Ethanol‐mediated carcinogenesis in the human esophagus implicates CYP2E1 induction and the generation of carcinogenic DNA‐lesions

Chronic alcohol consumption is a major risk factor for esophageal cancer. Various mechanisms may mediate carcinogenesis including the genotoxic effect of acetaldehyde and oxidative stress. Ethanol exerts its carcinogenic effect in the liver among others via the induction of cytochrome P450 2E1 (CYP2E1) and the generation of carcinogenic etheno‐DNA adducts. Here we investigated if such effects can also be observed in the human esophagus. We studied nontumorous esophageal biopsies of 37 patients with upper aerodigestive tract cancer and alcohol consumption of 102.3 ± 131.4 g/day (range: 15–600 g) as well as 16 controls without tumors (12 teetotalers and 4 subjects with a maximum of 25 g ethanol/day). CYP2E1, etheno‐DNA adducts and Ki67 as a marker for cell proliferation were determined immunohistologically. Chronic alcohol ingestion resulted in a significant induction of CYP2E1 (p = 0.015) which correlated with the amount of alcohol consumed (r = 0.6, p < 0.001). Furthermore, a significant correlation between CYP2E1 and the generation of the carcinogenic exocyclic etheno‐DNA adducts 1,N⁶‐ethenodeoxyadenosine (r = 0.93, p < 0.001) and 3,N⁴‐ethenodeoxycytidine (r = 0.92, p < 0.001) was observed. Etheno‐DNA adducts also correlated significantly with cell proliferation (p < 0.01), which was especially enhanced in patients who both drank and smoked (p < 0.001). Nonsmokers and nondrinkers had the lowest rate of cell proliferation, CYP2E1 expression and DNA lesions. Our data demonstrate for the first time an induction of CYP2E1 in the esophageal mucosa by ethanol in a dose dependent manner in man and may explain, at least in part, the generation of carcinogenic DNA lesions in this target organ.     

A therapeutic approach with combination of interferon-gamma and autophagy inhibitor for oral squamous cell carcinoma

Former clinical trials and experimental research have indicated that Interferon-gamma therapy does not achieve an ideal effect in solid tumors. Autophagy has been associated with tumor chemoresistance. The aim of this study was to explore the efficacy of Interferon-gamma and autophagy inhibitor in the combination treatment of oral squamous cell carcinoma. Interferon-gamma-induced apoptosis was evaluated by the expression of relative proteins (cleaved-PARP and caspase-3) and flow cytometry. Interferon-gamma induced autophagy was assessed by the expression of Beclin1, LC3B, and P62. The synergistic effect of interferon-gamma and autophagy inhibitor (chloroquine) was evaluated in vitro and in vivo. Interferon-gamma induced anti-proliferation, apoptosis, and autophagy in oral squamous cell carcinoma cells. Autophagy-related protein 5 was a key feature in Interferon-gamma-induced autophagy flux. Interferon-gamma and chloroquine had obvious synergistic effects on cellular growth inhibition and apoptosis promotion in oral squamous cell carcinoma cells and xenograft models. Our findings suggest that Interferon-gamma-induced autophagy plays a cellular protective role, and blocking autophagy flux can promote Interferon-gamma mediated oral squamous cell carcinoma cell apoptosis. The combination of Interferon-gamma and autophagy inhibitors represents a novel strategy for oral squamous cell carcinoma therapy.     

In vitro assessment of cytochrome P450 inhibition and induction potential of azacitidine

Purpose

To assess the potential inhibitory and inductive effects of azacitidine on cytochrome P450 isozymes in vitro.

Methods

The inhibitory effects of azacitidine on various CYP isozymes were determined in human liver microsomes. In addition, the ability of azacitidine to induce CYP enzymes in cultured human hepatocytes was evaluated.

Results

Azacitidine did not inhibit CYP2B6-, CYP2C8-, CYP2C9-, CYP2C19-, CYP2D6-, and CYP3A4-mediated activities in human liver microsomes up to a concentration of 100 μM, while weak inhibition (<30% inhibition) of CYP1A2 and CYP2E1 activities was observed at 100 μM azacitidine. In vitro azacitidine did not induce CYP1A2, CYP2C19, or CYP3A4/5 activities in cultured human hepatocytes.

Conclusions

Azacitidine is not an inhibitor or inducer of the cytochrome P450 isozymes tested; therefore, clinically relevant pharmacokinetic drug–drug interactions are unlikely to occur between azacitidine and co-administered substrates of these CYP isozymes.     

CYP1A1 and CYP2E1 genotypes and risk of esophageal squamous cell carcinoma in a high-incidence region,Kashmir

The study analyzed the relationship between genetic polymorphisms of phase I xenobiotic metabolizing enzymes, cytochromes P450 (CYP) 1A1 and CYP2E1 and esophageal squamous cell carcinoma (ESCC) in Kashmir, India. The different genotypes of CYP1A1 and CYP2E1 were analyzed by polymerase chain reaction and restriction fragment length polymorphism in 526 ESCC cases and equal number of matched controls. Conditional logistic regression models were used to assess the association of various genotypes with ESCC, gene–gene, and gene–environment interactions. High risk of ESCC was found in participants who carried CYP1A1 Val/Val genotype (OR?=?2.87; 95 % CI?=?1.00–8.44) and the risk increased in such individuals when c1/c1 of CYP2E1 genotype was also present (OR?=?5.68; 95 % CI?=?1.09–29.52). Risk due to CYP1A1 Val/Val genotype was further enhanced (OR?=?8.55; 95 % CI?=?1.86–42.20) when the analysis was limited to ever smokers. Participants who carried CYP2E1 c1/c2 genotype showed an inverse relation (OR?=?0.27; 95 % CI?=?0.17–0.43) with ESCC. The inverse association of CYP2E1 c1/c2 genotype was retained when CYP1A1 Ile/Ile was also present (OR?=?0.18; 95 % CI?=?0.09–0.32), as well as when analysis was limited to ever smokers (OR?=?0.45; 95 % CI?=?0.23–0.90). Significant interaction was found between CYP1A1 (Val/Val) and CYP2E1 (c1/c1) genotypes (OR?=?1.30; 95 % CI?=?1.12–1.51; P?=?0.001) and between CYP1A1 (Val/Val) and smoking (OR?=?1.31; 95 % CI?=?1.01–1.69; P?=?0.043). The study suggests CYP1A1 Val/Val and CYP2E1 c1/c1 genotypes are significantly associated with ESCC risk.     

Rat liver epithelial cells express functional cytochrome P450 2E1

Rat liver epithelial cells (RLECs) isolated by trypsinizationof the livers of normal 10 day old rats are largely used inco-culture with primary hepatocytes. The aim of the presentstudy was to investigate the expression of biotransformationenzyme-encoding genes in three preparations of RLEC lines. Althoughno expression of cytochrome P450 1A1/2 (CYP1A1/2), CYP2B1/2,CYP2C6 or CYP3A mRNAs could be detected, we found that all ofthe different preparations of RLECs expressed a high level ofCYP2E1 mRNA. We demonstrated the presence of the CYP2E1 apoproteinin microsomes of RLECs by immunoblot analyses, together withchlorzoxazone 6-hydroxylation, an activity known to be mainlycatalyzed by CYP2E1. In addition, acetone treatment of thesecells resulted in an increase in both CYP2E1 apoprotein andchlorzoxazone 6-hydroxylation activity levels. Finally, we showedthe susceptibility of RLECs to N-methyl formamide- and diethylnitrosamine-inducedtoxicity, suggesting metabolic activation by CYP2E1. Thus, RLECsmay cooperate with hepatocytes to CYP2El-mediated metabolismin the co-culture model. In addition, transfection experimentswith a CYP2E1 promoter construct, in which the proximal 539bp containing the binding site for HNF1     

MicroRNA-25 regulates chemoresistance-associated autophagy in breast cancer cells,a process modulated by the natural autophagy inducer isoliquiritigenin

Recent findings have revealed that dysregulated miRNAs contribute significantly to autophagy and chemoresistance. Pharmacologically targeting autophagy-related miRNAs is a novel strategy to reverse drug resistance. Here, we report a novel function of isoliquiritigenin (ISL) as a natural inhibitor of autophagy-related miR-25 in killing drug-resistant breast cancer cells. ISL induced chemosensitization, cell cycle arrest and autophagy, but not apoptosis, in MCF-7/ADR cells. ISL also promoted the degradation of the ATP-binding cassette (ABC) protein ABCG2 primarily via the autophagy-lysosome pathway. More importantly, miRNA 3.0 array experiments identified miR-25 as the main target of ISL in triggering autophagy flux. A mechanistic study validated that miR-25 inhibition led to autophagic cell death by directly increasing ULK1 expression, an early regulator in the autophagy induction phase. miR-25 overexpression was demonstrated to block ISL-induced autophagy and chemosensitization. Subsequent in vivo experiments showed that ISL had chemosensitizing potency, as revealed by an increase in LC3-II staining, the downregulation of ABCG2, a reduction in miR-25 expression and the activation of the miR-25 target ULK1. Overall, our results not only indicate that ISL acts as a natural autophagy inducer to increase breast cancer chemosensitivity, but also reveal that miR-25 functions as a novel regulator of autophagy by targeting ULK1.     

Chemical mechanism of the radiation potentiating effects of 2,2-dimethylaziridine-type antitumor agents

Three 2,2-dimethyl phosphoraziridines, bis(2,2-dimethylaziridinyl)phosphinyl urethane (AB-132, NSC 51325), ethyl bis(2,2-dimethylaziridinyi)phosphinate (AB-163 NSC 108878) and bis(2,2-dimethylaziridinyl)-N-hydroxyurethane (AB-182, NSC 200724) which have shown synergistic antitumor effects in conjunction with X-irradiation, are characterized by their rapid hydrolysis via S_N1-mechanism and via the formation of transient 5-membered cyclic intermediates exhibiting phosphorylating activities. It was shown that the formation of the latter correlates with the observed maxima in the cholinesterase inhibitory effects of these compounds. It is proposed that the same intermediate hydrolysis products may block the repair of X-irradiation induced breaks in the DNA strands by phosphorylating their free YOH end groups. Additional mechanisms of the radiation potentiating effects of these compounds (not shown by their C-unsubstituted analogs), involving free radical-ion formation, are currently under investigation.     

Ras inhibition enhances autophagy,which partially protects cells from death

Autophagy, a process of regulated turnover of cellular constituents, is essential for normal growth control but may be defective under pathological conditions. The Ras/PI3K/mTOR signaling pathway negatively regulates autophagy. Ras signaling has been documented in a large number of human cancers. In this in-vitro study we examined the effect of the Ras inhibitor Salirasib (S-trans, trans-farnesylthiosalicylic acid; FTS) on autophagy induction and cell viability. We show that Ras inhibition by FTS induced autophagy in several cell lines, including mouse embryonic fibroblasts and the human cancer cell lines HeLa, HCT-116 and DLD-1. The autophagy induced by FTS seems to inhibit the cell death induced by FTS, since in the absence of autophagy the death of FTS-treated cells was enhanced. Therefore, inhibition of autophagy may promote the inhibition of tumor cell growth and the cell death mediated by FTS.     

Combining AKT inhibition with chloroquine and gefitinib prevents compensatory autophagy and induces cell death in EGFR mutated NSCLC cells

Although non-small cell lung cancer (NSCLC) patients with EGFR mutation positive (EGFR M+) tumors initially respond well to EGFR tyrosine kinase inhibitor (TKI) monotherapy, the responses are usually incomplete. In this study we show that AKT inhibition, most importantly AKT2 inhibition, synergises with EGFR TKI inhibition to increase cell killing in EGFR M+ NSCLC cells. However, our data also suggest that the synergistic pro-apoptotic effects may be stunted due to a prosurvival autophagy response induced by AKT inhibition. Consequently, inhibiting autophagy with chloroquine significantly enhanced tumor cell death induced by gefitinib and AKT inhibitors in EGFR M+ cells in vitro, and produced greater tumor shrinkage in EGFR M+ xenografts in vivo. Together, our findings suggest that adding chloroquine to EGFR and AKT inhibition has the potential to improve tumor responses in EGFR M+ NSCLC, and that selective targeting of AKT2 may provide a new treatment option in NSCLC.     

Liver cancer-related gene CYP2E1 expression in HBV transgenic mice with acute liver injury

The objective of this research was to study the CYP2E1 gene expression in carbon tetrachloride (CCl₄)-induced acute liver injury in hepatitis B virus (HBV) transgenic mice. Twenty-four HBV (?) and 24 HBV (+) transgenic mice aged 8 to 10 weeks were selected for the present study. Intraperitoneal injection of 1.0 μL/g of CCl₄ (1:4 dissolved in olive oil) to mice was performed to induce acute liver injury model. Eight normal clean-grade C57BL/6 mice were taken as the control group. The control group received saline intraperitoneally. The mice in each group were killed 3, 6, 12, 24, 48, and 72 h after injection. The liver tissue samples of mice were collected. The liver histological changes at different time points in each group were observed under light microscope. The quantitative PCR methods were utilized to measure the relative mRNA levels of CYP2E1 gene in liver tissues. Immunohistochemistry and Western blot techniques were used to observe tissue expression levels of CYP2E1 in each group. Compared with that of the control group, mRNA and protein expression levels of CYP2E1 significantly increased both in the HBV (?) group and in the HBV (+) group after the CCl₄ induced the acute liver injury, and it reached the peak at 72 h after the CCl₄ injection. Compared with the HBV (?) group, the mice in the HBV (+) group had severe liver damage and significantly increased CYP2E1 gene and protein expression levels. In the CCl₄-induced acute liver injury of HBV transgenic mice, the CYP2E1 gene expression significantly increased. The results provided evidence for the HBV-induced liver damage and liver cancer pathogenesis.     

Influence of NQO1, ALDH2, and CYP2E1 genetic polymorphisms, smoking, and alcohol drinking on the risk of lung cancer in Koreans

Objectives  We investigated the association of genetic polymorphisms of NQO1, ALDH2, CYP2E1, and the combined genotype of these genes on lung cancer risk, and also evaluated the association after stratification by cumulative smoking amounts and alcohol drinking levels. Methods  The case–control study was performed in 387 lung cancer patients and 387 age- and sex-matched cancer-free controls. Direct interview was conducted and the genotypes of NQO1, ALDH2, and CYP2E1 were investigated using PCR-RFLP or 5′-nuclease activity assay. Results  The proportion of individuals with occupational history of mining was significantly higher in cases than in controls. The risk of lung cancer was significantly lower in light-drinkers (<108 g/week) than non-drinkers. The NQO1 Pro/Ser + Ser/Ser genotype showed an increased risk for lung cancer with a marginal significance (OR = 1.35, 95% CI = 0.99–1.86) compared with NQO1 Pro/Pro genotype. In heavy-smokers, the combination of NQO1 Pro/Ser + Ser/Ser and CYP2E1 c1/c1 genotype was associated with a significantly increased risk for lung cancer (OR = 2.25, 95% CI = 1.14–4.43) compared with those of NQO1 Pro/Pro and CYP2E1 c1/c2 + c2/c2 genotype. We found a significant interaction between alcohol drinking level and the CYP2E1 genotype (P = 0.0227). Conclusions  Our result suggests that the risk of lung cancer is affected by smoking, alcohol drinking, and the genetic polymorphism of NQO1. In particular, genetic polymorphisms for NQO1, CYP2E1, and ALDH2 synergistically with cumulative smoking amounts and alcohol drinking levels interact in the carcinogenesis of lung cancer in Koreans.     

Expression of cytochrome P450 2A5 in preneoplastic and neoplastic mouse liver lesions

Cytochrome P450 (CYP) 2A5 is involved in the metabolism of carcinogens like aflatoxin B₁ and N-nitrosodiethylamine (NDEA), and CYP2A5 levels are increased in some pathological states of the liver (e.g., infectious hepatitis and porphyria). We analyzed the expression of CYP2A5 during experimental liver carcinogenesis in three different mouse strains (C3H/He, C57BL/6J, and B6C3F1) with immunohistochemical techniques and in situ hybridization. In normal liver, CYP2A5 protein and mRNA were detected in centrilobular hepatocytes only. Phenobarbital treatment increased the number of CYP2A5-positive centrilobular hepatocytes and the CYP2A5-positive areas were extended into the middle zone in all strains, but periportal hepatocytes remained negative. Fifty percent of the spontaneous foci in untreated mice, over 90% of the foci in mice treated with NDEA or phenobarbital and all of the hepatocellular adenomas and carcinomas displayed positive immunostaining and a strong CYP2A5 mRNA signal by in situ hybridization. In the liver tumors metastasized to the lung, expression of CYP2A5 had largely disappeared. CYP2A5 expression in neoplastic and putative preneoplastic lesions, although sometimes heterogeneous, was apparently independent of the typical zonal expression pattern in normal tissue. As expected, the C57BL/6J mice developed fewer foci and tumors than the C3H/He and B6C3F1 mice, but the phenotype of CYP2A5 overexpression was similar in all the strains. Our data suggest that the increased expression of CYP2A5 may play an important role in the development of liver cancer in mice and may be used as a novel marker for spontaneous and NDEA-induced mouse liver foci. Mol. Carcinog. 22:229–234, 1998. © 1998 Wiley-Liss, Inc.     

Interaction of genetic polymorphisms in cytochrome P450 2E1 and glutathione S-transferase M1 to breast cancer in Taiwanese woman without smoking and drinking habits

P450 (CYP) and glutathione S-transferase (GST) are involved in the activation and detoxification of many potential carcinogens. Although, the interaction between environmental exposure and genetic polymorphisms of cytochrome P450 2E1 (CYP2E1) and glutathione S-transferase M1 (GSTM1) in breast cancer has been assessed, the gene–gene interactions between CYP2E1 and GSTM1 related to breast cancer have not been focused on and reported. We conducted a hospital-based case-control study to investigate whether the genetic interaction effects of CYP2E1 and GSTM1 modify the risk of developing breast cancer independent of the effect of cigarette smoking and alcohol consumption. Individuals with the C2/C2 genotype of CYP2E1 had a lower risk (OR = 0.24, 95% CI = 0.08–0.74) when compared with those with the C1/C1 genotype. However, there was no significant difference (OR = 1.05, 95% CI = 0.73–1.50) in the GSTM1 genotype frequency between the cases with breast cancer and that of the controls. When individuals with the genotype of C1/C1 or C1/C2 of CYP2E1 and the wild-type of GSTM1 were compared with those of C2/C2 of CYP2E1 and the null-type of GSTM1 however, we found a significantly increased risk (OR = 3.50, 95% CI = 1.01–16.55) in the breast cancer patients. Our findings indicated a gene–gene interaction between CYP2E1 and GSTM1 was accessible to developing breast cancer in Taiwanese women without the habits of cigarette smoking and alcohol consumption even though independent effects of CYP2E1 and GSTM1 were weak or non-significant and suggest that environmental carcinogen besides cigarette and alcohol consumption could induce breast cancer.Szu-Hsien Wu and Shih-Meng Tsai contributed equally to this work.     

Genetic polymorphism of cytochrome P450 (CYP) 1A1, CYP1A2, and CYP2E1 genes modulate susceptibility to gastric cancer in patients with Helicobacter pylori infection

Background

Activity of cytochrome P450 (CYP), a polymorphic carcinogen-activating enzyme, is exaggerated following Helicobacter pylori infection. We studied the role of CYP2E1, CYP1A2 (rs762551), and CYP1A1 (rs4646903) polymorphisms in association with H. pylori infection in gastric carcinogenesis.

Methods

Genotyping of CYP2E1 (96-bp insertion), CYP1A2 (164A to C), and CYP1A1 (3801C to T) was carried out in 88, 76, 53, and 170 patients with gastric cancer (GC), functional dyspepsia (FD), peptic ulcer (PU), and healthy controls (HC), respectively. Serum IgG antibody (all subjects), rapid urease test, and histology (GC, FD, and PU patients) were used to test for H. pylori.

Results

CYP2E1 gene polymorphism was more common among patients with GC than HC and PU [48/88 (54.5 %) vs. 67/170 (39.4 %); OR 1.9, 95 % CI 1.1–3.2, p = 0.016) and [PU 18/53 (34 %); OR 2.3 (1–4.7), p = 0.02]. CYP1A2 CC or CT genotypes was lower among patients with GC than HC [50/88 (56.8 %) vs. 120/170 (70.6 %); OR 0.54 (0.31–0.92), p = 0.023]. CYP1A1 polymorphism and CYP1A1–CYP1A2 haplotypes were comparable among different groups. CYP2E1 was also more common in patients with GC than HC and PU in the absence of H. pylori [33/60 (55 %) vs. 19/52 (36.5 %); OR 4 (1.5–11.4), p = 0.007 and PU 7/22 (31.8 %); OR 3.4 (1–11.6), p = 0.05]. CYP1A1 (CT + TT) was more common in patients with GC than PU in presence of H. pylori [17/26 (65.4 %) vs. 11/29 (38 %); OR 3.0 (1.03–9.3), p = 0.045].

Conclusions

The presence of CYP2E1 (96-bp insertion) is associated with increased risk of GC even in absence of H. pylori. CYP1A2 CC or CT is associated with reduced risk of GC.     

Associations of CYP2E1 rs2031920 and rs3813867 polymorphisms with colorectal cancer risk: a systemic review and meta-analysis

Cytochrome P450 2E1 (CYP2E1) is a natural enzyme involved in the metabolic activation of many carcinogens, and the functional polymorphisms in the CYP2E1 gene might have impacts on colorectal cancer risk. Many studies were published to assess the associations of CYP2E1 rs2031920 and rs3813867 polymorphisms with colorectal cancer risk, but no consistent findings were reported. A systemic review and meta-analysis of eligible studies was performed to comprehensively assess the associations above. Odds ratios (ORs) with 95 % confidence interval (95 % CIs) were used to assess the strength of the associations. Seventeen studies from 15 publications with 17,082 individuals were finally included into this meta-analysis. Meta-analysis of the 13 studies on CYP2E1 rs2031920 polymorphism showed that there was a significant association between CYP2E1 rs2031920 polymorphism and colorectal cancer risk under two genetic models (c2 versus c1: OR?=?1.19, 95 % CI 1.03–1.37, P?=?0.022; c2c2/c2c1 versus c1c1: OR?=?1.16, 95 % CI 1.00–1.35, P?=?0.046). Meta-analysis of those four case–control studies on CYP2E1 rs3813867 polymorphism showed that there was no significant association between CYP2E1 rs3813867 polymorphism and colorectal cancer risk under all contrast models (c2 versus c1: OR?=?0.96, 95 % CI 0.80–1.16, P?=?0.672; c2c2 versus c1c1: OR?=?1.26, 95 % CI 0.43–3.67, P?=?0.672; c2c2/c1c2 versus c1c1: OR?=?0.95, 95 % CI 0.78–1.16, P?=?0.114; and c2c2 versus c1c2/c1c1: OR?=?1.17, 95 % CI 0.41–3.36, P?=?0.775). Therefore, the findings from this meta-analysis suggest that CYP2E1 rs2031920 polymorphism is associated with colorectal cancer risk, but CYP2E1 rs3813867 polymorphism is not associated with colorectal cancer risk. In addition, more well-designed studies with large sample size are needed to provide a more precise evaluation on the associations above.