| Colorectal cancer,also known as large intestine cancer,is one of the common malignant tumors of the digestive tract.Since 1998,the morbidity of colorectal cancer has continued to increase in China,and in 2014,the morbidity ranked fourth among all cancers.Due to the lack of early screening,most patients have been diagnosed with middle or late stage of colorectal cancer.Therefore,the surgery plus chemoradiotherapy is widely used in Chinese colorectal cancer patients for the treatment.Capecitabine(Cap)is one of the main drugs for colorectal cancer treatment,which significantly improved the therapeutic effect and reduced the overall adverse reactions,but the adverse reactions in hand and foot(morbidity 31.7%),digestive tract(morbidity>50%)and the heart(morbidity 33.8%)still seriously hindered its clinical application.Clinical targeted treatments are often prescribed after serious adverse reactions' occurrence,while preventive prescriptions are lack of selectivity.The metabolic process of Cap is complicated,and in vivo exposures of its metabolites are largely different,so the discovery of biomarkers based on metabolic enzyme polymorphism is difficult because of the interferences,and there aren't any reliable biomarkers discovered for the Cap.Pharmacometabonomics is a branch of metabolomics which can quantitatively analyze the metabolic profile of all small molecule metabolites in organisms before drug administration,and correlates them with physiological,pathological and external disturbances,and finally analyzes the results and influences of drug administration.Therefore,it is possible to discover biomarkers of adverse reactions by pharmacometabonomic and identify patients with serious adverse reactions to Cap before prescription.It will greatly improve the compliance of patients and reduce the economic and physical burden of patients.Stromal cell-mediated drug resistance is one of the important mechanisms of cancer drug resistance,and the mechanisms of cell interactions between cancer cells and stromal cells are complex.It was found that activated stromal cells could be transformed into cancer-associated stromal cells after triggering a series of variations such as gene expression.As a result,over or reduced expression of many cytokines and proteins will be carried out to reconstitute the extracellular matrix of cancer cells,which can provide favorable microenvironment for cancer cell growth,invasion,metastasis,drug resistance,immune escape and other processes.Some studies have demonstrated the part role of stromal cells in drug resistance,but whether the number of stromal cells affects drug resistance is not known.The genes expression variations after activation of stromal cells and cancer cells is still unclear.Whether there is direct competition between stromal cells and cancer cells and affecting the drug resistance needs further exploration.Based on the above questions,this paper first established an ultra high-performance liquid chromatography tandem mass spectrometry(UHPLC-MS/MS)method to determinate Cap and its five metabolites,which requires only 100 ?L of plasma sample and one-step liquid-liquid extraction for the pretreatment.The method has a good selectivity and high mass spectrometric responses of Cap and its five metabolites.The analytical time is 5 min,and the lower limit of quantification is about 20 ng/mL,and the linear range is approximately 20-5000 ng/mL with a good linearity of Cap and its five metabolites.This method is superior to most of the reported methods.The precision and accuracy,stability,recovery and matrix effect,residual effect,dilution effect and so on all meet the requirements of the pharmacopoeia and the practicality of the method was also validated on clinical samples.In addition,the UHPLC-MS/MS method for determination of the final metabolite,fluoro-?-alanine(FBAL),in urinary was established.FBAL is closely related to the cardiotoxicity of Cap.This method requires only 10 ?L of urine sample,and the pretreatment can be completed within 15 minutes based on sample dilution.The linear range is 20-10000 ng/mL with a good linearity of FBAL,and the other items,for instance,precision and accuracy,stability,extraction recovery and matrix effect,residual effect,dilution effect all meet the requirements of the pharmacopoeia.For the pharmacometabolomics study,55 colorectal cancer patients were enrolled after treated with Cap.Preoperative plasmas were collected and adverse reactions after administration of Cap were detailedly recorded.The morbidity of hand-foot syndrome in Chinese population was 5.5%.No patients suffered from ? grade 3 hand-foot syndrome,while the morbidity of ? grade 2 vomit was 21.8%,and the morbidity of ? grade 2 diarrhea was 27.3%,and 43.6% of the patients were diagnosed with ? grade 2 nausea,and the morbidity of ? grade 2 liver injury was 14.5%.No other adverse reactions at ? grade 2 were found.And then,metabolomics analysis was performed on the preoperative plasma from all the patients,and the metabolic profiles between the patients with severe adverse reactions(? grade 2)and those who did not occurred(<grade 2)were compared.The results showed that patients with severe diarrhea and vomit have lower content of selenoadenosine homocysteine while the content of sphingosine-1-phosphate and glycocholic acid in vomit patients increased significantly,and the sphingosine-1-phosphate didn't make a sense to diarrhea.The glycocholic acid declined in the patients with ? grade 2 liver injury.The compounds reflected the different metabolism of selenium-containing amino acid,sphingolipid,and cholic acid in patients,and the selenoadenosine homocysteine,glycocholic acid and sphingosine-1-phosphate may be candidate biomarkers for clinical screening of patients suffering from serious adverse reactions to Cap in advance.To solve the problem that some of the patients still suffered from Cap induced liver injury even though the liver protective agent glutathione was prescribed,this paper compared the liver-protecting effects of magnesium isoglycyrrhizinate and glutathione in rat model.The results showed that Cap can cause purpura-like liver injury in rats,and low doses of magnesium isoglycyrrhizinate and glutathione have a compromised liver-protecting effect with slightly decrease of the ALT and AST and other indicators,while medium and high doses of magnesium isoglycyrrhizinate have better liver protection effect,and liver indicators decreased significantly.The experimental results provide a reference for clinical application of liver protection agents.In addition,we explored that if the magnesium isoglycyrrhizinate would suppress the FBAL excretion which may cause a further heart adverse reaction.The effects of the liver-protecting agent,magnesium isoglycyrrhizinate,on FBAL excretion in rat model were investigated.The rats were randomly designated to control group,the different doses of magnesium isoglycyrrhizinate groups and the positive drug group(glutathione).The urine samples of rats were collected in 24 hours and transferred to measurement process of FBAL.the results showed that magnesium isoglycyrrhizinate wouldn't inhibit the excretion of FBAL.To explore the cell interactions between colorectal cancer cells and the corresponding stromal cells and drug resistance of cancer cells influenced by stromal cells,the basic models of human cell and rat cell were firstly established based on the exchange of conditioned medium(CM)between cancer cells and stromal fibroblasts.The CM of fibroblasts could promote the growth of cancer cells,and its effect is positively correlated with the number of fibroblasts,while the influence of CM from cancer cell on fibroblasts is not quantitatively dependent.When the ratio is 1:5 between cancer cells and fibroblasts,fibroblasts have the best survival.Based on the contact co-culture of cancer cells and fibroblasts,the cell competitions between cancer cells and fibroblasts were explored.The results showed that there wasn't a directly competition between fibroblasts and cancer cells,and fibroblasts could promote the growth of cancer cells and slowed down or even stopped the growth of themselves.The addition of monocytes in the co-culture system of cancer cells and fibroblasts could slightly suppress the growth rate of cancer cells,and improved the growth rate of fibroblasts.Based on the above model,the non-contact co-culture experiment of cancer cells and fibroblasts firstly revealed the expressive variations of some cytokines such as CCL5,TIMP-1,CXCL2/3,IFN-?,CCL5,At the same time,western-blot and PCR results showed that co-culture can promote cancer metastasis and fibroblast activation.The cytokines may be related to the activation of fibroblasts and epithelial to mesenchymal transitions of cancer cells.In addition,our results showed that fibroblasts could significantly improve drug resistance and promote cancer growth rate under 5-FU treatment by exchange the CM if the number of fibroblasts was more than half of the cancer cells.Finally,the 5-FU and monocyte were added simultaneously to the contact co-culture system,the growth rate of cancer cells was still higher than the other cancer cells cultured alone,but the fibroblasts grew faster.The results demonstrated that the ratio of cancer cell and fibroblast may be a biomarker for drug resistance.In summary,this study established two rapid quantitative methods for the determination of Cap and its metabolites in biological matrices,and found differential endogenous metabolites in patients with digestive tract adverse reactions caused by Cap and the better effect of magnesium isoglycyrrhizinate as a liver-protecting drug,and finally we explored the cell interactions between colorectal cancer cells and corresponding stromal cells and its effects on cancer cell growth and drug resistance.The experimental results are of great significance for further improving the therapeutic effect of colorectal cancer. |
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