Efficacy and Quality of Life with the Modified Versus the Traditional Thoraco-Laparoscopic McKeown Procedure for Esophageal Cancer: A Multicenter Propensity Score-Matched Study.

BACKGROUND: This study aimed to compare the efficacy and postoperative quality of life for patients with esophageal cancer treated by either the modified or the traditional thoracolaparoscopic McKeown procedure. METHODS: This retrospective case-control study included 269 patients with esophageal cancer admitted to three medical centers in China from February 2020 to August 2022. The patients were divided according to surgical method into the layered hand-sewn end-to-end invagination anastomosis group (modified group) and the traditional hand anastomosis group (traditional group). Propensity score-matching (PSM) was used to maintain balance and comparability between the two groups. RESULTS: The differences in age and tumor location between the patients in the traditional and modified groups were statistically significant. After PSM, the aforementioned factors were statistically insignificant. After PSM, each group had 101 patients. The modified group showed the greater advantage in terms of postoperative hospital stay (P = 0.036), incidence of anastomotic leak (P = 0.009), and incidence of gastroesophageal reflux (P < 0.001), and the difference was statistically significant. The results of the Quality of Life Questionnaire Core 30 (QLQ-C30) and Quality of Life Questionnaire Oesophageal Cancer Module 18 (QLQ-OES18) scales showed that the modified group also had the advantage over the traditional group in terms of physical function, overall health status, loss of appetite, eating, reflux, obstruction, and loss of appetite scores at the first and third months after surgery. CONCLUSION: The modified thoraco-laparoscopic McKeown procedure is a safe and effective surgical approach that can significantly reduce the incidence of postoperative anastomotic leak and gastroesophageal reflux, shorten the postoperative hospital stay, and improve the postoperative quality of life for patients with esophageal cancer.

Identification and Analysis of bZIP Family Genes in Sedum plumbizincicola and Their Potential Roles in Response to Cadmium Stress.

Sedum plumbizincicola (Crassulaceae), a cadmium (Cd)/zinc (Zn)/lead (Pb) hyperaccumulator native to Southeast China, is potentially useful for the phytoremediation of heavy metal-contaminated soil. Basic leucine zipper (bZIP) transcription factors play vital roles in plant growth, development, and abiotic stress responses. However, there has been minimal research on the effects of Cd stress on the bZIP gene family in S. plumbizincicola. In this study, 92 SpbZIP genes were identified in the S. plumbizincicola genome and then classified into 12 subgroups according to their similarity to bZIP genes in Arabidopsis. Gene structure and conserved motif analyses showed that SpbZIP genes within the same subgroup shared similar intron-exon structures and motif compositions. In total, eight pairs of segmentally duplicated SpbZIP genes were identified, but there were no tandemly duplicated SpbZIP genes. Additionally, the duplicated SpbZIP genes were mainly under purifying selection pressure. Hormone-responsive, abiotic and biotic stress-responsive, and plant development-related cis-acting elements were detected in the SpbZIP promoter sequences. Expression profiles derived from RNA-seq and quantitative real-time PCR analyses indicated that the expression levels of most SpbZIP genes were upregulated under Cd stress conditions. Furthermore, a gene co-expression network analysis revealed that most edge genes regulated by hub genes were related to metal transport, responses to stimuli, and transcriptional regulation. Because its expression was significantly upregulated by Cd stress, the hub gene SpbZIP60 was selected for a functional characterization to elucidate its role in the root response to Cd stress. In a transient gene expression analysis involving Nicotiana benthamiana leaves, SpbZIP60 was localized in the nucleus. The overexpression of SpbZIP60 enhanced the Cd tolerance of transgenic Arabidopsis plants by inhibiting ROS accumulation, protecting the photosynthetic apparatus, and decreasing the Cd content. These findings may provide insights into the potential roles of the bZIP family genes during the S. plumbizincicola response to Cd stress.

Integrative analysis of transcriptome and proteome provides insights into adaptation to cadmium stress in Sedum plumbizincicola.

Sedum plumbizincicola, a cadmium (Cd) hyperaccumulating herbaceous plant, can accumulate large amounts of Cd in the above-ground tissues without being poisoned. However, the molecular mechanisms regulating the processes are not fully understood. In this study, Transcriptional and proteomic analyses were integrated to investigate the response of S. plumbizincicola plants to Cd stress and to identify key pathways that are potentially responsible for Cd tolerance and accumulation. A total of 630 DAPs (differentially abundant proteins, using fold change >1.5 and adjusted p-value <0.05) were identified from Tandem Mass Tag (TMT)- based quantitative proteomic profiling, which were enriched in processes including phenylpropanoid biosynthesis, protein processing in endoplasmic reticulum, and biosynthesis of secondary metabolites. Combined with the previous transcriptomic study, 209 genes and their corresponding proteins showed the identical expression pattern. The identified genes/proteins revealed the potential roles of several metabolism pathways, including phenylpropanoid biosynthesis, oxidative phosphorylation, phagosome, and glutathione metabolism, in mediating Cd tolerance and accumulation. Lignin staining and Cd accumulation assay of the transgenic lines over-expressing a selected Cd up-regulated gene SpFAOMT (Flavonoid 3',5'-methyltransferase) showed its functions in adapting to Cd stress, and provided insight into its role in lignin biosynthesis and Cd accumulation in S. plumbizincicola during Cd stress.

Viral Entry Inhibitors Targeting Six-Helical Bundle Core against Highly Pathogenic Enveloped Viruses with Class I Fusion Proteins.

Type Ⅰ enveloped viruses bind to cell receptors through surface glycoproteins to initiate infection or undergo receptor-mediated endocytosis and initiate membrane fusion in the acidic environment of endocytic compartments, releasing genetic material into the cell. In the process of membrane fusion, envelope protein exposes fusion peptide, followed by an insertion into the cell membrane or endosomal membrane. Further conformational changes ensue in which the type 1 envelope protein forms a typical six-helix bundle structure, shortening the distance between viral and cell membranes so that fusion can occur. Entry inhibitors targeting viral envelope proteins, or host factors, are effective antiviral agents and have been widely studied. Some have been used clinically, such as T20 and Maraviroc for human immunodeficiency virus 1 (HIV-1) or Myrcludex B for hepatitis D virus (HDV). This review focuses on entry inhibitors that target the six-helical bundle core against highly pathogenic enveloped viruses with class I fusion proteins, including retroviruses, coronaviruses, influenza A viruses, paramyxoviruses, and filoviruses.

Analysis of the short-term outcomes of biportal robot-assisted lobectomy.

BACKGROUND: The present study aimed to assess the short-term consequences of biportal robot-assisted lobectomy, validating its safety and effectiveness. METHODS: A retrospective analysis evaluated the clinical data and short-term results of 18 patients in the single medical group of the centre who underwent biportal robot-assisted lobectomy plus lymph node dissection from November 2020 to March 2021. RESULTS: Lobectomy and lymph node dissection could be successfully accomplished in all 18 patients with the assistance of a biportal robot; there was no conversion to thoracotomy during the operation. There were 10 males and 8 females with their ages ranging from 37 to 73 (58.83 ± 9.07) years. The total operation time was 74-146 (105.06 ± 18.22) min. Punching time was 2-9 (5.11 ± 1.74) min. Docking time was 8-16 (11.94 ± 2.41) min. Console time was 50-104 (78.06 ± 17.40) min. Chest closing time was 8-17 (10.28 ± 2.74) min. Blood loss was 60-132 (94.11 ± 41.41) ml. The number of lymph nodes dissected was 16-30 (21.78 ± 4.13). Chest tube duration was 2-10 (4.06 ± 1.98) days. Drainage on the first day following surgery was 100-500 (337.22 ± 117.01) ml. Total drainage was 370-1100 (692.78 ± 161.01) ml. Duration of hospital stay was 4-12 (5.89 ± 1.94) days. The median 24 and 72 h visual analogue score scores were 4 (3-7) and 3 (2-5). Total cost (¥) was 51 000-85 000 (68 000 ± 10 000), respectively. There was one case of atrial fibrillation and one case of pulmonary infection. The complication rate was 11.11%. No serious complications were recorded after surgery, and no deaths occurred within 30 days post-surgery. The final pathological diagnosis revealed 10 cases of squamous cell carcinoma, 7 cases of adenocarcinoma and 1 case of benign disease. CONCLUSION: The biportal robot-assisted lobectomy was found to be safe and effective in the treatment of lung cancer.

Brahma-related gene-1 promotes tubular senescence and renal fibrosis through Wnt/ß-catenin/autophagy axis.

Although accelerated cellular senescence is closely related to the progression of chronic kidney disease (CKD) and renal fibrosis, the underlying mechanisms remain largely unknown. Here, we reported that tubular aberrant expression of Brahma-related gene 1 (BRG1), an enzymatic subunit of the SWItch/Sucrose Non-Fermentable complex, is critically involved in tubular senescence and renal fibrosis. BRG1 was significantly up-regulated in the kidneys, predominantly in tubular epithelial cells, of both CKD patients and unilateral ureteral obstruction (UUO) mice. In vivo, shRNA-mediated knockdown of BRG1 significantly ameliorated renal fibrosis, improved tubular senescence, and inhibited UUO-induced activation of Wnt/ß-catenin pathway. In mouse renal tubular epithelial cells (mTECs) and primary renal tubular cells, inhibition of BRG1 diminished transforming growth factor-ß1 (TGF-ß1)-induced cellular senescence and fibrotic responses. Correspondingly, ectopic expression of BRG1 in mTECs or normal kidneys increased p16INK4a, p19ARF, and p21 expression and senescence-associated ß-galactosidase (SA-ß-gal) activity, indicating accelerated tubular senescence. Additionally, BRG1-mediated pro-fibrotic responses were largely abolished by small interfering RNA (siRNA)-mediated p16INK4a silencing in vitro or continuous senolytic treatment with ABT-263 in vivo. Moreover, BRG1 activated the Wnt/ß-catenin pathway, which further inhibited autophagy. Pharmacologic inhibition of the Wnt/ß-catenin pathway (ICG-001) or rapamycin (RAPA)-mediated activation of autophagy effectively blocked BRG1-induced tubular senescence and fibrotic responses, while bafilomycin A1 (Baf A1)-mediated inhibition of autophagy abolished the effects of ICG-001. Further, BRG1 altered the secretome of senescent tubular cells, which promoted proliferation and activation of fibroblasts. Taken together, our results indicate that BRG1 induces tubular senescence by inhibiting autophagy via the Wnt/ß-catenin pathway, which ultimately contributes to the development of renal fibrosis.

Antiviral activity of Mulberroside C against enterovirus A71 in vitro and in vivo.

Enterovirus A71 (EV-A71) is one of the main causative agents of hand, foot and mouth disease which seriously threatens young children's health and lives. However, there is no effective therapy currently available for treating these infections. Therefore, effective drugs to prevent and treat EV-A71 infections are urgently needed. Here, we identified Mulberroside C potently against the proliferation of EV-A71. The in-vitro anti-EV-A71 activity of Mulberroside C was assessed by cytopathic effect inhibition and viral plaque reduction assays, and the results showed that Mulberroside C significantly inhibited EV-A71 infection. The downstream assays affirmed that Mulberroside C inhibited viral protein and RNA synthesis. Furthermore, Mulberroside C effectively reduced clinical symptoms in EV-A71 infected mice and reduced mortality at higher concentrations. The mechanism study indicated that Mulberroside C bound to the hydrophobic pocket of viral capsid protein VP1, thereby preventing viral uncoating and genome release. Taken together, our study indicated that Mulberroside C could be a promising EV-A71 inhibitor and worth extensive preclinical investigation as a lead compound.

FOXO3a accumulation and activation accelerate oxidative stress-induced podocyte injury.

Podocyte injury is the primary cause of glomerular injury in diabetic nephropathy (DN). Advanced oxidation protein products (AOPPs), the triggers and markers of oxidative stress in DN, have been linked to podocyte damage. However, the underlying mechanism is not yet clear. Here, we investigated the potential role of FOXO3a, a key transcription factor in the response to stress, in mediating AOPPs-induced podocyte injury. We found that FOXO3a expression was increased in the glomeruli of kidney biopsies from patients with DN and it was positively correlated with proteinuria. The serum from patients with DN significantly increased FOXO3a and its downstream genes FasL and Bim, thereby inducing the high level of cleaved caspase3 and the loss of nephrin and podocin expressions in podocytes. Blockade of AOPPs signaling by a neutralizing antibody against the receptor of advanced glycation end products (αRAGE) abolished the effect of DN serum on podocytes, confirming the pathogenic role of AOPPs in DN serum. Downregulation of FOXO3a decreased AOPPs-induced podocyte apoptosis and restored the levels of podocyte markers nephrin and podocin, and upregulation of FOXO3a exacerbated these changes in podocytes after AOPPs treatment. Furthermore, FOXO3a specifically activated proapoptotic genes in podocytes only in the presence of AOPPs. Mechanistically, AOPPs increased the FOXO3a protein levels by inhibiting their autophagic degradation in a ROS/mTOR-dependent manner. Moreover AOPPs activated the accumulated FOXO3a by maintaining FOXO3a in the nucleus, and this process was dependent on ROS-mediated AKT signaling deactivation. These studies suggest that FOXO3a plays a critical role in mediating AOPPs-induced podocyte injury and reveal a new mechanistic linkage of oxidative stress, FOXO3a activation and podocyte injury in DN.

Scaffold Hopping-Driven Optimization of 4-(Quinazolin-4-yl)-3,4-dihydroquinoxalin-2(1H)-ones as Novel Tubulin Inhibitors.

Scaffold hopping-driven lead optimizations were performed based on our prior lead 7-methoxy-4-(2-methylquinazolin-4-yl)-3,4-dihydroquinoxalin-2(1H)-one (2a) by C-ring expansion and isometric replacement of the A/B-ring, successively, aimed at finding new potential alternative drug candidates with different scaffold(s), high antitumor activity, and other improved properties to replace prior, once promising drug candidates that failed in further studies. Two series of new compounds 7 (a-d) and 13 (a-j) were synthesized and evaluated for antitumor activity, leading to the discovery of three highly potent compounds 13c, 13d, and 13e with different scaffolds. They exhibited similar high antitumor activity with single digital low nanomolar GI50 values (4.6-9.6 nM) in cellular assays, comparable to lead 2a, clinical drug candidate CA-4, and paclitaxel in the same assays. Further biological evaluations identified new active compounds as tubulin polymerization inhibitors targeting the colchicine binding site. Moreover, 13d showed better aqueous solubility than 2a and a similar log P value.

Flavonoids of Rosa roxburghii Tratt exhibit radioprotection and anti-apoptosis properties via the Bcl-2(Ca(2+))/Caspase-3/PARP-1 pathway.

The objective of our study was to assess the radioprotective effect of flavonoids extracted from Rosa roxburghii Tratt (FRT) and investigate the role of Bcl-2(Ca(2+))/Caspase-3/PARP-1 pathway in radiation-induced apoptosis. Cells and mice were exposed to (60)Co γ-rays at a dose of 6 Gy. The radiation treatment induced significant effects on tissue pathological changes, apoptosis, Ca(2+), ROS, DNA damage, and expression levels of Bcl-2, Caspase-3 (C-Caspase-3), and PARP-1. The results showed that FRT acted as an antioxidant, reduced DNA damage, corrected the pathological changes of the tissue induced by radiation, promoted the formation of spleen nodules, resisted sperm aberration, and protected the thymus. FRT significantly reduced cell apoptosis compared with the irradiation group. The expression of Ca(2+) and C-Caspase-3 was decreased after FRT treatment compared with the radiation-treated group. At the same time, expression of prototype PARP-1 and Bcl-2 increased, leading to a decrease in the percentage of apoptosis cells in FRT treatment groups. We conclude that FRT acts as a radioprotector. Apoptosis signals were activated via the Bcl-2(Ca(2+))/Caspase-3/PARP-1 pathway in irradiated cells and FRT inhibited this pathway of apoptosis by down-regulation of C-Caspase-3 and Ca(2+) and up-regulation of prototype PARP-1 and Bcl-2.

NADP(+)-IDH Mutations Promote Hypersuccinylation that Impairs Mitochondria Respiration and Induces Apoptosis Resistance.

Elucidating the tumorigenic mechanism of R-2-hydroxyglutarate (R-2HG) is critical for determining how NADP(+)-IDH mutations cause cancer. Here we report that R-2HG induces cancerous metabolism and apoptosis resistance through promoting hypersuccinylation. By competitive inhibition of the mitochondrial tricarboxylic acid cycle enzyme succinate dehydrogenase (SDH), R-2HG preferentially induced succinyl-CoA accumulation and hypersuccinylation in the mitochondria. IDH1 mutation-bearing glioma samples and cells were hypersuccinylated in the mitochondria. IDH1 mutation or SDH inactivation resulted in hypersuccinylation, causing respiration inhibition and inducing cancerous metabolism and mitochondrial depolarization. These mitochondrial dysfunctions induced BCL-2 accumulation at the mitochondrial membrane, leading to apoptosis resistance of hypersuccinylated cells. Relief of hypersuccinylation by overexpressing the desuccinylase SIRT5 or supplementing glycine rescued mitochondrial dysfunctions, reversed BCL-2 accumulation, and slowed the oncogenic growth of hypersuccinylated IDH1(R132C)-harboring HT1080 cells. Thus, R-2HG-induced hypersuccinylation contributes to the tumorigenicity of NADP(+)-IDH mutations, suggesting the potential of hypersuccinylation inhibition as an intervention for hypersuccinylation-related tumors.

Flavonoids of Rosa roxburghii Tratt act as radioprotectors.

BACKGROUND: To study the radioprotective effects of flavonoids from Rosa roxburghii Tratt (FRT). MATERIALS AND METHODS: The radioprotective effects of FRT were investigated by examining cell viability, 30-day survival of mice and the number of colony-forming units in spleen (CFU-S) after total-body 60Co irradiation. RESULTS: The survival rates of irradiated cells gradually increased with increasing concentrations of FRT. The survival rate was the highest at 87% with a concentration of 30 µg/mL. Pretreatment with FRT was needed to realize its radioprotective activity in mice at the dose of 60 mg/kg. With the increasing doses of 30 mg/kg, 60 mg/kg and 120 mg/kg, the numbers of CFU-S increased, and were significantly different compared with the control group. CONCLUSIONS: Pretreatment with FRT prior to irradiation resulted in significantly higher cell survival at 24 h after 5 Gy radiation, increased 30-day survival in mice after exposure to a potentially lethal dose of 8 Gy, and resulted in a higher number of CFU-S in mice after exposure to a dose of 6 Gy. These results collectively indicate that FRT is an effective radioprotective agent.

A novel synthetic dibenzocyclooctadiene lignan analog XLYF-104-6 attenuates lipopolysaccharide-induced inflammatory response in RAW264.7 macrophage cells and protects BALB/c mice from sepsis.

The wide range of inflammation mechanisms under control by NF-κB makes this pathway as an attractive target for new anti-inflammatory drugs. Herein, we showed that a new dibenzocyclooctadiene lignan analog XLYF-104-6, with a chemical name of 1,2,3,10,11-pentamethoxydibenzocycloocta-6,7-[c] pyrrole-1,3-dione, inhibited lipopolysaccharide (LPS)-induced NF-κB activation in RAW264.7 cells through preventing IκBα degradation and p65 nuclear translocation. The inhibitory activity of this compound on NF-κB activation contributes to the reduction of LPS-induced TNF-α and IL-6 productions. Notably, XLYF-104-6 suppressed LPS-induced iNOS expression and NO production in a NF-κB independent manner, since IKK inhibitor BAY 11-7082 has failed to exert similar inhibitory effect on iNOS expression and NO production. In addition, XLFY-104-6 also exerted anti-inflammatory action in endotoxemic mice by decreasing plasma LPS-induced TNF-α and IL-1ß levels as well as increasing plasma LPS-induced IL-10 concentrations. These findings suggest XLYF-104-6 could act as a leading compound for developing a potential anti-inflammatory drug.

Discovery of novel antitumor dibenzocyclooctatetraene derivatives and related biphenyls as potent inhibitors of NF-κB signaling pathway.

Several dibenzocyclooctatetraene derivatives (5-7) and related biphenyls (8-11) were designed, synthesized, and evaluated for inhibition of cancer cell growth and the NF-κB signaling pathway. Compound 5a, a dibenzocyclooctatetraene succinimide, was discovered as a potent inhibitor of the NF-κB signaling pathway with significant antitumor activity against several human tumor cell lines (GI50 1.38-1.45 µM) and was more potent than paclitaxel against the drug-resistant KBvin cell line. Compound 5a also inhibited LPS-induced NF-κB activation in RAW264.7 cells with an IC50 value of 0.52 µM, prevented IκB-α degradation and p65 nuclear translocation, and suppressed LPS-induced NO production in a dose-dependent manner. The antitumor data in cellular assays indicated that relative positions and types of substituents on the dibenzocyclooctatetraene or acyclic biphenyl as well as torsional angles between the two phenyls are of primary importance to antitumor activity.

Epidemiology of drug-induced liver injury in China: a systematic analysis of the Chinese literature including 21,789 patients.

BACKGROUND: The epidemiology of drug-induced liver injury (DILI) in China has rarely been studied before. The aim of the present study was to determine the etiology of DILI in a Chinese population by reporting a systematic analysis of the Chinese literature published from 1994 to 2011. METHODS: A comprehensive database search of the Chinese literature was performed to obtain all the relevant studies. The data, including the drug names and patients' sex, age, clinical classification, and prognosis, were collected and analyzed. RESULTS: In this research, we found 279 studies including 24 112 patients. There were 265 studies that reported the sex of 21 789 patients, 11 787 men and 10 002 women. The therapeutics included (but were not limited to) tuberculostatics, complementary and alternative medicines (CAMs), antibiotics, NSAIDs, antineoplastics, central nervous system agents, antithyroid drugs, and immunomodulators. Of these drugs, tuberculostatics and CAMs were the most common etiologies of DILI in China. CONCLUSION: DILI in China has a different etiology from that in Europe and USA. NSAIDs, which are the most common causes of DILI in western populations, are uncommon in China. Therefore, government, physicians, and patients should pay more attention to these drugs in DILI.

Development of a UGT1A1 reporter gene assay for induction studies: correlation between reporter gene data and regulation of UGT1A1 in human hepatocytes.

The present manuscript describes the development of a cell-based reporter transcriptional activation assay for evaluating induction of UGT1A1. A reporter construct (pGL-UGT1A1-Luc) encompassing the proximal promoter (nucleotide -254 to +38) and distal enhancer (-3483 to -3194) regions of the human UGT1A1 gene was generated by PCR cloning, and co-transfected with a previously generated PXR construct (pSG5-PXR) into HepG2 cells. The system was then validated using known ligands of PXR, rifampicin (RIF), clotrimazole (CLOT) sulfinpyrazone (SPZ) and phenobarbital (PB), which produced dose dependent induction of UGT1A1 luciferase activity by 4.4, 5.3, 4.7 and 3.7 fold, respectively, relative to the vehicle control, 0.1 % dimethylsulfoxide (DMSO). Aryl hydrocarbon receptor (AhR) ligands a-naphthoflavone (a-Naph), b-naphthoflavone (b-Naph) and 3-methylchloranthene (3-MC) increased UGT1A1 luciferase activity in a concentration dependent manner resulting in 17.2, 11.3 and 6.1 fold, respectively, at their highest concentrations, suggesting that endogenous AhR is also involved in the regulation of the UGT1A1 reporter construct in HepG2 cells. For comparison with transcriptional regulation of endogenous UGT1A1, 10 mM RIF, 50 mM SPZ, 10 mM CLOT, 4 mM 3-MC, 10 mM b-Naph and 25 mM a-Naph also induced UGT1A1 mRNA in human primary hepatocytes by 2.5, 2.8, 3.2, 3.7, 3.9 and 4.3 fold, respectively. In summary, by co-transfecting the UGT1A1 reporter and PXR constructs into HepG2 cells, we have developed a cellular model for evaluating induction of UGT1A1. Data from the reporter gene assay correlated with that generated in human primary hepatocytes. Based on these data, we suggest that this reporter gene assay can be used as a screening tool in the early stages of drug discovery, to evaluate potential induction of UGT1A1 by new chemical entities and to aid in lead selection and optimization.

Metabolism of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) by human CYP1B1 genetic variants.

Human cytochrome P450 1B1 (CYP1B1) plays a critical role in the metabolic activation of a variety of procarcinogens, including 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). The existence of human CYP1B1 missense genetic variants has been demonstrated, but their activities in metabolizing PhIP are unknown. In this study, we expressed 15 naturally occurring CYP1B1 variants (with either single or multiple amino acid substitutions) and determined their activity changes in metabolizing PhIP to its two major metabolites, 2-hydroxyamino-PhIP and 4'-hydroxy-PhIP. Although the PhIP-metabolizing activities of four variants (Ala(119)Ser, Pro(379)Leu, Ala(443)Gly, Arg(48)Gly/Leu(432)Val) were comparable with that of the expressed wild-type CYP1B1, five variants (Trp(57)Cys, Gly(61)Glu, Arg(48)Gly/Ala(119)Ser, Arg(48)Gly/Ala(119)Ser/Leu(432)Val, Arg(48)Gly/Ala(119)Ser/Leu(432)Val/Ala(443)Gly) exhibited more than 2-fold decrease in activity and a reduction in the catalytic efficiency (V(max)/K(m)) for both N- and 4-hydroxylation of PhIP. Six variants (Gly(365)Trp, Glu(387)Lys, Arg(390)His, Pro(437)Leu, Asn(453)Ser, Arg(469)Trp) showed little activity in PhIP metabolism, but the molecular mechanisms involved are apparently different. The microsomal CYP1B1 protein level was significantly decreased for the Trp(365), Lys(387), and His(390) variants and was not detectable for the Ser(453) variant. In contrast, there was no difference between the Trp(469) variant and the wild-type in the microsomal CYP1B1 protein level and P450 content but the Trp(469) variant totally lost its metabolic activity toward PhIP. The Leu(437) variant also had a substantial amount of CYP1B1 protein in the microsomes, but there was a lack of detectable P450 peak and activity. Our results should be useful in selecting appropriate CYP1B1 variants as cancer susceptibility biomarkers for human population studies related to PhIP exposure.

Genetic variation of human cytochrome p450 reductase as a potential biomarker for mitomycin C-induced cytotoxicity.

The importance of genetic variation in clinical response to various drugs is now well recognized. Identification of genetic biomarkers that can predict efficacy and toxicity of chemotherapeutic drugs in cancer patients holds great promise in treatment improvement and cost reduction. Mitomycin C (MMC) is a common anticancer drug used for the treatment of numerous types of tumors. Metabolism-mediated activation, by either one-electron or two-electron reduction, plays a critical role in the chemotherapeutic action of MMC. NADPH-cytochrome P450 (oxido)reductase (POR) is a major enzyme responsible for MMC activation through the one-electron reductive pathway, which leads to the production of semiquinone anion radicals and subsequent DNA damage in the cells. Recently, a total of six naturally occurring human POR variants with single amino acid changes (Y181D, A287P, R457H, V492E, C569Y, and V608F) have been identified. Although the catalytic efficiency of these variants in reduction of cytochrome c was reported to be altered, their capability in activating MMC, a direct substrate of POR, has not been examined. In the present study, we demonstrated that except for the C569Y variant, MMC-induced toxicity assayed as cell viability and proliferative capability was significantly decreased in the Flp-In Chinese hamster ovary cells stably expressing all the other POR variants in comparison with the cells expressing wild-type human POR. Cells expressing the V608F and Y181D variants had a complete loss of the capability to activate MMC. Our finding suggests that these functional POR genetic variations may serve as a potential biomarker to predict the chemotherapeutic response to MMC.

The missense genetic polymorphisms of human CYP2A13: functional significance in carcinogen activation and identification of a null allelic variant.

Cytochrome P450 2A13 (CYP2A13), an enzyme predominantly expressed in human respiratory tissues, is highly efficient for the metabolic activation of two suspected human lung carcinogens 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and aflatoxin B1 (AFB1). Functional genetic polymorphisms of CYP2A13 may therefore be an important factor in human susceptibility to related lung cancers. Among the reported CYP2A13 polymorphisms with missense variations, only CYP2A13*2 variant (containing either a single or double variation of R25Q and R257C) was studied for its NNK-metabolizing activity. The present study demonstrated that there was no remarkable difference in AFB1- and NNK-induced toxicity between the Flp-In Chinese Hamster Ovary (CHO) cells stably expressing wild-type CYP2A13 and the cells expressing the individual polymorphic variants R25Q, D158E, R257C, R25Q/R257C, V323L, F453Y, and R494C. In contrast, cells transfected with R101Q variant complementary DNA (cDNA), same as the vector control cells, showed no significant death even at highest concentrations of AFB1 (10microM) and NNK (200microM). This result correlated with the lack of CYP2A13 protein in the R101Q-CHO cells, although the genomic integration of transfected R101Q cDNA and the expression of R101Q messenger RNA were clearly demonstrated in these stable transfectants. Consistent with the possibility that the variation might reduce the protein stability, R101Q variant protein expressed in insect cells showed a loss of P450 peak and coumarin 7-hydroxylase activity as well as an increased susceptibility to limited protein digestion. Thus, the R101Q polymorphic change results in a null allelic variant of CYP2A13. Our results should be useful in designing and interpreting molecular epidemiological studies related to CYP2A13 genetic polymorphisms.