Inhibitory effect of flavonoids on multidrug and toxin extrusion protein 1 function: Implications for food/herb-drug interaction and drug-induced kidney injury.

Multidrug and toxin extrusion protein 1 (MATE1), an efflux transporter mainly expressed in renal proximal tubules, mediates the renal secretion of organic cationic drugs. The inhibition of MATE1 will impair the excretion of drugs into the tubular lumen, leading to the accumulation of nephrotoxic drugs in the kidney and consequently potentiating nephrotoxicity. Screening and identifying potent MATE1 inhibitors can predict or minimize the risk of drug-induced kidney injury. Flavonoids, a group of polyphenols commonly found in foodstuffs and herbal products, have been reported to cause transporter-mediated food/herb-drug interactions. Our objective was to investigate the inhibitory effects of flavonoids on MATE1 in vitro and in vivo and to assess the effects of flavonoids on cisplatin-induced kidney injury. Thirteen flavonoids exhibited significant transport activity inhibition (>50%) on MATE1 in MATE1-MDCK cells. Among them, the six strongest flavonoid inhibitors, including irisflorentin, silymarin, isosilybin, sinensetin, tangeretin, and nobiletin, markedly increased cisplatin cytotoxicity in these cells. In cisplatin-induced in vivo renal injury models, irisflorentin, isosilybin, and sinensetin also increased serum creatinine and blood urea nitrogen levels to different degrees, especially irisflorentin, which exhibited the most potent nephrotoxicity with cisplatin. The pharmacophore model indicated that the hydrogen bond acceptors at the 3, 5, and 7 positions may play a critical role in the inhibitory effect of flavonoids on MATE1. Our findings provide helpful information for predicting the potential risks of flavonoid-containing food/herb-drug interactions and avoiding the exacerbation of drug-induced kidney injury via MATE1 mediation.

Inhibition of hepatocellular carcinoma cell proliferation through regulation of the Cell Cycle, AGE-RAGE, and Leptin signaling pathways by a compound formulation comprised of andrographolide, wogonin, and oroxylin A derived from Andrographis Paniculata(Burm.f.) Nees.

ETHNOPHARMACOLOGICAL RELEVANCE: In 2020, liver cancer contributed to approximately 0.9 million new cases and 0.83 million deaths, making it the third leading cause of mortality worldwide. Andrographis paniculata (Burm.f.) Nees(APN), a traditional Chinese or ethnic medicine extensively utilized in Asia, has been historically employed for treating hepatitis and liver cancer. However, the precise molecular mechanism responsible for its therapeutic efficacy remains unclear. AIM OF THE STUDY: To identify and replace the active components of APN on liver cancer, which is investigate the potential of a Multi-Component Chinese Medicine derived from Andrographis paniculata (Burm.f.) Nees(APN-MCCN) for the treatment of liver cancer. MATERIALS AND METHODS: Firstly, the TCMSP database and two liver cancer disease databases were utilized to optimize the chemical constituents of APN and the disease-related targets of liver cancer. The network was constructed using Cytoscape to visualize the relationships between them. Subsequently, the optimal combination of components in APN-MCCN for the treatment of liver cancer was determined using the contribution index method. HPLC analysis was performed to measure the content of each component. Pathway enrichment and gene annotation were conducted using the ClueGo plugin. In vivo efficacy was evaluated by transplanting S180 and H22 tumor-bearing mouse models. In vitro efficacy was determined through MTT assay, morphological observations, flow cytometry analysis, and scratch tests. Western blotting was used to validate the protein expression. The transfection techniques were employed to knockdown the expressions of key protein in different pathway. RESULTS: We obtained 24 effective compounds, with andrographolide contributing 20.78%, wogonin contributing 41.85%, and oroxylin A contributing 30.26% to the overall composition. Based on the predicted enrichment degree and correlation with liver cancer, we identified a total of 27 pathways, among which the Leptin signaling pathway, AGE-RAGE signaling pathway, and Cell Cycle signaling pathway were selected for further investigation. The content of andrographolide, oroxylin A, and wogonin in APN was found to be 0.104%, 0.0024%, and 0.0052%, respectively. In vivo experiments demonstrated that APN-MCCM significantly reduced tumor weight in S180 tumor-bearing mice and prolonged the survival time of H22 liver cancer-bearing mice. APN-MCCM exhibited inhibitory effects on the proliferation, apoptosis, and migration of liver cancer cells while arresting them in the G2/M phase. Furthermore, APN-MCCM down-regulated the protein expression of NCOA1, PTPN1, and GSK3B in the Leptin signaling pathway, NOS2 and NOS3 in the AGE-RAGE signaling pathway, CCNA2, CDK1, CDK2, and CDK7 in the Cell Cycle signaling pathway. Additionally, it upregulated the protein phosphorylation of p-P38 and p-JUN in the AGE-RAGE signaling pathway. Knockout experiments revealed that the inhibitory effect of APN-MCCM on liver cancer cell migration was prevented when the MAPK or NCOA1 genes were knocked out. Similarly, knocking out the CDK7 gene blocked the G2/M phase arrest induced by APN-MCCM in liver cancer cells. CONCLUSIONS: APN-MCCM, consisting of andrographolide, wogonin, and oroxylin A, exhibits inhibitory effects on the cell proliferation of liver cancer cells by targeting the cell cycle pathway. Additionally, it suppresses the migration of liver cancer cells through the AGE-RAGE and Leptin signaling pathways.

Inhibitory interaction of flavonoids with organic anion transporter 3 and their structure-activity relationships for predicting nephroprotective effects.

The organic anion transporter 3 (OAT3), an important renal uptake transporter, is associated with drug-induced acute kidney injury (AKI). Screening and identifying potent OAT3 inhibitors with little toxicity in natural products, especially flavonoids, in reducing OAT3-mediated AKI is of great value. The five strongest OAT3 inhibitors from the 97 flavonoids markedly decreased aristolochic acid I-induced cytotoxicity and alleviated methotrexate-induced nephrotoxicity. The pharmacophore model clarified hydrogen bond acceptors and hydrophobic groups are the critical pharmacophores. These findings would provide valuable information in predicting the potential risks of flavonoid-containing food/herb-drug interactions and optimizing flavonoid structure to alleviate OAT3-related AKI.

Oridonin suppresses gastric cancer SGC-7901 cell proliferation by targeting the TNF-alpha/androgen receptor/TGF-beta signalling pathway axis.

Statistics provided by GLOBOCAN list gastric cancer as the sixth most common, with a mortality ranking of third highest for the year 2020. In China, a herb called Rabdosia rubescens (Hemsl.) H.Hara, has been used by local residents for the treatment of digestive tract cancer for hundreds of years. Oridonin, the main ingredient of the herb, has a curative effect for gastric cancer, but the mechanism has not been previously clarified. This study mainly aimed to investigate the role of TNF-alpha/Androgen receptor/TGF-beta signalling pathway axis in mediating the proliferation inhibition of oridonin on gastric cancer SGC-7901 cells. MTT assay, cell morphology observation assay and fluorescence assay were adopted to study the efficacy of oridonin on cell proliferation. The network pharmacology was used to predict the pathway axis regulated by oridonin. Western blot assay was adopted to verify the TNF-α/Androgen receptor/TGF-ß signalling pathway axis regulation on gastric cancer by oridonin. The results showed Oridonin could inhibit the proliferation of gastric cancer cells, change cell morphology and cause cell nuclear fragmentation. A total of 11signaling pathways were annotated by the network pharmacology, among them, Tumour necrosis factor alpha (TNF-α) signalling pathway, androgen receptor (AR) signalling pathway and transforming growth factor (TGF-ß) signalling pathway account for the largest proportion. Oridonin can regulate the protein expression of the three signalling pathways, which is consistent with the results predicted by network pharmacology. These findings indicated that oridonin can inhibit the proliferation of gastric cancer SGC-7901 cells by regulating the TNF-α /AR /TGF-ß signalling pathway axis.

Inhibitory effects of flavonoids on glucose transporter 1 (GLUT1): From library screening to biological evaluation to structure-activity relationship.

Glucose transporter 1 (GLUT1) is mainly responsible for glucose uptake and energy metabolism, especially in the aerobic glycolysis process of tumor cells, which is closely associated with the advancement of tumors. Numerous studies have demonstrated that the inhibition of GLUT1 can decrease the growth of tumor cells and enhance drug sensitivity, so GLUT1 is considered to be a promising therapeutic target for cancer treatment. Flavonoids are a group of phenolic secondary metabolites present in vegetables, fruits, and herbal products, some of which were reported to increase cancer cells' sensitivity to sorafenib by inhibiting GLUT1. Our objective was to screen potential inhibitors of GLUT1 from 98 flavonoids and assess the sensitizing effect of sorafenib on cancer cells. and illuminate the structure-activity relationships of flavonoids with GLUT1. Eight flavonoids, including apigenin, kaempferol, eupatilin, luteolin, hispidulin, isosinensetin, sinensetin, and nobiletin exhibited significant inhibition (>50%) on GLUT1 in GLUT1-HEK293T cells. Among them, sinensetin and nobiletin showed stronger sensitizing effects and caused a sharp downward shift of the cell viability curves in HepG2 cells, illustrating these two flavonoids might become sensitizers to enhance the efficacy of sorafenib by inhibiting GLUT1. Molecular docking analysis elucidated inhibitory effect of flavonoids on GLUT1 was related to conventional hydrogen bonds, but not Pi interactions. The pharmacophore model clarified the critical pharmacophores of flavonoids inhibitors are hydrophobic groups in 3'positions and hydrogen bond acceptors. Thus, our findings would provide useful information for optimizing flavonoid structure to design novel GLUT1 inhibitors and overcome drug resistance in cancer treatment.

Simultaneous determination of NTB-3119, a novel anti-tuberculosis agent, and its major metabolites in mouse plasma by LC-MS/MS and its application in preclinical pharmacokinetics study.

NTB-3119, a novel benzothiopyranone derivative, has been developed as a potential anti-tuberculosis(TB) drug with strong activity. In this study, three major metabolites of NTB-3119 were firstly identified in vitro and in vivo. A sensitive and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was established for the quantitative analysis of NTB-3119 and its major metabolites NTB-3190, NTB-3202 and NTB-3204 in mouse plasma. The plasma samples were processed by protein precipitation with organic solvent. NTB-3119, NTB-3190, NTB-3202, NTB-3204 and NTB-4A (Internal Standard, IS) were separated by a Zorbax-SB C18 column (100 mm × 2.1 mm, 3.5 µm) with a gradient mobile phase of acetonitrile/water at a flow rate of 0.25 mL/min. The analytes were detected by electrospray positive ion mode in Parallel Reaction Monitoring (PRM) mode on a high resolution mass spectrum (HRMS, Thermo Q Executive). The monitored transitions were m/z 456.15632 → 360.06137 for NTB-3119, m/z 426.18214 → 246.01891 for NTB-3190, m/z 472.15124 → 360.06143 for NTB-3202, m/z 442.17706 → 246.01903 for NTB-3204 and m/z 337.13691 → 163.02081 for NTB-4A, respectively. Good linearity was conducted in the range of 5-2000 ng/mL for NTB-3119, NTB-3202 and NTB-3204 as well as 2.5-1000 ng/mL for NTB-3190. The inter- and intra-batch precision (RSD%) were both lower than 13.3 %, with the accuracy ranged from 88.0 % to 108.1 %. The analytes were proved to be stable during all samples storage, preparation and analytic procedures. The validated method was successfully applied to study the pharmacokinetics and bioavailability of NTB-3119 after oral treatment in Balb/c mouse.

Quantification of 2-NBDG, a probe for glucose uptake, in GLUT1 overexpression in HEK293T cells by LC-MS/MS.

The growth and proliferation of most cancer cells involve the excessive uptake of glucose mediated by glucose transporters. An effective strategy for cancer therapy has been to inhibit the GLUTs that are usually overexpressed in a variety of tumor cells. 2-NBDG is a GLUT1 substrate that can be used as a probe for GLUT1 inhibitors. An accurate and simple assay for 2-NBDG in a HEK293T cell model overexpressing GLUT1 was developed using liquid chromatography-tandem mass spectrometry. Chromatographic separation was achieved using a Xbridge® Amide column (3.5 µm, 2.1 mm × 150 mm, Waters) with acetonitrile-water containing 2 µM ammonium acetate (80:20, v/v) at a flow rate of 0.25 mL/min. Mass detection was conducted in the parallel reaction monitoring (PRM) mode. The calibration curve for 2-NBDG showed good linearity in the concentration range of 5-500 ng/mL with satisfactory precision, a relative standard deviation ranging from 2.92 to 9.59% and accuracy with a relative error ranging from -13.14 to 7.34%. This method was successfully applied to quantify the uptake of GLUT1-mediated 2-NBDG, and the results clearly indicated inhibition of GLUT1 by WZB117 and quercetin (two potent glucose transporter inhibitors) in the GLUT1-HEK293T cell model. This study provides a convenient and accurate method for high-throughput screening of selective and promising GLUT1 inhibitors.

Computational analysis for identification of early diagnostic biomarkers and prognostic biomarkers of liver cancer based on GEO and TCGA databases and studies on pathways and biological functions affecting the survival time of liver cancer.

BACKGROUND: Liver cancer is the sixth most commonly diagnosed cancer and the fourth most common cause of cancer death. The purpose of this work is to find new diagnostic biomarkers or prognostic biomarkers and explore the biological functions related to the prognosis of liver cancer. METHODS: GSE25097 datasets were firstly obtained and compared with TCGA LICA datasets and an analysis of the overlapping differentially expressed genes (DEGs) was conducted. Cytoscape was used to screen out the Hub Genes among the DEGs. ROC curve analysis was used to screen the Hub Genes to determine the genes that could be used as diagnostic biomarkers. Kaplan-Meier analysis and Cox proportional hazards model screened genes associated with prognosis biomarkers, and further Gene Set Enrichment Analysis was performed on the prognosis genes to explore the mechanism affecting the survival and prognosis of liver cancer patients. RESULTS: 790 DEGs and 2162 DEGs were obtained respectively from the GSE25097 and TCGA LIHC data sets, and 102 Common DEGs were identified by overlapping the two DEGs. Further screening identified 22 Hub Genes from 102 Common DEGs. ROC and survival curves were used to analyze these 22 Hub Genes and it was found that there were 16 genes with a value of AUC > 90%. Among these, the expression levels of ESR1,SPP1 and FOSB genes were closely related to the survival time of liver cancer patients. Three common pathways of ESR1, FOBS and SPP1 genes were identified along with seven common pathways of ESR1 and SPP1 genes and four common pathways of ESR1 and FOSB genes. CONCLUSIONS: SPP1, AURKA, NUSAP1, TOP2A, UBE2C, AFP, GMNN, PTTG1, RRM2, SPARCL1, CXCL12, FOS, DCN, SOCS3, FOSB and PCK1 can be used as diagnostic biomarkers for liver cancer, among which FOBS and SPP1 genes can also be used as prognostic biomarkers. Activation of the cell cycle-related pathway, pancreas beta cells pathway, and the estrogen signaling pathway, while on the other hand inhibition of the hallmark heme metabolism pathway, hallmark coagulation pathway, and the fat metabolism pathway may promote prognosis in liver cancer patients.

The prognostic value of preoperative inflammatory indexes in gallbladder carcinoma with hepatic involvement.

BACKGROUND: Neutrophil-Lymphocyte Ratio (NLR) and Platelet-Lymphocyte Ratio (PLR) have been considered as indicators for prognosis in various cancers. However, the prognostic values of NLR and PLR have never been tested in gallbladder carcinoma (GBC) with hepatic involvement. OBJECTIVE: The aim of the current study was to assess the prognostic significance of NLR, PLR, and other candidate biomarkers in GBC with liver involvement. METHODS: Receiver operating characteristic (ROC) curve analyses were utilized to pinpoint the cut-off values for NLR, PLR, and Monocyte-Lymphocyte Ratio (MLR). Univariate analyses were employed to estimate the impact of NLR, PLR, MLR, and other inflammatory indexes on median survival. Multivariate analyses were used to verify the independent prognostic predictors. RESULTS: Eighty four patients were enrolled from 2009 to 2017. The cut-off values for NLR, PLR, and MLR were 3.20, 117.75, and 0.25, respectively. Univariate analyses revealed that TNM stage, NLR, PLR, MLR, lactate dehydrogenase, alkaline phosphatase, and carcinoembryonic antigen were significantly associated with decreased survival in GBC with hepatic involvement. Advanced TNM stage (P< 0.001) and elevated preoperative NLR (P= 0.002) were significantly associated with lower median survival periods, as revealed by multivariate analyses. CONCLUSIONS: These findings suggest that preoperative NLR may be an independent prognostic factor in evaluating prognosis in GBC with liver involvement.

The effect of α-solanine on the activity, gene expression, and kinetics of arylamine N-acetyltransferase in HepG2 cells.

α-solanine is one of the major components of Solanum nigrum Linn., the fruits of which are used in China for food. In the present study, α-solanine was selected to assess the inhibition of arylamine N-acetyltransferase (NAT) activity and mRNA expression of NAT and kinetics in HepG2 cells. NAT activity was examined by HPLC. The double-reciprocal plot was contrived to yield a regression equation to calculate Km and Vmax. NAT mRNA expression was determined by PCR. The results revealed that α-solanine could significantly decrease NAT activity in intact HepG2 cells or the cytoplasm. Km did not differ either for intact HepG2 cells or for the cytoplasm, however Vmax was significantly different. α-solanine could decrease the expression of NAT1 mRNA and NAT2 mRNA. In summary, α-solanine was a non-competitive inhibitor of NAT in HepG2 cells. It decreased NAT activity through non-competitive inhibition of NAT activity and by decreasing the expression of NAT1 mRNA and NAT2 mRNA.

R-Phycoerythrin Induces SGC-7901 Apoptosis by Arresting Cell Cycle at S Phase.

R-Phycoerythrin (R-PE), one of the chemical constituents of red algae, could produce singlet oxygen upon excitation with the appropriate radiation and possibly be used in photodynamic therapy (PDT) for cancer. Documents reported that R-PE could inhibit cell proliferation in HepG2 and A549 cells, which was significative for cancer therapy. This is due to the fact that R-PE could kill cancer cells directly as well as by PDT. However, little is known about the cytotoxicity of R-PE to the SGC-7901 cell. In this study, it has been found that R-PE could inhibit SGC-7901 proliferation and induce cell apoptosis, which was achieved by arresting the SGC-7901 cell at S phase. CyclinA, CDK2 and CDC25A are proteins associated with the S phase, and it was found that R-PE could increase the expression of cyclin A protein and decrease the expression of CDK2 and CDC25A proteins. Thus, it was concluded that R-PE reduced the CDK2 protein activated through decreasing the CDC25A factor, which reduced the formation of Cyclin-CDK complex. The reduction of Cyclin-CDK complex made the SGC-7901 cells arrest at the S phase. Therefore, R-PE induced apoptosis by arresting the SGC-7901 cell at S phase was successful, which was achieved by the expression of the CDC25A protein, which reduced the CDK2 protein actived and the formation of Cyclin-CDK complex.

Oridonin induces apoptosis through the mitochondrial pathway in human gastric cancer SGC-7901 cells.

Oridonin is one of the most important antitumor active ingredients of Rabdosia rubescens. Recently published studies from our laboratory have demonstrated that oridonin was able to arrest human gastric cancer SGC-7901 cells at G2/M phase. However, little is known about inducing apoptosis in gastric cancer. The aim of this study was to investigate the effect of oridonin on antineoplastic capability of SGC-7901 cells and the detailed molecular mechanism of oridonin-mediated intrinsic pathway of apoptosis. Cell proliferation was assessed by MTT assay while apoptosis induced by oridonin was determined by Hoechst 33342 staining assay and Annexin V/PI double staining assay. Early apoptotic rate was stained by Annexin V/PI and detected by flow cytometry. Apoptosis pathway was analyzed by western blot analysis of Bcl-2, Bax, cytochrome c and caspase-3 expression. The results showed that oridonin was able to inhibit the SGC-7901 cell proliferation, the 50% growth inhibition (IC50) was 22.74 µM. Oridonin could induce cell apoptosis of SGC-7901 cells and the early apoptotic rates induced by 0, 20, 40, 80 µmol/l oridonin were 1.53±0.67, 3.33±0.29, 84.80±0.82 and 96.43±0.51%, respectively. Western blot analysis revealed that oridonin downregulated Bcl-2 protein (the anti-apoptotic factor) and upregulated Bax protein (pro-apoptotic factor), eventually leading to a reduction in the ratio of Bcl-2/Bax proteins. Furthermore, oridonin induced the release of cytochrome c from the mitochondria to the cytosol and the activation of caspase-3. Taken together, the current study suggested that oridonin induced apoptosis in SGC-7901 cells via the mitochondrial signal pathway, which may represent one of the major mechanisms of oridonin-mediated apoptosis in SGC-7901 cells.