Screening Anti-breast Cancer Constituents From ’Clearing Heat-toxin And Resolving Masses’ TCM And Research On Their Pharmacological Mechanisms

Breast cancer remains the most common cancer among womenworldwide and the deaths of breast cancer were the leading cause ofcancer death in women.‘Clearing heat-toxin and resolving masses’ is thetherapy principle for breast hyperplasia and breast tumor in traditionalChinese medicine. Six Chinese herbs, namely, Pu-Gong-Ying, Gan-Cao,Chai-Hu, Mu-Xiang, Gua-Lou and Huang-Yao-Zi, are frequently used incomplex traditional Chinese medicing formulas for breast hyperplasia andbreast tumor therapy according to references. The pharmacologicaleffects of these Chinese herbs are all described as ‘clearing heat-toxin andresolving masses’ in traditional use. Therefore, we speculated that theseherbs may contain anti-breast cancer compounds.In the present work, a bioactivity-guided fractionation screening platform, including fractionation by semi-preparative high performanceliquid chromatography (semi-preparative HPLC), cytotoxity screening byMCF-7cell lines, and identification by ultraperformance liquidchromatography coupled to quadrupole-time-of-flight mass spectrometer(UPLC–Q-TOF/MS), was developed for fast screening and identifyingpotential anti-breast cancer constituents of the Chinese herbs. Twopotential anti-breast cancer compounds, namely, costunolide (Cos) anddehydrocostus lactone (Dehy), were discovered in VOSL (volatile oilextracted from Mu-Xiang), and interestingly, the combination of Cos andDehy (Cos-Dehy) showed a synergism on inhibiting the proliferation ofMCF-7cells in vitro, which exhibited a potential application prospect forbreast cancer therapy.The original constituents of traditional Chinese medicines (TCMs)were usually changed into bioactive metabolites in vivo bygastrointestinal or liver metabolism after oral administration. Bothoriginal constituents of TCMs and their bioactive metabolites can bescreened by serum pharmacological screening method. Therefore, basedon serum pharmacological screening method, the bioactivity-guidedfractionation screening platform was further developed for fast screeningand identifying potential anti-breast cancer constituents of the Chineseherbs. Three potential anti-breast cancer components from Pu-Gong-Ying,namely11,13-dihydrotaraxinic acid, taraxinic acid β-D-glucoside, and ainslioside, were identified on the basis of the fragmentation pattern andaccurate molecular data obtained by UPLC–Q-TOF MSE.11,13-Dihydrotaraxinic acid, which is the original component of Pu-Gong-Ying,exhibits anti-carcinogenic activity without biotransformation. However,hydrolyses of β-glucosidic bonds of taraxinic acid β-D-glucoside andainslioside mediated by intestinal flora are essential to theiranti-carcinogenic activity in vivo. Compared with conventional screening,this screening strategy not only can eliminate the interference of manyinactive compounds of TCMs and endogenous constituents of serum, butcan also screen original components of TCMs and their bioactivemetabolites. Furthermore, the strategy can clearly correlate the chemicalprofile of TCMs with their activities.We used a comprehensive two-dimensional gas chromatography–time-of-flight mass spectrometry (GC GC TOF/MS) and an ultraperformance liquid chromatography–quadrupole time-of-flight massspectrometry (UPLC–Q-TOF/MS) based metabolomic approach to gainpotential serum and urine biomarkers of breast cancer in MCF-7xenograft mice. We also used VOSL and Cos-Dehy treatments in thesame animal model to test their potential therapeutics and explore theiranti-tumor mechanisms in vivo. Our research demonstrated that VOSLcan significantly inhibit the growth of MCF-7xenograft tumors throughpharmacological mechanism of “multiple-components, multiple-targets and system therapy”. Significant variations in MCF-7xenograft mice,characterized by a total of118differentially expressed serum and urinemetabolites were observed based on GC and LC MS metabonomicstudy. These differential metabolites were mainly related to fivedysregulated metabolic pathways including glycolysis/pyruvatemetabolism, valine, leucine and isoleucine metabolism, phenylalanineand tyrosine metabolism, steroid hormone metabolism, and unsaturatedfatty acids metabolism. Seven representative metabolites, arachidonate,docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), linoleate,dihomo-γ-linolenate,20α-hydroxyprogesterone, and cortisone wereselected as a panel of candidate pathological biomarkers of MCF-7xenografts. Logistic regression was used to combine the7variables into amultivariable. The receiver operating characteristic (ROC) analysis basedon the multivariable yielded a very satisfactory result, which was able toabsolutely discriminate MCF-7xenograft mice from the healthy controls,with a100%sensitivity and specificity, moreover, the changes of thesebiomarkers levels in Cos-Dehy treated group and VOSL treated group,were obviously attenuated compared to NT group. Therefore, our studyhighlights the significance of the distinct urinary and serumal metabolicprofile of MCF-7xenograft mice, characterized by a panel of metabolitesthat may be further developed to be diagnostic markers for the earlydetection of breast cancer in the future. Both VOSL and Cos-Dehy can significantly inhibit the growth of MCF-7xenograft tumor throughregulating it’s dysregulated metabolic pathways, including phenylalanineand tyrosine metabolism, steroid hormone metabolism, and unsaturatedfatty acids metabolism, besides, VOSL also can regulate energy-relateddysregulated metabolic pathways of MCF-7xenograft mice, includingglycolysis/pyruvate metabolism and TCA cycle, to make the mice morehealthy, therefore, VOSL and Cos-Dehy both have potential implicationvalues for breast cancer therapy.The pharmacological mechanisms of Cos-Dehy and VOSL oninhibiting the proliferation of MCF-7cell lines were investigated byphosphoproteomics and cell cycle analysis. Our results demonstrated thatCos-Dehy and VOSL can arrest MCF-7cells at S phase, interfere theorganization of cytoskeleton, and induce apoptosis of MCF-7cells byAKAP protein and cAMP both mediated up-regulated activity of PKA(protein kinaseA).As Cos and Dehy were the main anti-carcinogenic constituents inVOSL, and combination treatment (Cos-Dehy) showed synergisms oninhibiting the proliferation of MCF-7cells in vitro and in vivo, therefore,the pharmacokinetics and metabolism of Cos and Dehy in rats wereinvestigated after a single intravenous administration. Our results showthat the VZand CLZof Cos were both smaller than those of Dehy, and theCmaxof Cos was higher than that of Dehy. As the t1/2zof Cos and Dehy were both very short and their VZwere both relatively large, wespeculated that these two drug candidates could easily be combined withhigh-affinity proteins of liver and then eliminated rapidly. Therefore,according to the pharmacokinetic analysis of Cos and Dehy in vivo, webelieve that the research and development of sustained-releasepreparations may be very important for the further development of thesedrug candidates. Phase II biotransformation was the main metabolicpathway of Cos and phase biotransformation was the main metabolicpathways of Dehy. Both compounds also had common metabolicpathways, namely, the two sequential desaturations and N-acetylcysteineconjugation. Through the comprehensive analysis of thepharmacokinetics and metabolites of Cos and Dehy in vivo, thisinvestigation may provide some fundamental information for the furtherdevelopment of the two drug candidates.