Research progress of traditional Chinese medicine in the treatment of breast cancer based on “Binding of Phlegm and Stasis”

Xue-Feng Jiang ,Ying-Chang Fu,Yi-Gui LaiJin ChenHui-Jie FanYe-Jian HuFang-Min ChenFang-Hua YangSu-Ying MoQiang Wang

11Department of Traditional Chinese Medicine,Yangjiang People's Hospital,Yangjiang 529500,China;2College of traditional Chinese Medicine,Southern Medical University,Guangzhou 510515,China.

Breast cancer is a common malignancy in female,which seriously endangers women's health.Chinese medicine believed that the incidence of breast cancer is related to “Stagnation of Liver-Qi” and “Phlegm,Disharmony of Thoroughfare and Conception Vessels”,“Stagnation of Qi and Blood Stasis”,constitution and other characteristics.Modern research believed that phlegm is related to abnormal energy metabolism of tumor cells,and stasis is the pathological change of tumor angiogenesis.How to combine tumor angiogenesis and energy replacement abnormality with the theory of “Binding of Phlegm and Stasis” has become a new direction of traditional Chinese medicine anti-tumor research.The drugs of “Activating Blood Circulation” and “Removing Blood Stasis”,“Resolving Phlegm” and “Dispersing Constipation” have a wide range of pharmacological effects and definite curative effect.They have the functions of anti-inflammatory,anti-tumor,anti-pathogenic microorganism and improving the immune ability of the body.In this paper,the effects and mechanisms of phlegm-reducing and stasis dispersing active ingredients and compound preparations in regulating angiogenesis and energy metabolism of breast cancer were reviewed,and the differences in efficacy,targets and pathways of Phlegm-Reducing and Stasis Dispersing drugs were summarized,so as to provide some reference for the research and development of traditional Chinese medicine for breast cancer.

Key words:Breast cancer,Binding of phlegm and stasis,Traditional Chinese medicine,Energy metabolism,Angiogenesis

Breast cancer (BC) is the most common malignancy tumor in women and the leading cause of cancer death in women worldwide.According to GLOBOCAN 2018 estimated,there were approximately 2,088,849 (11.6%)new cases and 626,679 (6.6%) deaths from BC occurred that year [1].In China,there were 367,900 cases of invasive BC and 97,900 deaths,ranking first in the incidence of malignant tumors among women [2].Recent studies have shown that tumor microenvironment is closely related to the occurrence and development of breast cancer,especially tumorrelated angiogenesis and energy metabolism,which play an important role in the development and evolution of breast cancer [3,4].This paper intends to explain the differentiation and treatment of breast cancer tumor microenvironment from the theory of Binding of Phlegm and Stasis.

Etiology and pathogenesis of breast cancer in TCM

BC belongs to the category of “Ruyan” in traditional Chinese medicine (TCM).Chinese medicine believed that the incidence of BC was related to “Stagnation of Liver-Qi and Phlegm”,“Disharmony of Thoroughfare and Conception Vessels”,“Stagnation of Qi and Blood Stasis”,constitution and other characteristics.Its pathogenesis basically follows the rule of “Qi Stagnation-Phlegm Coagulation-Blood Stasis-Stasis-Phlegm Type” [5].At the end of the 20th century,researchers proposed a “multistage development model for BC.” The development of BC had experienced the continuous process of “usual ductal hyperplasiaatypical ductal epithelial hyperplasia-ductal carcinoma in situ-invasive breast cancer” [6].This is consistent with the occurrence and development pattern of BC in Chinese medicine.Therefore,the treatment of BC with the use of herbs for Resolving Phlegm and Dispersing Knot and Promoting Blood Circulation and Removing Blood Stasis.

Phlegm syndromes and tumor energy metabolism

TCM believe that “Phlegm” is a pathological product formed by abnormal metabolism of body fluid.It can be divided into “tangible phlegm” and “intangible phlegm”[7].The body “Qi” machine runs not smoothly,causes the body fluid to accumulate in the body,if stays in the respiratory organ,the cough can come out,then forms the tangible phlegm;if you stay in the “Zang-Fu”organs,channels and collaterals,it is invisible sputum.Modern research has proposed a hypothesis on the nature of “Phlegm Turbidity” based on the pathological products formed by metabolic disorders of 3 nutrients(sugar,fat and protein) [8].Abnormal energy metabolism has been considered as one of the ten characteristics of tumors.Even under aerobic conditions,high levels of glucose are absorbed for glycolysis and large amounts of lactic acid are produced.This metabolic transformation is often referred to as the Warburg effect or aerobic glycolysis[9].However,the genes in the metabolic network are recombined so that not only aerobic glycolysis can occur,but also oxidative phosphorylation can occur,and they coordinate with each other through the lactic acid shuttle pathway to produce metabolic symbiosis.At the same time,it was found that the fatty acids needed for tumor cell proliferation were mainly derived from de novo synthesis pathway,and the important enzymes related to fatty acid synthesis and oxidative metabolism were overexpressed in tumor tissues [10].Abnormal fatty acid metabolism is associated with tumor growth,survival and invasion,indicating that abnormal fatty acid metabolism provides a vital material and energy source for tumor growth.Using H nuclear magnetic resonance based metabonomic analysis,compared with healthy subjects,various amino acids including leucine,alanine,citrulline,tyrosine,histidine,arginine,methionine,isoleucine,valine,acetylcysteine,etc.in the plasma of patients of the phlegm-stasis syndrome were significantly lowered.Glucose,glycoprotein,glutamine,myo-inositol,lactic acid,choline,creatine also significantly decreased.But the plasmal formic acid,acetone,acetic acid,acetoacetate,pyruvate,βhydrocxy butyrate,carnitine,malonic acid,and unsaturated fatty acid,very low density lipoprotein cholesterol,low density lipoprotein cholesterol increased in tumor patients of the phlegm-stasis syndrome [11].Therefore,phlegm may be closely associated with abnormal energy metabolism.

“Stasis Syndromes” and tumor angiogenesis

“Stasis” refers to the pathological product formed by the stagnation of blood in the body,which is also the pathological factor leading to a variety of diseases [12].Tumor angiogenesis is a process in which tumor cells induce capillary growth and establish blood circulation in new tumor tissues [13].A lot of vascular growth factors stimulation can cause tumor endothelial cell activation,endothelial cell proliferation,proteolytic enzyme causing vascular local extracellular matrix and basement membrane degradation,sprout of endothelial cells,proliferation and migration,formation of new blood vessels,new blood vessels endothelial cells arranged in a tubular,internal,can form new tumors.When tumor cells invade and metastasize,it is also necessary to induce new blood vessels at the metastasis site to construct an independent nutritional network to promote tumor growth.This process is very similar to the formation of “Blood Stasis Syndrome” in TCM.Current studies have confirmed that the abnormal function of vascular endothelial cells is one of the pathological foundations of the pathogenesis of “Blood Stasis”.At the same time,tumor cells can directly activate the body's coagulation system;it can also express plasminogen activator inhibitors,so that the fibrinolytic function of the body is inhibited and the blood coagulation activity is enhanced.It can also indirectly mediate platelet adhesion and coagulation,form platelet embolus,stimulate endogenous blood coagulation pathway,etc,so that the tumor is in the state of high coagulation in the process of occurrence and development,and the blood circulation in the state of high coagulation in the thick,sticky,cohesion,collection state,and TCM “Blood Stasis Syndrome” has a very similar pathological basis [14,15].In this regard,researchers believe that the essence of “Blood Stasis Syndrome” is the oxygen utilization obstacle of tissues and cells.Hypoxia prompts cells to secrete vascular endothelial growth factor,induces angiogenesis,and promotes tumor growth,while tumor volume increases and aggravates hypoxia,both of which are vicious cycles [16].Therefore,“Stagnant Internal Knot” is the pathological change of tumor angiogenesis.How to combine tumor angiogenesis and energy replacement abnormality with the theory of “Binding of Phlegm and Stasis” has become a new direction of TCM anti-tumor research.

The TCM approach to the diagnosis and treatment of BC is to emphasize the holistic concept and syndrome differentiation,running through the ideas of treating the disease at the root and treating the disease without the disease.The combination of syndrome differentiation and disease differentiation,treatment of both symptoms and root causes,and treatment of both attack and supplement,can strengthen the body and dispel the pathogenic factors,reduce toxicity and increase the effect,improve the quality of life of patients,and prolong the survival time of patients [17].The drugs of“Activating Blood Circulation and Removing Blood Stasis”,Resolving Phlegm and Dispersing Constipation have a wide range of pharmacological effects and definite curative effect.They have the functions of antiinflammatory,anti-tumor,anti-pathogenic microorganism and improving the immune ability of the body [18].

Regulation of BC angiogenesis by drugs that Promote Blood Circulation and Remove Blood Stasis,Dissipate Phlegm and Resolve Masses

VEGF is found that the strongest angiogenesis factor,it induced by the accumulation of mononuclear cells inhibit apoptosis of endothelial cells and vascular endothelial cells fiber protease,and the expression of matrix metalloproteinases,thus promotes the growth of endothelial cells and vascular matrix of mature,accelerate the formation of new blood vessels,conducive to the growth of tumor [19].The experimental study showed that the prescriptions of“Activating Blood and Eliminating Phlegm” had a significant down-regulation effect on angiogenic factors.Xu found in vivo and in vitro studies that matrine against BC by downregulating the VEGF via the Wnt/β-catenin pathway [20].Peng proved that the potential anti-cancer effects of andro,indicating that andro could inhibit COX-2 expression through attenuating p300 HAT activity and suppress angiogenesis via VEGF pathway [21].Kaempferol inhibited angiogenic ability by targeting VEGF receptor-2 and downregulating the PI3K/AKT,MEK and ERK pathways in VEGF-stimulated human umbilical vein endothelial cells [22].In addition to the active ingredients of TCM,the compound drugs of“Resolving Phlegm and Promoting Blood” circulation can also affect the related factors and pathways of blood vessel regulation of BC and play a role in inhibiting the growth of BC.Qingdu granule (hesperidin,rutin,saikosaponins a,saikosaponins d),a traditional Chinese herbal prescription,treatment significantly suppressed tumor growth,inhibited VEGF release,and decreased microvessel density.And it reduced protein expressions of VEGF,VEGFR2 and NFATc3 [23].Sun's study found that compound Sanjin Capsule (Jiubuying,Yanhusuo,Banbianlian,Baihuasheshecao,etc) an empirical prescription for the treatment of breast cancer,regulates angiogenesis and inhibits the growth and metastasis of breast tumor cells by down-regulating VEGF and bFGF [24].In addition,drugs that Promote Blood Circulation and Remove Blood Stasis can also improve hemodynamics and hemorheology of BC patients.Peng observed the effects of drugs (astragalus membranaceus,ginseng,salvia miltiorrhiza miltiorrhiza,rhubarb,turcumic,angelica sinensis,peach kernel,poria cocos,tangerine peel,red peoniae peoniae,safflower,atractyloplasty,licorice) for promoting blood circulation and removing blood stasis on blood viscosity,Th1/Th2 cytokine drift and cancerous fatigue in patients with BC after surgery and chemotherapy.The results showed that in the observation group,the whole blood viscosity,plasma viscosity and erythrocytosis equation K value were significantly decreased,the serum IL-2,TNF-α,IFN-γ and Th1/Th2 cell ratios were significantly increased,while the levels of IL-4,IL-6 and IL-10 were significantly decreased [25].Song established a rat model of precancerous BC,and found that zedoary turmeric oil can reduce the viscosity of the whole blood in the middle section,the viscosity of the whole blood in the low section,the viscosity of the plasma,and the viscosity of reduction,and restore the perfusion of microcirculation,thus improving the hemorheology of the rats in the model of precancerous BC [26].

Regulation of abnormal energy metabolism by drugs that Dissipate Phlegm and Disperse Masses,Activating Blood Circulation in breast cancer

Tumor cells are characterized by abnormally active glycolytic pathways,active glutamine metabolism and abnormal lipid metabolism.This abnormal metabolic pattern is named energy metabolic reprogramming of tumor cells.Some “Phlegm-Reducing” and “Stasis Dispersion”-related drugs can regulate the abnormal energy metabolism of BC cells.Treatment with Eu significantly reduced intracellular ATP levels in MCF10A-ras cells.This effect was mediated mainly through inhibiting oxidative phosphorylation complexs and the expression of fatty acid oxidation proteins including PPARα,MCAD and CPT1C by downregulating c-Myc/PGC-1β/ERRα pathway and decreasing oxidative stress in MCF10A-ras cells [27].Lee's research showed that external use of Ruyanneixiao cream (Dingxiang,Dahuang,Ruxiang,Moyao,Yanhusuo,Wangbuliuxing,Meiguihua,Bingpian) efficiently blocked precancerous mammary lesions by interfering with glycolysis induced by inhibition of hypoxia inducible factor-1α,hexokinase 2,phosphofructokinase,and pyruvate kinase M2 expression [28].Lipid can provide lipid components of biofilm through energy supply,regulate cell membrane fluidity and lipid molecular signal transduction,and promote tumor cell proliferation,invasion and metastasis and other malignant biological activities.Dr.Li evaluated inhibitory activities of 47 species of drug ethanol extracts on fatty acid synthase (FAS) and MDAMB-231 cells,showed evidence of Koelreuteria integrifoliola.And other 4 plants had the potential to be developed to FAS inhibitors and even anti-tumor drug precursors.The inhibitory effect of flavonoids from bamboo leaves on FAS was confirmed and very strong inhibitory activity of isovitexin of FAS in vitro was found [29].The results has important reference value for the utilization ofKoelreuteriaintegrifoliolaresources and use of bamboo leaf flavonoids in FAS inhibitor development.Therefore,the anti-tumor strategy is to interfere with the energy metabolism of aerobic glycolysis of tumor cells,destroy the suitable microenvironment of tumor cells,and affect the biological behaviors of tumor cells,such as proliferation,adhesion,movement and migration,so as to achieve the purpose of retarding tumor growth or inhibiting tumor metastasis.

TCM regulates energy metabolism and angiogenesis in BC

At the same time,some studies have shown that“Phlegm-Reducing” and “Stasis Dissipating” drugs can not only inhibit the angiogenesis of breast tumors,but also regulate the abnormal energy metabolism of tumor cells.Cantharidin a natural compound from an arthropod insect cantharis exerts potent anticancer activity.Pan found cantharidin inhibits the PKM2 nuclear translocation and breaks GLUT1/PKM2 glycolytic loop,resulting in aerobic glycolysis transformation to oxidation and subsequent reversing the metastases in BC [30].Upon tube formation and rat aortic ring assay,it was found that cantharidin can decrease the number of completely formed tubes and reduce the density and length of vascular sprouting,indicating that angiogenesis can also be inhibited.The mechanism of action might be that cantharidin inhibits angiogenesis by suppressing VEGF induced JAK1/STAT3,ERK and AKT signaling pathways [31].Tetrameric transformation and nuclear translocation of PKM2 are essential for cancer metastasis,and βelemene inhibited BC metastasis via blocking aerobic glycolysis mediated by dimeric PKM2 transformation and nuclear translocation [32].β-elemene also can enhance apoptosis of the endothelial cells cultured in vitro,inhibit their angiogenic ability,reduce the number of tube formation of ECV304 cells on matrigel,and inhibit the activity of MMP-2 and MMP-9 [33].Quercetin,a flavonoid known to possess antiangiogenesis and antitumor properties,inhibited calcineurin activity in vitro.the result of research shows that quercetin inhibit angiogenesis of human BC xenograft in nude mice,which was associated with suppressing calcineurin activity and calcineurin/NFAT pathway [34].The further cells experiments exhibited that quercetin successfully blocked cell glycolysis by inhibiting the level of glucose uptake and the production of lactic acid,and also decreased the level of glycolysisrelated PKM2,GLUT1 and LDHA [35].

Compared with normal cells,tumor cells have the ability of infinite replication potential,resistance to apoptosis,shielding anti-growth signals,continuous angiogenesis,tissue invasion and metastasis,etc.The microenvironmental requirements,energy supply and metabolic pathways required to support the above characteristics of tumor cells are the core of tumor characteristics research.Based on the syndrome differentiation of mutual association of phlegm and blood stasis,the drugs of “Resolving Phlegm and Dispersing Constipation,Promoting Blood Circulation and Removing Blood Stasis” have been used to treat tumors with good results.It is still controversial whether the drugs that “Promote Blood Circulation and Dissipate Blood Stasis” have anti-tumor effect.Currently,some clinical studies have found that the drugs that “Promote Blood Circulation and Dissipate Blood Stasis” may promote tumor metastasis,but most basic studies shown that the drugs can inhibit tumor metastasis.By combining the modern medical theory,is clear about “phlegm fights,promoting blood circulation to remove blood stasis drugs inhibiting tumor and tumor metastasis,and further puts forward the potential molecular basis of the role,namely” phlegm fights,promoting blood circulation to remove blood stasis drugs may by interfering with tumor angiogenesis,regulation of energy metabolism,inhibit tumor growth and metastasis (Figure 1).From “abnormal new blood vessels-stasis” and “metabolic abnormalities-phlegm”theory in the study of scattered stasis drugs in antitumor and tumor metastasis,reveal “Phlegm Fights,Promoting Blood Circulation to Remove Blood Stasis”method is one of the modern molecular basis for the treatment of tumor,anti-tumor and tumor metastasis for TCM research provides a new train of thought,and to provide basis for clinical antitumor application of TCM.

Figure 1 Traditional Chinese medicine for “Promoting Blood Circulation and Removing Blood Stasis”,dissipating phlegm and resolving masses regulate abnormal energy metabolism and angiogenesis of breast cancer