Huaier Polysaccharides Inhibit Invasion And Metastasis Of Triple-negative Breast Cancer

Breast cancer is the most common malignant tumor in China,which poses a serious threat to women's health and life.The annual incidence of new cases is about 1.38 million,and the death rate is about 450,000 [1].At present,the conventional treatment of breast cancer is mainly surgical resection,concurrent postoperative chemotherapy,radiotherapy,endocrine therapy,molecular targeted therapy.Although current research suggests that postoperative adjuvant therapy is an important means of preventing recurrence and improving the prognosis of breast cancer patients,conventional adjuvant therapy does not benefit all breast cancer patients [2].15% of breast cancer patients are triple-negative breast cancer(TNBC),that is,estrogen receptor(ER),progesterone receptor(PR),and human epidermal growth factor receptor 2(HER-2)are negative.This part of patients cannot undergo endocrine therapy,targeted therapy and other adjuvant treatment after routine surgery and chemotherapy,often prone to local recurrence and distant metastasis,their prognosis is often poor [3].Therefore,finding new adjuvant treatments for patients with triple-negative breast cancer is an urgent problem that needs to be solved [4].Traditional Chinese Medicine(TCM)and its extracts have unique advantages in preventing tumorigenesis,inhibiting tumor formation,and preventing recurrence and metastasis.Huaier(Trametes robiniophila Murr)is a kind of fungus used as TCM,and its main active ingredient is polysaccharides(PS-T)[15].At present,Huaier granules(national first class new medicine)are used in the adjuvant treatment of a variety of clinical malignant tumors,which have anti-tumor effects and improve the quality of patients` life.Studies have confirmed that PS-T has inhibitory and killing effects on a variety of breast cancer cell lines.However,it is not clear whether Huaier can improve DFS and OS in TNBC patients,and the underlying mechanism needs further study.Experimental methods and results:1.Clinical study of postoperative treatment for triple negative breast cancer with Huaier granulesMETHODS:A total of 201 patients with triple-negative breast cancer treated at the Breast Disease Center of Southwest Hospital of Army Military Medical University from October 2010 to September 2014 were included.The pathological stage was from stage ? to stage ?.The patients were randomly divided into the Huaier granules group(experimental group)and the Huaier granules group(control group).The median follow-up was 46 months,and the DFS and OS of patients were observed.RESULTS:Among the 101 patients in the experimental group,13(12.9%)patients had disease progression,including 10 deaths(9.9%).Except one patient died of abnormal causes,the other patients died due to disease progression.Of the 100 patients in the control group,18(18.0%)had disease progression,of which 14(14.0%)died.The 5-year DFS between the experimental group and the control group was 87.1% vs 82%.There was no significant difference between the two groups(P=0.396),but the 5-year DFS of stage III patients was 81.3% vs 53.8%,which was statistically different.(P=0.03);the 5-year OS of the experimental group and the control group was 90.1% vs 86%,there was no statistical difference between the two groups(P=0.396),but the 5-year OS of the stage III patients was 87.5% VS 65.4.%,there was a statistical difference(P=0.046).In addition,among the 13 patients in the experimental group,10 patients(76.9%)had disease progression in the 6-month group and 3 patients(23.1%)had disease progression in the 18-month group.There was a statistical difference between the two groups(P=0.028)CONCLUSION:Huaier granules can effectively improve 5-year DFS and OS in patients with stage III triple-negative breast cancer;2.Huaier polysaccharides inhibit invasion and migration of triple negative breast cancer cell linesMETHODS:In this study,the effects of Huaier polysaccharides on the invasion and migration of breast cancer cell line 4T-1 in vitro,and mouse lung metastasis model experiments in vivo were conducted to investigate the ability of Huaier polysaccharides to inhibit the invasion and migration of triple negative breast cancer cell lines.RESULTS:1.Through the Transwell experiment,the number of cells treated with polysaccharides was significantly decreased(control group 243±21,PS-T treatment group 52±8,p=0.000);2.The scratch width of the control group and the 1?g/ml,5?g/ml polysaccharides treatment group was observed after 12 h and 24 h.The scratch width of the control group was significantly smaller than that of the experimental group;3.In the metastatic model experiment,the lung metastasis nodules were scored and counted,grade I: nodule diameter <0.5 mm;grade II: 0.5 mm ? nodule diameter <1 mm;grade III: 1 mm ? nodule diameter <2 mm;grade IV: Nodule diameter ? 2mm.The formula for calculating the metastatic nodule score is: I × 1 + II × 2 + III × 3 + IV × 4.The results showed that the lung surface nodule score of the control group was 64.7±6.2,which was significantly higher than that of the high dose(13.7±4.1,p=0.008)and low dose(42.7±5.3,p=0.026)polysaccharides treatment group;4.Western blottings were used to detect EMT-associated epidermal markers: E-cadherin(CDH1),Occludin(OCLN),and Cytokeratin(CK8);interstitial markers: N-cadherin(CDH2),Vimentin,and Fibronectin(FN1)levels.The results showed that the marker expression of epithelial cells in 4T-1 cells was significantly increased and the expression of mesenchymal marker was significantly decreased.CONCLUSION:1.Huaier polysaccharides inhibits the invasion and migration of 4T-1 cells,and can significantly inhibit the occurrence of EMT in 4T-1 cells;2.Huaier polysaccharides can significantly inhibit lung metastasis of breast cancer in the metastatic model experiment.3.Effect of autophagy on EMT inhibition of triple negative breast cancer cells by PS-TMETHODS:This section firstly used the technique of confocal and Western blotting to observe the effect of PS-T on the autophagy of breast cancer cell line MDA-MB-231.Then we synthesized the ATG5 interference to stabilize the lentivirus.The ATG5 stable interfering breast cancer cell line was constructed,and the invasion and migration ability of the cells were observed.Western blotting was used to detect the effect of autophagy on the inhibition of EMT in MDA-MB-231 cells by PS-T.RESULTS:1.By Western blotting,the results showed that after 5?g/ml PS-T treatment for 24 h,the autophagy marker proteins LC3-I/II increased significantly,while the P62 protein decreased and Beclin-1 protein increased.MDA-MB-231 cells were infected by the RFP-GFP two-color adenovirus and observed by two-photon confocal microscopy.LC3-GFP and LC3-RFP increased simultaneously,and the number of LC3-RFP increased significantly;2.Western blotting was used to detect EMT-relation proteins.It was found that after treatment with LY294002,the expression of epidermal marker of EMT were significantly inhibited while the expression of mesenchymal marker of EMT were significantly reduced;3.Construction of autophagy key protein ATG5 stable interference cell line.Three siRNAs targeting ATG5 mRNA at positions 393,443 and 853 were designed.The interference efficiency of targeting small fragments was analyzed by qPCR.The results showed that siRNA targeting 853 was the most efficient,reaching 70%.A small fragment of siRNA was inserted into the p LVX-ShRNA2-Puro plasmid to construct a lentiviral recombinant plasmid.The constructed plasmid was named p LVX-ShRNA2-Puro-Homo-ATG5-853,and it was packaged and transferred into HEK293 cells.The transfection efficiency was observed at 24 h and 72 h after transfection,and the lentivirus pLVX-ATG5 titer was measured.It is: 8 × 107 TU / ml.MDA-MB-231 was infected,and the infection efficiency was observed after 48 hours.We then screened positive cells using the optimal concentration of 0.2 ?g/ml Puromycin.Monoclonal cells were obtained by limiting dilution;4 Transwell chamber assay was used to detect the migration ability of MDA-MB-231-siNC and MDA-MB-231-siATG5.The results showed that the number of cells was significantly decreased after treatment with polysaccharides from scorpion(siNC group 283±21,siNC+PS-T)Treatment group 98±8,p=0.015).siATG5 group 271±25,siATG5+PS-T treatment group 238±18,p=0.082.It can be seen that the inhibition of MDA-MB-231 migration by PS-T after siATG5 was significantly weakened,p=0.0076,and the scratch test also verified the result;5.The effect of PS-T on the invasion of MDA-MB-231 was detected by transwell invasion assay.The results showed that the number of cells was significantly decreased after treatment with polysaccharides from the ear polysaccharides(37±6 in the siNC group and 14±4 in the siNC+PS-T treatment group,p=0.011).39±9 in the siATG5 group and 35±18 in the siATG5+PS-T treatment group,p=0.064.It can be seen that the inhibition of MDA-MB-231 migration by PS-T after siATG5 was significantly weakened,p=0.0081;6.Western blotting experiments observed changes in EMT-related proteins after ATG5 interference.After the cells were treated with culture medium or 5 ?g / ml PS-T for 24 hours,the cells were lysed and proteins were collected,and the levels of EMT-related markers were detected by western detection.The results showed that the expression of epithelial markers on MDA-MB-231-siNC cells were significantly increased and the expression of mesenchymal markers were significantly reduced after the treatment with PS-T.This effect was reversed after disturbing ATG5.CONCLUSION:1.PS-T significantly induced up-regulation of autophagy in MDA-MB-231 cells;2.The inhibitions of autophagy can down-regulate the inhibitory effect of PS-T on EMT transformation in MDA-MB-231 breast cancer cells;3.ATG5 interference down-regulated the inhibitory effect of PS-T on MDA-MB-231 migration and invasion;4.ATG5 interference down-regulated the inhibition of EMT in MDA-MB-231 by PS-T.4.Huaier polysaccharides induced autophagy inhibit EMT in triple negative breast cancer cellsMETHODS:In this section,Western blotting technology was used to confirm the expression level of Snail,a key transcription factor for EMT,during the process of huaier polysaccharides-induced autophagy inhibiting EMT transformation in breast cancer cells.At the same time,it was confirmed by double luciferase experiments that sophora polysaccharides induced autophagy by degrading Snail,thereby regulating EMT-related proteins at the transcription level.The expression levels of LC3 and Snail protein in MDA-MB-231 or MDA-MB-231-siATG5 cell line were detected by immunofluorescence assay.Finally,Transwell experiments were used to reversely verify that Snail overexpression can reverse the inhibition of breast cancer cell invasion by PS-T.RESULTS:1.The protein level of Snail was detected by western blotting.The results showed that PS-T treatment could significantly reduce the amount of Snail protein in the cell,but after interfering with ATG5,this effect was significantly weakened and had almost no effect on Snail;2.The E-cad protein promoter reporter plasmid was constructed and subsequently detected.The response to PS-T treatment showed that the fluorescence ratio of the 5 ?g/ml PS-T treatment group was significantly up-regulated compared to the control group,which was nearly 5-fold,and this effect almost disappeared in cells interfering with ATG5(p =0.0008);3.The immunofluorescence test was used to detect the expression level of LC3 and Snail protein of MDA-MB-231 or MDA-MB-231-siATG5 cell line.The results showed that the expression of LC3 protein increased after PS-T treatment,and the amount of intracellular Snail protein was significantly reduced.However,this effect was significantly weakened after ATG5 was interfered with and had almost no effect on Snail;4.MDA-MB-231 cells were first transfected with pcDNA3.1-snail,and transwell assay was performed 24 hours after transfection.The results showed that the number of cells treated with polysaccharides was significantly decreased(Vector group 37.3±1.5),Vector+PS-T treatment group 13.0±1.2,p=0.0001),overexpression snail group 54.3±2.8,overexpression snail+PS-T treatment group 49.7±1.5,p=0.2182.It can be seen that the inhibitory effect of PS-T on MDA-MB-231 migration was significantly attenuated after overexpression of snail,p=0.001.CONCLUSION:Huaier polysaccharides-induced autophagy is involved in the inhibition of EMT in breast cancer cells by degrading Snail.