| Background and Purpose:Colorectal cancer(CRC)is a common malignant tumor in the digestive system.In2020,the incidence of colorectal cancer is 10.0%,or about 1.93 million cases,and is the second largest cause of cancer-related mortality in the world.5-FU is the first-line chemotherapy drug for patients in clinical CRC.According to the investigation statistics,after long-term use of adjuvant chemotherapy drugs containing 5-FU in patients with colorectal cancer,about 50% of advanced patients are resistant to 5-FU and its metabolites.The anticancer effect of 5-FU is only 10%-17%,and the decreased exposure concentration of 5-FU in cells is one of the reasons for the decreased efficacy and the chemical resistance of tumor cells.Therefore,there is a need to develop multiple combination therapy regimens to improve its therapeutic efficacy and reduce its toxicity.Based on our research group’s previous literature research,Schisandrin B(Sch.B)combined with antitumor drugs has the effect of enhancing efficacy and reducing toxicity.This study studied the mechanism of Sch.B increasing the exposure level of 5-FU in colorectal cancer cells and coordinating the anti-colorectal cancer effect of 5-FU.Methods:First,A CCK-8 assay was used to assess the effects of 5-FU,Sch.B alone and in combination on the proliferation of Caco-2,HT-29 and SW480 cells and primary CRC cells,the Tunel cell apoptosis assay was used to investigate the effects of 5-FU,Sch.B alone and in combination on the apoptosis of HCT116 cells.Secondly,Sch.B was studied to increased intracellular 5-FU exposure in HCT116 cells.RT-PCR assay was used to investigate the influence of Sch.B on ABC efflux transporters.The changes of small molecules and endogenous substrates of ABC efflux transporters in HCT116 cells treated with Sch.B were profiled and quantified by metabolomics assay.The effect of Sch.B on ABC efflux transporter expression was verified by Western blot assay.To further study the effects of Sch.B on the transporters of ABCC2,ABCC5 and ABCC11.Adenovirus transfection assay was used to induce high expression of ABCC2,ABCC5 and ABCC11 proteins,western blot assay was performed to investigate the effect of Sch.B on overexpression of ABCC2,ABCC5 and ABCC11 proteins,and CCK-8 assay was performed to investigate the effect of Sch.B combined with5-FU on the proliferation of up-regulation ABCC5 and ABCC11 in HCT116 cells and changes in the concentration of 5-FU in overexpressed ABCC5 and ABCC11 in cells.Thirdly,a direct,sensitive and effective LC-MS/MS method for the determination of Sch.B content changes in cells was established and verified.The pre-treatment method was as follows: the protein was precipitated by methanol containing internal standard(V:V=1:3)and then the sample was injected into the LC/MS-MS system.Chromatographic conditions:Atlantis T3-C18 column(2.1*100 mm,3 μm),mobile phase: water(containing 0.2%formic acid)-methanol,gradient elution program was: 0 min,0% B;0-0.5 min,0%-90% B;0.51-53 min,100%B.The flow rate of mobile phase was 0.4 m L/min,and injection volume was 5 μL.Mass spectrometry conditions: ESI ionization source was used for detection in positive ion mode,data was collected in multiple reaction monitoring mode(MRM).Finally,transcriptome assay was used to study the molecular mechanism of inhibition of ABCC2,ABCC5 and ABCC11 transporters and study the molecular mechanism of inhibition of ABC transporter by Sch.B.Results:First,the combination of 5-FU and Sch.B significantly inhibited the proliferation of Caco-2,HT-29 and SW480 cells and primary CRC cells compared with the control group or the 5-FU group alone,and the results of the Tunel apoptosis assay demonstrated that Sch.B enhanced the effect of 5-FU in promoting apoptosis of HCT116 cells.Secondly,Sch.B was studied to increased intracellular 5-FU exposure in HCT116 cells.The results of RT-PCR showed that Sch.B inhibited the mRNA expression of ABCC2,ABCC5 and ABCC11.The results of metabolomics revealed that Sch.B inhibited the expression of ABCC11,and the efflux of leukotriene metabolites and caused the accumulation of leukotriene metabolites in the cells.To further study the effects of Sch.B on the transporters of ABCC2,ABCC5 and ABCC11.The results of RT-PCR and Western blot verified the high expression of ABCC2,ABCC5 and ABCC11 proteins.CCK-8 assay revealed that the combination of 5-FU and Sch.B inhibited the proliferation of expressed ABCC5 and ABCC11 cells.LC-MS/MS quantification of intracellular 5-FU concentration levels showed that Sch.B inhibited the expression of ABCC11 and contributed to accumulate intracellular 5-FU concentration.Thirdly,the linearity of Sch.B was good in the range of 20-1000 ng/m L,and the methodologies were in accordance with the pharmacopoeial requirements.Finally,to study the molecular mechanism of inhibition of ABCC2,ABCC5 and ABCC11 transporters by Sch.B.After treatment with Sch.B,20 genes such as ABCC2,ABCC5 and ABCC11 and 26 genes such as CCN2 and PLPPR3 were significantly down-regulated and up-regulated,respectively.GO enrichment analysis showed that the glutathione transmembrane transport and leukotriene B4 catabolic process were significantly inhibited and down-regulated.GO level2 and KEGG pathway enrichment of differentially expressed genes showed that differentially expressed genes were enriched in the transporter pathway and the activity of the transporter was significantly inhibited.Conclusions:The combination of the two drugs significantly suppressed the proliferation of colorectal cancer cell lines(Caco-2,HT-29,SW480)and primary CRC cells and promoted the apoptosis.Secondly,Sch.B was studied to increased intracellular 5-FU exposure in HCT116 cells,the results showed that Sch.B significantly inhibited the mRNA expression of ABCC2,ABCC5 and ABCC11 proteins,Sch.B down-regulated the efflux of ABCC11 protein,which resulted in decreased efflux of its specific endogenous substrate leukotriene metabolites and caused intracellular leukotriene metabolites accumulation.To further study the effects of Sch.B on the transporters of ABCC2,ABCC5 and ABCC11.The high expression of ABCC2,ABCC5 and ABCC11 proteins was significantly suppressed by Sch.B,and the inhibition of ABCC5 and ABCC11 protein expression decreased the efflux of 5-FU drug from the HCT116 cells,increased its intracellular exposure concentration and enhanced the antitumor effect.Thirdly,a simple,rapid and sensitive UHPLC-MS/MS method was developed and validated to measure the concentration of Sch.B in cells.The spatial distribution of Sch.B in nucleus,which may be helpful in investigating the effects of Sch.B on cancer cells and its synergistic effect with 5-FU.Finally,to study the molecular mechanism of inhibition of ABCC2,ABCC5 and ABCC11 transporter by Sch.B.260 and 73 genes were up-regulated and down-regulated in HCT116 cells,respectively.After Sch.B treatment,20 and 26 differentially expressed genes were significantly down-regulated and up-regulated.Sch.B inhibits the function of ABCC2,ABCC5 and ABCC11 transporters by inhibiting the activity of the transporter pathway,and may inhibit glutathione transmembrane transport and leukotriene B4 catabolic process. |
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