The Interventional Effect And Mechanism Of Traditional Chinese Medicine For Replenishing Qi, Activating Blood And Detoxification On Triple-negative Breast Cancer

Background:Breast cancer is the leading cause of cancer death in women globally.Triple-negative breast cancer(TNBC),a heterogeneous collection of breast cancers lacking estrogen receptor(ER),progesterone receptor(PR),and human epidermal growth factor receptor 2(HER2)expression,represents approximately 10-20%of all breast cancers.Comparing to the other subtypes of breast cancer,TNBC has a poorer prognosis due to lacking efficient treatment methods.Heparanase(HPSE)is an endo-glucuronidase with the function of cleaving heparan sulfate(HS)chains of proteoglycans.This function alters the extracellular matrix(ECM)structure and releases bioactive saccharide fragments,HS-bound growth factors,and cytokines into the tumor microenvironment.In this way,HPSE facilitates tumor proliferation,angiogenesis,invasion and metastasis.HPSE is highly expressed in multiple cancer types and associated with the grim clinical outcome of cancer patients.Recently,some studies found that HPSE modulated autophagy in malignant cells.Autophagy is an evolutionarily conserved membrane-trafficking process,by means of lysosomal degrading harmful cytoplasmic entities.In the case of tumorigenesis,autophagy eliminates genomic instability and degrades endogenous pro-inflammatory factors in pre-malignant cells to avoid malignant transformation.On the other hand,autophagy supports tumor progression by maintaining metabolic and redox homeostasis in cancer cells under severe micro-environments.Herbal products have been used in traditional medicine in different cultures for thousands of years.Nature compounds from herbal medicine have anti-tumorigenic properties to optimal therapeutic efficiency and overcome drug resistance.We previously reported the effect of some formulas based on the therapy theory of Yiqi(tonify energy),Huoxuehuayu(promoting blood circulation),and Jiedusanjie(eliminating tumor-induced toxicity and tumor remission).The formula has been confirmed with anti-tumor and anti-metastatic effects in breast cancerTo simplify and optimize the drug combination,we selected astragalus membranaceus(stands for Yiqi),solanum nigrum Linn,lotus plumule(on behalf of Jiedusanjie),ligusticum(on behalf of Huoxuehuayu)according to the clinical experience of Professor Xiao-Min Wang.Astragalus membranaceus var.mongholicus(Bunge)P.K.Hsiao,also called Huangqi,is predominantly used to tonify Qi(promoting the energy)according to TCM theoryAim of the study:This study is aimed at evaluating the anti-tumor property of SANT in HPSE related TNBC,both in-vitro and in-vivo,and analysis the underlying mechanisms.Materials and Methods:In this study,we explored HPSE expression and survival of breast cancer patients in databases.We performed MTS,trans-well and wound scratch assays to assess the effect of SANT on cell proliferation and migration.Confocal microscopy and western blot were applied to verify the autophagy flux induced by SANT.Mice models were used to evaluate the efficacy and safety of SANT in-vivo by tumor weights and volumes or serum index,respectively.To analyze the underlying mechanisms of SANT,we conducted Human autophagy PCR Array and Angiogenesis Proteome Profiler on tumor tissues.Results:1.HPSE mRNA expression levels were upregulated in multi-type cancers compared to that in normal tissue.In the case of breast cancer,HPSE expression in cancer tissue was more than two-fold increase than that in normal tissue.Patients with a high level of HPSE expression associated with despairing outcomes in both RFS(P=1.7e-12)and OS(P=0.00016)in breast cancer patients.HPSE DNA copy number is lower,while mRNA expression is higher in TNBC than that in other breast cancer subtypes.These results indicated that HPSE transcription rate is upregulated in TNBC2.231-Hpa cells with markedly elevated HPSE expression were verified by western blot.LC3B was used to detect basal autophagy levels in both cell lines.Comparing the ratio of LC3B-?/-? conversion,we found that 231-Hpa cells with a significantly higher LC3B-? to-? conversion rate(P=0.0003).We established orthotopic breast cancer models to assess the impact of HPSE in tumor growth.The immunofluorescent staining of tumors demonstrated prominently improved HPSE expression in 231-Hpa tumor tissues.In the 231-Hpa group,higher tumor burden(0.608g vs.0.437g,n>6 in each group,P=0.0242),more massive lung metastasis(0.167g vs.0.148g,n>6 in each group,P=0.0164)were found compared to 231-Mock group.Higher cell reproductive capacity presented by Ki-67 staining and more vascular endothelial cells shown by CD31 positive staining were detected in 231-Hpa tumor tissue3.In vitro,we examined the inhibitory effect of these medicines individually or in combination by MTS assay.?-solanine,in a concentration range of 0-64 ?M,reduced nearly 50%of cell viability in 231-Mock cells while approximately 60%of that in 231-Hpa cells for 24 h treatment.After 48 h exposure,?-solanine exhibited an inhibition effect of 68.93%in 231-Mock cells and 81.06%in 231-Hpa cells.NEF also showed a dose-and time-dependent inhibitory efficacy reaching an inhibition ratio of 60-80%for 24-48 h exposure.AS-? and TMP marginally affected the cell viability.Besides,we investigated the metastatic potentials of 231-Mock/Hpa cells altered by SANT treatment.The quantitative analysis of stained cells revealed that SANT significantly suppressed the migration in both cell lines.In 231-Hpa cells,SANT decreased the migration cells by 79%compared to the control.While for 231-Mock cells,SANT also reduced the migrated cells by 72%.Wound scratch assay further confirmed these findings.After 24 h SANT treatment,the relative wound areas were decreased by 51.5%in 231-Hpa cells and 40.6%in 231-Mock cells.In addition,we conducted western blots to examine HPSE expression under SANT administration.SANT could slightly reduce the active form(50kDa)of HPSE but exert almost no effect on the latent form(65kDa)of HPSE in 231-Hpa cells.We also used PI-88,an inhibitor of HPSE,as a positive control and found that SANT,PI-88,and the combination of SANT with PI-88 all reduced the active form of HPSE.To investigate the influence of SANT on autophagy,we transfected 231-Hpa cells with the GFP-RFP-LC3B fusion protein.There was an enhancement of LC3B punctate formation in a time-dependent manner indicating of autophagosome accumulation.To figure out whether SANT induced or inhibited autophagy flux,we treated 231-Hpa cells with SANT individually or in combination with the autophagy late-stage inhibitor chloroquine(CQ).As expected,the CQ combination induced an increase conversion from LC3B-I to-II,which reflected that the autophagy process in 231-Hpa cells was indeed promoted by SANT.4.In-vivo,SANT,or PI-88 distinctly suppress the tumor growth(SANT vs Control,360.59 vs.598.32mm3,P=0.0381;PI-88 vs.Control,364.93 vs.598.32mm3,P=0.0344).The tumor weight were also decreased in SANT and PI-88 groups(SANT vs.Control,0.29 vs.0.54g,P=0.0046;PI-88 vs.Control,0.37vs.0.54g,P=0.0262)Compared to control group,SANT(17.76±3.41%vs.7.52±2.02%,P<0.0001)or PI-88(17.76±3.41%vs.10.17±2.76%,P<0.001)evidently reduced the proportion of Ki-67-positive cells,while SANT had better performance than PI-88 on the inhibiting effect.In our research,SANT prominently suppressed the formation of MVD and VM by 57%(P<0.0001)and 33%(P=0.0019),respectively,which demonstrated that SANT partly worked through antagonism of angiogenesis.While PI-88 exhibited better inhibition effect in decreasing MVD and VM than SANT.We assessed liver function with ALT,AST,CHO,and TG,while evaluated kidney function by CRE and BUN.All these indicators were within normal ranges,indicating the biosafety of SANT.5.SANT administration upregulated the expression of genes involved in the autophagy process,including ATG10,ATG16L1,ATG16L2,ATG4B,ATG4D,ATG9A.PI-88 treatment down-regulated genes involved in autophagy process such as AMBRA1,ATG16L1,ATG4D,BAD,BCL2L1,DAPK1,GABARAPL1,LAMP1 and ULK2.SANT reduced the protein levels of HB-EGF,thrombospondin-2,amphiregulin,leptin,IGFBP-9,EGF,coagulation factor ?,MMP-9(pro and active form),and elevated proteins levels of serpin E1,platelet factor 4Conclusions:Heparanase high expression was related to inferior OS,RFS,or invasive breast carcinoma patients.Heparanase promotes tumorigenicity and autophagy flux in-vitro.SANT suppressed tumor cell proliferation and migration,and enhanced autophagy flux in vitro,and exerted inhibition effect of SANT on tumor growth and angiogenesis in vivo.SANT regulated gene expression of molecules involved in the autophagy process and proteins related to tumor angiogenesis These findings indicated that herbal compounds SANT might be a promising candidate in adjunctive onco-therapy discovery for TNBC.This study also provides novel strategies of applying natural compounds combination for clinical purposes.