Adenosine Triphosphate Binding Cassette Transporter G2 Research Articles

An ATP13A1-assisted topogenesis pathway for folding multi-spanning membrane proteins

Many multi-spanning membrane proteins contain poorly hydrophobic transmembrane domains (pTMDs) protected from phospholipid in mature structure. Nascent pTMDs are difficult for translocon to recognize and insert. How pTMDs are discerned and packed into mature, muti-spanning configuration remains unclear. Here, we report that pTMD elicits a post-translational topogenesis pathway for its recognition and integration. Using six-spanning protein adenosine triphosphate-binding cassette transporter G2 (ABCG2) and cultured human cells as models, we show that ABCG2's pTMD2 can pass through translocon into the endoplasmic reticulum (ER) lumen, yielding an intermediate with inserted yet mis-oriented downstream TMDs. After translation, the intermediate recruits P5A-ATPase ATP13A1, which facilitates TMD re-orientation, allowing further folding and the integration of the remaining lumen-exposed pTMD2. Depleting ATP13A1 or disrupting pTMD-characteristic residues arrests intermediates with mis-oriented and exposed TMDs. Our results explain how a "difficult" pTMD is co-translationally skipped for insertion and post-translationally buried into the final correct structure at the late folding stage to avoid excessive lipid exposure.

Efficacy, Safety, and Population Pharmacokinetics of Eltrombopag in Children with Different Severities of Aplastic Anemia.

Eltrombopag was approved as a first-line treatment for patients older than 2 years old with severe aplastic anemia (SAA). However, data on eltrombopag in children with different types of aplastic anemia (AA), especially non-severe AA (NSAA), are limited. We performed a prospective, single-arm, and observational study to investigate eltrombopag's efficacy, safety, and pharmacokinetics in children with NSAA, SAA, and very severe AA (VSAA). The efficacy and safety were assessed every 3 months. The population pharmacokinetic (PPK) model was used to depict the pharmacokinetic profile of eltrombopag. Twenty-three AA children with an average age of 7.9 (range of 3.0-14.0) years were enrolled. The response (complete and partial response) rate was 12.5%, 50.0%, and 100.0% after 3, 6, and 12 months in patients with NSAA. For patients with SAA and VSAA, these response rates were 46.7%, 61.5%, and 87.5%. Hepatotoxicity occurred in one patient. Fifty-three blood samples were used to build the PPK model. Body weight was the only covariate for apparent clearance (CL/F) and volume of distribution. The allele-T carrier of adenosine triphosphate-binding cassette transporter G2 was found to increase eltrombopag's clearance. However, when normalized by weight, the clearance between the wild-type and variant showed no statistical difference. In patients with response, children with NSAA exhibited lower area under the curve from time zero to infinity, higher CL/F, and higher weight-adjusted CL/F than those with SAA or VSAA. However, the differences were not statistically significant. The results may support further individualized treatment of eltrombopag in children with AA.

Effect of application at Shu-Mu acupoint on serum uric acid in hyperuricemic rats

To observe the effect of application at Back-Shu with Front-Mu acupoints on serum uric acid (SUA) and kidney uric acid transport related proteins in hyperuricemia rats, so as to explore the mechanism of Shu-Mu acupoint application on treatment of hyperuricemia. SD rats were randomly divided into blank control, model, vaseline application and medication application groups, with 8 rats in each group. The hyperuricemia rat model was established by gavage of potassium oxonate. Rats in the vaseline application group received application of vaseline at bilateral "Ganshu"(BL18) and "Qimen"(LI14), "Pishu"(BL20) and "Zhangmen"(LR13), "Shenshu" (BL23) and "Jingmen"(GB25). Rats in the medication application group received application of traditional Chinese medicine at the same acupoints. The contents of SUA and creatinine (SCr) were detected by automatic biochemical analyzer. H.E. staining was used to observe the pathological changes of kidney. And the protein expression levels of organic anion transporter 1(OAT1) and adenosine triphosphate binding cassette transporter G2(ABCG2) were detected by immunohistochemistry. Rats in the model group showed symptoms such as polydipsia, polyuria, loose stools, fatigue, weakness, etc. The renal tubules atrophied, and urate crystals can be seen in the lumen. Compared with the control group. the SUA content in the model group increased (P<0.01)and the expressions of OAT1 and ABCG2 protein in kidney decreased (P<0.01). After intervention and in comparison with the model group showed that, the diet, excretion function, and mental state of the rats in the medication application group returned to normal, and the pathological changes of the kidney tissue were alleviated, the SUA content was down-regulated(P<0.01)and the expression levels of OAT1 and ABCG2 in the kidney up-regulated (P<0.01). There was no statistically significant difference in the SCr content among the 4 groups (P＞0.05). Medication application at Shu-Mu points can effectively reduce the SUA level of hyperuricemia rats, which may be related to its effects in up-regulating the protein expressions of OAT1 and ABCG2 in the kidney and reducing the damage to the kidneys.

Correlation between adenosine triphosphate (ATP)-binding cassette transporter G2 (ABCG2) and drug resistance of esophageal cancer and reversal of drug resistance by artesunate

The present study investigated the correlation between the abnormal expression of adenosine triphosphate (ATP)-binding cassette transporter G2 (ABCG2) in esophageal cancer and the drug resistance of esophageal cancer, the reversal effect in drug resistance of artesunate (Art), and the mechanism underlying esophageal cancer using nude mice with subcutaneous xenograft as an animal model. The gene and protein expression of ABCG2 was detected in 80 cases of esophageal cancer, atypical dysplasia, and normal mucosa. A subcutaneous xenograft tumor mouse model was established by subcutaneous inoculation of esophageal cancer cell line Eca109/ABCG2 into BALB/c nu/nu nude mice. The reversal of drug resistance by Art in esophageal cancer was studied in vivo. The mice model was injected intraperitoneally with Art and/or doxorubicin (ADM). The animals were divided into six groups: high dose Art (50 mg/kg), low dose Art (25 mg/kg), ADM (1 mg/kg), ADM and high-dose Art, ADM and low-dose Art, and physiological saline as a control. ABCG2 protein expression and cellular ADM concentration were detected by flow cytometry. The mRNA and protein expressions of ABCG2 in esophageal cancer were significantly higher than that in the normal esophagus (P < 0.01). The volume and mass of the subcutaneously implanted tumors in the ADM+Art high- and low-dose groups were significantly decreased than that in the control group (P < 0.05), while the apoptosis rate of tumor cells and the cellular concentration of ADM were increased significantly (P < 0.05), and the ABCG2 protein expression in the tumor cells decreased significantly (P < 0.05). Abnormally high expression of ABCG2 in esophageal cancer tissues is involved in the multidrug resistance of esophageal cancer. In vivo studies showed that Art could reverse the esophageal cancer drug resistance by regulating the ABCG2 expression in tumor cells.

Effects of CO2 Lasers on Dental Pulp Biology in Rats.

The purpose of this study was to investigate the effects of CO2 lasers on the proliferation and differentiation of dental pulp cells, and their latent self-recovery in connection with their stemness using reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry. The first molars from male Sprague-Dawley rats, each weighing ∼150-200 g, were used for this study. The upper first molars were irradiated with a 10,600 nm wavelength CO2 laser under identical parameters (2 W CO2 laser, energy 4J, energy density 203.84 J/cm(2) for 8.8 sec) through the dentin of the occlusal surface. The molars were extracted immediately, or at 1, 3 or 5 days after the laser irradiation. RT-PCR analysis using primers specific for heat shock protein 70 (Hsp70), adenosine triphosphate (ATP)-binding cassette transporter G2 (ABCG2), dentin sialophosphoprotein (DSPP), and dentin matrix protein 1 (DMP1), and immunohistochemistry using antibodies specific for proliferating cell nuclear antigen (PCNA), ABCG2, CD34, and CD44 were performed. RT-PCR analysis revealed that Hsp70 mRNA expression in the immediate group and ABCG2 mRNA expression at day 1 were the highest. DSPP and DMP1 mRNA expression in the laser-irradiated groups increased gradually, reaching its peak on the 5th day of the experiment, although no significant difference found among groups with regard to DMP1 expression. Immunohistochemically, PCNA-positive cells were observed at all times after the laser irradiation; however, they were most evident on day 3. CD44-positive cells were observed strongly on day 1 and day 3, while ABCG2-positive cells were the most evident on day 3. These results demonstrate that CO2 laser irradiation induces degeneration in the pulp tissue, which is then repaired by newly formed odontoblast-like cells.

Isolation, culture and identification of cancer stem-like cells from human thyroid carcinoma tissues

Objective To isolate and culture human thyroid cancer stem-like cells side population(SP) from human thyroid carcinoma specimens and compare the differences in octamer-binding transcription factor 4(Oct-4)and adenosine triphosphate binding cassette transporter G2(ABCG2)expression between SP cells and non-SP cells. Methods SP and non-SP cells were sorted from human thyroid carcinoma cells by fluorescence-activated cell sorting(FACS). The expression of Oct-4 and ABCG2 in SP cells and non-SP cells was detected by Western blotting. Results SP cells with stem cell potency were isolated from human thyroid carcinoma cells. The content of sorted SP cells in experimental group and control group was 1.40% and 0.155% respectively(P< 0.05). The expression of Oct-4 and ABCG2 was significantly higher in SP cells than in non-SP cells. Conclusion SP cells, which have the potency of stem cells, exist in human thyroid carcinoma cells. Key words: Thyroid carcinoma; Side population cells; Cancer stem cells

Docetaxel-induced human breast ccarcinoma drug-resistant BT549/Docetaxel stem cell isolation and adenosine triphosphate binding cassette transporter G2 gene expression level

Objective To isolate docetaxel-induced human breast carcinoma drug-resistant stem cells MCF7/Docetaxel and study its biological characteristics. Methods By suspension culture combined with docetaxel resistant cell line sorting method, BT549/Docetaxel stem cells were isolated, and BT549, and BT549/Docetaxel stem cells were enriched by suspension culture method. The cells were labeled with CD44 and CD24, and detected by flow cytometry. Reverse transcriptase-polymerase chain reaction(RT-PCR) was used to detect the expression of adenosine triphosphate binding cassette transporter G2(ABCG2)in BT549 and BT549/Docetaxel stem cells. Results The population doubling time of BT549/Docetaxel and BT549 stem cells was(39.22±0.65)and(29.20 ±0.50) h with the difference being statistically significant(P< 0.05). Flow cytometric analysis showed CD44+/CD24-/low ratio in BT549 microspheres cells was(13.8±0.5)%, higher than that of BT549 cells in monolayer culture[(2.8±0.6)%, P<0.01].CD44+/CD24-/low ratio in BT549/Docetaxel microspheres cells was(19.1±0.3)%, higher than that in the monolayer culture[(5.0± 0.4)%, P< 0.01].CD44+/CD24-/low ratio in BT549/Docetaxel microspheres cells was significantly higher than that in BT549 microspheres cells(P<0.05). The drug content in BT549 stem cells was (46.9±0.5) ng/106 cells, and that in BT549/Docetaxel stem cells was(7.1±0.3) ng/106 cells (P< 0.01). After culture with normal medium for 4 h, the drug content in BT549 cells was(28.3± 0.6)ng/106 cells, and that in BT549/Docetaxel cells was(3.5±0.1)ng/106 cells(P< 0.01). The expression ABCG2 m RNA in BT549/Docetaxel stem cells was significantly higher than that in BT549 stem cells(P< 0.01). Conclusion The BT549/Docetaxel stem cells were successfully isolated with stable growth and drug resistance. ABCG2 gene expression in BT549/Docetaxel stem cells was significantly increased. Key words: Docetaxel; Drug resistance; Breast cancer stem cell

Low raltegravir concentration in cerebrospinal fluid in patients with ABCG2 genetic variants.

Adenosine triphosphate-binding cassette transporter G2 (ABCG2) is expressed on the cerebrospinal fluid (CSF) side of choroid plexus epithelial cells, which form the blood-CSF barrier. Raltegravir was recently identified as a substrate of ABCG2. In the present study, we analyzed the relationship between single-nucleotide polymorphisms of ABCB1 and ABCG2 genes and raltegravir concentrations in 31 plasma and 14 CSF samples of HIV-infected patients treated with raltegravir-containing regimens. The mean CSF raltegravir concentration was significantly lower in CA (25.5 ng/mL) and AA (<10 ng/mL) genotypes at position 421 in ABCG2 gene compared with CC (103.6 ng/mL) genotype holders (P = 0.016).

Significantly increased expression of OCT4 and ABCG2 in spheroid body-forming cells of the human gastric cancer MKN-45 cell line

The cancer stem cell (CSC) theory hypothesizes that CSCs are the cause of tumor formation, recurrence and metastasis. Key to the study of CSCs is their isolation and identification. The present study investigated whether spheroid body-forming cells in the human gastric cancer (GC) MKN-45 cell line are enriched for CSC properties, and also assessed the expression of the candidate CSC markers, octamer-binding transcription factor-4 (OCT4) and adenosine triphosphate-binding cassette transporter G2 (ABCG2) in the MKN-45 spheroid body cells. The MKN-45 cells were plated in a stem cell-conditioned culture system to allow for spheroid body formation. The expression levels of OCT4 and ABCG2 in the spheroid body cells were assessed by qPCR, western blot analysis and immunofluorescence staining, while the tumorigenicity of the spheroid body-forming cells was assessed by in vivo xenograft studies in nude mice. The MKN-45 cells were able to form spheroid bodies when cultured in stem cell-conditioned medium. The spheroid body-forming cells showed a significantly higher (P<0.01) expression of OCT4 and ABCG2 compared with the parental cells. These data suggest that the spheroid body cells from the MKN-45 GC cell line cultured in stem cell-conditioned medium possessed gastric CSC properties. The co-expression of OCT4 and ABCG2 by these cells may represent the presence of a subpopulation of gastric CSCs.

Co-expression of CD44 and ABCG2 in spheroid body-forming cells of gastric cancer cell line MKN45.

The cancer stem cell (CSC) theory hypothesizes that CSCs are regarded as the cause of tumor formation, recurrence and metastasis. This study aimed to investigate whether spheroid body-forming cells in human gastric cancer cell were enriched for CSC properties, and to assess the expression of candidate CSC markers, cluster of differentiation 44 (CD44) and adenosine triphosphate binding cassette transporter G 2 (ABCG2) in the MKN45 spheroid body cells. Human gastric cancer cell line MKN45 were plated in stem cell conditioned culture system allowed for spheroid body forming. The expression levels of CD44 and ABCG2 in the spheroid body cells were assessed by quantitative real-time PCR, western blot analysis and immunofluorescence staining, and the tumorigenicity of the spheroid body-forming cells were assessed by in vivo xenograft studies in nude mice. The MKN45 cells could form spheroid bodies cultured in stem cell conditioned medium. The spheroid body-forming cells showed a significantly greater (p <0.05) expression of CD44 and ABCG2 than the parental cells. Spheroid body cells from gastric cancer cell line MKN45 cultured in stem cell conditioned medium possessed gastric CSC properties. The cells co-expressed of CD44 and ABCG2 might represent a subpopulation of gastric CSCs.

The Bovine ATP-Binding Cassette Transporter ABCG2 Tyr581Ser Single-Nucleotide Polymorphism Increases Milk Secretion of the Fluoroquinolone Danofloxacin

The bovine adenosine triphosphate-binding cassette transporter G2 (ABCG2/breast cancer resistance protein) polymorphism Tyr581Ser (Y581S) has recently been shown to increase in vitro transepithelial transport of antibiotics. Since this transporter has been extensively related to the active secretion of drugs into milk, the potential in vivo effect of this polymorphism on secretion of xenobiotics in livestock could have striking consequences for milk production, the dairy industry, and public health. Our purpose was to study the in vivo effect of this polymorphism on the secretion of danofloxacin, a widely used veterinary antibiotic, into milk. Danofloxacin (1.25 mg/kg) was administered to six Y/Y 581 homozygous and six Y/S 581 heterozygous lactating cows, and plasma and milk samples were collected and analyzed by high-performance liquid chromatography. No differences were found in the pharmacokinetic parameters of danofloxacin in plasma between the two groups of animals. In contrast, Y/S heterozygous cows showed a 2-fold increase in danofloxacin levels in milk. In addition, the pharmacokinetic elimination parameters, mean residence time and elimination half-life, were significantly lower in the milk of the animals carrying the Y/S polymorphism. These in vivo results are in agreement with our previously published in vitro data, which showed a greater capacity of the S581 variant in accumulation assays, and demonstrate, for the first time, an important effect of the Y581S single-nucleotide polymorphism on antibiotic secretion into cow milk. These findings could be extended to other ABCG2 substrates, and may be relevant for the treatment of mastitis and for the design of accurate and novel strategies to handle milk residues.

Adenosine triphosphate-binding cassette transporter G2 expression in endometriosis and in endometrium from patients with and without endometriosis

To investigate adenosine triphosphate (ATP)-binding cassette transporter G2 (ABCG2) expression in endometriosis and in samples of endometrium from patients with and without endometriosis. Prospective study. University hospital. Patients with and without endometriosis. Endometrial and endometriotic tissues obtained throughout the menstrual cycle. Density of ABCG2(+) microvessels, density of CD31(+) microvessels. No statistically significant differences in the density of ABCG2(+) microvessels were observed between endometrium of patients with and without endometriosis in the proliferative phase and early, middle, and late secretory phases. The density of ABCG2(+) microvessels was statistically significantly higher in the menstrual endometrium of patients with endometriosis compared with patients without endometriosis. The density of ABCG2(+) microvessels was reduced in the ectopic endometrium compared with matched eutopic endometrium except in cases of deep infiltrating endometriosis. The density of ABCG2(+) microvessels was statistically significantly higher in deep infiltrating endometriosis compared with ovarian endometriosis and red and black peritoneal lesions throughout the menstrual cycle. ABCG2 is strongly expressed in the endothelial cells of microvessels of eutopic endometrium, and the density of ABCG2(+) microvessels is reduced in ectopic endometrium except in cases of deep infiltrating endometriosis. ABCG2(+) microvessels may represent an integral part of the pathophysiology of deep infiltrating endometriosis.

Analysis of the effect of the bovine adenosine triphosphate-binding cassette transporter G2 single nucleotide polymorphism Y581S on transcellular transport of veterinary drugs using new cell culture models1

In commercial dairy production, the risk of drug residues and environmental pollutants in milk from ruminants has become an outstanding problem. One of the main determinants of active drug secretion into milk is the ATP-binding cassette transporter G2/breast cancer resistance protein (ABCG2/BCRP). It is located in several organs associated with drug absorption, metabolism, and excretion, and its expression is highly induced during lactation in the mammary gland of ruminants, mice, and humans. As a consequence, potential contamination of milk could expose suckling infants to xenotoxins. In cows, a SNP for this protein affecting quality and quantity of milk production has been described previously (Y581S). In this study, our main purpose was to determine whether this polymorphism has an effect on transcellular transport of veterinary drugs because this could alter substrate pharmacokinetics and milk residues. We stably expressed the wild-type bovine ABCG2 and the Y581S variant in Madin-Darby canine kidney epithelial cells (MDCKII) and MEF3.8 cell lines generating cell models in which the functionality of the bovine transporter could be addressed. Functional studies confirmed the greater functional activity in mitoxantrone accumulation assays for the Y581S variant with a greater relative V(MAX) value (P = 0.040) and showed for the first time that the Y581S variant presents greater transcellular transport of the model ABCG2 substrate nitrofurantoin (P = 0.024) and of 3 veterinary antibiotics, the fluoroquinolone agents enrofloxacin (P = 0.035), danofloxacin (P = 0.001), and difloxacin (P = 0.008), identified as new substrates of the bovine ABCG2. In addition, the inhibitory effect of the macrocyclic lactone ivermectin on the activity of wild-type bovine ABCG2 and the Y581S variant was also confirmed, showing a greater inhibitory potency on the wild-type protein at all the concentrations tested (5 μM, P = 0.017; 10 μM, P = 0.001; 25 μM, P = 0.008; and 50 μM, P = 0.003). Differential transport activity depending on the genotype together with the differential inhibition pattern might have clinical consequences, including changes in substrate pharmacokinetics (and subsequently pharmacodynamics) and more specifically, changes in secretion of ABCG2 substrates into milk, potentially implying important consequences to veterinary therapeutics.