OBJECTIVE: To explore the genetic etiology in four patients with hyperbilirubinemia, and discuss the correlation between clinical characteristics and molecular basis. METHODS: The data of clinical manifestation and auxiliary examinations were collected. Genomic DNA of the four patients was extracted and analyzed by next-generation sequencing using the panel including genes involved in hereditary metabolic liver diseases. Suspected variants were verified by Sanger sequencing. RESULTS: All of the four patients were males with normal liver enzymes. It was revealed that all the patients had heterozygous variants, among which c.3011C>T, c.2443C>T and c.2556del were the variants which have not been reported previously. CONCLUSION All of the patients were diagnosed as Dubin-Johnson syndrome (DJS) caused by ABCC2 gene variants. The novel variants add to the spectrum of genetic variants of the disease. Because of the favorite prognosis, precise diagnosis can greatly reduce the psychological pressure of patients and avoid excessive treatments. At the same time, it could provide pertinent genetic counseling for the families.
DNA ; Female ; Heterozygote ; Humans ; Jaundice, Chronic Idiopathic/genetics* ; Male ; Multidrug Resistance-Associated Protein 2 ; Multidrug Resistance-Associated Proteins/genetics* ; Phenotype

OBJECTIVE: To explore the genetic etiology and differential diagnosis for a patient with jaundice. METHODS: Clinical data of the patient and his parents were collected. Genes associated with metabolic liver diseases were subjected to high-throughput sequencing. The pathogenicity of the candidate variants was predicted by using bioinformatics software. RESULTS: High-throughput sequencing revealed that the proband has harbored two variants of the ABCC2 gene (NM_000392) including c.3011C>T (p.T1004I) and c.3541C>T (p.R1181X), which were respectively inherited from his father and mother. Both variants have been previously unreported and predicted to be pathogenic by bioinformatics analysis. CONCLUSION The proband was diagnosed with Dubin-Johnson syndrome due to the compound heterozygous variants of the ABCC2 gene. Genetic testing has enabled accurate differential diagnosis of Dubin-Johnson syndrome in this patient.
Genetic Testing ; High-Throughput Nucleotide Sequencing ; Humans ; Jaundice, Chronic Idiopathic/pathology* ; Multidrug Resistance-Associated Protein 2 ; Multidrug Resistance-Associated Proteins/genetics* ; Mutation

OBJECTIVE: To investigate evodiamine (EVO)-induced hepatotoxicity and the underlying mechanism. METHODS: HepG2 cells were treated with EVO (0.04-25 μmol/L) for different time intervals, and the cell survival rate was examined by cell counting kit-8 (CCK-8) method. After HepG2 cells were treated with EVO (0.2, 1 and 5 μmol/L) for 48 h, the alanine transaminase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), alkaline phosphatase (ALP) activities and total bilirubin (TBIL) content of supernatant were detected. A multifunctional microplate reader was used to detect the intracellular superoxide dismutase (SOD) activity and malondialdehyde (MDA) content in HepG2 cells to evaluate the level of cell lipid peroxidation damage. The interactions between EVO and apoptosis, autophagy or ferroptosis-associated proteins were simulated by molecular docking. The HepG2 cells were stained by mitochondrial membrane potential (MMP) fluorescent probe (JC-10) and annexin V-fluorescein isothiocyanate/propidium iodide (Annexin V-FITC/PI), and MMP and apoptosis in HepG2 cells were detected by flow cytometry. The protein expression levels of caspase-9, caspase-3, bile salt export pump (BSEP) and multidrug resistance-associated protein 2 (MRP2) were detected by Western blot. RESULTS: The cell survival rate was significantly reduced after the HepG2 cells were exposed to EVO (0.04-25 μmol/L) in a time- and dose-dependent manner. The half maximal inhibitory concentration (IC50) of the HepG2 cells treated with EVO for 24, 48 and 72 h were 85.3, 6.6 and 4.7 μmol/L, respectively. After exposure to EVO (0.2, 1 and 5 μmol/L) for 48 h, the ALT, AST, LDH, ALP activities and TBIL content in the HepG2 cell culture supernatant, and the MDA content in the cells were increased, and SOD enzyme activity was decreased. Molecular docking results showed that EVO interacted with apoptosis-associated proteins (caspase-9 and caspase-3) better. JC-10 and Annexin V-FITC/PI staining assays demonstrated that EVO could decrease MMP and promote apoptosis in the HepG2 cells. Western blot results indicated that the protein expressions of cleaved caspase-9 and cleaved caspase-3 were upregulated in the HepG2 cell treated with EVO for 48 h. In contrast, the protein expressions of pro-caspase-3, BSEP and MRP2 were downregulated. CONCLUSION These results suggested that 0.2, 1 and 5 μmol/L EVO had the potential hepatotoxicity, and the possible mechanism involved lipid peroxidation damage, cell apoptosis, and cholestasis.
ATP Binding Cassette Transporter, Subfamily B, Member 11 ; Apoptosis ; Caspase 3 ; Caspase 9 ; Cholestasis ; Hep G2 Cells/drug effects* ; Humans ; Lipid Peroxidation ; Liver/drug effects* ; Molecular Docking Simulation ; Multidrug Resistance-Associated Protein 2 ; Quinazolines/toxicity*

BACKGROUND AND OBJECTIVEMultidrug resistance (MDR) is a major obstacle in the chemotherapy of cancer patients. The aim of this study was to establish a mutidrug-resistant cell line SK-Hep1/DDP and explore its molecular mechanism of the MDR.

METHODSSK-Hep1/DDP cell line was induced by pulse treatment using a high concentration of cisplatin (DDP) in vitro. The chemoresistance indexes of cells were evaluated by CCK-8 assays. The protein of MDR1 (ABCB1), MRP1 (ABCC1), MRP2 (ABCC2) and Bax were detected by Western blotting, and the effect of MDR1 inhibitor cyclosporine A (CsA) on expression of MDR1 proteins in SK-Hep1 and SK-Hep1/DDP cell lines. Flow cytometry was performed to determine the distribution of the cell cycle and cell apoptosis ratio.

RESULTSThe SK-Hep1/DDP cells were 13.76 times more resistant to DDP in comparison with SK-Hep1 cells, and SK-Hep1/DDP cells also exhibited cross-resistance to many other chemotherapeutic agents (adramycin and 5-fuorouracil). MDR1, MRP1, and MRP2 protein expressions were significantly higher in the SK-Hep1/DDP than in the SK-Hep1 (P < 0. 01), but Bax was lower in the SK-Hep1/DDP than in the SK-Hep1(P < 0. 01). There was no obvious influence between SK-Hep1 and SK-Hep1/DDP cells in the expression of MDR1 by MDR1 inhibtor CsA (P > 0. 05). The percentages of cells in G(2)/M and S phase were significantly increased in SK-Hep1/DDP in comparison with those in SK-Hep1 [(20.67 +/- 5.69)% vs. (12.14 +/- 3.36)%; (42.20 +/- 2.65)% vs. (27.91 +/- 2.16)%; P < 0. 01]. After the cells were exposed to 10 μg/mL DDP for 24 h, the cell apoptosis rate of SK-Hep1/DDP was decreased in comparison with SK-Hep1, but it was increased in those with pretreatment of MDR1 inhibitor CsA as compared with those without pretreatment.

CONCLUSIONSA reliable multi-drug resistant human hepatoma cell line SK-Hep1/DDP is successfully established. The MDR mechanisms of this cell lines are closely related to the over-expression of MDR1 MRP1 and MRP2, lower expression of Bax and the attenuated cell apoptosis induced by chemotherapeutic agents.

ATP-Binding Cassette, Sub-Family B, Member 1 ; antagonists & inhibitors ; metabolism ; Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cisplatin ; pharmacology ; Cyclosporine ; pharmacology ; Doxorubicin ; pharmacology ; Drug Resistance, Multiple ; Drug Resistance, Neoplasm ; Fluorouracil ; pharmacology ; Humans ; Liver Neoplasms ; metabolism ; pathology ; Multidrug Resistance-Associated Proteins ; metabolism ; Vincristine ; pharmacology ; bcl-2-Associated X Protein ; metabolism

The aim of this study was to investigate the mechanism to reverse the drug resistance of leukemia cells in tetrandrine (Tet) alone or in combination with droloxifen (Drol) by using protein chips and to lay the theoretical basis for the clinical applications. Three monoclonal antibodies against P-glycoprotein (P-gp), the multidrug resistance-associated protein (MRP1) and the breast cancer resistance protein (BCRP) were immobilized onto the agarose gel film-coated glass slides. Protein chips were prepared respectively from K562/A02 cells cultured for 12, 24 and 48 hours with Tet alone or in combination with Drol. The results showed that Tet alone or in combination with Drol could decrease only the expression of P-gp in a time-dependent manner, the effect for 48 hours as follows: Tet + Drol 82.620 +/- 3.227; Tet alone 86.440 +/- 2.906; Drol alone 87.230 +/- 2.049; control 93.670 +/- 2.748 (P < 0.05). However, down-regulation of P-gp by K562/A02 cells cultured with Tet alone or in combination with Drol began at 24 hours (Tet + Drol 85.270 +/- 3.095; control 93.670 +/- 2.748, P < 0.05). The results were coincident with that of FCM. It is concluded that Tet and Drol can downregulate the expression of P-gp in the time-dependent way. There is a significant difference between Tet alone and Tet combined with Drol at 24 hours (P < 0.05). The expression of MRP1 and BCRP are not closely correlated with the reversal mechanism of Tet and Drol, and which may be involved in the mechanism of this combination to reverse multidrug resistance in leukemia.
ATP Binding Cassette Transporter, Sub-Family G, Member 2 ; ATP-Binding Cassette Transporters ; biosynthesis ; ATP-Binding Cassette, Sub-Family B, Member 1 ; biosynthesis ; Antineoplastic Agents ; pharmacology ; Benzylisoquinolines ; pharmacology ; Drug Resistance, Multiple ; Drug Resistance, Neoplasm ; drug effects ; Drug Synergism ; Humans ; K562 Cells ; Leukemia ; metabolism ; pathology ; Multidrug Resistance-Associated Proteins ; biosynthesis ; Neoplasm Proteins ; biosynthesis ; Protein Array Analysis ; Tamoxifen ; analogs & derivatives ; pharmacology

OBJECTIVETo evaluate the use of protein array chips in detection of multidrug-resistance proteins.

METHODSHuman erythroleukemic cell line K562 and its doxorubicin-resistant counterpart K562/A02 were used in the study. Monoclonal antibodies against P-glycoprotein (P-gP), multidrug resistance-associated protein (MRP1) and breast cancer resistance protein (BCRP) were immobilized onto agarose film-coated glass. The antibody-cell binding was assessed by capturing K562 and K562/A02 cells. The protein array was observed under a microscope and the image was captured with a CCD camera. The expression levels of the three proteins were also measured by flow cytometry (FCM).

RESULTSThe expression of P-gP and BCRP in K562 was very low. However, MRP1 expression was high. P-gP and MRP1 were highly expressed in K562/A02, while the expression of BCRP was low. FCM results showed that the expression rate of P-gP, MRP1 and BCRP in K562 cells was 5.98% +/- 2.19%, 95.80% +/- 3.98%, 1.03% +/- 0.45%, respectively, while that in K562/A02 cells was 92.67% +/- 1.80%, 97.18% +/- 1.02%, 3.98% +/- 0.37%, respectively. The results of protein array method are consistent with those of FCM (P > 0.05).

CONCLUSIONIt is feasible to develop a new protein array technique and to provide a novel method for multi-drug resistant cell detection, with a high throughput, high specificity, simple procedure and low cost.

ATP Binding Cassette Transporter, Sub-Family G, Member 2 ; ATP-Binding Cassette Transporters ; analysis ; ATP-Binding Cassette, Sub-Family B, Member 1 ; analysis ; Drug Resistance, Multiple ; genetics ; Drug Resistance, Neoplasm ; genetics ; Humans ; K562 Cells ; Multidrug Resistance-Associated Proteins ; analysis ; Neoplasm Proteins ; analysis ; Protein Array Analysis

Multidrug resistance (MDR) in cancer cells can significantly attenuate the response to chemotherapy and increase the likelihood of mortality. The major mechanism involved in conferring MDR is the overexpression of ATP-binding cassette (ABC) transporters, which can increase efflux of drugs from cancer cells, thereby decreasing intracellular drug concentration. Modulators of ABC transporters have the potential to augment the efficacy of anticancer drugs. This editorial highlights some major findings related to ABC transporters and current strategies to overcome MDR.
ATP Binding Cassette Transporter, Sub-Family G, Member 2 ; ATP-Binding Cassette Transporters ; antagonists & inhibitors ; metabolism ; ATP-Binding Cassette, Sub-Family B, Member 1 ; antagonists & inhibitors ; metabolism ; Antineoplastic Agents ; therapeutic use ; Drug Resistance, Multiple ; Drug Resistance, Neoplasm ; Humans ; Molecular Targeted Therapy ; Multidrug Resistance-Associated Proteins ; antagonists & inhibitors ; metabolism ; Nanomedicine ; Neoplasm Proteins ; antagonists & inhibitors ; metabolism ; Neoplasms ; drug therapy ; metabolism ; Protein-Tyrosine Kinases ; antagonists & inhibitors

OBJECTIVETo study the expression of miRNA-106a gene in esophageal squamous cell carcinoma (ESCC) and its association with clinicopathological features and prognosis of ESCC patients.

METHODSReal-time fluorescence quantitative polymerase chain reaction (PCR) assay was used to determine the expression of miRNA-106a gene in esophageal cancer tissue and corresponding normal mucosa of 81 cases. Immunohistochemical technique was applied to detect the expression of p53, human epidermal growth factor receptor 2 (HER-2), DNA topoisomerase II (Topo II) and multidrug resistance-associated protein (MRP). The association of miRNA-106a expression with clinicopathological features, expression of related proteins, and prognosis of the patients was analyzed.

RESULTSAmong the 81 cases, under-expression of miRNA-106a gene was found in 48 cases (59.3%), normal expression in 22 cases (27.2%), and overexpression in 11 cases (13.6%). The expression of miRNA-106 gene was significantly associated with lymph node metastasis, pathological stage, and nerve invasion (all P < 0.05), significantly associated with expression of p53 (P = 0.006), and not significantly associated with expressions of HER-2, Topo II and MRP proteins (all P > 0.05). The expression of miRNA-106a gene was also significantly associated with progression-free survival (PFS, P = 0.032), but not significantly with overall survival (OS, P = 0.486). The results of Cox multivariate regression analysis showed that the PFS of ESCC patients was significantly correlated with lymph node metastasis (P = 0.029), but not correlated with the age, gender, tumor length, T stage, degree of differentiation, nerve invasion, and miRNA-106a expression (all P > 0.05).

CONCLUSIONSIn esophageal squamous cell carcinomas, the miRNA-106a gene is under-expressed, with tumor suppressor function, and may be regarded as a biological marker to assess the prognosis in patients with esophageal squamous cell carcinoma.

Adult ; Aged ; Biomarkers, Tumor ; metabolism ; Carcinoma, Squamous Cell ; genetics ; metabolism ; pathology ; Cell Line, Tumor ; DNA Topoisomerases, Type II ; metabolism ; Disease-Free Survival ; Esophageal Neoplasms ; genetics ; metabolism ; pathology ; Female ; Humans ; Immunohistochemistry ; Lymphatic Metastasis ; Male ; MicroRNAs ; metabolism ; Middle Aged ; Multidrug Resistance-Associated Proteins ; metabolism ; Neoplasm Invasiveness ; Neoplasm Staging ; Proportional Hazards Models ; Real-Time Polymerase Chain Reaction ; Receptor, ErbB-2 ; metabolism ; Survival Rate ; Tumor Suppressor Protein p53 ; metabolism