Hemantkumar Chavan

The ATP-Binding Cassette Transporter ABCB6 Is Induced by Arsenic and Protects against Arsenic Cytotoxicity

Arsenic, an environmental carcinogen, remains a major public health problem. Arsenic damages biological systems through multiple mechanisms, including the generation of reactive oxygen species. ABCB6 is an ATP-binding cassette transporter that is highly expressed in cells resistant to arsenic. We have recently demonstrated that ABCB6 expression protects against cellular stressors. In the present study, we evaluated the significance of ABCB6 expression to arsenic toxicity both in mice and in cell culture. We show that sodium arsenite induces ABCB6 expression in a dose-dependent manner both in mice fed sodium arsenite in drinking water and in cells exposed to sodium arsenite in vitro. Arsenite-induced ABCB6 expression was transcriptionally regulated, but this induction was not mediated by the redox-sensitive transcription factor nuclear factor-erythroid 2-related factor 2 (Nrf2). We demonstrate that, in HepG2 and Hep3B cells, knockdown of ABCB6 expression using ABCB6-specific small interfering RNA sensitized the cells to arsenite toxicity. In contrast, stable overexpression of ABCB6 conferred a strong survival advantage toward arsenite-induced oxidative stress. Collectively, these results, obtained by both loss of function and gain of function analysis, suggest that ABCB6 expression in response to sodium arsenite might be an endogenous protective mechanism activated to protect cells against arsenite-induced oxidative stress.

Functional significance of the ATP-binding cassette transporter B6 in hepatocellular carcinoma

ABCB6 is a mitochondrial transporter that regulates porphyrin biosynthesis. ABCB6 expression is upregulated in hepatocellular carcinoma (HCC) but the significance of this upregulation to HCC is not known. In the present study, we investigated: 1) ABCB6 expression in 18 resected human hepatocellular carcinoma (HCC) tissues and 3 human hepatoma cell lines; 2) pattern of ABCB6 expression during liver disease progression; and 3) functional significance of ABCB6 expression to HCC using the hepatoma cell line Huh7. ABCB6 expression was determined by real‐time quantitative reverse transcription‐polymerase chain reaction and western blotting. ABCB6 expression was upregulated in all the HCC specimens and the three‐hepatoma cell lines. Increased ABCB6 expression correlated with liver disease progression with the pattern of expression being HCC > cirrhosis > steatosis. Small hairpin RNA (shRNA)‐mediated knockdown of ABCB6 in Huh7 cells lead to decreased cellular proliferation and colony formation. Attenuation of ABCB6 expression did not affect Huh7 apoptosis but lead to a delay in G2/M phase of the cell cycle. In contrast, ABCB6 overexpression resulted in increased growth and proliferation of Huh7 cells. Since ABCB6 expression is induced in multiple tumor types we explored the role of ABCB6 in other cancer cells. ShRNA mediated knockdown of ABCB6 in HEK293 and K562 cells reduced cellular proliferation leading to a delay in G2/M phase, while ABCB6 overexpression promoted cell growth and proliferation. Collectively, these findings, obtained by loss of function and gain of function analysis, suggest that ABCB6 plays a role in cell growth and proliferation by targeting the cell cycle.

Metabolomics reveals the formation of aldehydes and iminium in gefitinib metabolism.

Gefitinib (GEF), an inhibitor of epidermal growth factor receptor (EGFR) tyrosine kinase, is widely used for the treatment of cancers, particularly non-small cell lung cancer. However, its clinical use is limited by multiple adverse effects associated with GEF, such as liver and lung injuries, severe nausea, and diarrhea. Although, the exact mechanism of GEF adverse effects are still unknown, xenobiotic-induced bioactivation is thought to play a significant role in GEF induced toxicity. Using a metabolomic approach, we investigated the metabolic pathways of GEF in human and mouse liver microsomes. Thirty four GEF metabolites and adducts were identified and half of them are novel. The potential reactive metabolites, two aldehydes and one iminium, were identified for the first time. The previously reported GSH adducts and primary amines were observed as well. The aldehyde and iminium pathways were further confirmed by using methoxylamine and potassium cyanide as trapping reagents. Using recombinant CYP450 isoforms, CYP3A4 inhibitor, and S9 from Cyp3a-null mice, we confirmed CYP3A is the major enzyme contributing to the formation of aldehydes, GSH adducts, and primary amines in liver. Multiple enzymes contribute to the formation of iminium. This study provided us more knowledge of GEF bioactivation and enzymes involved in metabolic pathways, which can be utilized for understanding the mechanism of adverse effects associated with GEF and predicting possible drug-drug interactions. Further studies are suggested to determine the roles of these bioactivation pathways in GEF toxicity.

Efficient Purification and Reconstitution of ATP Binding Cassette Transporter B6 (ABCB6) for Functional and Structural Studies

Background: The ABCB6 protein is proposed to transport coproporphyrinogen from the cytoplasm into the mitochondria. Results: Purified ABCB6 reconstituted into liposomes demonstrates coproporphyrinogen-stimulated ATP hydrolysis and coproporphyrinogen transport. Conclusion: ABCB6 does not require additional components for substrate-stimulated ATPase activity and substrate transport. Significance: Development of an in vitro system with pure and active ABCB6 for structure and functional studies is indicated. The mitochondrial ATP binding cassette transporter ABCB6 has been associated with a broad range of physiological functions, including growth and development, therapy-related drug resistance, and the new blood group system Langereis. ABCB6 has been proposed to regulate heme synthesis by shuttling coproporphyrinogen III from the cytoplasm into the mitochondria. However, direct functional information of the transport complex is not known. To understand the role of ABCB6 in mitochondrial transport, we developed an in vitro system with pure and active protein. ABCB6 overexpressed in HEK293 cells was solubilized from mitochondrial membranes and purified to homogeneity. Purified ABCB6 showed a high binding affinity for MgATP (Kd = 0.18 μm) and an ATPase activity with a Km of 0.99 mm. Reconstitution of ABCB6 into liposomes allowed biochemical characterization of the ATPase including (i) substrate-stimulated ATPase activity, (ii) transport kinetics of its proposed endogenous substrate coproporphyrinogen III, and (iii) transport kinetics of substrates identified using a high throughput screening assay. Mutagenesis of the conserved lysine to alanine (K629A) in the Walker A motif abolished ATP hydrolysis and substrate transport. These results suggest a direct interaction between mitochondrial ABCB6 and its transport substrates that is critical for the activity of the transporter. Furthermore, the simple immunoaffinity purification of ABCB6 to near homogeneity and efficient reconstitution of ABCB6 into liposomes might provide the basis for future studies on the structure/function of ABCB6.

Polycyclic Aromatic Hydrocarbons (PAHs) Mediate Transcriptional Activation of the ATP Binding Cassette Transporter ABCB6 Gene via the Aryl Hydrocarbon Receptor (AhR)

Background: ABCB6 is an ATP binding cassette transporter that regulates heme biosynthesis. Results: Polyaromatic hydrocarbons increase heme synthesis in liver by activating ABCB6 expression via the aryl hydrocarbon receptor. Conclusion: ABCB6 is required for PAH-mediated induction of heme biosynthesis. Significance: ABCB6 expression might be clinically relevant in polyaromatic hydrocarbon-induced porphyrias and carcinogenesis. Liver is endowed with a mechanism to induce hepatic cytochromes P450 (CYP450s) in response to therapeutic drugs and environmental contaminants, leading to increased detoxification and elimination of the xenobiotics. Each CYP450 is composed of an apoprotein moiety and a heme prosthetic group, which is required for CYP450 activity. Thus, under conditions of CYP450 induction, there is a coordinate increase in heme biosynthesis to compensate for the increased expression of CYP450s. ABCB6, a mitochondrial ATP binding cassette transporter, which regulates coproporphyrinogen transport from the cytoplasm into the mitochondria to complete heme biosynthesis, represents a previously unrecognized rate-limiting step in heme biosynthesis. However, it is not known if exposure to drugs and environmental contaminants induces ABCB6 expression, to assure an adequate and apparently coordinated supply of heme for the generation of functional cytochrome holoprotein. In the present study, we demonstrate that polycyclic aromatic hydrocarbons (PAHs), the widely distributed environmental toxicants shown to induce porphyrin accumulation causing hepatic porphyria, up-regulate ABCB6 expression in both mice and humans. Using siRNA technology and Abcb6 knock-out mice, we demonstrate that PAH-mediated increase in hepatic porphyrins is compromised in the absence of ABCB6. Moreover, in vivo studies in aryl hydrocarbon receptor (AhR) knock-out mice demonstrate that PAH induction of ABCB6 is mediated by AhR. Promoter activation studies combined with electrophoretic mobility shift assay and chromatin immunoprecipitation assay demonstrate direct interactions between the AhR binding sites in the ABCB6 promoter and the AhR receptor, implicating drug activation mechanisms for ABCB6 similar to those found in inducible cytochrome P450s. These studies are the first to describe direct transcriptional activation of both mouse and human ABCB6 by xenobiotics.

Proteomic analysis of miltefosine-resistant Leishmania reveals the possible involvement of eukaryotic initiation factor 4A (eIF4A)☆

Another explanation for recovery is that she was septic nd improved with antibiotic therapy, although the evience seems to be against this. SVR was initially low, hich is consistent with septic shock; however, her clincal picture never appeared to be one of sepsis. A rapid ecovery was seen with withdrawal of liposomal amphoericin B and flucytosine, strengthening the conclusion hat the condition was solely related to an adverse drug eaction. Reversible cardiomyopathy with flucytosine and amphoericin B is rare. It was crucial for the medical team to ecognise the potential for cardiac toxicity and to discontinue oth medications. The patient should never be re-challenged ith either drug. Funding: No funding sources. Competing interests: None declared. Ethical approval: Not required.

Targeting the Enterohepatic Bile Acid Signaling Induces Hepatic Autophagy via a CYP7A1–AKT–mTOR Axis in Mice

Background & Aims Hepatic cholesterol accumulation and autophagy defects contribute to hepatocyte injury in fatty liver disease. Bile acid synthesis is a major pathway for cholesterol catabolism in the liver. This study aims to understand the molecular link between cholesterol and bile acid metabolism and hepatic autophagy activity. Methods The effects of cholesterol and cholesterol 7α-hydroxylase (CYP7A1) expression on autophagy and lysosome function were studied in cell models. The effects and mechanism of disrupting enterohepatic bile acid circulation on hepatic autophagy were studied in mice. Results The results first showed differential regulation of hepatic autophagy by free cholesterol and cholesterol ester, whereby a modest increase of cellular free cholesterol, but not cholesterol ester, impaired lysosome function and caused marked autolysosome accumulation. We found that CYP7A1 induction, either by cholestyramine feeding in mice or adenovirus-mediated CYP7A1 expression in hepatocytes, caused strong autophagy induction. Mechanistically, we showed that CYP7A1 expression markedly attenuated growth factor/AKT signaling activation of mechanistic target of rapamycin (mTOR), but not amino acid signaling to mTOR in vitro and in vivo. Metabolomics analysis further found that CYP7A1 induction not only decreased hepatic cholesterol but also altered phospholipid and sphingolipid compositions. Collectively, these results suggest that CYP7A1 induction interferes with growth factor activation of AKT/mTOR signaling possibly by altering membrane lipid composition. Finally, we showed that cholestyramine feeding restored impaired hepatic autophagy and improved metabolic homeostasis in Western diet–fed mice. Conclusions This study identified a novel CYP7A1–AKT–mTOR signaling axis that selectively induces hepatic autophagy, which helps improve hepatocellular integrity and metabolic homeostasis.

A Novel Flow Cytometric HTS Assay Reveals Functional Modulators of ATP Binding Cassette Transporter ABCB6

ABCB6 is a member of the adenosine triphosphate (ATP)-binding cassette family of transporter proteins that is increasingly recognized as a relevant physiological and therapeutic target. Evaluation of modulators of ABCB6 activity would pave the way toward a more complete understanding of the significance of this transport process in tumor cell growth, proliferation and therapy-related drug resistance. In addition, this effort would improve our understanding of the function of ABCB6 in normal physiology with respect to heme biosynthesis, and cellular adaptation to metabolic demand and stress responses. To search for modulators of ABCB6, we developed a novel cell-based approach that, in combination with flow cytometric high-throughput screening (HTS), can be used to identify functional modulators of ABCB6. Accumulation of protoporphyrin, a fluorescent molecule, in wild-type ABCB6 expressing K562 cells, forms the basis of the HTS assay. Screening the Prestwick Chemical Library employing the HTS assay identified four compounds, benzethonium chloride, verteporfin, tomatine hydrochloride and piperlongumine, that reduced ABCB6 mediated cellular porphyrin levels. Validation of the identified compounds employing the hemin-agarose affinity chromatography and mitochondrial transport assays demonstrated that three out of the four compounds were capable of inhibiting ABCB6 mediated hemin transport into isolated mitochondria. However, only verteporfin and tomatine hydrochloride inhibited ABCB6’s ability to compete with hemin as an ABCB6 substrate. This assay is therefore sensitive, robust, and suitable for automation in a high-throughput environment as demonstrated by our identification of selective functional modulators of ABCB6. Application of this assay to other libraries of synthetic compounds and natural products is expected to identify novel modulators of ABCB6 activity.

Proteomic analysis of wild type and arsenite-resistant Leishmania donovani

Leishmania donovani, causative organism for visceral leishmaniasis, is responsible for considerable mortality and morbidity worldwide. Generation of drug-resistant variants continue to challenge the chemotherapy, the mainstay to fight the disease. The aim of current study was proteomic profiling of wild type (Ld-Wt) and arsenite-resistant (Ld-As20) L. donovani. Significant differences in protein profiles were observed between Ld-As20 and its parent Ld-Wt strain. Proteomic analysis of 158 spots from Ld-Wt and 144 spots from, Ld-As20 identified 77 and 74 protein entries, respectively, through MALDI-TOF/TOF based mass spectrometry and database search. A shift in the isoelectric point of few proteins was observed both in Ld-Wt and Ld-As20, which raises the possibility of continuous arsenite stress, resulting in the differences in the protein profiles of drug-resistant strain from its parent wild type strain. The comparative proteomic data holds the key for elucidation of the multifactorial and complex drug resistance mechanism, like arsenite resistance, in the parasite.

Sortilin 1 modulates hepatic cholesterol lipotoxicity in mice via functional interaction with liver carboxylesterase 1

The liver plays a key role in cholesterol metabolism. Impaired hepatic cholesterol homeostasis causes intracellular free cholesterol accumulation and hepatocyte injury. Sortilin 1 (SORT1) is a lysosomal trafficking receptor that was identified by genome-wide association studies (GWAS) as a novel regulator of cholesterol metabolism in humans. Here we report that SORT1 deficiency protected against cholesterol accumulation-induced liver injury and inflammation in mice. Using an LC-MS/MS-based proteomics approach, we identified liver carboxylesterase 1 (CES1) as a novel SORT1-interacting protein. Mechanistic studies further showed that SORT1 may regulate CES1 lysosomal targeting and degradation and that SORT1 deficiency resulted in higher liver CES1 protein abundance. Previous studies have established an important role of hepatic CES1 in promoting intracellular cholesterol mobilization, cholesterol efflux, and bile acid synthesis. Consistently, high cholesterol atherogenic diet-challenged Sort1 knock-out mice showed less hepatic free cholesterol accumulation, increased bile acid synthesis, decreased biliary cholesterol secretion, and the absence of gallstone formation. SORT1 deficiency did not alter hepatic ceramide and fatty acid metabolism in high cholesterol atherogenic diet-fed mice. Finally, knockdown of liver CES1 in mice markedly increased the susceptibility to high cholesterol diet-induced liver injury and abolished the protective effect against cholesterol lipotoxicity in Sort1 knock-out mice. In summary, this study identified a novel SORT1-CES1 axis that regulates cholesterol-induced liver injury, which provides novel insights that improve our current understanding of the molecular links between SORT1 and cholesterol metabolism. This study further suggests that therapeutic inhibition of SORT1 may be beneficial in improving hepatic cholesterol homeostasis in metabolic and inflammatory liver diseases.