Development Of P-gp High/low Expression MCF7 Cell Membrane Chromatographic System And Its Application For Screening Anti-breast-cancer Components From Traditional Chinese Medicines

Breast cancer(BC)is the most common malignant tumor in women with a high lethal rate.The treatment of BC has drawn worldwide attention.Conventional treatments include surgery,radiotherapy and chemotherapy.However,the consequential adverse effects of these methods,such as the decreased immune function and subsequent multidrug resistance(MDR),which have very serious adverse effects on the health and quality of life of patients,and give the treatment of BC caused a lot of trouble.Traditional Chinese medicine(TCM)has a long history and a wide range of applications,TCM with its adverse reactions,multi-target role,syndrome differentiation,overall conditioning and other characteristics plays an important supporting role in the treatment of BC.There are already a large number of TCM monomer or compound used in clinical treatment process.However,it is still mainly dependent on the clinical experience of the majority of doctors.There is no clear norms as a reference,and the mechanism by which Chinese medicine exerts its efficacy remains unclear.Therefore,it is a very important task to quickly screen out the active ingredients which have the efficacy from traditional Chinese medicine and study its mechanism.Cell membrane chromatography(CMC)is a bio-affinity chromatography technology with a rapid development in recent years.Using the cell membrane and silica gel as the stationary phase,CMC can be used to simulate the interaction between the drug molecule and the cell membrane receptor in vitro.CMC combines the separation of components and active screening,with the advantages of convenient operation,fast and stable,high sensitivity,especially suitable for the active screening of complex components such as TCM.The main research content of this topic is divided into the following three parts:1.The establishment of a new comprehensive two-dimensional(2D)high/low expression P-gp of APTES-decorated MCF7,MCF7/ADR-cell membrane chromatography(CMC)/capcell-C18 column/time-of-flight mass spectrometry systemIn this study,two MCF7 and MCF7 / ADR cells were selected.The expression of p-gp in cells was verified by the method of flow cytometry and Western Blot.The expression of P-gp on MCF7 cells was low,while the expression of MCF7 / ADR cells was high,and there was no significant change after 2 weeks cultured without Doxorubicin.A new comprehensive two-dimensional(2D)high/low expression P-gp of APTES-decorated MCF7,MCF7/ADR-cell membrane chromatography(CMC)/capcell-C18 column/time-of-flight mass spectrometry system was established.In order to examine its validity and specificity,four comprehensive two-dimensiona analysis systems were constructed,they are silica gel column and APTES-decorated silica gel column,APTES-decorated MCF7 and APTES-decorated MCF7 / ADR membrane column.The system effectiveness study showed that gefitinib had no retention behavior on silica gel column and APTES-decorated silica gel column,but showed significant retention behavior on APTES-decorated MCF7 CMC columns and APTES-decorated MCF7 / ADR CMC columns.Correspondingly,dexamethasone did not show any retention behavior on all four columns,and thus the system was proved to be effective and could be used for screening anti-breast cancer potential active compounds in complex TCM.The system specificity study showed that Tariquidar had no significant retention behavior on silica gel column,APTES-decorated silica gel column and APTES-decorated MCF7 CMC column but there was a significant retention behavior on APTES-decorated MCF7 / ADR CMC column.The system presents a certain degree of specificity.Through investigation of the effectiveness and specificity of the system,it provides a platform for the next screening of potential active ingredients against BC from the complex system of TCM and lay a foundation for the study of the relationship between the active ingredient and P-gp.2.Screening anti-breast cancer active ingredients from TCM based on comprehensive 2D breast cancer cell membrane chromatographyFirstly,eight kinds of common TCMs,which have been reported in the literature to have anti-cancer effects,were selected as objects,the chemical composition library was constructed.The extracts of TCM were prepared and analyzed by HPLC/ TOF-MS system.The preliminary identification of the components and the second-dimensional separation for comprehensive two-dimensional analysis of the optimal conditions were guaranteed.A new crehensive two-dimensional APTES-decorated MCF7,MCF7/ADR-cell membrane chromatography(CMC)/capcell-C18 column/time-of-flight mass spectrometry system was established for screening potential active components of eight kinds of TCMs.Our screen results showed that Dahuang and Huangqin have more retention components and a better retention behavior,therefore,these two kinds of TCMs were chosen to continue the study.Completed the comprehensive 2D contour map,the identification results and some of the standard compounds of these compounds were validated.The results showed that Dahuang extract in MCF7 CMC has 14 retention components and in MCF7 / ADR CMC has 15 retention components,10 of the components are the same,in addition,six components including rhapontigenin,piceatannol,emodin,rhaponticin,rhapontin,chrysophanol,physcion were validated.The results showed that Huangqin extract in MCF7 CMC has 15 retention components and in MCF7 / ADR CMC has 14 retention components,10 of the components are the same,in addition,five components including wogonin,baicalin,oroxylin A,baicalein,wogonoside were validated.The above research has laid a good foundation for the next experiment such as pharmacodynamic validation of potential active ingredients and study of the relationship between active ingredient and P-gp.3.Potential active ingredients activity against breast cancer and its relationship with P-gpAccording to the screening results of Dahuang and Huangqin based on comprehensive two-dimensional MCF7 and MCF7 / ADR CMC systems previously established,eleven compounds which were obtainable were selected for the next study.All of the compounds have been tested for potential anti-breast cancer efficacy in vitro by inhibiting proliferation.The results showed that rhapontigenin,piceatannol,emodin from Dahuang and wogonin,baicalin,oroxylin A,baicalein from Huangqin have certain killing effect to both MCF7 and MCF7 / ADR cells,and the sensitivity between the two cells is different.Flow cytometry was used to examine whether the killing effect was apoptosis.After that,in order to study whether the same compound on the killing effect of two cells is related to the action of P-gp,the effects of the above seven components on P-gp activity were investigated,results show that baicalin is an inhibitor of P-gp enzyme activity,while the other six components are P-gp enzyme activity agonists.In order to study the relationship between baicalin and P-gp,the effects of different concentrations of baicalin on the expression of P-gp in MCF7 / ADR cells were detected by flow cytometry and Western blot,results showed that baicalin had no significant effect on the expression of P-gp in MCF7 / ADR cells.In addition,the synergistic effect between baicalin and DOX was examined to reverse the resistance of doxorubicin to MCF7 / ADR cells,the results showed that baicalin could reverse the resistance of MCF7/ADR cells to DOX.