and ALP in the serum of mice pre-and post-administration,suggesting that Lupeol had no significant hepatorenal toxicity to mice at the dose of 40mg/kg（P>0.05）.In addition,H&E staining of the major organs and tissues showed no obvious toxicity of Lupeol to all organs in mice,and no significant difference in organ weight（P>0.05）.The lung metastasis model showed a significant decrease in the number of lung tumor nodules in mice after lupine administration（P<0.05）.Western-blot test results showed an increase in the expression of E-cadherin protein（P<0.05）,and a decrease in the expression of N-cadherin and Twist protein（P<0.05）following lupin administration.Immunohistochemical staining also demonstrated an increase in the expression of autophagy marker LC3B in the lung tissue with an increase in drug concentration,while the expression of Twist protein decreased,with a noteworthy difference compared with the blank group（P<0.05）.The results of in vivo experiments are in accordance with the results of in vitro experiments.ConclusionThe findings of the experiment suggest that Lupeol has the potential to impede the proliferation and metastasis of triple negative breast cancer.The underlying mechanism is thought to be the promotion of autophagy through the AKT-m TOR pathway,which leads to a decrease in the migration of triple negative breast cancer cells and a suppression of the expression of the EMT transcription factor Twist1,thus inhibiting the EMT process.