Songhee Han

A cancer-specific variant of the slco1b3 gene encodes a novel human organic anion transporting polypeptide 1B3 (OATP1B3) localized mainly in the cytoplasm of colon and pancreatic cancer cells

OATP1B3 is a member of the OATP (organic anion transporting polypeptides) superfamily, responsible for mediating the transport of numerous endogenous and xenobiotic substances. Although initially reported to be exclusively expressed in the liver, several studies reported that OATP1B3 is frequently expressed in multiple types of cancers and may be associated with differing clinical outcomes. However, a detailed investigation on the expression and function of OATP1B3 protein in cancer has been lacking. In this study, we confirmed that colon and pancreatic cancer cells express variant forms of OATP1B3, different from OATP1B3 wild-type (WT) expressed in the normal liver. OATP1B3 variant 1 (V1), the most prevalent form among the variants, contains alternative exonic sequences (exon 2a) instead of exons 1 and 2 present in OATP1B3 WT. The translated product of OATP1B3 V1 is almost identical to OATP1B3 WT, with exception to the first 28 amino acids at the N-terminus. Exogenous expression of OATP1B3 V1 revealed that OATP1B3 V1 undergoes post-translational modifications and proteasomal degradation to a differing extent compared to OATP1B3 WT. OATP1B3 V1 showed only modest transport activity toward cholecystokin-8 (CCK-8, a prototype OATP1B3 substrate) in contrast to OATP1B3 WT showing a markedly efficient uptake of CCK-8. Consistent with these results, OATP1B3 V1 was localized mainly in the cytoplasm with a much lower extent of trafficking to the surface membrane compared to OATP1B3 WT. In summary, our results demonstrate that colon and pancreatic cancer cells express variant forms of OATP1B3 with only limited transport activity and different subcellular localization compared to OATP1B3 WT. These observed differences at the molecular and functional levels will be important considerations for further investigations of the biological and clinical significance of OATP1B3 expression in cancer.

Role of hypoxia inducible factor-1α in the regulation of the cancer-specific variant of organic anion transporting polypeptide 1B3 (OATP1B3), in colon and pancreatic cancer.

Organic anion transporting polypeptide 1B3 (OATP1B3) was initially considered to be a liver-specific transporter, mediating the uptake of a variety of endogenous and xenobiotic substances. Over the past decade, several investigations reported that OATP1B3 is also expressed across multiple types of cancers. Only recently, our laboratory and others demonstrated the identity of cancer-specific OATP1B3 variants (csOATP1B3) arising from the use of an alternative transcription initiation site, different from the wildtype (WT) OATP1B3 expressed in the normal liver. However, the mechanisms regulating the expression of csOATP1B3 remained unknown. In our current study, we investigated the role of hypoxia and the involvement of hypoxia inducible factor-1α (HIF-1α) in regulating the transcription of csOATP1B3. Our RT-PCR and immunoblotting results indicated that csOATP1B3, but not WT OATP1B3, can be induced in response to ambient or chemical hypoxia (upon exposure to 1% O₂ or cobalt chloride). Reporter assays with deletion and mutated constructs of the csOATP1B3 promoter revealed a functional hypoxia response element (HRE) located in the proximal upstream region. Constructs harboring the HRE displayed the upregulated reporter gene expression in response to hypoxia, but not when mutated. Electrophoretic mobility shift assays using a biotin-labeled csOATP1B3 promoter HRE probe indicated the binding of HIF-1α, which was blocked by an excess of unlabeled csOATP1B3 probe. Furthermore, siRNA-based knockdown of HIF-1α caused a substantial decrease in the expression level of csOATP1B3. Taken together, these findings demonstrate that the transcription of csOATP1B3 is actively engaged during hypoxia, through a commonly utilized pathway involving HIF-1α.

Functional characterization of CYP107W1 from Streptomyces avermitilis and biosynthesis of macrolide oligomycin A

Streptomyces avermitilis contains 33 cytochrome P450 genes in its genome, many of which play important roles in the biosynthesis process of antimicrobial agents. Here, we characterized the biochemical function and structure of CYP107W1 from S. avermitilis, which is responsible for the 12-hydroxylation reaction of oligomycin C. CYP107W1 was expressed and purified from Escherichia coli. Purified proteins exhibited the typical CO-binding spectrum of P450. Interaction of oligomycin C and oligomycin A (12-hydroxylated oligomycin C) with purified CYP107W1 resulted in a type I binding with Kd values of 14.4 ± 0.7 μM and 2.0 ± 0.1 μM, respectively. LC-mass spectrometry analysis showed that CYP107W1 produced oligomycin A by regioselectively hydroxylating C12 of oligomycin C. Steady-state kinetic analysis yielded a kcat value of 0.2 min(-1) and a Km value of 18 μM. The crystal structure of CYP107W1 was determined at 2.1 Å resolution. The overall P450 folding conformations are well conserved, and the open access binding pocket for the large macrolide oligomycin C was observed above the distal side of heme. This study of CYP107W1 can help a better understanding of clinically important P450 enzymes as well as their optimization and engineering for synthesizing novel antibacterial agents and other pharmaceutically important compounds.

Evaluation of Luminescent P450 Analysis for Directed Evolution of Human CYP4A11

Cytochrome P450 4A11 (CYP4A11) is a fatty acid hydroxylase enzyme expressed in human liver. It catalyzes not only the hydroxylation of saturated and unsaturated fatty acids, but the conversion of arachidonic acid to 20-hydroxyeicosatetraenoic acid (20-HETE), a regulator of blood pressure. In this study, we performed a directed evolution analysis of CYP4A11 using the luminogenic assay system. A random mutant library of CYP4A11, in which mutations were made throughout the entire coding region, was screened with luciferase activity to detect the demethylation of luciferin-4A (2-[6-methoxyquinolin-2-yl]-4,5-dihydrothiazole-4-carboxylic acid) of CYP4A11 mutants in Escherichia coli. Consecutive rounds of random mutagenesis and screening yielded three improved CYP4A11 mutants, CP2600 (A24T/T263A), CP2601 (T263A), and CP2616 (A24T/T263A/V430E) with ~3-fold increase in whole cells and >10-fold increase in purified proteins on the luminescence assay. However, the steady state kinetic analysis for lauric acid hydroxylation showed the significant reductions in enzymatic activities in all three mutants. A mutant, CP2600, showed a 51% decrease in catalytic efficiency (kcat/Km) for lauric acid hydroxylation mainly due to an increase in Km. CP2601 and CP2616 showed much greater reductions (>75%) in the catalytic efficiency due to both a decrease in kcat and an increase in Km. These decreased catalytic activities of CP2601 and CP2616 can be partially attributed to the changes in substrate affinities. These results suggest that the enzymatic activities of CYP4A11 mutants selected from directed evolution using a luminogenic P450 substrate may not demonstrate a direct correlation with the hydroxylation activities of lauric acid.

Expression and Characterization of Truncated Recombinant Human Cytochrome P450 2J2

The human cytochrome P450 2J2 catalyzes an epoxygenase reaction to oxidize various fatty acids including arachidonic acid. In this study, three recombinant enzyme constructs of P450 2J2 were heterologously expressed in Escherichia coli and their P450 proteins were successfully purified using a Ni2+-NTA affinity column. Deletion of 34 amino acid residues in N-terminus of P450 2J2 enzyme (2J2-D) produced the soluble enzyme located in the cytosolic fraction. The enzymatic analysis of this truncated protein indicated the typical spectral characteristics and functional properties of P450 2J2 enzyme. P450 2J2-D enzymes from soluble fraction catalyzed the oxidation reaction of terfenadine to the hydroxylated product. However, P450 2J2-D enzymes from membrane fraction did not support the P450 oxidation reaction although it displayed the characteristic CO-binding spectrum of P450. Our finding of these features in the N-terminal modified P450 2J2 enzyme could help understand the biological functions and the metabolic roles of P450 2J2 enzyme and make the crystallographic analysis of the P450 2J2 structure feasible for future studies.

Bioactivation of Aromatic Amines by Human CYP2W1, An Orphan Cytochrome P450 Enzyme

The human genome contains approximately 13 orphan cytochrome P450 (P450, CYP) genes, of which the apparent function or substrate has not been identified. However, they seem to possess their own biological relevance in some tissues or developmental stages. Here, we characterized the heterologously expressed CYP2W1, an orphan P450 enzyme. The recombinant CYP2W1 protein containing a 6 × (His) -tag at Nterminus has been expressed in Escherichia coli and purified. Expression level of CYP2W1 holoenzyme was around 500 nmol P450 holoenzyme per liter culture medium. The reduced CO difference spectrum of CYP2W1 showed a maximum absorption at 449 nm. CYP2W1 indicated the significant induction to bioactivate Trp-P-1, MeIQ, and IQ in E. coli DJ701 tester strain. However, the bioactivation of B[α]P, and NNK by CYP2W1 was relatively low. The model structure of CYP2W1 suggested the characteristic P450 folds with the lengths and orientations of the individual secondary elements. The F-G loop is situated on the distal side of heme to accommodate the flexibility of active site of CYP2W1. These studies can provide useful information for the finding of its biological roles and structure-function relationships of an orphan CYP2W1 enzyme.

Structural Analysis of the Streptomyces avermitilis CYP107W1-Oligomycin A Complex and Role of the Tryptophan 178 Residue

CYP107W1 from Streptomyces avermitilis is a cytochrome P450 enzyme involved in the biosynthesis of macrolide oligomycin A. A previous study reported that CYP107W1 regioselectively hydroxylated C12 of oligomycin C to produce oligomycin A, and the crystal structure of ligand free CYP107W1 was determined. Here, we analyzed the structural properties of the CYP107W1-oligomycin A complex and characterized the functional role of the Trp178 residue in CYP107W1. The crystal structure of the CYP107W1 complex with oligomycin A was determined at a resolution of 2.6 Å. Oligomycin A is bound in the substrate access channel on the upper side of the prosthetic heme mainly by hydrophobic interactions. In particular, the Trp178 residue in the active site intercalates into the large macrolide ring, thereby guiding the substrate into the correct binding orientation for a productive P450 reaction. A Trp178 to Gly mutation resulted in the distortion of binding titration spectra with oligomycin A, whereas binding spectra with azoles were not affected. The Gly178 mutant’s catalytic turnover number for the 12-hydroxylation reaction of oligomycin C was highly reduced. These results indicate that Trp178, located in the open pocket of the active site, may be a critical residue for the productive binding conformation of large macrolide substrates.

Polymer Micelle Formulation for the Proteasome Inhibitor Drug Carfilzomib: Anticancer Efficacy and Pharmacokinetic Studies in Mice

Carfilzomib (CFZ) is a peptide epoxyketone proteasome inhibitor approved for the treatment of multiple myeloma (MM). Despite the remarkable efficacy of CFZ against MM, the clinical trials in patients with solid cancers yielded rather disappointing results with minimal clinical benefits. Rapid degradation of CFZ in vivo and its poor penetration to tumor sites are considered to be major factors limiting its efficacy against solid cancers. We previously reported that polymer micelles (PMs) composed of biodegradable block copolymers poly(ethylene glycol) (PEG) and poly(caprolactone) (PCL) can improve the metabolic stability of CFZ in vitro. Here, we prepared the CFZ-loaded PM, PEG-PCL-deoxycholic acid (CFZ-PM) and assessed its in vivo anticancer efficacy and pharmacokinetic profiles. Despite in vitro metabolic protection of CFZ, CFZ-PM did not display in vivo anticancer efficacy in mice bearing human lung cancer xenograft (H460) superior to that of the clinically used cyclodextrin-based CFZ (CFZ-CD) formulation. The plasma pharmacokinetic profiles of CFZ-PM were also comparable to those of CFZ-CD and the residual tumors that persisted in xenograft mice receiving CFZ-PM displayed an incomplete proteasome inhibition. In summary, our results showed that despite its favorable in vitro performances, the current CFZ-PM formulation did not improve in vivo anticancer efficacy and accessibility of active CFZ to solid cancer tissues over CFZ-CD. Careful consideration of the current results and potential confounding factors may provide valuable insights into the future efforts to validate the potential of CFZ-based therapy for solid cancer and to develop effective CFZ delivery strategies that can be used to treat solid cancers.

Enhanced Purification of Recombinant Rat NADPH-P450 Reductase by Using a Hexahistidine-Tag

NADPH-P450 reductase (NPR) transfers electrons from NADPH to cytochrome P450 and heme oxygenase enzymes to support their catalytic activities. This protein is localized within the endoplasmic reticulum membrane and utilizes FMN, FAD, and NADPH as cofactors. Although NPR is essential toward enabling the biochemical and pharmacological analyses of P450 enzymes, its production as a recombinant purified protein requires a series of tedious efforts and a high cost due to the use of NADP+ in the affinity chromatography process. In the present study, the rat NPR clone containing a 6× Histidine-tag (NPR-His) was constructed and heterologously expressed. The NPR-His protein was purified using Ni2+-affinity chromatography, and its functional features were characterized. A single band at 78 kDa was observed from SDS-PAGE and the purified protein displayed a maximum absorbance at 455 nm, indicating the presence of an oxidized flavin cofactor. Cytochrome c and nitroblue tetrazolium were reduced by purified NPR-His in an NADPH-dependent manner. The purified NPR-His successfully supported the catalytic activities of human P450 1A2 and 2A6 and fungal CYP52A21, yielding results similar to those obtained using conventional purified rat reductase. This study will facilitate the use of recombinant NPR-His protein in the various fields of P450 research.

Functional Significance of Cytochrome P450 1A2 Allelic Variants, P450 1A2*8, *15, and *16 (R456H, P42R, and R377Q).

P450 1A2 is responsible for the metabolism of clinically important drugs and the metabolic activation of environmental chemicals. Genetic variations of P450 1A2 can influence its ability to perform these functions, and thus, this study aimed to characterize the functional significance of three P450 1A2 allelic variants containing nonsynonymous single nucleotide polymorphisms (P450 1A2*8, R456H; *15, P42R; *16, R377Q). Variants containing these SNPs were constructed and the recombinant enzymes were expressed and purified in Escherichia coli. Only the P42R variant displayed the typical CO-binding spectrum indicating a P450 holoenzyme with an expression level of ∼ 170 nmol per liter culture, but no P450 spectra were observed for the two other variants. Western blot analysis revealed that the level of expression for the P42R variant was lower than that of the wild type, however the expression of variants R456H and R377Q was not detected. Enzyme kinetic analyses indicated that the P42R mutation in P450 1A2 resulted in significant changes in catalytic activities. The P42R variant displayed an increased catalytic turnover numbers (kcat) in both of methoxyresorufin O-demethylation and phenacetin O-deethylation. In the case of phenacetin O-deethylation analysis, the overall catalytic efficiency (kcat/Km) increased up to 2.5 fold with a slight increase of its Km value. This study indicated that the substitution P42R in the N-terminal proline-rich region of P450 contributed to the improvement of catalytic activity albeit the reduction of P450 structural stability or the decrease of substrate affinity. Characterization of these polymorphisms should be carefully examined in terms of the metabolism of many clinical drugs and environmental chemicals.