| Malignant tumor is a serious threat to human health.Multidrug resistance(MDR)and invasion and metastasis are the main causes for the failure of tumor treatment.It is one of the important topics in anti-tumor research to find the active components with high efficiency and low toxicity from traditional Chinese medicine based on MDR and metastasis mechanism.In this paper,the potential anti-tumor active components were screened by ultra-high performance liquid chromatography-mass spectrometry(UPLC-MS)at the molecular and cellular levels.The mechanism of the reversal and inhibiting tumor metastasis of the active components are studied based on the combination of metabolomics and cell biology.Lactate dehydrogenase(LDH),a key rate limiting enzyme in glycolysis,is overexpressed in many tumor cells and is considered to be an important target of invasive cancer.LDH is used as a target molecule to screen its inhibitors from traditional Chinese medicine.At the same time,P-glycoprotein(P-gp)mediated MDR reversal agents were screened based on MDCK-MDRI cell model.1.Screening LDH inhibitors from traditional Chinese MedicineThe immobilized LDH method was established to screen potential LDH inhibitors from Rhubarb and Polygonum cuspidata.Firstly,LDH was immobilized onto the surface of amino-modified magnetic nanoparticles via covalent binding.In order to obtain the maximum enzyme activity,the immobilization conditions including pH,time and LDH concentration were optimized by combining single factor and response surface.When the pH value was 5.85,the reaction time was 3.14 h and the LDH concentration was 0.37 mg/ml,the activity of immobilized LDH reached the maximum.The amount of LDH immobilized on magnetic nanoparticles was about 49 ?g enzyme/mg carrier under the optimal conditions.Subsequently,the ligand fishing assay was performed to validate the specificity and selectivity of immobilized LDH using a model mixture,which consisted of galloflavin,chlorogenic acid and verbascoside.Finally,the immobilized LDH approach combined with ultra-high performance liquid chromatography-tandem mass spectrometry technique(UHPLC-MS/MS)was applied to screen potential LDH inhibitors from two anthraquinone-rich natural products(Rhubarb and Polygonum cuspidatum).Nine and six compounds were identified from Rhubarb and Polygonum cuspidatum extracts respectively,of which three compounds were common to both.This result further confirmed the feasibility of the immobilized LDH approach.2.Screening P-gp inhibitors based on MDCK-MDR1 cellsMDCK-MDR1 cells with overexpression of P-gp were used to screen P-gp inhibitors from Polygonum cuspidatum and Chelidonium majus and their main components based on four aspects:cytotoxicity,rhodamine-123(Rh-123)accumulation and efflux,western blot and transport experiment.The IC20 values of every drug were determined by cytotoxicity.Anthraquinone and polyphenols were selected 40,20,10?M for follow-up study.The Rh-123 accumulation and efflux results showed that Polygonum cuspidatum had better inhibition on P-gp than Chelidonium majus.Anthraquinones had the best inhibitory effect on P-gp,especially aloe emodin and rhubarb,followed by polyphenols and three alkaloids with high toxicity.In addition,it could be preliminarily speculated that other compounds could inhibit the function and expression of P-gp except polydatin,trimethoxy stilbene and chelerythrine,through the results of different treatment time on Rh-123 accumulation.The western blot results indicated that the compounds could inhibit the expression of P-gp except polydatin and chelerythrine.Aloe emodin,chrysophanol and resveratrol were selected for transport studies.The results showed that aloe emodin and resveratrol could significantly inhibit the efflux of digoxin,but chrysophanol could not.In addition,we added five alkaloids with similar structure and less toxicity to study.It was found that matrine,oxymatrine,sophocarpine,oxysophocarpine and sophoridine had better inhibition on P-gp,and they were expected to be reversal agents of MDR.3.The mechanisms of aloe emodin(AE)on reversing MDRThe reversal effect and mechanism of AE were studied based on the drug resistance model of breast cancer MCF-7/ADR cells.AE could significantly reverse the ADR resistance in MCF-7/ADR cells and its reversal index was 4.86.The combination of 20 ?M AE and 4 ?M ADR had no effect on the P-gp level,but notably promoted the accumulation of ADR in drug-resistant cells.The results of fluorescence microscopy showed that AE could make more ADR enter the nucleus where ADR could play an anti-tumor role.The efflux function of P-gp required ATP,but AE reduced the intracellular ATP level.AE played a reversal role might through inhibiting the efflux function of P-gp.The research results of energy metabolism pathways indicated that the combination of AE and ADR could inhibit glycolysis,TCA cycle,glutamine metabolism and related amino acid synthesis pathways.Moreover,we found that AE not only reversed ADR-induced resistance,but also induced autophagy as a defense mechanism,and inhibition of this process can enhance the reversal activity of AE.In addition,the combination of AE and ADR arrested G2/M cell cycle and induced apoptosis through DNA damage,ROS generation,caspase-3 activation.4.The mechanisms of aloe emodin(AE)and emodin(EMD)on inhibiting invasion and metastasis ability of breast cancerThe co-culture model of breast cancer cells(MCF-7)and human umbilical vein endothelial cells(HUVEC)was investigated by metabolomics approach based on UPLC-Q-TOF/MS.We screened and identified 25 biomarkers,which involved in glutathione metabolism,methionine cycle,purine metabolism and TCA cycle etc.These metabolic pathways were related to the malignant phenotype of tumor.The inhibition of invasion and metastasis of MCF-7 cells by AE and EMD was studied by co-culture model.The results showed that AE could inhibit the adhesion,invasion and angiogenesis of MCF-7 cells,while EMD mainly inhibited the adhesion process.Next,the metabolomics approach was applied to investigate the mechanism of AE and EMD inhibiting invasion and metastasis of MCF-7 cells.27 and 13 biomarkers were respectively identified in AE and EMD groups,involving polyamine metabolism,vitamin B6 metabolism,methionine cycle,TCA cycle,glutathione metabolism,purine metabolism and aspartic acid synthesis etc.The typical metabolites in these pathways were selected for quantitative analysis.It was found that AE could significantly reduce the content of these metabolites and the effect was significantly better than EMD. |
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