Structural Features of the NAD-Dependent In Situ Retinoic Acid Supply System in Esophageal Mucosa

Background: We previously reported that an NAD-dependent in situ retinoic acid supply system, which comprises some isoforms of alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) and provides retinoic acid from retinol via a 2-step oxidation process, exists in the rat esophagus. Herein, their isoforms responsible for the pathway and its localization in the rat esophagus was examined. Methods: The expressions of mRNAs of various isoforms of ADH and ALDH were examined in the fraction mainly comprising mucosal layer of the rat esophagus by RT-PCR. Expression levels of Class IV ADH and ALDH 1A1 were compared between the fractions and that mainly comprising muscle layer of the rat esophagus by quantitative PCR. The catalytic activities producing retinoic acid from retinal were compared between the 2 fractions and its optimum pH was also determined. Results: Classes I, III, and IV ADHs and ALDHs 1A1 and 3A1 were predominant isoforms in the rat esophageal mucosa. The expression levels of mRNA of Class IV ADH and ALDH 3A1 were significantly higher in the mucosal than in the muscle layer. Consistently, the catalytic activities producing retinoic acid from retinal were significantly higher in the former than the latter. The optimum pH of the process was 9.0. Conclusions: Considering the affinities for retinol and retinal of ADHs and ALDHs expressed in the rat esophagus, the NAD-dependent in situ retinoic acid supply system in the rat esophagus is thought to comprise Class IV ADH and ALDH 1A1. In the rat esophagus, the system exists predominantly in the mucosal layer.     

Nicotinamide Adenine Dinucleotide-Dependent Retinoic Acid Formation From Retinol in the Human Gastric Mucosa: Inhibition by Ethanol, Acetaldehyde, and H2 Blockers

All-trans retinoic acid formation from all-trans retinol (vitamin A) in the human gastric mucosa was studied. When all-trans retinol and the human gastric mucosa were incubated together, all-trans retinoic acid was formed in the presence of nicotinamide adenine dinucleotide (NAD). When the NAD was not added, hardly any formation was observed. The formation of all-trans retinoic acid tended to be attenuated by 10 mM ethanol. Moreover, it was significantly attenuated in a concentration-dependent manner by ethanol at concentrations of 100 mM and above. Acetaldehyde at concentrations of 50 μM and above also significantly attenuated its formation in a concentration-dependent manner. Some H2 blockers, which include ranitidine hydrochloride and cimetidine, significantly attenuated the formation of all-trans retinoic acid, whereas famotidine failed to suppress it. There is an NAD-dependent pathway by which all-trans retinoic acid is produced from all-trans retinol in the human gastric mucosa. Inhibitors of alcohol dehydrogenase, which include ethanol and some H2 blockers, and of aldehyde dehydrogenase, which include acetaldehyde, inhibit its production.     

维甲酸诱导的人食管癌EC—109细胞分化相关基因的克隆

目的：克隆维甲酸诱导的人食管癌ＥＣ－１０９细胞分化相关基因。方法：在全反式维甲酸诱导人食管癌ＥＣ－１０９细胞系分化的基础上，采用ｍＲＮＡ差异显示技术（ｍＲＮＡｄｉｆｆｅｒｅｎｔｉａｌｄｉｓｐｌａｙ，ＤＤ）对ＥＣ－１０９细胞全反式维甲酸诱导前后的基因表达差异情况进行了分析。结果：全反式维甲酸诱导前后的细胞之间存在明显的基因表达差异，经克隆筛选获得了一些经全反式维甲酸诱导激活或抑制的差异表达基因片段。对其中一个片段（ＲＡａｌ２）的序列分析及同源性比较结果表明其与人的ａｌｐｈａ－ｃａｔｅｎｉｎ基因（Ｄ１３８６６，ＨＵＭＡＣＡ，２４３９ｂｐ）１００％同源。结论：全反式维甲酸具有调控分化相关基因表达的重要作用，在全反式维甲酸诱导前后的细胞之间存在明显的基因表达差异     

复方青黛片联合全反式维甲酸治疗急性早幼粒细胞白血病的近期临床疗效观察

目的:观察复方青黛片联合全反式维甲酸治疗急性早幼粒细胞白血病(APL)的临床疗效、作用特点及不良反应。方法:将APL患者随机分22例,21例及18例3组,分别给予全反式维甲酸(ATRA),复方青黛片及ATRA联合复方青黛片治疗,观察APL的缓解率,无病生存期及药物不良反应。结果:联合治疗组可减少达缓解所需时间,较快速恢复凝血功能。结论:复方青黛片联合全反式维甲酸治疗APL是一种更为有效及合理的方法,是切实可行的,值得推广。     

全反式维甲酸对COPD大鼠肺组织中基质金属蛋白酶-2,9表达的影响

目的:探讨MMP-2,MMP-9及TIMP-1在肺损伤和修复过程中的作用及全反式维甲酸(ATRA)拮抗COPD的机制。方法:100只健康雄性SD大鼠被随机分为4组:正常对照组、模型组、ATRA预防组、ATRA治疗组。采用烟熏加内毒素致大鼠COPD模型,观察病理组织形态学改变,应用免疫组化检测肺组织中MMP及TIMP的表达,及ATRA对它们的影响。结果:正常肺组织MMP-2、MMP-9、TIMP-1有少量染色阳性,模型组比正常对照组表达明显增强。ATRA预防和治疗组比模型组表达明显下降,但仍比正常对照组增高。结论:MMP-2、MMP-9表达增强,使细胞外降解增加,可能是肺气肿形成机制之一。ATRA可以降低COPD大鼠的MMP-2和MMP-9表达。     

Chinese alcoholic patients with esophageal cancer are genetically different from alcoholics with acute pancreatitis and liver cirrhosis

OBJECTIVE: It is a mystery why some alcoholic patients acquire certain organ-specific complications of alcoholism, whereas other alcoholic patients acquire different ones. The aim of this study was to investigate the differences among Chinese alcoholic patients with esophageal cancer, acute pancreatitis, and liver cirrhosis by studying the genetic polymorphisms of ADH2, ADH3, ALDH2, and P4502E1. METHODS: Liver alcohol dehydrogenase (ADH), aldehyde dehydrogenase (ALDH), and cytochrome P4502E1 (P4502E1) are polymorphic at the ADH2, ADH3, and ALDH2 loci and the 5'-flanking region of the P4502E1. Using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method, we determined the polymorphism of the above-mentioned alcohol metabolizing genes in 59 alcoholics with carcinoma of the esophagus (alcoholic esophageal Ca), 87 acute alcoholic pancreatitis patients, 116 alcoholics with liver cirrhosis (alcoholic cirrhosis), 19 alcoholics with both liver cirrhosis and acute pancreatitis (alcoholic P plus C), and 241 nonalcoholic patients. RESULTS: The results showed that the allele frequency of ALDH2*2 was significantly higher in the alcoholic esophageal Ca group than in the alcoholic pancreatitis and alcoholic cirrhosis groups. The allele frequency of ADH2*1 was significantly higher in the alcoholic esophageal Ca patients than in nonalcoholic control groups. The ALDH2*2 was significantly lower in alcoholic groups (except the alcoholic esophageal Ca group) than in nonalcoholic control groups. The allele frequencies of ADH2*1 and ALDH2*2 are higher in alcoholic patients with esophageal Ca than alcoholic patients without it. The genotype distribution of P4502E1, detected by RsaI and PstI, was not different among alcoholic patients with different organ diseases. CONCLUSIONS: The allele frequency of ADH2*1 and ALDH2*1 are different among subpopulations of alcoholics, suggesting that alcoholic patients with different specific types of organ damage are genetically different. The Chinese alcoholic patients with the ADH2*1 and ALDH2*2 allele are more susceptible to esophageal Ca.     

Aldehyde Dehydrogenases of the Rat Colon: Comparison with Other Tissues of the Alimentary Tract and the Liver

Intracolonic bacteria have previously been shown to produce substantial amounts of acetaldehyde during ethanol oxidation, and it has been suggested that this acetaldehyde might be associated with alcohol-related colonic disorders, as well as other alcohol-induced organ injuries. The capacity of colonic mucosa to remove this bacterial acetaldehyde by aldehyde dehydrogenase (ALDH) is, however, poorly known. We therefore measured ALDH activities and determined ALDH isoenzyme profiles from different subcellular fractions of rat colonic mucosa. For comparison, hepatic, gastric, and small intestinal samples were studied similarly. Alcohol dehydrogenase (ADH) activities were also measured from all of these tissues. Rat colonic mucosa was found to possess detectable amounts of ALDH activity with both micromolar and millimolar acetaldehyde concentrations and in all subcellular fractions. The ALDH activities of co-Ionic mucosa were, however, generally low when compared with the liver and stomach, and they also tended to be lower than in small intestine. Mitochondrial low K_m ALDH2 and cytosolic ALDH with low K_m for acetaldehyde were expressed in the colonic mucosa, whereas some cytosolic high K_m isoenzymes found in the small intestine and stomach were not detectable in colonic samples. Cytosolic ADH activity corresponded well to ALDH activity in different tissues: in co-Ionic mucosa, it was ～6 times lower than in the liver and about one-half of gastric ADH activity. ALDH activity of the colonic mucosa should, thus, be sufficient for the removal of acetaldehyde produced by colonic mucosal ADH during ethanol oxidation. It may, however, be insufficient for the removal of the acetaldehyde produced by intracolonic bacteria. This may lead to the accumulation of acetaldehyde in the colon and colonic mucosa after ingestion of ethanol that might, at least after chronic heavy alcohol consumption, contribute to the development of alcohol-related colonic morbidity, diarrhea, and cancer.     

Inhibitory effect of all-trans retinoic acid on human hepatocellular carcinoma cell proliferation

AIM: To study the inhibitory effect of all-trans retinoic acid on human hepatocellular carcinoma cell line SMMC-7721 and to explore the mechanism of its effect. METHODS: SMMC-7721 cells were divided into two groups, one treated with all-trans retinoic acid (ATRA) for 5 days and the other as a control group. Light microscope and electron microscope were used to observe the morphological changes. Telomerase activity was analyzed with silver-stained telomere repeated assay protocal (TRAP). Expression of Caspase-3 was demonstrated with western blot. RESULTS: ATRA-treated cells showed differentiation features including small and pyknotic nuclei, densely stained chromatin and fewer microvilli. Besides, ATRA could inhibit the activity of telomerase, promote the expression of Caspase-3 and its activation. CONCLUSION: Telomerase activity and Caspase-3 expression are changed in human hepatocellular carcinoma cell line SMMC-7721 treated with all-trans retinioc acid. The inhibition of telomerase activity and the activation of Caspase-3 may be the key steps through which ATRA inhibits the proliferation of SMMC-7721 cell line.     

全反式维甲酸对血管平滑肌细胞增生和细胞周期素依赖激酶抑制蛋白P27蛋白表达的影响

目的：探讨全反式维甲酸对细胞周期素依赖激酶抑制蛋白P27蛋白表达，血管平滑肌细胞DNA合成和增生的影响。方法：取Wistar大鼠胸主动脉血管平滑肌细胞培养，用^3H—TdR掺入率检测血管平滑肌细胞DNA合成和增生，用western—blotting印迹和图像分析方法检测细胞周期素依赖激酶抑制蛋白P27蛋白表达。结果：全反式维甲酸（2．5μmol／L）明显抑制胎牛血清（20％FBS）诱导的血管平滑肌细胞P27蛋白表达；全反式维甲酸明显抑制胎牛血清诱导的血管平滑肌细胞增生和DNA合成。结论：全反式维甲酸具有抗血管平滑肌细胞增生作用，其机制与促进P27蛋白表达有关。     

The environmental light influences the circulatory levels of retinoic acid and associates with hepatic lipid metabolism

Environmental light is involved in the regulation of photochemical reaction in mouse retina. It remains unclear whether light-mediated increase in all-trans retinoic acid (ATRA) synthesis in retina will result in altering the circulatory levels of ATRA and regulating downstream gene expression and physiological function. Here we showed circulatory levels of ATRA decreased in mice under constant darkness and elevated by light exposure. Fat gene pancreatic lipase-related protein 2 (mPlrp2) and its partner procolipase (mClps), but not hepatic lipase (mHl), activated in livers for responding to lack of light illuminating. Light-triggered alterations in circulatory ATRA levels regulated ecto-5'-nucleotidase gene expression by retinoic acid receptor retinoic acid receptor-alpha and modulated 5'-AMP levels in blood and were associated with mPlrp2 and mClps expression in the livers. Mice deficient in adenosine receptors displayed mPlrp2 and mClps expression in livers under 12-h light, 12-h dark cycles. Caffeine blocked adenosine receptors and induced hepatic mPlrp2 and mClps expression in wild-type mice. Mice activated in hepatic mPlrp2 and mClps expression lowered hepatic and serum lipid levels and markedly elevated circulatory levels of all-trans retinol. Our results suggest environmental light influence hepatic lipid homeostasis by light-modulated retinoic acid signaling associated with mPlrp2 and mClps gene expression in livers.     

Alcohol and Aldehyde Dehydrogenases in Human Esophagus: Comparison With the Stomach Enzyme Activities

Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) isoenzymes from surgical esophageal and gastric mucosa were compared by agarose isoelectric focusing. Two prominent ADH forms, designated μ1 (equivalent to the recently reported μ-form) and μ2, were expressed in all the 15 esophagus specimens studied, whereas only four of seven examined gastric specimens exhibited a weak to moderately strong μ1-ADH activity band on the isoelectric focusing gels. p l values of the esophageal μ1-ADH and μ2-ADH, and the liver π-ADH were determined to be 8.61, 8.13, and 8.90, respectively. μ-ADHs exhibited high K_m for ethanol (12 mM) and low sensitivity to 4-methylpyrazole inhibition. ALDH3 (BB form) and ALDH1 were the major high- and low-K_m aldehyde dehydrogenase in the esophagus, respectively. The ADH and ALDH activities were determined at pH 7.5 to be 751 ± 78 and 29.9 ± 3.0 nmol/min/g tissue, respectively (measured at 500 mM ethanol or at 200 μM acetaldehyde; mean ± sem ; N = 15). The esophageal ADH activity was approximately 4-fold and the ALDH activity 20% that of the stomach enzyme. Because the presence of high activity and high K_mμ-ADHs as well as low-activity ALDH1 were found in human esophageal mucosa, it is suggested that there may exist an accumulation of intracellular acetaldehyde during alcohol ingestion. This reactive and toxic metabolite may be involved in the pathogenesis of alcohol-induced esophageal disorders.     

Acute myeloid leukemia with myelodysplasia-related changes mimicking acute promyelocytic leukemia

We describe a patient with acute myeloid leukemia with myelodysplasia-related changes (AML-MRC) that clinically resembled acute promyelocytic leukemia (APL). The karyotype of his leukemic cells was 46, XY, del (3) (q?) and did not include a chromosomal translocation involving the retinoic acid receptor-α gene. However, retinoic acid syndrome developed, and partial remission was achieved after treatment with all-trans retinoic acid (ATRA) followed by chemotherapy. Our case might provide new insights into the mechanism of the growth inhibitory effect of ATRA on APL-like cells.     

全反式维甲酸对肝癌细胞HepG2的分化、侵袭迁移的影响

目的:探讨全反式维甲酸(ATRA)对肝癌细胞HepG2的诱导分化作用及侵袭迁移的影响.方法:MTT法测定ATRA对肝癌细胞HepG2的抑制作用;平皿集落形成法检测ATRA对HepG2细胞锚定依赖性生长作用;ELISA法检测ATRA作用前后HepG2 AFP分泌量的变化;RT-PCR检测ATRA对HepG2细胞MMP-9及Nanog的mRNA表达的影响;Western blot检测ATRA对HepG2细胞MMP-9及Nanog蛋白表达的影响;Transwell及划痕实验检测其对侵袭及迁移能力的影响.结果:MTT法显示ATRA抑制体外培养人肝癌细胞HepG2细胞生长,呈剂量和时间依赖性;ELISA显示ATRA诱导HepG2细胞分化和凋亡,使代表肝细胞恶变的AFP分泌量明显下降(P<0.05);平皿克隆形成实验结果表明未经ATRA处理的HepG2可形成克隆,但经ATRA刺激后,其形成克隆变小且数目明显减少;ATRA呈时间及浓度依赖性下调Nanog mRNA及蛋白的表达,并在24h呈浓度依赖性下调MMP-9 mRNA及蛋白表达;Transwell实验和划痕实验结果显示ATRA能抑制HepG2细胞的侵袭和迁移能力.结论...     

抑癌基因p16对维A酸诱导肺癌细胞分化作用的影响

目的探讨抑癌基因p16在维A酸(RA)对肺癌细胞的增殖抑制和分化调控过程中的作用.方法运用基因转染技术将抑癌基因p16转入人肺癌细胞A549中,进而以浓度为5×10-6mol/L的全反式维A酸(ATRA)处理转染和未转染p16基因的肺癌A549细胞1～4d,观察A549细胞生长,流式细胞仪进行细胞周期分析,应用免疫组化分析肺组织分化标志物粘蛋白(MUC1)的变化,同时Westernblot观察ATRA对P16蛋白表达的影响.结果转染p16抑癌基因的A549肺癌细胞经ARTA处理后,细胞增殖能力明显下降,更多的细胞被阻滞于G1/G0期,MUC1的表达下调,Westernblot表明经ATRA处理后P16蛋白表达增加.结论抑癌基因p16能协同RA抑制肺癌A549细胞的恶性增殖及诱导其分化.     

全反式维甲酸对培养的大鼠动脉平滑肌细胞增殖的影响

目的 研究全反式维甲酸(ATRA)对培养的大鼠动脉平滑肌细胞(VSMCs)的影响。方法 血管平滑肌细胞采用组织贴块法培养。ATRA(2.5×10-6mol/L)分别作用于经PDGF—BB(10 ng/ml)和20％FCS刺激后的VSM—Cs,作用24h后分别行3H—TdR,3H—Leucine掺入实验测定。结果 ATRA明显抑制由PDGF—BB(10ng/ml)和20％FCS促进的VSMCs的DNA和蛋白质合成。结论ATRA具有抑制VSMCs增殖的作用。     

Polymorphisms of Alcohol Dehydrogenase‐1B and Aldehyde Dehydrogenase‐2 and the Blood and Salivary Ethanol and Acetaldehyde Concentrations of Japanese Alcoholic Men

Background: The effects of genetic polymorphism of aldehyde dehydrogenase‐2 (ALDH2) on alcohol metabolism are striking in nonalcoholics, and the effects of genetic polymorphism of alcohol dehydrogenase‐1B (ADH1B) are modest at most, whereas genetic polymorphisms of both strongly affect the susceptibility to alcoholism and upper aerodigestive tract (UADT) cancer of drinkers. Methods: We evaluated associations between ADH1B/ADH1C/ALDH2 genotypes and the blood and salivary ethanol and acetaldehyde levels of 168 Japanese alcoholic men who came to our hospital for the first time in the morning and had been drinking until the day before. Results: The ethanol levels in their blood and saliva were similar, but the acetaldehyde levels in their saliva were much higher than in their blood, probably because of acetaldehyde production by oral bacteria. Blood and salivary ethanol and acetaldehyde levels were both significantly higher in the subjects with the less active ADH1B*1/*1 genotype than in the ADH1B*2 carriers, but none of the levels differed according to ALDH2 genotype. Significant linkage disequilibrium was detected between the ADH1B and ADH1C genotypes, but ADH1C genotype did not affect the blood or salivary ethanol or acetaldehyde levels. High blood acetaldehyde levels were found even in the active ALDH2*1/*1 alcoholics, which were comparable with the levels of the inactive heterozygous ALDH2*1/*2 alcoholics with less active ADH1B*1/*1. The slope of the increase in blood acetaldehyde level as the blood ethanol level increased was significantly steeper in alcoholics with inactive heterozygous ALDH2*1/*2 plus ADH1B*2 allele than with any other genotype combinations, but the slopes of the increase in salivary acetaldehyde level as the salivary ethanol level increased did not differ between the groups of subjects with any combinations of ALDH2 and ADH1B genotypes. Conclusions: The ADH1B/ALDH2 genotype affected the blood and salivary ethanol and acetaldehyde levels of nonabstinent alcoholics in a different manner from nonalcoholics, and clear effects of ADH1B genotype and less clear effects of ALDH2 were observed in the alcoholics. Alterations in alcohol metabolism as a result of alcoholism may modify the gene effects, and these findings provide some clues in regard to associations between the genotypes and the risks of alcoholism and UADT cancer.     

全反式维甲酸治疗急性早幼粒细胞性白血病致Sweet综合征--病例报告及文献复习

目的:提高对全反式维甲酸(ATRA)治疗急性早幼粒细胞性白血病(APL)少见副作用的认识。方法:报告1例ATRA治疗APL致Sweet综合征的病例及治疗过程,并对相关文献进行复习总结。结果:ATRA治疗APL可以导致Sweet's综合征,采用糖皮质激素治疗有效。结论:Sweet综合征是维甲酸的少见副作用,临床上应提高对该综合征的早期诊断。     

Retinoid-induced apoptosis in B-cell chronic lymphocytic leukaemia cells is mediated through caspase-3 activation and is independent of p53, the retinoic acid receptor,and differentiation

The aim of this study was to investigate the effects of all-trans retinoic acid (ATRA) on apoptosis induction, Bcl-2 family protein expression, and differentiation in B-cell chronic lymphocytic leukaemia (B-CLL) cells. ATRA induced apoptosis in all the B-CLL samples tested, and this was accompanied by a specific reduction in Bcl-2 and Mcl-1 protein expression in the apoptotic cells. In contrast, Bax, p21, and p53 expression was not altered in either the viable or apoptotic B-CLL cells, inferring that ATRA utilises a p53-independent cell death pathway. Caspase-3 activation was shown to be a prerequisite for ATRA-induced apoptosis, which was inhibited by the pan-caspase inhibitor Z-VAD-FMK and the caspase-9 inhibitor Z-LEHD-FMK. In addition, the retinoic acid receptor (RAR) antagonist AGN194310 failed to abrogate the apoptotic effects of ATRA, indicating that RAR binding was not necessary for ATRA-induced apoptosis. Furthermore, there was no evidence of ATRA-induced differentiation of the B-CLL cells in this study either in terms of altered morphology or immunophenotype. In summary these data indicate that ATRA induces apoptosis via the intrinsic apoptotic pathway, and this is independent of RAR binding, p53 activation, and cellular differentiation in B-CLL cells.