CYP Family Members Research Articles

Abstract 7277: Discovery of INV-9956, an orally available and highly selective Cyp11A1 inhibitor for the treatment of castration-resistant prostate cancer with drug resistant AR mutations

Abstract Androgen Receptor (AR) signaling plays a profound role in prostate cancer and development. Efforts in harnessing androgen and AR axis for castration-resistant prostate cancer treatment have led to discovery of several therapeutics, including AR antagonists and Cyp17 inhibitors. However, resistance to these approved therapeutics has emerged, and remained a significant unmet medical need. Among the prominent resistance mechanisms of action are restored androgen signaling via AR amplification and overexpression, and gain-of-function mutations occurring at AR ligand binding domain that can be agonized by existing AR antagonists and/or non-androgenic steroids such as glucocorticoids. Targeting Cyp11A1 that catalyzes the formation of pregnenolone is effective to ablate biosynthesis of androgens and all steroids and thus holds promise to deliver coverage of a significant percentage of drug resistant AR mutations and benefit CRPC patients post treatment with AR antagonists or Cyp17 inhibitors. Here we present discovery and characterization of INV-9956, an orally available, selective, and potent Cyp11A1 inhibitor with well-acceptable pharmacokinetic properties. INV-9956 potently inhibits biosynthesis of pregnenolone and other androgens in H295R cells with IC50 at single-digit nanomolar, suppresses proliferation of prostate cancer cell lines harboring major clinically acquired drug resistant AR LBD mutations, such as AR L702H using coculture assays. Orally administered INV-9956 showed consistent dose dependent PK/PD correlations across animal species. Particularly, INV-9956 displayed pronounced tumor inhibition superior to known competitors in a CRPC VCaP xenograft model and in a “humanized” animal model. Furthermore, INV-9956 displayed a good tox profile in 14-day repeated dosing rat and dog studies, supported by a high selectivity to Cyp11A1 over other Cyp family members, clean hERG and mini-Ames, and no significant off-target activity in a safety panel screen. The promising preclinical profiles of INV-9956 lend strong support to its potentials in addressing unmet needs in CRPC treatments and warrant clinical development. Citation Format: Chang Bai, Danqi Chen, Zilei Xia, Shujuan Huang, LIshan Lin, Zhaopie Zeng, Jing Su, Yongkui Sun. Discovery of INV-9956, an orally available and highly selective Cyp11A1 inhibitor for the treatment of castration-resistant prostate cancer with drug resistant AR mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7277.

Detrimental impacts of concomitant application of cadmium and pesticides are ameliorated by 24-epibrassinolide through alteration in oxidative status and CYP genes expression in Zea mays L.

The ameliorative impact of 24-epibrassinolide (EBL) was assessed under toxicity of cadmium (Cd) and three different pesticides including diazinon (DZ), imidacloprid (IMI), and chlorpyrifos (CPS) in maize plants. Maize seeds were pre-treated with 0.01 μM of EBL and grown in soil contaminated with 100 μM Cd for one month. Then, at five fully expanded leaf stage, plants received DZ, IMI, and CPS pesticides separately by foliar spray and were harvested five days later. The EBL seed primed plants, grown in soil contaminated with Cd and exposed to pesticides, exhibited higher deleterious photosynthesis (Pn) and stomatal conductivity (Gs) compared to un-EBL-treated plants. A considerable decrease was observed in the pesticide's residue and Cd, MDA and H2O2 contents of EBL + Cd + Pesticide treated plants compared to the Cd + Pesticide groups plants. Higher phenol, flavonoid and protein contents, as well as increment in SOD, CAT, POD, APX, PAL, GST, and GR enzymes activity were detected in EBL + Cd + Pesticide groups compared to the plants imposed with Cd + Pesticides. The expression analyses of four CYP family members including CYP71C1, CYP71C3v2, CYP72A5, and CYP81A9 revealed downregulation under Cd and Cd + pesticides treatments, while EBL seed priming highly induced transcript abundance of CYP71C1 (EBL + Cd + IMI); CYP71C3v2 (EBL + Cd + DZ & EBL + Cd + IMI) and CYP81A9 (EBL + Cd + DZ & EBL + Cd + CPS), specifically. The higher mRNA accumulation of CYP72A5 gene was exhibited under all three different EBL + Cd + pesticides treatments. The 24-epibrassinolide alleviates the adverse impacts of Cd and pesticides via the activation of enzymatic and non-enzymatic components to eliminate ROS through the up-regulation of CYP genes. This facilitates conjugation of glutathione to pesticides residue, leading to their rapid transportation into the vacuole or out of the membrane.

Involvement of glyphosate in disruption of biotransformation P450 enzymes and hepatic lipid metabolism in chicken

The current study investigated the potentially harmful consequences of pure glyphosate or Roundup® on CYP family members and lipid metabolism in newly hatched chicks. On the sixth day, 225 fertilized eggs were randomly divided into three treatments: (1) the control group injected with deionized water, (2) the glyphosate group injected with 10 mg pure glyphosate/Kg egg mass and (3) the Roundup group injected 10 mg the active ingredient glyphosate in Roundup®/Kg egg. The results of the study revealed a reduction in hatchability in chicks treated with Roundup®. Moreover, change of Lipid concentration in serum and the liver-treated groups. Additionally, increased liver function enzymes and increased oxidative stress in the glyphosate and Roundup® groups. Furthermore, liver tissues showed histological changes and several lipid deposits in glyphosate-treated groups. Hepatic CYP1A2 and CYP1A4 expressions were significantly increased (p < .05) after glyphosate exposure, and suppression of CYP1C1 mRNA expression was significant (p < .05) after Roundup® exposure. The pro-inflammatory cytokines genes IFN-γ and IL-1β expression were significantly increased (p < .05) after Roundup® exposure. In addition, there were significant differences in the levels of expression genes which are related to lipid synthesis or catabolism in the liver. In conclusion, in ovo glyphosate exposure caused disruption of biotransformation, pro-inflammatory and lipid metabolism in chicks.

Identification of Potential Cytochrome P450 3A5 Inhibitors: An Extensive Virtual Screening through Molecular Docking, Negative Image-Based Screening, Machine Learning and Molecular Dynamics Simulation Studies.

Cytochrome P450 3A5 (CYP3A5) is one of the crucial CYP family members and has already proven to be an important drug target for cardiovascular diseases. In the current study, the PubChem database was screened through molecular docking and high-affinity molecules were adopted for further assessment. A negative image-based (NIB) model was used for a similarity search by considering the complementary shape and electrostatics of the target and small molecules. Further, the molecules were segregated into active and inactive groups through six machine learning (ML) matrices. The active molecules found in each ML model were used for in silico pharmacokinetics and toxicity assessments. A total of five molecules followed the acceptable pharmacokinetics and toxicity profiles. Several potential binding interactions between the proposed molecules and CYP3A5 were observed. The dynamic behavior of the selected molecules in the CYP3A5 was explored through a molecular dynamics (MD) simulation study. Several parameters obtained from the MD simulation trajectory explained the stability of the protein–ligand complexes in dynamic states. The high binding affinity of each molecule was revealed by the binding free energy calculation through the MM-GBSA methods. Therefore, it can be concluded that the proposed molecules might be potential CYP3A5 molecules for therapeutic application in cardiovascular diseases subjected to in vitro/in vivo validations.

Correlation between CYP1A1 polymorphisms and susceptibility to glyphosate-induced reduction of serum cholinesterase: A case-control study of a Chinese population

Glyphosate (GLP) is one of the most common herbicides worldwide. The serum cholinesterase (ChE) may be affected when exposed to glyphosate. Reduction of serum ChE by herbicides is probably related to cytochrome P450 (CYP450) family polymorphisms. We suspect that the abnormal ChE caused by GLP could be correlated with the CYP family members. To determine whether CYP1B1 (rs1056827 and rs1056836) and CYP1A1 (rs1048943) gene polymorphisms and individual susceptibility to GLP-induced ChE abnormalities were interrelated in the Chinese Han population, we performed this genetic association study on a total of 230 workers previously exposed to GLP, including 115 cases with reduced serum ChE and 115 controls with normal serum ChE. Two even groups of cases and controls were enrolled. The CYP1A1 and CYP1B1 polymorphisms in both groups were genotyped using TaqMan. Subjects with the CYP1A1 rs619586 genotypes showed an increased risk of GLP-induced reduction of serum ChE, which was more evident in the following subgroups: female, > 35 years old, history of GLP exposure time <10 years and >10 years, nonsmoker and nondrinker. The results show that CYP1A1 rs619586 was significantly associated with the GLP-induced reduction in serum ChE and could be a biomarker of susceptibility for Chinese GLP exposed workers. Because of a large number of people exposed to glyphosate, this study has a significance in protecting their health.

Transcriptome analysis of self- and cross-pollinated pistils revealing candidate unigenes of self-incompatibility in Camellia oleifera

ABSTRACTSelf-incompatibility (SI) is a mechanism used to prevent inbreeding and promote outcrossing in a wide variety of angiosperms. Camellia oleifera (Theaceae), is one of the most prominent wood oil plants. However, self-incompatibility results in a relatively low ratio of fruit/seed sets to flowers in C. oleifera. In this study, fluorescence microscopy and confocal microscopy were used to observe how pollen tube growth differed between cross- vs. self-pollination events. We found that self-pollen tubes appear twisted and clustered when growing to the base of the style and it is impossible for self-pollen tubes to reach the embryo sac to complete double fertilization. In contrast, pollen tubes from crossing events grow normally and reach the ovary to complete fertilization. We characterised candidate genes related to SI and investigated the underlying mechanisms regulating fertilization by performing RNA-seq on C. oleifera. In addition, we also conducted qRT-PCR on 20 unigenes to validate our RNA-seq data. We identified genes encoding protein kinases and phosphatases, Ca2+ signaling proteins, transcription factors, CYP family members, and defense-related proteins as candidate genes related to SI in C. oleifera. With these genes identified, more thorough studies of SI mechanisms in C. oleifera have become possible.

Analysis and preliminary characterisation of the cytochrome P450 monooxygenases from Frankia sp. EuI1c (Frankia inefficax sp.)

Frankia bacteria are nitrogen fixing species from the Actinobacterium phylum which live on the root nodules of plants. They have been hypothesised to have significant potential for natural product biosynthesis. The cytochrome P450 monooxygenase complement of Frankia sp. EuI1c (Frankia inefficax sp.), which comprises 68 members, was analysed. Several members belonged to previously uncharacterised bacterial P450 families. There was an unusually high number of CYP189 family members (21) suggesting that this family has undergone gene duplication events which are classified as “blooms”. The likely electron transfer partners for the P450 enzymes were also identified and analysed. These consisted of predominantly [3Fe–4S] cluster containing ferredoxins (eight), a single [2Fe–2S] ferredoxin and a couple of ferredoxin reductases. Three of these CYP family members were produced and purified, using Escherichia coli as a host, and their substrate range was characterised. CYP1027H1 and CYP150A20 bound a broad range of norisoprenoids and terpenoids. CYP1074A2 was highly specific for certain steroids including testosterone, progesterone, stanolone and 4-androstene-3,17-dione. It is likely that steroids are the physiological substrates of CYP1074A2. These results also give an indication that terpenoids are the likely substrates of CYP1027H1 and CYP150A2. The large number of P450s belonging to distinct families as well as the associated electron transfer partners found in different Frankia strains highlights the importance of this family of enzymes has in the secondary metabolism of these bacteria.

Abstract LB-272: Discovery and characterization of a novel FGFR4 Inhibitor for the treatment of hepatocellular carcinoma

Abstract Introduction: Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related deaths worldwide and has one of the fastest growing incidence among cancers. Aberrant FGF19-FGFR4 signaling occurs in 10-30% of HCC patients and drives tumor development and progression. Pan-FGFR inhibitors are associated with hyperphosphatemia in clinic due to inhibition of FGFR1. Therefore, selectively targeting FGFR4 may render a safer treatment approach with improved clinical benefits. Using advanced computation-aided structural analysis and medicinal chemistry design, we have discovered a novel and highly selective small molecular FGFR4 inhibitor, ABSK-011, demonstrating robust anti-tumor activity in hepatocellular carcinoma models. Method: ABSK-011 was evaluated in biochemical Caliper assay and Celltiter-Glo assay for its inhibition on FGFR4 enzymatic and cellular activities. Its selectivity against other FGFR family members was analyzed through related biochemical, cellular, and KinomeScan profiling assays. The pharmacokinetics (PK) profile of ABSK-011 was characterized across various species of animals including mouse, rat and dog. Efficacy studies were conducted in two HCC xenograft models, and preliminary toxicity profile was also evaluated. Results: ABSK-011 inhibits FGFR4 with IC50 &amp;lt; 10 nM in biochemical assay and exhibits at least 50 fold selectivity against other FGFR kinases. The strong selectivity arises from the ability of ABSK-011 to irreversibly modify Cys552, a residue within the active site of FGFR4 that is not conserved among other FGFR kinases. ABSK-011 also shows greater than 800-fold selectivity against other RTK members. In HCC cells with autocrine production of FGF19, ABSK-011 inhibits the auto-phosphorylation of FGFR4 and blocks signal transduction from FGFR4 to downstream pathway activation. HCC cell lines harboring amplification and overexpression of FGF19 are uniquely sensitive to growth inhibition by ABSK-011 (EC50 &amp;lt; 50 nM) compared with cancer cell lines with FGFR1 dependence (EC50 &amp;gt; 2000 nM), confirming selectivity for FGFR4 over FGFR1. In vivo PK study demonstrated high exposure and superior DMPK profile of ABSK-011 in mouse, rat and dog. In preclinical in vivo studies, oral administration of ABSK-011 strongly inhibits the growth and induces regressions of subcutaneous xenograft tumors dependent upon FGFR4 activity. Suppression of tumor growth is dose dependent and well correlated with pharmacodynamic inhibition of FGFR4 signaling. Safety studies showed that ABSK-011 has no inhibition on CYP family members, hERG, etc. Conclusion: ABSK-011, presented here by Abbisko Therapeutics, is a highly potent, selective, and oral-available small molecular FGFR4 inhibitor. Its superior profile supports fast-track preclinical development. Citation Format: Zhui Chen. Discovery and characterization of a novel FGFR4 Inhibitor for the treatment of hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-272.

Abstract 3888: The NantOmics Pharmacogenomics Test: An integrative panomic approach to pharmacogenomics screening

Abstract Numerous oncology drugs have pharmacogenomics warnings on their FDA labels, yet pharmacogenomics screening is not routinely applied in clinical practice. Pharmacogenomics testing is used to reduce the chance of drug-induced toxicities, improve patient outcomes, and reduce treatment costs by tailoring therapies to the patient's genotype. Here we present the NantOmics pharmacogenomics test, which uses whole genome (DNA) and whole exome sequencing data from FFPE tumors and matched normal samples to predict and advise physicians of possible drug-induced toxicities. Using FDA labels and Clinical Pharmacogenomics Implementation Consortium (CPIC) guidelines, we developed a clinical pharmacogenomics panel including 31 markers in 11 genes linked to toxicities from 14 cancer therapies, as well as a research panel of 13 gene-drug pairs reported in primary literature. The test screens for both germline and somatic variants to predict how an oncology patient may respond to routinely-used chemotherapies. Several genes in our panel, including CYP2D6 and other CYP family members, prove challenging to properly genotype due to high levels of polymorphism, multiple similar haplotypes, and structural variations. We have established a kmer-based approach to accurately determine haplotypes for these genes, as well as coverage-based methods to infer hybridizations and copy number variations. For pharmacogenes with ambiguous genotypes comprising multiple heterozygous SNPs, we utilize any allele fraction imbalance observed at these loci in the tumor DNA and/or RNA sequencing data to disambiguate the correct genotype. The test was run on 1,172 patient samples, 95.8% of which contained a variant in at least one gene from our panel. Furthermore, 7.5% of patients had genomic variants associated with severe or life-threatening drug toxicities. For all alleles in our clinical panel, we observed similar allele frequencies to those reported in the ExAC database. Our test was validated on several cell lines from the CDC GeT-RM, and was able to accurately determine the genotypes for all genes present in our test panels in each of the cell lines tested. To further validate our test, we ran it on a cohort of patients previously genotyped by an independent CLIA-validated PCR-based panel, as well as on a set of synthetic data. In all validation studies, we were able to demonstrate that the test detects each variant in our panel, and correctly determines patient genotype in all studied cases. Given the high percentage of patients with potentially treatment-altering genomic variants, these results underscore the need for more routine pharmacogenomics screening in the oncological setting. Citation Format: Camille Schwartz, John Little, Charles Vaske, Stephen Benz, Patrick Soon-Shiong, Shahrooz Rabizadeh, J. Zachary Sanborn. The NantOmics Pharmacogenomics Test: An integrative panomic approach to pharmacogenomics screening [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3888.

Abstract LB-288: A highly selective small molecule CSF-1R inhibitor demonstrates strong immunomodulatory activity in syngeneic models

Abstract Introduction: CSF-1R is the receptor for Colony-Stimulating Factor 1 (CSF-1), which belongs to the type III protein tyrosine kinase family. CSF-1R signaling is critical for macrophage/myeloid-derived suppressing cells (MDSC) recruitment, proliferation, and survival. Inhibition of CSF-1R leads to the reduction and repolarization of macrophages and MDSCs in tumor microenvironment, offering potential combinatory synergy with immune-oncology checkpoint antibodies for the treatment of multiple cancers. By using state-of-art computational structure analysis and medicinal chemistry design, we have discovered a novel, highly selective, and potent small molecule CSF-1R inhibitor, ABSK-021. Method: ABSK-021 was evaluated in biochemical ADP-Glo assay and Cell-Titer Glo assay for its inhibition on CSF-1R enzymatic activity and cellular proliferation. Its selectivity against other receptor tyrosine kinases was analyzed by ELISA, cellular proliferation, and KinomeScan assays. The pharmacokinetics (PK) profile of ABSK-021 was characterized in four species of animals (mouse, rat, dog, monkey). Efficacy studies were conducted in two syngeneic models, and flow cytometry analysis was performed in tumor-bearing mice after short term dosing. Its preliminary toxicity profile was also generated by several classical safety assays. Results: ABSK-021 has potent biochemical and cellular activities against CSF-1R (IC50&amp;lt;30 nM), resulting in strong inhibition of CSF-1R phosphorylation and the proliferation of bone-marrow derived macrophage at low nM concentration. ABSK-021 has over 40-fold selectivity against KIT/FLT3/PDGFR-alpha/VEGFR2, which are in the same tyrosine kinase family with CSF-1R. KinomeScan also revealed its excellent selectivity against other receptor tyrosine kinases. In vivo PK study demonstrated high exposure and superior DMPK profile of ABSK-021 in mouse, rat, dog, and monkey. Efficacy studies showed that ABSK-021 strongly enhanced anti-tumor efficacy of anti-PD-1 antibody in syngeneic models. Reduction of tumor-associated macrophage and MDSC after ABSK-021 dosing was confirmed by flow cytometry analysis. Safety studies showed that ABSK-021 has no inhibition on CYP family members, hERG, and other 43 safety targets in CEREP's SafetyScreen panel (IC50&amp;gt;10 μM). Conclusion: ABSK-021, presented here by Abbisko Therapeutics, is a highly potent, selective, and oral-available small molecule CSF-1R inhibitor. Its superior profile supports fast-track preclinical development. Citation Format: Shuqun Yang, Baowei Zhao, Mingming Zhang, Yuan Zhao, Xin Chen, Lei Zhang, Hongping Yu, Zhui Chen, Yaochang Xu. A highly selective small molecule CSF-1R inhibitor demonstrates strong immunomodulatory activity in syngeneic models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-288.

Sex‐dependent regulation of cytochrome P450 family members Cyp1a1, Cyp2e1, and Cyp7b1 by methylation of DNA

Sexual differences are only partially attributable to hormones. Cultured male or female cells, even from embryos before sexual differentiation, differ in gene expression and sensitivity to toxins, and these differences persist in isolated primary cells. Male and female cells from Swiss Webster CWF mice manifest sex-distinct patterns of DNA methylation for X-ist and for cytochrome P450 (CYP; family members 1a1, 2e1m, and 7b1. Dnmt3l is differentially expressed but not differentially methylated, and Gapdh is neither differentially methylated nor expressed. CYP family genes differ in expression in whole tissue homogenates and cell cultures, with female Cyp expression 2- to 355-fold higher and Dnmt3l 12- to 32-fold higher in males. DNA methylation in the promoters of these genes is sex dimorphic; reducing methylation differences reduces to 1- to 6-fold differences in the expression of these genes. Stress or estradiol alters both methylation and gene expression. We conclude that different methylation patterns partially explain the sex-based differences in expression of CYP family members and X-ist, which potentially leads to inborn differences between males and females and their different responses to chronic and acute changes. Sex-differential methylation may have medical effects.

Three novel cytochrome P450 genes identified in the marine polychaete Perinereis nuntia and their transcriptional response to xenobiotics

Polychaetes have previously been used as bioindicators of environmental pollution. Their ability to eliminate organic pollutants such as polycyclic aromatic hydrocarbons (PAH) has been extensively analyzed. However, the cytochrome P450 monooxygenases (CYP) genes in polychaetes, which catalyze the first step of oxidative degradation of PAHs, have received little attention. Based on the partial sequences of three CYP genes that were enriched by subtractive cDNA libraries of the polychaete Perinereis nuntia, we amplified and sequenced the full-length cDNA of these novel CYP genes. These genes were named CYP4BB2, CYP423A1 and CYP424A1 by the Cytochrome P450 Nomenclature Committee. The deduced amino acid sequence of CYP4BB2 in P. nuntia showed 68% sequence identity to CYP4BB1 in Nereis virens, and was listed as a new member of the CYP4BB subfamily. The sequence of CYP423A1 and CYP424A1 both share less than 40% sequence identity to all known CYP enzymes and were classed into new CYP families. CYP family members are composite parts of a larger group called a clan. CYP4BB2 and CYP424A1 are listed as CYP4 clan members, while CYP423A1 is of the CYP2 clan. The 3D structures of these P. nuntia CYPs were successfully predicted by homology-modeling using the SWISS-MODEL workspace. The models of CYP424A1 and CYP4BB2 were created using 1jpzB (CYP102A) as a template, while CYP423A1 utilized 3czhB (CYP2R1) as its template. The presence of characteristic CYP superfamily motifs, such as the F-G⋯C-G amino acid sequence, and the conservation of the three-dimensional CYP structure shown by the modeling, suggested that these novel P. nuntia CYP genes may contain conserved functional domains of CYP monooxygenases. To examine the effect of xenobiotics on living organisms, we analyzed the transcriptional levels of these three new CYP genes in sandworms (P. nuntia) exposed to seawater artificially contaminated with benzo[a]pyrene (BaP). We also exposed individuals to industrial wastewater collected from Quanzhou Bay, Fujian, China, which was known to be contaminated with PAHs. Worms exposed to BaP had significantly higher levels of CYP4BB2, CYP423A1 and CYP424A1 mRNA. Transcription was up-regulated 5.9-, 5.3- and 12.3-folds respectively compared with the control worms living in clean seawater. The transcriptional levels of CYPs in worms cultured in the diluted wastewater collected from Quanzhou Bay, all positively correlated with the levels of PAHs detected in the water. The transcriptional up-regulation of the three CYP genes observed in this study, suggest the monooxygenases encoded by these CYP genes may play an important role in the detoxification of PAHs in this polychaete worm. These CYPs maybe essential for the adaptation of worms to contaminated environments and may be useful in the assessment of xenobiotic exposure.

Cyclophilin Inhibitors Block Arterivirus Replication by Interfering with Viral RNA Synthesis

Virus replication strongly depends on cellular factors, in particular, on host proteins. Here we report that the replication of the arteriviruses equine arteritis virus (EAV) and porcine reproductive and respiratory syndrome virus (PRRSV) is strongly affected by low-micromolar concentrations of cyclosporine A (CsA), an inhibitor of members of the cyclophilin (Cyp) family. In infected cells, the expression of a green fluorescent protein (GFP) reporter gene inserted into the PRRSV genome was inhibited with a half-maximal inhibitory concentration (IC(50)) of 5.2 μM, whereas the GFP expression of an EAV-GFP reporter virus was inhibited with an IC(50) of 0.95 μM. Debio-064, a CsA analog that lacks its undesirable immunosuppressive properties, inhibited EAV replication with an IC(50) that was 3-fold lower than that of CsA, whereas PRRSV-GFP replication was inhibited with an IC(50) similar to that of CsA. The addition of 4 μM CsA after infection prevented viral RNA and protein synthesis in EAV-infected cells, and CsA treatment resulted in a 2.5- to 4-log-unit reduction of PRRSV or EAV infectious progeny. A complete block of EAV RNA synthesis was also observed in an in vitro assay using isolated viral replication structures. The small interfering RNA-mediated knockdown of Cyp family members revealed that EAV replication strongly depends on the expression of CypA but not CypB. Furthermore, upon fractionation of intracellular membranes in density gradients, CypA was found to cosediment with membranous EAV replication structures, which could be prevented by CsA treatment. This suggests that CypA is an essential component of the viral RNA-synthesizing machinery.

Research on relationship between cyclophilin and carcinomas

It has been demonstrated that certain members of cyclophilins (CyP) family are related to tomurigenesis, which might result from the role for Cyp in cell proliferation. Most members of Cyp family express specifically in tumor tissue in contrast to in normaltissue and cancer-brink tissue. It is possible that a novel approach to cancer therapy and diagnosis would be appeared through exploring the relationship between Cyp and tumor. Key words: Neoplasms; Cyclophilins

In utero tobacco exposure epigenetically modifies placental CYP1A1 expression

The metabolic pathways used by higher-eukaryotic organisms to deal with potentially carcinogenic xenobiotic compounds from tobacco smoke have been well characterized. Carcinogenic compounds such as polycyclic aromatic hydrocarbons are metabolized sequentially in 2 phases: in phase I, CYP1A1 catalyzes conversion into harmful hydrophilic DNA adducts, whereas in phase II, GSTT1 enables excretion via conjugation into polar electrophiles. In an effort to understand susceptibility to in utero tobacco exposure, we previously characterized known metabolic functional polymorphisms and demonstrated that although deletion of fetal GSTT1 significantly modified birth weight in smokers, no polymorphism fully accounted for fetal growth restriction. Because smoking up-regulates CYP1A1 expression, we hypothesized that nonallelic (epigenetic) dysregulation of placental CYP1A1 expression via alterations in DNA methylation (meCpG) may further modify fetal growth. In the present article, we compared placental expression of multiple CYP family members among gravidae and observed significantly increased CYP1A1 expression among smokers relative to controls (4.4-fold, P < .05). To fully characterize CYP1A1 meCpG status, bisulfite modification and sequencing of the entire proximal 1-kilobase promoter (containing 59 CpG sites) were performed. CpG sites immediately proximal to the 5′-xenobiotic response element transcription factor binding element were significantly hypomethylated among smokers (55.6% vs 45.9% meCpG, P = .027), a finding that uniquely correlated with placental gene expression ( r = 0.737, P = .007). Thus, in utero tobacco exposure significantly increases placental CYP1A1 expression in association with differential methylation at a critical xenobiotic response element.

Identification of differential expression of genes in hepatocellular carcinoma by suppression subtractive hybridization combined cDNA microarray

The genetic background of hepatocellular carcinoma (HCC) has yet to be completely understood. Here, we describe the application of suppression subtractive hybridization (SSH) coupled with cDNA microarray analysis for the isolation and identification of differential expression of genes in HCC. Twenty-six known genes were validated as up-regulated and 19 known genes as down-regulated in HCC. The known genes identified were found to have diverse functions. In addition to the overexpression of AFP, these genes (increased in the presence of HCC) are involved in many processes, such as transcription and protein biosynthesis (HNRPDL, PABPC1, POLR2K, SRP9, SNRPA, and six ribosomal protein genes including RPL8, RPL14, RPL41, RPS5, RPS17, RPS24), the metabolism of lipids and proteins (FADS1, ApoA-II, ApoM, FTL), cell proliferation (Syndecan-2, and Annexin A2), and signal transduction (LRRC28 and FMR1). Additionally, a glutathione-binding protein involved in the detoxification of methylglyoxal known as GLO1 and an enzyme which increases the formation of prostaglandin E(2) known as PLA2G10 were up-regulated in HCC. Among the underexpressed genes discovered in HCC, most were responsible for liver-synthesized proteins (fibrinogen, complement species, amyloid, albumin, haptoglobin, hemopexin and orosomucoid). The enzyme implicated in the biotransformation of CYP family members (LOC644587) was decreased. The genes coding enzymes ADH1C, ALDH6A1, ALDOB, Arginase and CES1 were also found. Additionally, we isolated a zinc transporter (Zip14) and a function-unknown gene named ZBTB11 (Zinc finger and BTB domain containing 11) which were underexpressed, and seven expression sequence tags deregulated in HCC without significant homology reported in the public database. Essentially, by using SSH combined with a cDNA microarray we have identified a number of genes associated with HCC, most of which have not been previously reported. Further characterization of these differentially expressed genes will provide information useful in understanding the genes responsible for the development of HCC.