FDX1 as a novel biomarker and treatment target for stomach adenocarcinoma.

BACKGROUND: Stomach adenocarcinoma (STAD) is one of the main reasons for cancer-related deaths worldwide. This investigation aimed to define the connection between STAD and Cuproptosis-related genes (CRGs). Cuproptosis is a newly identified form of mitochondrial cell death triggered by copper. AIM: To explore the identification of potential biomarkers for STAD disease based on cuproptosis. METHODS: A predictive model using Gene Ontology (GO), Least Absolute Shrinkage and Selection Operator (LASSO), Kyoto Encyclopedia of Genes and Genomes (KEGG), Gene Set Variation Analysis (GSVA), and Gene Set Enrichment Analysis analyzed gene interconnections, focusing on 3 copper-related genes and their expression in The Cancer Genome Atlas-STAD. Networks for mRNA-miRNA and mRNA-transcription factor interactions were constructed. The prognostic significance of CRG scores was evaluated using time-receiver operating characteristic, Kaplan-Meier curves, and COX regression analysis. Validation was conducted with datasets GSE26942, GSE54129, and GSE66229. Expression of copper-related differentially expressed genes was also analyzed in various human tissues and gastric cancer subpopulations using the human protein atlas. RESULTS: Three significant genes (FDX1, LIAS, MTF1) were identified and selected via LASSO analysis to predict and classify individuals with STAD into high and low CRG score subgroups. These genes were down-regulated in both risk categories. GO and KEGG analyses highlighted their involvement mainly in the electron transport chain. After validating their differential expression, FDX1 emerged as the most accurate diagnostic marker for gastric cancer. Additionally, the RCircos package localized FDX1 on chromosome 11. CONCLUSION: Our study revealed that FDX1 could be a potential biomarker and treatment target for gastric malignancy, providing new ideas for further scientific research.

Hydroxysafflor yellow A inhibits the hyperactivation of rat platelets by regulating the miR-9a-5p/SRC axis.

Pathological platelet activation plays a vital role in the prevalence of cardiovascular diseases. Hydroxysafflor yellow A (HSYA) has been shown to have significant anti-platelet aggregation and anti-activation effects, but its mechanism of action is unclear. Our study showed that HSYA inhibited the expression of platelet surface glycoproteins IIß/III α (GPIIß/III α) and thromboxane A2 (TXA2) during platelet activation and reduced platelet Ca2+ accumulation. HSYA significantly reduced the number of platelets and inhibited adrenaline-induced platelet hyperaggregation in rats. Transcriptomic analysis of platelets suggested that HSYA significantly suppressed SRC and MAPK3 (ERK1/2) gene expression. YEEI peptide, an SRC activator, could significantly reverse the inhibition of HSYA on the phosphorylation of SRC/PLCγ2/PKCδ/MEK/ERK1/2 pathway proteins and reverse the effect of HSYA on platelet activation-related markers GPIIß/IIIα protein, TXA2 and cAMP. The SRC genes were further predicted by transcriptome analysis of HSYA-regulated miRNAs combined with bioinformatics techniques. The results suggested that HSYA could significantly upregulate the expression level of the miR-9a-5p gene and further confirmed that miR-9a-5p had a targeted regulatory relationship with SRC by dual-luciferase activity reporter and cell transfection experiments. The inhibitory effect of HSYA on the SRC/PLCγ2/PKCδ/MEK/ERK1/2 pathway was significantly reversed after platelets were transfected with the miR-9a inhibitor, while SRC siRNA attenuated the effect of the miR-9a inhibitor. SRC siRNA was able to attenuate the effect of the miR-9a inhibitor. In conclusion, this study suggests that HSYA can inhibit the activation of the SRC/PLCγ2/PKC δ/MEK/ERK1/2 axis by upregulating platelet miR-9a-5p, thereby reducing the activation of platelets and inhibiting platelet aggregation.

Efficacy of L-Carnitine for Dilated Cardiomyopathy: A Meta-Analysis of Randomized Controlled Trials.

BACKGROUND: L-carnitine mediates the utilization of fatty acids and glucose in the myocardium. The potential of L-carnitine in managing dilated cardiomyopathy (DCM) in patients has been extensively reported, with additional benefits. OBJECTIVE: This meta-analysis purposed to explore the clinical efficacy of L-carnitine therapy on DCM patients. METHODS: We searched publications up to May 2020 from several databases including PubMed, Embase, Cochrane Library, Chinese Biomedical (CBM) database, Chinese Science and Technology Periodicals database (VIP), Chinese National Knowledge Infrastructure (CNKI) database, and Wanfang database. Subsequently, publications that met the inclusion criteria were systematically evaluated by two independent reviewers. RESULTS: A total of 23 RCTs conducted in China with 1455 DCM patients were included in this study. In the meta-analysis, L-carnitine therapy was associated with a considerable improvement in the overall efficacy (RR = 1.28, 95% CI (1.21-1.36), P < 0.0001), left ventricular ejection fraction (LVEF) (MD = 6.16%, 95% CI (4.50, 7.83), P < 0.0001), and cardiac output (CO) (MD = 0.88 L/min, 95% CI (0.51, 1.25), P < 0.0001) as compared to the control group. Moreover, L-carnitine therapy significantly decreased left ventricular end-diastolic dimension (LVEDD) (MD = -2.53, 95% CI (-3.95, -1.12), P = 0.0005), brain natriuretic peptide (BNP) (SMD = -1.71 ng/L, 95% CI (-3.02, -0.40), P = 0.01), and the transforming growth factor-beta (TGF-ß1) (MD = -56.78 ng/L, 95% CI (-66.02, -47.53), P < 0.0001). CONCLUSIONS: L-carnitine potentially enhanced the therapeutic efficiency in DCM patients. Following weaknesses in the evidence due to low methodological quality and high clinical heterogeneity in the included studies, well-designed trials are recommended.

Hypofibrinogenemia induced by high-dose tigecycline-case report and review of literature.

RATIONALE: Extensive off-label use may affect the safety profile of tigecycline. Tigecycline-associated hypofibrinogenemia is potentially life threatening, although the frequency of life-threatening reactions is unknown and their incidence is easily overlooked. We report a case of 2 instances of treatment with high-dose tigecycline, each of which presented with hypofibrinogenemia. PATIENT CONCERNS: An 86-year-old male patient was treated twice with high-dose tigecycline and presented with hypofibrinogenemia both times. The decrease in fibrinogen occurred within 3 to 7 days of tigecycline treatment. Other coagulation parameters had slightly prolonged values. DIAGNOSES: Coagulopathy and hypofibrinogenemia. INTERVENTIONS: We discontinued the tigecycline. OUTCOMES: The fibrinogen level normalized within 5 days after the withdrawal of tigecycline. Following 80 days of hospitalization, the patient was transferred to the rehabilitation hospital for further treatment. LESSONS: We suggest routine strict monitoring of coagulation parameters, particularly fibrinogen. Attention should be paid to below-normal fibrinogen levels due to increased bleeding risk and severity of reaction at fibrinogen levels below 1 g/L.

Substrate Transport Properties of the Human Peptide/Histidine Transporter PHT2 in Transfected MDCK Cells.

PHT2, a member of the proton-coupled oligopeptide transporter family, participates in the transportation of small peptides and histidine from lysosomes to the cytosol. It facilitates maintenance of intracellular peptide homeostasis. However, it remains a challenge to elucidate the functional properties of PHT2 due to its localization in the lysosomal membrane. The aim of this study was to explore the transport function and substrate properties of human PHT2 (hPHT2) by transfecting Madin-Darby canine kidney cells with hPHT2 mutants to obtain stably expressed protein in the cell membrane. Using this cell model, we found that the transport activity of hPHT2 reached a maximum capacity when the extracellular pH was 5.5. hPHT2 showed relatively low affinity for Gly-Sar and relatively high affinity for d3-L-histidine, with Km values of 428 ± 88 µM and 66.9 ± 5.7 µM, respectively. Several typical substrates or inhibitors of PEPT1 and PEPT2, including valacyclovir, Gly-Gly-Gly, and cefadroxil but not 5-aminolevulinic acid or captopril, were proven to be substrates of hPHT2. However, hPHT2 showed low affinity for valacyclovir with a Km value of 5350 ± 1234 µM. In conclusion, this study established a suitable and efficient cell model to explore the function of hPHT2 in vitro and provided important information on the transport activity and substrate properties of hPHT2.

Expression and regulation of proton-coupled oligopeptide transporters in colonic tissue and immune cells of mice.

A number of studies have implicated proton-coupled oligopeptide transporters (POTs) in the initiation and/or progression of inflammatory bowel disease and immune cell signaling. With this in mind, the aim of this study was to delineate the expression of POTs in mouse colonic tissues and immune cells, and characterize the potential role of these transporters in nucleotide-binding oligomerization domain (NOD) signaling. Using a dextran sodium sulfate (DSS)-induced colitis mouse model, we found that DSS down regulated Pht1 gene expression and up regulated Pht2 gene expression in colonic tissue and immune cells. In contrast, PEPT1 protein was absent from the colonic tissue and immune cells of normal and DSS-treated mice. NOD ligands, muramyl dipeptide (MDP) and l-Ala-γ-d-Glu-meso-diaminopimelic acid (tri-DAP), were shown to be substrates of PHT2 in MDCK-hPHT219,20AA cells. Subsequent studies revealed that the immune response of lamina propia mononuclear cells may be regulated by PHT1 and PHT2, and that PHT2 facilitated the NOD-dependent immune response in RAW264.7 macrophages. These results clarified the expression of POTs in mouse colonic segments, cells and subtypes, and the role of increased Pht2 expression during chemically-induced colitis in facilitating NOD-dependent immune response. The findings further suggest that intestinal PHT2 may serve as a therapeutic target for IBD therapy.

Multiple organic cation transporters contribute to the renal transport of sulpiride.

Sulpiride, a selective dopamine D2 receptor blocker, is used widely for the treatment of schizophrenia, depression and gastric/duodenal ulcers. Because the great majority of sulpiride is positively charged at physiological pH 7.4, and ~70% of the dose recovered in urine is in the unchanged form after human intravenous administration of sulpiride, it is believed that transporters play an important role in the renal excretion of sulpiride. The aim of the present study was to explore which transporters contribute to the renal disposition of sulpiride. The results demonstrated that sulpiride was a substrate of human carnitine/organic cation transporter 1 (hOCTN1) and 2 (hOCTN2), human organic cation transporter 2 (hOCT2), human multidrug and toxin efflux extrusion protein 1 (hMATE1) and 2-K (hMATE2-K). Sulpiride accumulation from the basolateral (BL) to the apical (AP) side in MDCK-hOCT2/pcDNA3.1 cell monolayers was much greater than that in MDCK-hOCT2/hMATE1 cells, and cimetidine dramatically reduced the intracellular accumulation of sulpiride from BL to AP. In addition, the accumulation of sulpiride in mouse primary renal tubular cells (mPRTCs) was markedly reduced by inhibitors of Oct2 and Octns. The results implied that OCTN1, OCTN2, OCT2, MATE1 and MATE2-K probably contributed to the renal transfer of sulpiride, in which OCT2 mediated the uptake of sulpiride from the bloodstream to the proximal tubular cells, while MATEs contributed to the sulpiride efflux from the proximal tubular cells to the renal lumen, and OCTNs participated in both renal secretion and reabsorption.

Multiple drug transporters mediate the placental transport of sulpiride.

Sulpiride is a typical antipsychotic drug for the treatment of schizophrenia, depression and other psychological disorders. It has been proven that a small amount of sulpiride could cross the human placenta using an ex vivo placental perfusion model. However, the placental transfer mechanism has not been elucidated. Considering the structure of sulpiride, we speculated that the transporters expressed in placenta might be involved in sulpiride uptake across the blood-placenta barrier. The aim of our study was to determine which transporters contributed to the placental transfer of sulpiride. Our results revealed that sulpiride was a substrate of human organic cation transporter (hOCT) 3, human multidrug resistance protein (hMDR) 1 and human breast cancer resistance protein (hBCRP) using transfected cells expressing respective transporters. In addition, the accumulation of sulpiride in BeWo cells (a human choriocarcinoma cell line) was obviously affected by inhibitors of carnitine/organic cation transporter (OCTN) 2, MDR1 and BCRP. The accumulation of sulpiride in primary human trophoblast cells was obviously affected by inhibitors of OCT3, OCTN1 and OCTN2. The above results indicate that hOCTN1 and hOCTN2 likely contribute to the sulpiride uptake from maternal circulation to trophoblast cells, while hMDR1 and hBCRP mediate the efflux from trophoblast cells to maternal circulation, and hOCT3 probably is involved in the bidirectional transport of sulpiride between the placenta and fetal blood.

Multiple Drug Transporters Are Involved in Renal Secretion of Entecavir.

Entecavir (ETV) is a first-line antiviral agent for the treatment of chronic hepatitis B virus infection. Renal excretion is the major elimination path of ETV, in which tubular secretion plays the key role. However, the secretion mechanism has not been clarified. We speculated that renal transporters mediated the secretion of ETV. Therefore, the aim of our study was to elucidate which transporters contribute to the renal disposition of ETV. Our results revealed that ETV (50 µM) remarkably reduced the accumulation of probe substrates in MDCK cells stably expressing human multidrug and toxin efflux extrusion proteins (hMATE1/2-K), organic cation transporter 2 (hOCT2), and carnitine/organic cation transporters (hOCTNs) and increased the substrate accumulation in cells transfected with multidrug resistance-associated protein 2 (hMRP2) or multidrug resistance protein 1 (hMDR1). Moreover, ETV was proved to be a substrate of the above-described transporters. In transwell studies, the transport of ETV in MDCK-hOCT2-hMATE1 showed a distinct directionality from BL (hOCT2) to AP (hMATE1), and the cellular accumulation of ETV in cells expressing hMATE1 was dramatically lower than that of the mock-treated cells. The accumulation of ETV in mouse primary renal tubular cells was obviously affected by inhibitors of organic anion transporter 1/3 (Oat1/3), Oct2, Octn1/2, and Mrp2. Therefore, the renal uptake of ETV is likely mediated by OAT1/3 and OCT2 while the efflux is mediated by MATEs, MDR1, and MRP2, and OCTN1/2 may participate in both renal secretion and reabsorption.

Interaction of six protoberberine alkaloids with human organic cation transporters 1, 2 and 3.

1. Organic cation transporters (OCTs) play an important role in drug safety and efficacy. Protoberberine alkaloids are ubiquitous organic cations or weak bases with remarkable biological actives. This study was to elucidate the potential interaction of alkaloids (coptisine, jatrorrhizine, epiberberine, berberrubine, palmatine and corydaline) with OCTs using Madin-Darby canine kidney (MDCK) cells stably expressing human OCT1, OCT2 and OCT3. 2. All the tested alkaloids significantly inhibited the uptake of MPP(+), a model OCT substrate, in MDCK-hOCTs cells with the IC50 of 0.931-9.65 µM. Additionally, coptisine, jatrorrhizine and epiberberine were substrates of all the hOCTs with the Km of 0.273-5.80 µM, whereas berberrubine was a substrate for hOCT1 and hOCT2, but not for hOCT3, the Km values were 1.27 and 1.66 µM, respectively. The transport capacity of coptisine in MDCK cells expressing the variants of hOCT1-P341L or hOCT2-A270S was significantly higher than that in wild-type (WT) cells with the Clint (Vmax/Km) of 379 ± 7.4 and 433 ± 5.7 µl/mg protein/min, respectively. 3. The above data indicate that the tested alkaloids are potent inhibitors, and coptisine, jatrorrhizine, epiberberine and berberrubine are substrates of hOCT1, hOCT2 and/or hOCT3 with high affinity. In addition, the variants (OCT1-P341L and OCT2-A270S) possess higher transport capacity to coptisine than WT hOCTs.

[Establishment and application of cell models with stable expression of hOCTN1/2].

Human carnitine/organic cation transporter 1 and 2（hOCTN1 and hOCTN2） mediate transport of endogenous and exogenous compounds. The present study aimed to establish cell models with stable expression of hOCTN1 or hOCTN2 to study interactions with compounds and transporters. MDCK cells were transfected with pcDNA3.1 (+) plasmid vector containing hOCTN1 or hOCTN2（pcDNA3.1(+)-hOCTN1/2), several stable transfected clones were obtained after G418 screening. hOCTN1 and hOCTN2 clones were screened with ergothioneine and mildronate respectively as substrates to identify the best candidates. We explored interactions of endogenous substances, alkaloids, flavonoids and ACEIs with hOCTN1/2. As a result, the cellular accumulation of ergothioneine in MDCK-hOCTN1 or mildronate in MDCK-hOCTN2 was 122 and 108 folds of the control cells, respectively. The kinetic parameters, K(m) and V(max) of ergothioneine, mediated by MDCK-hOCTN1, were 8.19 ± 0.61 µmol·L-1 and 1 427 ± 49 pmol·mg(-1)(protein)·min(-1); while K(m) and V(max) of mildronate by MDCK- hOCTN2 were 52.3 ± 4.3 µmol·L(-1) and 2 454 ± 64 pmol·mg(-1)(protein)·min(-1). Dopamine, glutamine, piperine, berberine, nuciferine, lisinopril and fosinopril could inhibit ergothioneine or mildronate uptake by MDCK- hOCTN1/2. In conclusion, cell models with good stable hOCTN1 and hOCTN2 functions have been established successfully, which can be applied to the study of interactions between compounds and transporters of hOCTN1 and hOCTN2.