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Abstract Background Rhein can significantly delay the progression of chronic nephropathy. However, its mechanism of action has not been adequately elaborated, which hinders its extensive clinical application. In this work, the effects of rhein on models of TGF-β-induced NRK-49F cellular fibrosis and rat renal ischemia–reperfusion fibrosis were evaluated using metabolomics and western blotting. Methods The metabolic profiles of NRK-49F cells and rat urine, serum, and kidney tissues in the control, model, and rhein groups were investigated using UPLC-QTOF-MS. The levels of p-P65, p-IKK, p-AKT, p-P38, p-JNK and AP-1 in NRK-49F cells were measured using western blotting and immunofluorescence methods. Molecular docking and network pharmacology methods were employed to explore the relationship between the potential targets of rhein and key proteins in the NF-κB and MAPK signaling pathways. Results Various potential metabolites, including sphingolipids, ceramides, phosphatidylcholine, and lysophosphatidylcholine,14-hydroxy-E4-neuroprostane E, and 5-HPETE, were present in the cell, tissue, urine, and serum samples; however, few metabolites matches exactly among the four type of biological samples. These differential metabolites can effectively differentiated between the control, model, and rhein groups. Pathway enrichment analysis of differential metabolites unveiled that sphingolipid metabolism, arachidonic acid metabolism, and glycerophospholipid metabolism were closely related to nephropathy. Phosphorylation levels of AKT, IKK, P65 and AP-1 in NRK-49F cells was reduced by rhein treatment. Network pharmacology and molecular docking showed that the potential targets of rhein might regulated the expression of MAPK and AKT in the NF-κB and MAPK signaling pathways. Conclusion In brief, rhein might delays the progression of chronic nephropathy via the metabolic pathways, NF-κB and MAPKs signaling pathways, which provides the foundation for its development and clinical application.

The endoplasmic reticulum (ER) plays a pivotal role in protein folding and secretion, Ca2+ storage, and lipid synthesis in eukaryotic cells. When the burden of protein synthesis and folding required to be handled exceeds the processing capacity of the ER, the accumulation of misfolded/unfolded proteins triggers ER stress. In response to short-term ER stress, the unfolded protein response (UPR) is activated to allow cells to survive. When ER stress is severe and sustained, it typically provokes cell death through multiple approaches. It is well documented that ER stress and metabolic deregulation are functionally intertwined, both are considered contributing factors to the pathogenesis of liver diseases, including non-alcoholic fatty liver disease (NAFLD), alcoholic liver disease (ALD), ischemia/reperfusion (I/R) injury, viral hepatitis, liver fibrosis, and hepatocellular carcinoma (HCC). Hepatocytes are rich in smooth and rough ER, which harbor metabolic enzymes that are capable of sensing alterations in various nutritional status and external stimuli. Extensive research has focused on the molecular mechanism linking ER stress with metabolic enzymes. The purpose of this review is to summarize the current knowledge regarding the effects of ER stress on metabolic enzymes in various liver diseases and to provide potential therapeutic strategies for chronic liver diseases via targeting UPR.

In recent years, scientific researchers have increasingly become interested in noninvasive sampling methods for therapeutic drug monitoring and disease diagnosis. As a result, dried saliva spot (DSS), which is a sampling technique for collecting dried saliva samples, has been widely used as an alternative matrix to serum for the detection of target molecules. Coupling the DSS method with a highly sensitive detection instrument improves the efficiency of the preparation and analysis of biological samples. Furthermore, dried blood spots, dried plasma spots, and dried matrix spots, which are similar to those of the DSS method, are discussed. Compared with alternative biological fluids used in dried spot methods, including serum, tears, urine, and plasma, saliva has the advantage of convenience in terms of sample collection from children or persons with disabilities. This review aims to provide integral strategies and guidelines for dried spot methods to analyze biological samples by illustrating several dried spot methods. Herein, we summarize recent advancements in DSS methods from June 2014 to March 2021 and discuss the advantages and disadvantages of the key aspects of this method, including sample preparation and method validation. Finally, we outline the challenges and prospects of such methods in practical applications.

The tumor microenvironment (TME) is a crucial driving factor for tumor progression and it can hinder the body’s immune response by altering the metabolic activity of immune cells. Both tumor and immune cells maintain their proliferative characteristics and physiological functions through transporter-mediated regulation of nutrient acquisition and metabolite efflux. Transporters also play an important role in modulating immune responses in the TME. In this review, we outline the metabolic characteristics of the TME and systematically elaborate on the effects of abundant metabolites on immune cell function and transporter expression. We also discuss the mechanism of tumor immune escape due to transporter dysfunction. Finally, we introduce some transporter-targeted antitumor therapeutic strategies, with the aim of providing new insights into the development of antitumor drugs and rational drug usage for clinical cancer therapy.

The aim of this study was to develop a diagnostic strategy for esophageal squamous cell carcinoma (ESCC) that combines plasma metabolomics with machine learning algorithms. Plasma-based untargeted metabolomics analysis was performed with samples derived from 88 ESCC patients and 52 healthy controls. The dataset was split into a training set and a test set. After identification of differential metabolites in training set, single-metabolite-based receiver operating characteristic (ROC) curves and multiple-metabolite-based machine learning models were used to distinguish between ESCC patients and healthy controls. Kaplan-Meier survival analysis and Cox proportional hazards regression analysis were performed to investigate the prognostic significance of the plasma metabolites. Finally, twelve differential plasma metabolites (six up-regulated and six down-regulated) were annotated. The predictive performance of the six most prevalent diagnostic metabolites through the diagnostic models in the test set were as follows: arachidonic acid (accuracy: 0.887), sebacic acid (accuracy: 0.867), indoxyl sulfate (accuracy: 0.850), phosphatidylcholine (PC) (14:0/0:0) (accuracy: 0.825), deoxycholic acid (accuracy: 0.773), and trimethylamine N-oxide (accuracy: 0.653). The prediction accuracies of the machine learning models in the test set were partial least-square (accuracy: 0.947), random forest (accuracy: 0.947), gradient boosting machine (accuracy: 0.960), and support vector machine (accuracy: 0.980). Additionally, survival analysis demonstrated that acetoacetic acid was an unfavorable prognostic factor (hazard ratio (HR): 1.752), while PC (14:0/0:0) (HR: 0.577) was a favorable prognostic factor for ESCC. This study devised an innovative strategy for ESCC diagnosis by combining plasma metabolomics with machine learning algorithms and revealed its potential to become a novel screening test for ESCC.

Human UDP-glucuronosyltransferase (UGT) 2B7 is a crucial phase II metabolic enzyme that transfers glucuronic acid from UDP-glucuronic acid (UDPGA) to endobiotic and xenobiotic substrates. Biophysical and biochemical investigations of UGT2B7 are hampered by the challenge of the integral membrane protein purification. This study focused on the expression and purification of recombinant UGT2B7 by optimizing the insertion sites for the thermostabilized fusion protein apocytochrome b562RIL (BRIL) and various mutations to improve the protein yields and homogeneity. Preparation of the recombinant proteins with high purity accelerated the measurement of pharmacokinetic parameters of UGT2B7. The dissociation constants (KD) of two classical substrates (zidovudine and androsterone) and two inhibitors (schisanhenol and hesperetin) of UGT2B7 were determined using the surface plasmon resonance spectroscopy (SPR) for the first time. Using negative-staining transmission electron microscopy (TEM), UGT2B7 protein particles were characterized, which could be useful for further exploring its three-dimensional structure. The methods described in this study could be broadly applied to other UGTs and are expected to provide the basis for the exploration of metabolic enzyme kinetics, the mechanisms of drug metabolisms and drug interactions, changes in pharmacokinetics, and pharmacodynamics studies in vitro.

Abstract Human intestinal peptide transporter PEPT1 is commonly repressed in human colorectal cancer (CRC), yet its relationship with sensitivity to the common CRC treatment ubenimex has not previously been elucidated. In this study, we confirmed PEPT1 suppression in CRC using real-time quantitative polymerase chain reaction and western blotting and then investigated the underlying epigenetic pathways involved using bisulfite sequencing, chromatin immunoprecipitation, siRNA knockdown, and reporter gene assays. We found that PEPT1 transcriptional repression was due to both DNMT1-mediated DNA methylation of the proximal promoter region and HDAC1-mediated histone deacetylation, which blocked P300-mediated H3K18/27Ac at the PEPT1 distal promoter. Finally, the effects of the epigenetic activation of PEPT1 on the CRC response to ubenimex were evaluated using sequential combination therapy of decitabine and ubenimex both in vitro and in xenografts. In conclusion, epigenetic silencing of PEPT1 due to increased DNMT1 and HDAC1 expression plays a vital role in the poor response of CRC to ubenimex.

Colorectal cancer (CRC) is the third most commonly diagnosed cancer and the second leading cause of cancer-related deaths in the world. The downregulation of farnesoid X receptor (FXR) is frequently founded in CRC patients. The current study found that the decreased expression of FXR in colorectal cancer leads to disorders of bile acids (BAs) metabolism. The altered BAs profile shaped distinct intestinal flora and positively regulated secretory immunoglobulin A (sIgA). The dual regulation of BAs and sIgA enhanced adhesion and biofilm formation of enterotoxigenic Bacteroides fragilis (ETBF), which has a colorectal tumorigenesis effect. The abundance of ETBF increased significantly in intestinal mucosa of colitis-associated cancer (CAC) mice, and finally promoted the development of colorectal cancer. This study suggests that downregulation of FXR in CRC results in BAs dysregulation, and BAs have strong effects on sIgA and gut flora. The elevated BAs concentration and altered gut microbiome are risk factors for CRC.

An appropriate therapeutic index is crucial for drug discovery and development since narrow therapeutic index (NTI) drugs with slight dosage variation may induce severe adverse drug reactions or potential treatment failure. To date, the shared characteristics underlying the targets of NTI drugs have been explored by several studies, which have been applied to identify potential drug targets. However, the association between the drug therapeutic index and the related disease has not been dissected, which is important for revealing the NTI drug mechanism and optimizing drug design. Therefore, in this study, two classes of disease (cancers and cardiovascular disorders) with the largest number of NTI drugs were selected, and the target property of the corresponding NTI drugs was analyzed. By calculating the biological system profiles and human protein–protein interaction (PPI) network properties of drug targets and adopting an AI-based algorithm, differentiated features between two diseases were discovered to reveal the distinct underlying mechanisms of NTI drugs in different diseases. Consequently, ten shared features and four unique features were identified for both diseases to distinguish NTI from NNTI drug targets. These computational discoveries, as well as the newly found features, suggest that in the clinical study of avoiding narrow therapeutic index in those diseases, the ability of target to be a hub and the efficiency of target signaling in the human PPI network should be considered, and it could thus provide novel guidance in the drug discovery and clinical research process and help to estimate the drug safety of cancer and cardiovascular disease.

In Korea and China, ilaprazole is a widely used proton pump inhibitor in the treatment of gastric ulcers. In this study, a specific and sensitive LC-MS/MS method has been developed and validated for the quantification of ilaprazole enantiomers in the rat plasma, using R-lansoprazole as the internal standard. The enantioseparation was achieved on a CHIRALPAK AS-RH column (4.6 mm × 150 mm, i.d. 5 μm), with a mobile phase composed of 10 mM ammonium acetate aqueous solution and acetonitrile (60:40, V/V), at a flow-rate of 0.5 mL/min. The method was validated over the concentration range of 0.5–300 ng/mL for both, R- and S -ilaprazole. The lower limit of quantification was 0.5 ng/mL for both enantiomers. The relative standard deviation (RSD) of intra- and inter-day precision of R-ilaprazole and S-ilaprazole was less than 10.9%, and the relative error accuracy (RE) ranged from −0.5%–2.0%. Finally, the method was successfully evaluated in rats in a stereoselective pharmacokinetic study of the ilaprazole racemate.

Therapeutic monoclonal antibodies have become one of the central components of the healthcare system and continuous efforts are made to bring innovative antibody therapeutics to patients in need. It is equally critical to acquire sufficient knowledge of their molecular structure and biological functions to ensure the efficacy and safety by incorporating new detection approaches since new challenges like individual differences and resistance are presented. Conventional techniques for determining antibody disposition including plasma drug concentration measurements using LC-MS or ELISA, and tissue distribution using immunohistochemistry and immunofluorescence are now complemented with molecular imaging modalities like positron emission tomography and near-infrared fluorescence imaging to obtain more dynamic information, while methods for characterization of antibody’s interaction with the target antigen as well as visualization of its cellular and intercellular behavior are still under development. Recent progress in detecting therapeutic antibodies, in particular, the development of methods suitable for illustrating the molecular dynamics, is described here.

The recent pneumonia outbreak caused by a novel coronavirus (SARS-CoV-2) is posing a great threat to global public health. Therefore, rapid and accurate identification of pathogenic viruses plays a vital role in selecting appropriate treatments, saving people’s lives and preventing epidemics. It is important to establish a quick standard diagnostic test for the detection of the infectious disease (COVID-19) to prevent subsequent secondary spread. Polymerase chain reaction (PCR) is regarded as a gold standard test for the molecular diagnosis of viral and bacterial infections with high sensitivity and specificity. Isothermal nucleic acid amplification is considered to be a highly promising candidate method due to its fundamental advantage in quick procedure time at constant temperature without thermocycler operation. A variety of improved or new approaches also have been developed. This review summarizes the currently available detection methods for coronavirus nucleic acid. It is anticipated that this will assist researchers and clinicians in developing better techniques for timely and effective detection of coronavirus infection. Keywords: Coronavirus, Nucleic acid detection, PCR-Based methods, Isothermal nucleic acid amplification-based methods, Microarray-based methods

In recent years, the continuous occurrence of multi-drug resistance in the clinic has made people pay more attention to the transporter. Changes in the expression and activity of transporters can cause corresponding changes in drug pharmacokinetics and pharmacodynamics. The drug-drug interactions (DDI) caused by transporters can seriously affect drug effectiveness and toxicity. In the development of pharmaceutical preparations, people have increasingly concerned about the effects and regulation of transporters in drug effects. To improve the targeting and physicochemical properties of drugs, the development of targeted agents is very rapid. Among them, novel nano-formulations are the best. With the continuous innovation and development of nano-formulation, its application has become more and more extensive. Nano-formulation has exerted certain advantages in the drug development based on transporters, and is also involved in the combination of targeted transporters. This review focuses on the application of novel nano-agents targeting transporters and the introduction of drug-transporter-based nano-formulations. Keywords: Drug transporter, Nano-formulation, Drug delivery system, Bioavailability, P-gp

Renal cell carcinoma (RCC) is one of the most common malignant tumors affecting the urogenital system, accounting for 90% of renal malignancies. Traditional chemotherapy options are often the front-line choice of regimen in the treatment of patients with RCC, but responses may be modest or limited due to resistance of the tumor to anticarcinogen. Downregulated expression of organic cation transporter OCT2 is a possible mechanism underlying oxaliplatin resistance in RCC treatment. In this study, we observed that miR-489-3p and miR-630 suppress OCT2 expression by directly binding to the OCT2 3′-UTR. Meanwhile, via 786-O-OCT2-miRNAs stable expression cell models, we found that miRNAs could repress the classic substrate 1-methyl-4-phenylpyridinium (MPP+), fluorogenic substrate N,N-dimethyl-4-(2-pyridin-4-ylethenyl) aniline (ASP+), and oxaliplatin uptake by OCT2 both in vitro and in xenografts. In 33 clinical samples, miR-489-3p and miR-630 were significantly upregulated in RCC, negatively correlating with the OCT2 expression level compared to that in adjacent normal tissues, using tissue microarray analysis and qPCR validation. The increased binding of c-Myc to the promoter of pri-miR-630, responsible for the upregulation of miR-630 in RCC, was further evidenced by chromatin immunoprecipitation and dual-luciferase reporter assay. Overall, this study indicated that miR-489-3p and miR-630 function as oncotherapy-obstructing microRNAs by directly targeting OCT2 in RCC. KEY WORDS: OCT2, miRNA, Renal cell carcinoma, Epigenetic regulation, Oxaliplatin

Chiral drugs are very complex substances since individual enantiomers may differ in pharmacological and toxic effects, making it necessary to analyze enantiomers separately. In this study, we investigated the chiral differentiation of two ezetimibe enantiomers (i.e., SRS-EZM and RSR-EZM) and their mechanisms in complex with β-cyclodextrins (CDs) and metal ions as the auxiliary ligands. For this purpose, two complementary approaches have been employed: electrospray-tandem mass spectrometry (ESI-MS/MS) with collision induced dissociation (CID) and molecular modeling methods, including density functional theory (DFT) calculations and molecular dynamics (MD) simulations. The results showed a good agreement between experimental and theoretical data. It was demonstrated that SRS-EZM can be easily distinguished from RSR-EZM by applying CID in ESI-MS/MS. SRS-EZM is likely to form a more stable complex with β-CD and metal ions, and thus the [SRS-EZM]-Cu-[β-CD] cluster is more energetically difficult to separate from the SRS-EZM molecule compared with RSR-EZM. Such a difference may be attributed to the interactions between the drug molecule and the metal ion, as well as the cavity shape changes of the β-CDs upon complexation with molecular guests. Therefore, enantiomers in chiral drug can be recognized as ternary complexes of metal-analyte-β-CD by ESI-MS/MS with CID.

Renal cell carcinoma (RCC) is a common malignant tumour affecting the urinary system, and multidrug resistance is one of the major reasons why chemotherapy for this type of cancer often fails. Previous studies have shown that loss of the human organic cation transporter OCT2 is the main factor contributing to oxaliplatin resistance in RCC, and that DNA hypermethylation and histone methylation play important roles in the transcriptional repression of OCT2 in RCC. In this study, we found that histone acetylation also regulates OCT2 repression in RCC and elucidated the underlying mechanisms. In normal renal cells, HDAC7 combines with MYC at the OCT2 promoter, resulting in a decrease in free HDAC7, which in turn increases the levels of H3K18ac and H3K27ac at the OCT2 promotor and activates OCT2 expression. In RCC cells, however, the interaction between HDAC7 and MYC does not occur, which leads a high abundance of HDAC7 and low levels of H3K18ac and H3K27ac at the OCT2 promoter, thereby resulting in the inhibition of OCT2 transcription. We found that combined treatment using the DNA methylation inhibitor decitabine and the histone deacetylase inhibitor vorinostat significantly increased the expression of OCT2 in RCC cell lines, which sensitized these cells to oxaliplatin. We accordingly propose that the combination of anticancer agents and epigenetic drugs can provide a novel chemotherapeutic regimen.

MicroRNAs (miRNAs) are a family of endogenous, small (approximately 22 nucleotides in length), noncoding, functional RNAs. With the development of molecular biology, the research of miRNA biological function has attracted significant interest, as abnormal miRNA expression is identified to contribute to serious human diseases such as cancers. Traditional methods for miRNA detection do not meet current demands. In particular, nanomaterial-based methods, nucleic acid amplification-based methods such as rolling circle amplification (RCA), loop-mediated isothermal amplification (LAMP), strand-displacement amplification (SDA) and some enzyme-free amplifications have been employed widely for the highly sensitive detection of miRNA. MiRNA functional research and clinical diagnostics have been accelerated by these new techniques. Herein, we summarize and discuss the recent progress in the development of miRNA detection methods and new applications. This review will provide guidelines for the development of follow-up miRNA detection methods with high sensitivity and specificity, and applicability to disease diagnosis and therapy. Keywords: MicroRNAs, Detection method, Amplification strategies, Nanomaterial-based methods, Nucleic acid amplification-based methods

Luteolin is an important phytoestrogen with antidepressant effects found in many foods. We aimed to clarify the underlying antidepressant mechanisms of luteolin on PMAT associated with the monoamine hypothesis. We found rPmat had high expression in the limbic system, significant differences in mRNA expression between the pregnant and non-pregnant rats and an upward trend in mice with chronic unpredictable mild stress (CUMS). The results in the stably transfected cell models demonstrated that luteolin was the inhibitor of monoamine transporters. Further studies in C6 cells and primary hippocampal neurons revealed luteolin significantly downgraded PMAT mainly through the oestrogen β receptor (ERβ) and upgraded brain-derived neurotrophic factor (BDNF) expression. These results for PMAT have also been demonstrated in ovariectomized depressed rats. In conclusion, luteolin could directly and indirectly inhibit serotonin reuptake, resulting in an increase in monoamine neurotransmitters in the synaptic cleft. These findings manifest the antidepressant mechanisms of luteolin through PMAT.

Hypoxic microenvironment and metabolic dysregulation of tumor impairs the therapeutic efficacy of chemotherapeutic drugs, resulting in drug resistance and tumor metastasis, which has always been a challenge for the treatment of solid tumors, including renal cell carcinoma (RCC). Herein, starting from the evaluation of methionine metabolism in RCC cells, we demonstrated that the increased methionine accumulation in RCC cells was mediated by L-type amino acid transporter 1 (LAT1) under hypoxia. Glutathione (GSH), as a methionine metabolite, would attenuate the therapeutic efficacy of oxaliplatin through chemical chelation. Reducing methionine uptake by LAT1 inhibitor JPH203 significantly enhanced the sensitivity of RCC cells to oxaliplatin by reducing GSH production in vitro and in vivo. Therefore, we proposed an effective and stable therapeutic strategy based on the combination of oxaliplatin and LAT1 inhibitor, which is expected to solve the resistance of RCC to platinum-based drugs under hypoxia to a certain extent, providing a meaningful insight into the development of new therapeutic strategies and RCC treatment

Colorectal cancer (CRC) ranks third in incidence rate and second in mortality rate of malignancy worldwide, and the diagnosis and therapeutics of it remain to be further studied. With the emergence of noncoding RNAs (ncRNAs) and potential peptides derived from ncRNAs across various biological processes, we here aimed to identify a ncRNA-derived peptide possible for revealing the oncogenesis of CRC. Through combined predictive analysis of the coding potential of a batch of long noncoding RNAs (lncRNAs), the existence of an 85 amino-acid-peptide, named MEK1-binding oncopeptide (MBOP) and encoded from LINC01234 was confirmed. Mass spectrometry and Western blot assays indicated the overexpression of MBOP in CRC tissues and cell lines compared to adjacent noncancerous tissues and the normal colonic epithelial cell line. In vivo and in vitro migration and proliferation assays defined MBOP as an oncogenic peptide. Immunoprecipitation trials showed that MEK1 was the key interacting protein of MBOP, and MBOP promoted the MEK1/pERK/MMP2/MMP9 axis in CRC. Two E3-ligase enzymes MAEA and RMND5A mediated the ubiquitin–protease-system-related degradation of MBOP. This study indicates that MBOP might be a candidate prognostic indicator and a potential target for clinical therapy of CRC.

In the face of mounting global antibiotic resistance, which has become a critical clinical problem, antimicrobial peptides (AMPs) have received considerable interest as new therapeutics with the efficacy for the treatment of multidrug-resistant (MDR) infections due to their novel mechanism. However, certain inherent shortcomings such as instability seriously limit their systemic applications in the clinic. In this study, we intend to clarify the connection between three configurations of IK8 and their stability in plasma and liver S9 of various species by confirming the metabolites. The structural information of these metabolites was scanned and identified using HPLC and Q-TOF, respectively. The results found that IK8-D exhibits superior stability, compared with IK8-2D and IK8-L in plasma and liver S9 incubation, which indicated D- type amino acids could significantly increase the stability of antimicrobial peptides.

Background: Esophageal squamous cell carcinoma (ESCC) is a gastrointestinal malignancy with a poor prognosis. Although studies have shown metabolic reprogramming to be linked to ESCC development, no prognostic metabolic biomarkers or potential therapeutic metabolic targets have been identified.Method: The present study investigated some circulating metabolites associated with overall survival in 276 curatively resected ESCC patients using liquid chromatography/mass spectrometry metabolomics and Kaplan-Meier analysis. Tissue metabolomic analysis of 23-paired ESCC tissue samples was performed to discover metabolic dysregulation in ESCC cancerous tissue. A method consisting of support vector machine recursive feature elimination and LIMMA differential expression analysis was utilized to select promising feature genes within transcriptomic data from 179-paired ESCC tissue samples. Joint pathway analysis with genes and metabolites identified relevant metabolic pathways and targets for ESCC.Results: Four metabolites, kynurenine, 1-myristoyl-glycero-3-phosphocholine (LPC(14:0)sn-1), 2-piperidinone, and hippuric acid, were identified as prognostic factors in the preoperative plasma from ESCC patients. A risk score consisting of kynurenine and LPC(14:0)sn-1 significantly improved the prognostic performance of the tumor-node-metastasis staging system and was able to stratify risk for ESCC. Combined tissue metabolomic analysis and support vector machine recursive feature elimination gene selection revealed dysregulated kynurenine pathway as an important metabolic feature of ESCC, including accumulation of tryptophan, formylkynurenine, and kynurenine, as well as up-regulated indoleamine 2,3-dioxygenase 1 in ESCC cancerous tissue.Conclusions: This work identified for the first time four potential prognostic circulating metabolites. In addition, kynurenine pathway metabolism was shown to be up-regulated tryptophan-kynurenine metabolism in ESCC. Results not only provide a metabolite-based risk score system for prognosis, but also improve the understanding of the molecular basis of ESCC onset and progression, and as well as novel potential therapeutic targets for ESCC.

Renal cell carcinoma (RCC) is the most common type of kidney cancer. Increasingly evidences indicate that extracellular vesicles (EVs) orchestrate multiple processes in tumorigenesis, metastasis, immune evasion, and drug response of RCC. EVs are lipid membrane-bound vesicles in nanometer size and secreted by almost all cell types into the extracellular milieu. A myriad of bioactive molecules such as RNA, DNA, protein, and lipid are able to be delivered via EVs for the intercellular communication. Hence, the abundant content of EVs is appealing reservoir for biomarker identification through computational analysis and experimental validation. EVs with excellent biocompatibility and biodistribution are natural platforms that can be engineered to offer achievable drug delivery strategies for RCC therapies. Moreover, the multifaceted roles of EVs in RCC progression also provide substantial targets and facilitate EVs-based drug discovery, which will be accelerated by using artificial intelligence approaches. In this review, we summarized the vital roles of EVs in occurrence, metastasis, immune evasion, and drug resistance of RCC. Furthermore, we also recapitulated and prospected the EVs-based potential applications in RCC, including biomarker identification, drug vehicle development as well as drug target discovery.

Platinum-based anticancer drugs, including cisplatin, carboplatin, oxaliplatin, nedaplatin, and lobaplatin, are heavily applied in chemotherapy regimens. However, the intrinsic or acquired resistance severely limit the clinical application of platinum-based treatment. The underlying mechanisms are incredibly complicated. Multiple transporters participate in the active transport of platinum-based antitumor agents, and the altered expression level, localization, or activity may severely decrease the cellular platinum accumulation. Detoxification components, which are commonly increasing in resistant tumor cells, can efficiently bind to platinum agents and prevent the formation of platinum–DNA adducts, but the adducts production is the determinant step for the cytotoxicity of platinum-based antitumor agents. Even if adequate adducts have formed, tumor cells still manage to survive through increased DNA repair processes or elevated apoptosis threshold. In addition, autophagy has a profound influence on platinum resistance. This review summarizes the critical participators of platinum resistance mechanisms mentioned above and highlights the most potential therapeutic targets or predicted markers. With a deeper understanding of the underlying resistance mechanisms, new solutions would be produced to extend the clinical application of platinum-based antitumor agents largely.

Multidrug resistance-associated protein 4 (MRP4), a member of the adenosine triphosphate (ATP) binding cassette transporter family, pumps various molecules out of the cell and is involved in cell communication and drug distribution. Several studies have reported the role of miRNAs in downregulating the expression of MRP4. However, regulation of MRP4 by circular RNA (circRNA) is yet to be elucidated. In this study, MRP4 was significantly upregulated in hepatocellular carcinoma (HCC) tissues compared to the adjacent noncancerous tissues. Computational prediction, luciferase reporter assay and miRNA transfection were used to investigate the interaction between miRNAs and MRP4. miR-124-3p and miR-4524-5p reduced the expression of MRP4 at the protein but not mRNA level. Circular RNA in vivo precipitation and luciferase reporter assays demonstrated that circHIPK3, as a competitive endogenous RNA, binds with miR-124-3p and miR-4524-5p. Further, knockdown of circHIPK3 resulted in downregulation of MRP4 protein, whereas cotransfection of circHIPK3-siRNA and miR-124-3p or miR-4524-5p inhibitors restored its expression. In conclusion, we report that miR-4524-5p downregulates the expression of MRP4 and circHIPK3 regulates MRP4 expression by sponging miR-124-3p and miR-4524-5p for the first time. Our results may provide novel insights into the prevention of MRP4-related proliferation and multiple drug resistance in HCC.

The abundant miRNAs in urinary extracellular vesicles (EVs) represent ideal reservoirs for biomarker discovery, especially in renal cell carcinoma (RCC). However, the content and biological functions of microRNAs contained in urinary EVs in RCC remain ambiguous. In this study, urinary EVs were isolated and characterized from RCC patients and healthy volunteers. Differentially expressed microRNAs in urinary EVs were screened by small RNA sequencing. The target gene and biological functions of selected microRNAs were investigated through multifaceted methods. Results indicated that miR-224-5p was significantly upregulated in urinary EVs of RCC patients compared to healthy volunteers. The overexpression of miR-224-5p inhibited RCC cell proliferation and induced cell cycle arrest. The gene CCND1 encoding cyclin D1 was identified as a direct target of miR-224-5p via prediction and validation. Moreover, the invasive and metastatic abilities of RCC cells were enhanced by miR-224-5p. Interestingly, miR-224-5p also increased the stability of PD-L1 protein by inhibiting CCND1. This effect could be transmitted via EVs and further promoted the resistance of RCC cells to T cell-dependent toxicity. In summary, urinary EVs containing miR-224-5p were identified as a potential biomarker in RCC. Regulation of PD-L1 protein expression by miR-224-5p through suppressing CCND1 elucidates new roles of miR-224-5p in RCC progression.

Stereoselectivity in drug metabolism can not only influence the pharmacological activities, tolerability, safety, and bioavailability of drugs directly, but also cause different kinds of drug–drug interactions. Thus, assessing stereoselectivity in drug metabolism is of great significance for pharmaceutical research and development (R&D) and rational use in clinic. Although there are various methods available for assessing stereoselectivity in drug metabolism, many of them have shortcomings. The indirect method of chromatographic methods can only be applicable to specific samples with functional groups to be derivatized or form complex with a chiral selector, while the direct method achieved by chiral stationary phases (CSPs) is expensive. As a detector of chromatographic methods, mass spectrometry (MS) is highly sensitive and specific, whereas the matrix interference is still a challenge to overcome. In addition, the use of nuclear magnetic resonance (NMR) and immunoassay in chiral analysis are worth noting. This review presents several typical examples of drug stereoselective metabolism and provides a literature-based evaluation on current chiral analytical techniques to show the significance and challenges of stereoselectivity assessing methods in drug metabolism.

The antioxidant components of Coreopsis tinctoria have not been clearly identified; thus, here eleven constituents were isolated and identified from the capitula of Coreopsis tinctoria, in which taxifolin (7), 3,5-di-O-caffeoylquinic acid (9) and 4,5-di-O-caffeoylquinic acid (10) were identified for the first time. All eleven compounds possessed antioxidant activities based on DPPH assay and cellular antioxidant activity assay. Especially, okanin (11) and isookanin (6) exhibited excellent DPPH scavenging activities with the EC50 of 6.2 and 10.6 µM, respectively, which were stronger than those of ascorbic acid and butylated hydroxytoluene (positive controls) with the EC50 of 30.4 and 45.8 µM, respectively. Okanin and isookanin showed comparable cellular antioxidant activities to quercetin with the IC50 of 11.0 and 14.4 µM, respectively. Additionally, the two most potent antioxidants, okanin and isookanin, were detectable and reached maximal concentration of 10.5 and 10.6 µM in plasma at 20 min after oral administration of 100 mg/kg ethanol extract of Coreopsis tinctoria capitula. In conclusion, our results indicate that Coreopsis tinctoria capitula might be developed as antioxidant sources.

Stellera chamaejasme L. (Thymelaeaceae) is widely distributed in Mongolia, Tibet and the northern parts of China. Its roots are commonly used as “Langdu”, which is embodied in the Pharmacopoeia of the P.R. China (2010) as a toxic Traditional Chinese Medicine. It is claimed to have antivirus, antitumor and antibacterial properties in China and other Asian countries. Studies were carried out to characterize the inhibition of neochamaejasmin B (NCB) on P-glycoprotein (P-gp, ABCB1, MDR1). Rhodamine-123 (R-123) transport and accumulation studies were performed in MDCK-hMDR1 cells. ABCB1 (MDR1) mRNA gene expression and P-gp protein expression were analyzed. Binding selectivity studies based on molecular docking were explored. R-123 transport and accumulation studies in MDCK-hMDR1 cells indicated that NCB inhibited the P-gp-mediated efflux in a concentration-dependent manner. RT-PCR and Western blot demonstrated that the P-gp expression was suppressed by NCB. To investigate the inhibition type of NCB on P-gp, Ki and Ki’ values were determined by double-reciprocal plots in R-123 accumulation studies. Since Ki was greater than Ki’, the inhibition of NCB on P-gp was likely a mixed type of competitive and non-competitive inhibition. The results were confirmed by molecular docking in our current work. The docking data indicated that NCB had higher affinity to P-gp than to Lig1 ((S)-5,7-dihydroxy-2-(4-hydroxyphenyl)chroman-4-one).

RNA interference (RNAi) is useful for selective gene silencing. Cytochrome P450 3A4 (CYP3A4), which metabolizes approximately 50% of drugs in clinical use, plays an important role in drug metabolism. In this study, we aimed to develop a short hairpin RNA (shRNA) to modulate CYP3A4 expression. Three new shRNAs (S1, S2 and S3) were designed to target the coding sequence (CDS) of CYP3A4, cloned into a shRNA expression vector, and tested in different cells. The mixture of three shRNAs produced optimal reduction (55%) in CYP3A4 CDS-luciferase activity in both CHL and HEK293 cells. Endogenous CYP3A4 expression in HepG2 cells was decreased about 50% at both mRNA and protein level after transfection of the mixture of three shRNAs. In contrast, CYP3A5 gene expression was not altered by the shRNAs, supporting the selectivity of CYP3A4 shRNAs. In addition, HepG2 cells transfected with CYP3A4 shRNAs were less sensitive to Ginkgolic acids, whose toxic metabolites are produced by CYP3A4. These results demonstrate that vector-based shRNAs could modulate CYP3A4 expression in cells through their actions on CYP3A4 CDS, and CYP3A4 shRNAs may be utilized to define the role of CYP3A4 in drug metabolism and toxicity.

Lithocholic acid (LCA) deposited in human livers always induces drastic pains which need analgesic drug, like morphine to release. Our research showed that LCA can effectively inhibit uridine 5'-diphospho-glucuronosyltransferase 2B7 (UGT2B7) in morphine tolerance-like human normal liver cells, HL-7702, then increase μ-opioid receptor (MOR) and calcium-calmodulin dependent protein kinase IIα (CaMKIIα) expression. In vivo assay, UGT2B7 was significantly repressed in the livers of acute or chronic morphine tolerance mice pretreated with LCA (10, 50 and 100 mg/kg, p.o.). To investigate the connections between LCA function performance and changes of UGT2B7 enzymatic activity in mice livers, two morphine metabolites, morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G) were quantified by solid phase extraction (SPE)-HPLC-MS/MS. The result indicated no matter in acute or chronic morphine tolerance, the concentrations of M3G and M6G were all decreased, the later one fell even more. Besides that, 50mg/kg of LCA administration can prevent auto-phosphorylation of CaMKIIα at Thr286 in acute or chronic morphine tolerance mice prefrontal cortexes (mPFCs) due to synthesis increase of cyclic adenosine monophosphate (cAMP). As a consequence, UGT2B7 depression mediated by LCA can affect its selective catalysis ability to morphine, that may be responsible to acute or chronic morphine tolerance alleviation. These findings might assist to modify antinociception of morphine in clinic.

Isoliensinine, a natural phenolic bisbenzyltetrahydroisoquinoline alkaloid, has received considerable attention for its potential biological effects such as antioxidant and anti-HIV activities. From the dog hepatic microsomes of isoliensinine, three new N-demethylated and O-demethylated metabolites, 2-N-desmethyl-isoliensinine (M1), 2'-N-desmethylisoliensinine (M2), and 2'-N-6-O-didesmethylisoliensinine (M3), were identified by high-performance liquid chromatography and data-dependent electrospray ionization tandem mass spectrometry. Possible metabolic pathways for isoliensinine have been proposed. The result should prove very helpful for evaluation of the drug-like properties of isoliensinine and other bisbenzylisoquinoline alkaloids.

The stereoselective hydrolysis of esmolol in whole blood and in its separated components from rat, rabbit and human was investigated. Blood esterase activities were variable in different species in the order of rat>rabbit>human. Rat plasma showed the high esterase activity and had no stereoselectivity to enantiomers. Rabbit red blood cell (RBC) membrane, RBC cytosol and plasma all hydrolyzed esmolol but with different esterase activity, whereas the hydrolysis in RBC membrane and cytosol showed significant stereoselectivity towards R-(+)-esmolol. Esterase in RBC cytosol from human blood mainly contributed to the esmolol hydrolysis, which was demonstrated with no stereoselctivity. Esterase in human plasma showed a low activity, but a remarkable stereoselectivity with R-(+)-esmolol. In addition, the protein concentration affected the hydrolysis behavior of esmolol in RBC suspension. Protein binding of esmolol enantiomers in human plasma, human serum albumin (HSA) and α1-acid glycoprotein (AGP) revealed that there was a significant difference in bound fractions between two enantiomers, especially for AGP. Our results indicated that the stereoselective protein binding might play a role in the different hydrolysis rates of esmolol enantiomers in human plasma. Keywords: Esmolol enantiomers, Species-dependent, Stereoselective hydrolysis, Protein binding

This study aimed to determine the transepithelial transport characteristics of chiral drug enantiomers across Caco-2 cell monolayers, a model of human intestinal epithelial membrane. Six chiral aryloxy enantiomers (atenolol, sotalol, celiprolol, carvedilol, metoprolol and propafenone) were tested in bi-directional transport studies. The separation and quantitation of these enantiomers were performed by reversed-phase high-performance liquid chromatography (RP-HPLC) using 2, 3, 4, 6-tetra-O-acetyl-β-d-glucopyranosyl isothiocyanate (GITC) as a pre-column derivatizing agent. Bi-directional transport studies demonstrated that celiprolol and carvedilol exhibited significant enantioselectivity in polarized transport at the concentration range tested. The efflux ratio (ER) for (R)-(+)-celiprolol was 8.96, but it was much lower for (S)-(-)-celiprolol which is 3.42 at the concentration of 96.0 μM; carvedilol had the same transport behavior as celiprolol while the difference between the ER values of two enantiomers was not as significant as celiprolol at the concentration of 5.0 μM. They are 2.41 for (R)-(+)-carvedilol and 1.98 for (S)-(-)-carvedilol. But in the transport studies of racemic atenolol, sotalol, metoprolol and propafenone, no enantioselective transport were observed over the concentration range tested. Because P-glycoprotein (P-gp) is highly expressed in Caco-2 cells, we inferred that P-gp might participate in the transport processes of celiprolol and carvedilol in chirally discriminative ways.

The protein binding of non-steroidal anti-inflammatory drugs flurbiprofen, ketoprofen and etodolac with human serum albumin (HSA) was investigated using indirect chiral high performance liquid chromatography (HPLC) and ultrafiltration techniques. S-(â)-1-(1-naphthyl)-ethylamine (S-NEA) was utilized as chiral derivatization reagent and pre-column derivatization RP-HPLC method was established for the separation and assay of the three pairs of enantiomer. The method had good linear relationship over the investigated concentration range without interference. The average extraction efficiency was higher than 85% in different systems, and the intra-day and inter-day precisions were less than 15%. In serum albumin, the protein binding of etodolac enantiomers showed significant stereoselectivity that the affinity of S-enantiomer was stronger than R-enantiomer, and the stereoselectivity ratio reached 6.06; Flurbiprofen had only weak stereoselectivity in HSA, and ketoprofen had no stereoselectivity at all. Scatchard curves showed that all the three chiral drugs had two types of binding sites in HSA. Keywords: Protein binding, Non-steroidal anti-inflammatory drugs, Enantiomer, Stereoselectivity, Human serum albumin

The tuber of Corydalis yanhusuo is a famous traditional Chinese medicine and found to have potent pharmacological effects, such as antinociceptive, antitumor, antibacterial, anti-inflammatory, and anti-depressive activities. Although there are several methods to be developed for the analysis and detection of the bioactive ingredients’ alkaloids, so far, only few prominent alkaloids could be quantified, and in vitro and in vivo changes of comprehensive alkaloids after oral administration are still little known. In this study, we first developed a simple and sensitive high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) method to quantify the comprehensive alkaloids of extracts of C. yanhusuo in mouse plasma, using nitidine chloride as an internal standard. As results, at least fourteen alkaloids, including an aporphine (oxoglaucine), a protopine (protopine), five tertiary alkaloids (corydaline, tetrahydroberberine, tetrahydropalmatine, tetrahydrocolumbamine, and tetrahydrocoptisine) and seven quaternary alkaloids (columbamine, palmatine, berberine, epiberberine, coptisine, jatrorrhizine, and dehydrocorydaline) could be well quantified simultaneously in mouse plasma. The lower limits of quantification were greater than, or equal to, 0.67 ng/mL, and the average matrix effects ranged from 96.4% to 114.3%. The mean extraction recoveries of quality control samples were over 71.40%, and the precision and accuracy were within the acceptable limits. All the analytes were shown to be stable under different storage conditions. Then the established method was successfully applied to investigate the pharmacokinetics of these alkaloids after oral administration of the extract of Corydalis yanhusuo in mice. To the best of our knowledge, this is the first document to report the comprehensive and simultaneous analyses of alkaloids of C. yanhusuo in mouse plasma. It was efficient and useful for comprehensive pharmacokinetic and metabolomic analyses of these complex alkaloids after drug administration.

BYZX, [(E)-2-(4-((diethylamino)methyl)benzylidene)-5,6-dimethoxy-2,3-dihydroinden-one], belongs to a series of novel acetylcholinesterase inhibitors and has been synthesized as a new chemical entity for the treatment of Alzheimer's disease symptoms. When incubated with human liver microsomes (HLMs), BYZX was rapidly transformed into its metabolites M1, M2, and M3. The chemical structures of these metabolites were identified using liquid chromatography tandem mass spectrometry and nuclear magnetic resonance, which indicated that M1 was an N-desethylated and C = C hydrogenation metabolite of BYZX. M2 and M3 were 2 precursor metabolites, which resulted from the hydrogenation and desethylation of BYZX, respectively. Further studies with chemical inhibitors and human recombinant cytochrome P450s (CYPs), and correlation studies were performed. The results indicated that the N-desethylation of BYZX and M2 was mediated by CYP3A4 and CYP2C8. The reduced form of β-nicotinamide adenine dinucleotide 2'-phosphate was involved in the hydrogenation of BYZX and M3, and this reaction occurred in the HLMs and in the human liver cytosol. The hydrogenation reaction was not inhibited by any chemical inhibitors of CYPs, but it was significantly inhibited by some substrates of α,β-ketoalkene C = C reductases and their inhibitors such as benzylideneacetone, dicoumarol, and indomethacin. Our results suggest that α,β-ketoalkene C = C reductases may play a role in the hydrogenation reaction, but this issue requires further clarification.

Cytochrome P450 oxidoreductase (POR) is known as the sole electron donor in the metabolism of drugs by cytochrome P450 (CYP) enzymes in human. However, little is known about the effect of polymorphic variants of POR on drug metabolic activities of CYP3A4 and CYP2B6. In order to better understand the mechanism of the activity of CYPs affected by polymorphic variants of POR, six full-length mutants of POR (e.g., Y181D, A287P, K49N, A115V, S244C and G413S) were designed and then co-expressed with CYP3A4 and CYP2B6 in the baculovirus-Sf9 insect cells to determine their kinetic parameters. Surprisingly, both mutants, Y181D and A287P in POR completely inhibited the CYP3A4 activity with testosterone, while the catalytic activity of CYP2B6 with bupropion was reduced to approximately ~70% of wild-type activity by Y181D and A287P mutations. In addition, the mutant K49N of POR increased the CLint (Vmax/Km) of CYP3A4 up to more than 31% of wild-type, while it reduced the catalytic efficiency of CYP2B6 to 74% of wild-type. Moreover, CLint values of CYP3A4-POR (A115V, G413S) were increased up to 36% and 65% of wild-type respectively. However, there were no appreciable effects observed by the remaining two mutants of POR (i.e., A115V and G413S) on activities of CYP2B6. In conclusion, the extent to which the catalytic activities of CYP were altered did not only depend on the specific POR mutations but also on the isoforms of different CYP redox partners. Thereby, we proposed that the POR-mutant patients should be carefully monitored for the activity of CYP3A4 and CYP2B6 on the prescribed medication.

Cytochomosome P450 enzymes (CYP) are heme-containing monooxygenases responsible for oxidative metabolism of many exogenous and endogenous compounds including drugs. The species difference of CYP limits the extent to which data obtained from animals can be translated to humans in pharmacodynamics or pharmacokinetics studies. Transgenic expression of human CYP in animals lacking or with largely reduced endogenous CYP counterparts is recognized as an ideal strategy to correct CYP species difference. CYP3A is the most abundant CYP subfamily both in human and mammals. In this study, we designed a microRNA-based shRNA (miR-shRNA) simultaneously targeting four members of mouse CYP3A subfamily (CYP3A11, CYP3A16, CYP3A41 and CYP3A44), and transgenic mice expressing the designed miR-shRNA were generated by lentiviral transgenesis. Results showed that the CYP3A expression level in transgenic mice was markedly reduced compared to that in wild type or unrelated miR-shRNA transgenic mice, and was inversely correlated to the miR-shRNA expression level. The CYP3A expression levels in transgenic offspring of different generations were also remarkably lower compared to those of controls, and moreover the inhibition rate of CYP3A expression remained comparable over generations. The ratio of the targeted CYP3A transcriptional levels was comparable between knockdown and control mice of the same gender as detected by RT-PCR DGGE analysis. These data suggested that transgenic miR-shRNA suppressed CYP3A expression in a dose-dependent and inheritable manner, and transcriptional levels of the targeted CYP3As were suppressed to a similar extent. The observed knockdown efficacy was further confirmed by enzymatic activity analysis, and data showed that CYP3A activities in transgenic mice were markedly reduced compared to those in wild-type or unrelated miR-shRNA transgenic controls (1.11±0.71 vs 5.85±1.74, 5.9±2.4; P