The ATP-binding cassette sub-family B member 7 (ABCB7) is a membrane transport protein located on the inner membrane of mitochondria, which could be involved in the transport of heme from the mitochondria to the cytosol. ABCB7 also plays a central role in the maturation of cytosolic iron-sulfur (Fe/S) cluster-containing proteins, and mutations can cause a series of mitochondrial defects. X-linked sideroblastic anemia and ataxia (XLSA-A) is a rare cause of early onset ataxia, which may be overlooked due to the usually mild asymptomatic anemia. The genetic defect has been identified as a mutation in the ABCB7 gene at Xq12-q13. Here, we report the expression, purification and the 2D projections derived from negatively stained electron micrographs of recombinant H. sapiens ABCB7 (hABCB7), paving the way from an atomic structure determination of ABCB7.

The aim of the present study was to investigate the protein expression profiling of pregnane X receptor (PXR) and ATP-binding cassette sub-family B member 1 (ABCB1; also known as MDR1 or P-gp), present in the peripheral blood mononuclear cells (PBMCs) and cancerous tissues of cases of non-small cell lung cancer (NSCLC). Furthermore, the study aimed to assess the feasibility of predicting drug resistance through the medium of PBMCs. Of the subjects included in the study, 37 were histopathologically diagnosed with NSCLC and 17 were control patients without cancer. ThinPrep liquid-based smears with cytosine were applied in the examination of the PBMCs and proved quite effective in preserving the morphology and surface antigens of the lymphocytes. Measurements of expression levels in the PBMCs and cancerous tissues were obtained by immunohistochemical means. The results showed that, with the exception of the selective PXR expression in the normal lung tissues, the two types of proteins existed extensively throughout the PBMCs, normal tissues and tumors. Among the cancer patients, prior to chemotherapy, a significant rise in ABCB1 expression could be observed in the PBMCs, together with a similar rise in ABCB1 and PXR expression in the tumor specimens. Marked upregulation of the two proteins was detected in the PBMCs following 1 cycle of first-line chemotherapy. ABCB1 expression, correlated with PXR, persisted mostly in the PBMCs and tissue samples. When bound to and activated by ligands, PXR translocates from the cytoplasm to the nucleus of the cells. PXR subsequently binds to its DNA response elements as a heterodimer with the retinoid X receptor. A PXR translocation of moderate or low differentiation was identified in 3 cases of adenocarcinoma, which were co-expressing the two genes in the PBMCs prior to chemotherapy. During follow-up visits, tumor recurrence was observed within 3 months in 5 cases, which were characterized by PXR translocation. These findings indicate that the combined expression of PXR and ABCB1 in PBMCs may be used as a prospective indicator in diagnosis prior to histopathological diagnosis, and therefore may function as a novel biomarker for the prediction of drug resistance.

We developed a novel, erythropoietin-derived, non-erythropoiesis, cyclic helix B peptide (CHBP) that displays potent renoprotection against acute kidney injury (AKI). To determine the mechanism of CHBP-mediated protection, we investigated the proteomic profile of mice treated with CHBP in a kidney ischemia-reperfusion (IR) injury model. The isobaric tags for relative and absolute quantitation (iTRAQ)-labeled samples were analyzed using a QSTAR XL LC/MS system. In total, 38 differentially expressed proteins (DEPs) were shared by all experimental groups, while 3 DEPs were detected specifically in the IR + CHBP group. Eight significant pathways were identified, and oxidative phosphorylation was shown to be the most important pathway in CHBP-mediated renoprotection. The significant DEPs in the oxidative phosphorylation pathway elicited by CHBP are NADH-ubiquinone oxidoreductase Fe-S protein 6 (NDUFS6), alpha-aminoadipic semialdehyde synthase (AASS) and ATP-binding cassette sub-family D member 3 (ABCD3). The DEPs mentioned above were verified by RT-qPCR and immunostaining in mouse kidneys. We tested 6 DEPs in human biopsy samples from kidney transplant recipients. The trend of differential expression was consistent with that in the murine model. In conclusion, this study helps to elucidate the pharmacological mechanisms of CHBP before clinical translation.

Behavioral endophenotypes are determined by a multitude of counteracting but precisely balanced molecular and physiological mechanisms. In this study, we aim to identify potential novel molecular targets that contribute to the multigenic trait "anxiety". We used microarrays to investigate the gene expression profiles of different brain regions within the limbic system of mice which were selectively bred for either high (HAB) or low (LAB) anxiety-related behavior, and also show signs of comorbid depression-like behavior. We identified and confirmed sex-independent differences in the basal expression of 13 candidate genes, using tissue from the entire brain, including coronin 7 (Coro7), cathepsin B (Ctsb), muscleblind-like 1 (Mbnl1), metallothionein 1 (Mt1), solute carrier family 25 member 17 (Slc25a17), tribbles homolog 2 (Trib2), zinc finger protein 672 (Zfp672), syntaxin 3 (Stx3), ATP-binding cassette, sub-family A member 2 (Abca2), ectonucleotide pyrophosphatase/phosphodiesterase 5 (Enpp5), high mobility group nucleosomal binding domain 3 (Hmgn3) and pyruvate dehydrogenase beta (Pdhb). Additionally, we confirmed brain region-specific differences in the expression of synaptotagmin 4 (Syt4).Our identification of about 90 polymorphisms in Ctsb suggested that this gene might play a critical role in shaping our mouse model's behavioral endophenotypes. Indeed, the assessment of anxiety-related and depression-like behaviors of Ctsb knock-out mice revealed an increase in depression-like behavior in females. Altogether, our results suggest that Ctsb has significant effects on emotionality, irrespective of the tested mouse strain, making it a promising target for future pharmacotherapy.

The ATP-binding cassette, sub-family B member 4 knock-out mouse (Abcb4(-/-)) is a relevant model for chronic cholangiopathy in man. Due to the lack of this P-glycoprotein in the canalicular membrane of hepatocytes, the secretion of phospholipids into bile is absent, resulting in increased bile toxicity. Expression of insulin like growth factor binding protein 5 (Igfbp5) increases in time in the livers of these mice. It is unclear whether this induction is a consequence of or plays a role in the progression of liver pathology. The aim of this study was therefore to investigate the effect of IGFBP5 induction on the progression of liver fibrosis caused by chronic cholangiopathy. IGFBP5 and, as a control, green fluorescent protein were overexpressed in the hepatocytes of Abcb4(-/-) mice, using an adeno-associated viral vector (AAV). Progression of liver fibrosis was studied 3, 6, and 12 weeks after vector injection by analyzing serum parameters, collagen deposition, expression of pro-fibrotic genes, inflammation and oxidative stress. A single administration of the AAV vectors provided prolonged expression of IGFBP5 and GFP in the livers of Abcb4(-/-) mice. Compared to GFP control, fractional liver weight, extracellular matrix deposition and amount of activated hepatic stellate cells significantly decreased in IGFBP5 overexpressing mice even 12 weeks after treatment. This effect was not due to a change in bile composition, but driven by reduced inflammation, oxidative stress, and proliferation. Overexpression of IGFBP5 seems to have a protective effect on liver pathology in this model for chronic cholangiopathy.

Lysosomes are important for proper functioning of the retinal pigment epithelial (RPE) cells. RPE cells have a daily burden of phagocytosis of photoreceptor outer segments (POS) and also degrade cellular waste by autophagy. Here, we identified the role of Zinc-finger protein with KRAB and SCAN domains 3 (ZKSCAN3) in co-ordinate regulation of lysosomal function and autophagy in the RPE. Our studies show that in the RPE, ZKSCAN3 is predominantly nuclear in healthy cells and its nuclear expression is reduced upon nutrient deprivation. siRNA-mediated knockdown of ZKSCAN3 results in de-repression of some of the ZKSCAN3 target genes. Knockdown of ZKSCAN3 also resulted in an induction in autophagy flux, increase in the number of functional lysosomes and accompanied activation of lysosomal cathepsin B activity in ARPE-19 cells. We also demonstrated that inhibition of P38 mitogen-activated protein kinase (MAPK) retains ZKSCAN3 in the nucleus in nutrient-deprived cells. In summary, our studies elucidated the role of ZKSCAN3 as a transcriptional repressor of autophagy and lysosomal function in the RPE.

In this article, we describe the surprising non-specific reactivity in immunoblots of a rabbit polyclonal antibody (ref. Abcam 86222) expected to recognize the transporter associated with antigen processing like (TAP-L, ABCB9) protein. Although this antibody, according to company documentation, recognizes a band with the expected molecular weight of 84 kDa in HeLa, 293T and mouse NIH3T3 whole-cell lysates, we found that this band is also present in immunoblots of TAP-L deficient bone marrow-derived dendritic cell (BMDC) whole-cell lysates in three independent replicates. We performed extensive verification by multiple PCR tests to confirm the complete absence of the ABCB9 gene in our TAP-L deficient mice. We conclude that the antibody tested cross-reacts with an unidentified protein present in TAP-L knockout cells, which coincidentally runs at the same molecular weight as TAP-L. These findings underline the pitfalls of antibody specificity testing in the absence of cells lacking expression of the target protein.

There are interindividual differences in the adverse effects of atypical antipsychotics, which include autonomic nervous system (ANS) dysfunction. Accordingly, to clarify the interindividual differences in the adverse effects of specific atypical antipsychotics in schizophrenia, we investigated the association between ANS dysfunction and ATP-binding cassette transport sub-family B member 1 (ABCB1) gene polymorphisms in patients with schizophrenia.

Cancer stem cells (CSCs) play an important role in the progression of carcinoma and have a high potential for survival in stress environments. However, the mechanisms of survival potential of CSCs have been unclear. The aim of this study was to clarify the significance of autophagy systems of CSCs under stress environments. Four gastric cancer cell line were used. Side population (SP) cells were sorted from the parent cells, as CSC rich cells. The expression of stem cell markers was examined by RT-PCR. The viability of cancer cells under starvation and hypoxia was evaluated. The expression level of the autophagy molecule LC3B-II was examined by western blot. The numbers of autophagosomes and autolysosomes were counted by electron microscope. SP cells of OCUM-12 showed a higher expression of stem cell markers and higher viability in starvation and hypoxia. Western blot and electron microscope examinations indicated that the autophagy was more induced in SP cells than in parent cells. The autophagy inhibitor significantly decreased the viability under the stress environments. These findings suggested that Cancer stem cells of gastric cancer might maintain their viability via the autophagy system. Autophagy inhibitors might be a promising therapeutic agent for gastric cancer.

To evaluate retrospectively the association of cytochrome P450 3A (CYP3A) and ATP-binding cassette sub-family B member 1 (ABCB1) gene polymorphisms with the pharmacokinetics of cyclosporine A (CsA) in Chinese renal transplant patients.

Small cell lung cancer (SCLC) is a highly aggressive disease with a poor prognosis. Although most patients initially respond to topoisomerase inhibitors, resistance rapidly emerges. The aim, therefore, is to overcome resistance to topoisomerase I (irinotecan) or II (etoposide) inhibitors in SCLCs.

Recent studies have shown that microRNAs may regulate the ABCB1 gene (ATP-binding cassette, sub-family B [MDR/TAP], member 1). Computational programs have predicted that the 3'-untranslated region (3'-UTR) of ABCB1 contains a potential miRNA-binding site for miR-186. Here, we investigated the role of miR-186 in sensitizing ovarian cancer cells to paclitaxel and cisplatin.

In vitro studies suggest that liver fatty acid binding protein (L-FABP) and sterol carrier protein-2/sterol carrier protein-x (SCP2/SCPx) gene products facilitate uptake and metabolism and detoxification of dietary-derived phytol in mammals. However, concomitant upregulation of L-FABP in SCP2/SCPx null mice complicates interpretation of their physiological phenotype. Therefore, the impact of ablating both the L-FABP gene and SCP2/SCPx gene (L-FABP/SCP2/SCPx null or TKO) was examined in phytol-fed female wild-type (WT) and TKO mice. TKO increased hepatic total lipid accumulation, primarily phospholipid, by mechanisms involving increased hepatic levels of proteins in the phospholipid synthetic pathway. Concomitantly, TKO reduced expression of proteins in targeting fatty acids towards the triacylglycerol synthetic pathway. Increased hepatic lipid accumulation was not associated with any concomitant upregulation of membrane fatty acid transport/translocase proteins involved in fatty acid uptake (FATP2, FATP4, FATP5 or GOT) or cytosolic proteins involved in fatty acid intracellular targeting (ACBP). In addition, TKO exacerbated dietary phytol-induced whole body weight loss, especially lean tissue mass. Since individually ablating SCPx or SCP2/SCPx elicited concomitant upregulation of L-FABP, these findings with TKO mice help to resolve the contributions of SCP2/SCPx gene ablation on dietary phytol-induced whole body and hepatic lipid phenotype independent of concomitant upregulation of L-FABP.

Alzheimer's disease (AD) is the most common type of senile dementia affecting elderly people. The processing of amyloid precursor protein (APP) leading to the generation of β-amyloid (Aβ) peptide contributes to neurodegeneration and development of AD pathology. The endocytic trafficking pathway, which comprises of the endosomes and lysosomes, acts as an important site for Aβ generation, and endocytic dysfunction has been linked to increased Aβ production and loss of neurons in AD brains. Since insulin-like growth factor-II (IGF-II) receptor plays a critical role in the transport of lysosomal enzymes from the trans-Golgi network to endosomes, it is likely that the receptor may have a role in regulating Aβ metabolism in AD pathology. However, very little is known on how altered levels of the IGF-II receptor can influence the expression/function of various molecules involved in AD pathology. To address this issue, we evaluated the expression profiles of 87 selected genes related to AD pathology in mouse fibroblast MS cells that are deficient in murine IGF-II receptor and corresponding MS9II cells overexpressing ∼ 500 times the human IGF-II receptors. Our results reveal that an elevation in IGF-II receptor levels alters the expression profiles of a number of genes including APP as well as enzymes regulating Aβ production, degradation and clearance mechanisms. Additionally, it influences the expression of various lysosomal enzymes and protein kinases that are involved in Aβ toxicity. IGF-II receptor overexpression also alters expression of several genes involved in intracellular signalling as well as cholesterol metabolism, which play a critical role in AD pathology. The altered gene profiles observed in this study closely match with the corresponding protein levels, with a few exceptions. These results, taken together, suggest that an elevation in IGF-II receptor levels can influence the expression profiles of transcripts as well as proteins that are involved in AD pathogenesis.

Epigenetic silencing through promoter hypermethylation is an important hallmark for the inactivation of tumor-related genes in carcinogenesis. Here we identified the ATP-binding cassette sub-family B member 4 (ABCB4) as a novel epigenetically silenced target gene. We investigated the epigenetic regulation of ABCB4 in 26 human lung, breast, skin, liver, head and neck cancer cells lines and in primary cancers by methylation and expression analysis. Hypermethylation of the ABCB4 CpG island promoter occurred in 16 out of 26 (62%) human cancer cell lines. Aberrant methylation of ABCB4 was also revealed in 39% of primary lung cancer and in 20% of head and neck cancer tissues. In 37% of primary lung cancer samples, ABCB4 expression was absent. For breast cancer a significant hypermethylation occurred in tumor tissues (41%) compared to matching normal samples (0%, p = 0.002). Silencing of ABCB4 was reversed by 5-aza-2'-deoxycytidine and zebularine treatments leading to its reexpression in cancer cells. Overexpression of ABCB4 significantly suppressed colony formation and proliferation of lung cancer cells. Hypermethylation of Abcb4 occurred also in murine cancer, but was not found in normal tissues. Our findings suggest that ABCB4 is a frequently silenced gene in different cancers and it may act tumor suppressivly in lung cancer.

Due to poor water solubility and high susceptibility to chemical degradation, the applications of quercetin have been limited. This study investigated the effects of pH on the formation of quercetin-loaded nanoemulsion (NQ) and compared the hypocholesterolemic activity between quercetin and NQ to utilize the quercetin as functional food ingredient. NQ particle size exhibited a range of 207⁻289 nm with polydispersity index range (<0.47). The encapsulation efficiency increased stepwise from 56 to 92% as the pH increased from 4.0 to 9.0. Good stability of NQ was achieved in the pH range of 6.5⁻9.0 during 3-month storage at 21 and 37 °C. NQ displayed higher efficacy in reducing serum and hepatic cholesterol levels and increasing the release of bile acid into feces in rats fed high-cholesterol diet, compared to quercetin alone. NQ upregulated hepatic gene expression involved in bile acid synthesis and cholesterol efflux, such as cholesterol 7 alpha-hydroxylase (CYP7A1), liver X receptor alpha (LXRα), ATP-binding cassette transporter A1 (ABCA1) and ATP-binding cassette sub-family G member 1 (ABCG1). These results suggest at least partial involvement of hepatic bile acid synthesis and fecal cholesterol excretion in nanoemulsion quercetin-mediated beneficial effect on lipid abnormalities.

Secalonic acid D (SAD) could inhibit cell growth in not only sensitive cells but also multidrug resistant (MDR) cells. However, the molecular mechanisms need to be elucidated. Here, we identified that SAD possessed potent cytotoxicity in 3 pairs of MDR and their parental sensitive cells including S1-MI-80 and S1, H460/MX20 and H460, MCF-7/ADR and MCF-7 cells. Furthermore, SAD induced cell G2/M phase arrest via the downregulation of cyclin B1 and the increase of CDC2 phosphorylation. Importantly, JNK pathway upregulated the expression of c-Jun in protein level and increased c-Jun phosphorylation induced by SAD, which was linked to cell apoptosis via c-Jun/Src/STAT3 pathway. To investigate the mechanisms of upregulation of c-Jun protein by SAD, the mRNA expression level and degradation of c-Jun were examined. We found that SAD did not alter the mRNA level of c-Jun but inhibited its proteasome-dependent degradation. Taken together, these results implicate that SAD induces cancer cell death through c-Jun/Src/STAT3 signaling axis by inhibiting the proteasome-dependent degradation of c-Jun in both sensitive cells and ATP-binding cassette transporter sub-family G member 2 (ABCG2)-mediated MDR cells.

Most cancer patients receive radiotherapy in the course of their disease and the occurrence of radioresistance is associated with poor prognosis. The molecular pathways that drive enhanced tumorigenic potential during the development of radioresistance are poorly understood. Here, we demonstrate that aryl hydrocarbon receptor (AhR) plays a vital role in the maintenance of cancer stem-like properties. AhR promotes the cancer stem-like phenotype and drives metastasis by directly targeting the promoters of 'stemness' genes, such as the ATP-binding cassette sub-family G member 2 (ABCG2) gene. Moreover, the radioresistant sublines display high levels of oncometabolites including α-ketoglutarate, and treatment of cancer cells with α-ketoglutarate enhances their stem-like properties in an AhR activation-dependent manner. IKKα directly activates stemness-related genes through an interaction with AhR as a bone fide chromatin modifier. Thus, AhR is functionally linked with cancer stem-like properties, and it drives tumorigenesis in the occurrence of radioresistance.

Drug-metabolizing enzymes, transporters, and nuclear receptors are essential for the absorption, distribution, metabolism, and excretion (ADME) of drugs and xenobiotics. MicroRNAs participate in the regulation of ADME gene expression via imperfect complementary Watson-Crick base pairings with target transcripts. We have previously reported that Cytochrome P450 3A4 (CYP3A4) and ATP-binding cassette sub-family G member 2 (ABCG2) are regulated by miR-27b-3p and miR-328-3p, respectively. Here we employed our newly established RNA bioengineering technology to produce bioengineered RNA agents (BERA), namely BERA/miR-27b-3p and BERA/miR-328-3p, via fermentation. When introduced into human cells, BERA/miR-27b-3p and BERA/miR-328-3p were selectively processed to target miRNAs and thus knock down CYP3A4 and ABCG2 mRNA and their protein levels, respectively, as compared to cells treated with vehicle or control RNA. Consequently, BERA/miR-27b-3p led to a lower midazolam 1'-hydroxylase activity, indicating the reduction of CYP3A4 activity. Likewise, BERA/miR-328-3p treatment elevated the intracellular accumulation of anticancer drug mitoxantrone, a classic substrate of ABCG2, hence sensitized the cells to chemotherapy. The results indicate that biologic miRNA agents made by RNA biotechnology may be applied to research on miRNA functions in the regulation of drug metabolism and disposition that could provide insights into the development of more effective therapies.

ATP-binding cassette sub-family G member 2 (ABCG2) confers to the major phenotypes of side population (SP) cells, the cancer stem-like cells. In this study, the SP cells displayed a distinctly higher ABCG2 expression level, sphere formation efficiency (SFE) and growth rate even under hypoxia condition. CXCR4 overexpression by pcDNA-CXCR4 transfection robustly increased ABCG2 expression, and promoted SFE and growth of hypoxic SP cells, while CXCR4 inhibitor AMD3100 could suppress the promotion. Additionally, we found that CXCR4 promoted the expression of c-Jun, a major gene in the oncogenic JNK/c-Jun pathway. Our data on electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assays both showed that c-Jun directly bound with the ABCG2 promoter sequence. Moreover, overexpression of JNK/c-Jun promoted ABCG2 expression, SFE, and growth of hypoxic SP cells and the promotion could be rescued by c-Jun inhibitor SP600125. In conclusion, CXCR4 increases the growth and SFE of breast cancer SP cells under hypoxia through c-Jun-mediated transcriptional activation of ABCG2.