Inhibitory Effect Of Isotoosendanin On Growth And Metastasis Of Triple-negative Breast Cancer

Malignant tumors have been one of the top ten causes of death,which seriously threatens human life and health.The incidence and mortality of breast cancer is the highest in women with malignant tumors,which causes extreme harm to women's physical and mental health.Triple-negative breast cancer?TNBC?is a subtype of breast cancer with high metastasis and high recurrence.Its morbidity is around15?25%with no ideal treatment strategy till now,which causes a big problem.Toosendanin?TSN?is an active ingredient of traditional Chinese medicine Fructus Toosendan and Meliae Cortex.Previous studies indicated that TSN inhibited the growth and the metastasis of a variety of cancer cells including lung cancer,gastric cancer and pancreatic cancer.However,there is still no study about the inhibition of TSN on the growth and metastasis of TNBC.Our previous study found that TSN was converted into its low toxicity isomer isotoosendanin?ITSN?in stomach under the gastric acid environment after it was orally given to mice.There are few reports on the activity of ITSN at present.Does ITSN have anti-tumor activity?Can ITSN inhibit the growth and metastasis of TNBC?And what's the involved mechanism?All these above questions will be invstigated in this study.Firstly,ITSN was screened for anti-tumor activity,and it was found that TNBC cells had the lowest half maximal inhibitory concentration(IC₅₀)after ITSN treatment.It is suggested that TNBC cells might be the most sensitive cancer cell lines to ITSN-induced cytotoxicity.Next,transmission electron microscopy was used to verify whether ITSN will induced autophagy in TNBC cells.It was found that after ITSN treatment,there were obvious autophagosomes formed in the TNBC cells.Moreover,the expression of autophagy-related proteins showed that the ratio of LC3B II/LC3B I was significantly increase in TNBC cells treated with ITSN.The expression of Beclin1 was elevated,but the expression of autophagy substrate p62 was decreased in TNBC cells treated with ITSN.These data suggested that ITSN could induce autophagy in TNBC cells.Propidium Iodide?PI?staining was used to observe cell necrosis.It was found that the number of necrotic cells stained red in TNBC cells was increased in a time-dependent manner after ITSN treatment,suggesting necrosis was observed in TNBC.Further,flow cytometry experiments showed that the number of necrotic and apoptotic cells were both increased in TNBC cells treated with ITSN.Furthermore,apoptosis-related proteins are detected by using Western-blot assay.It was found that Caspase-3/9 was obviously cleaved and the expression of Bcl-x L had a significant decrease induced by ITSN in TNBC cells,suggesting the occurrence of apoptosis in TNBC cells treated with ITSN.The expression of pro-Caspase-1 and cleaved-Caspase-1 is also observed.There is no obvious change of pro-Caspase-1 and no expression of cleaved Caspase-1,suggesting that ITSN does not induce the pyroptosis of TNBC cells.High metastasis is the main cause of poor prognosis in patients with TNBC.So,next study was focused on the role of ITSN in inhibiting TNBC metastasis and its mechanism.Our results showed that ITSN inhibited the migration and invasion of TNBC cells including MDA-MB-231,BT549 and 4T1 cells in wound healing,migration and invasion assays.Epithelial-mesenchymal transition?EMT?has been reported to play an important role in the metastasis and invasion of malignant tumors.Next,the expression levels of EMT-related proteins in three TNBC cells were investigated by using Western-blot assay.The results showed that the expression of E-cadherin was increased,but the expression of Vimentin and?-SMA were decreased in TNBC cells treated with ITSN.To further determine the anti-metastasis activity of ITSN in vivo,we constructed a 4T1-luc-GFP in situ breast cancer metastasis model on Balb/c mice and a MDA-MB-231-luc-GFP in situ breast cancer metastasis model on nude mice.The selected doses of ITSN were 0.1 mg/kg and 1 mg/kg.In vivo imaging results showed that the metastasis rate of the ITSN administration group was significantly lower than that of the model group.The results of hematoxylin-eosin?H&E?staining showed that the number of TNBC metastases in the lung and liver of mice treated with ITSN was significantly reduced as compared with the model group in the Balb/c mice model.The number of TNBC metastases in the liver of the mice trated with ITSN was significantly decreased compared with the model group in the nude mice model,while no obvious lung metastasis and bone metastasis were found.At the same time,we found that the expression of E-cadherin was decreased in the tumor tissues of model group,but the expression of?-SMA and Vimentin was increased.However,this phenomenon was reversed after the administration of ITSN.The above experimental results imply that the inhibition of TNBC induced by ITSN might be due to its inhibition on EMT.Finally,ITSN was orally given to normal mice for five weeks to obverse whether it will have any hepatotoxicity.The results showed that serum alanine aminotransferase?AST?and aspartate aminotransferase?ALT?activities were not increased in the mice treated with ITSN,and the results of liver histological evaluation did not show any obvious damage in liver in mice treated with ITSN.These results suggest that ITSN had no obvious hepatotoxicity.In summary,ITSN has obvious anti-tumor activity.TNBC cells are the most sensitive tumor cells to ITSN-induced cytotoxicity.ITSN caused cell death by inducing autophagy,apoptosis and necrosis in TNBC cells.ITSN also suppressed TNBC metastasis,which may be exerted by inhibiting EMT of TNBC.This work lays a foundation for the research and development of ITSN as a potential anti-TNBC drug.