Role of CD44 in clear cell renal cell carcinoma invasiveness after antiangiogenic treatment

Treballs Finals de Grau de Farmàcia, Facultat de Farmàcia, Universitat de Barcelona, 2017. Tutor/a: Joan Carles Rodríguez Rubio.[eng] During last century, big effort to understand the biochemical basis of cancer was carried out. One of the principal branches of these cancer investigations used drugs to prevent the formation of new blood vessels –process called angiogenesis– responsible for the nutrients supply of the tumour. These drugs are generally called antiangiogenics. It was discovered that some types of tumour have or develop resistance to these drugs when treatment was long enough. For that reason, mechanisms of resistance, aggressiveness, invasion and/or metastasis after the treatment are nowadays relevant to study. Recently, a protein that could be involved in the increased invasiveness of tumour cells after the antiangiogenic treatment appeared. This project collects some evidence that indicates that this protein, called CD44, might play a role in the increased invasion after antiangiogenic treatment in mouse models of renal carcinoma.[cat] Durant l’últim segle, s’ha fet un gran esforç per aprofundir en la basant bioquímica de la investigació contra el càncer. Una de les branques principals d’aquesta investigació utilitza fàrmacs que prevenen la formació de nous vasos sanguinis –procés anomenat angiogènesis- encarregats de nodrir el tumor. Aquests fàrmacs es diuen generalment antiangiogènics. S’ha descobert que alguns tipus de tumor tenen o desenvolupen resistència a aquests fàrmacs quan el tractament és prou llarg. Per aquesta raó, actualment s’està investigant profundament quins són els mecanismes pels quals apareix aquesta resistència, així com també perquè els tumors es tornen més agressius, invasius i/o metastàtics després del tractament. Recentment s’ha descobert una proteïna que podria estar involucrada en l’augment de la invasivitat de les cèl·lules tumorals després del tractament antiangiogènic. Aquest treball recull algunes de les evidències que apunten cap al paper de la proteïna CD44 en l’increment de la invasió tumoral post-tractament amb fàrmacs antiangiogènics en models ratolins de càncer renal

In vitro bioactivation of N-hydroxy-2-amino-α-carboline

2-Amino-α-carboline (AαC) is a mutagenic and carcinogenic heterocyclic amine present in foods cooked at high temperature and in cigarette smoke. The mutagenic activity of AαC is dependent upon metabolic activation to N-hydroxy-AαC (N-OH-AαC); however, the metabolism of N-OH-AαC has not been studied. We have synthesized 2-nitro-α-carboline and N-OH-AαC and have examined in vitro bioactivation of N-OH-AαC by human and rodent liver cytosolic sulfotransferase(s) and acetyltransferase(s) and by recombinant human N-acetyltransferases, NAT1 and NAT2. The sulfotransferase-dependent bioactivation of N-OH-AαC by human liver cytosol exhibited large interindividual variation (0.5-75, n = 14) and was significantly higher than bioactivation of N-hydroxy-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-OH-PhIP), Correlation and inhibition studies suggested that the isoform of sulfotransferase primarily responsible for bioactivation of N-OH-AαC in human liver cytosol is SULT1A1. O-Acetyltransferase-dependent bioactivation of N-OH-AαC by human liver cytosol also exhibited large inter-individual variation (16-192, n = 18). In contrast to other N-hydroxy heterocyclic amines, which are primarily substrates only for NAT2, both NAT1 and NAT2 catalyzed bioactivation of N-OH-AαC. The rate of bioactivation of N-OH-AαC by both NAT1 and NAT2 was significantly higher than that for N-OH-PhIP. In rat and mouse liver cytosols, the level of sulfotransferase-dependent bioactivation of N-OH-AαC was similar to the level in the high sulfotransferase activity human liver cytosol. The level of O-acetyltransferase-dependent bioactivation of N-OH-AαC in rat liver cytosol was also comparable with that in the high acetyltransferase activity human liver cytosol, However, the level of O-acetyltransferase-dependent bioactivation of N-OH-AαC in mouse liver cytosol was comparable with that in the low acetyltransferase activity human liver cytosol. In contrast to N-OH-PhIP, bioactivation of N-OH-AαC was not inhibited by glutathione S-transferase activity; however, DNA binding of N-acetoxy-AαC was inhibited 20% in the presence of GSH. These results suggest that bioactivation of N-OH-AαC may be a significant source of DNA damage in human tissues after dietary exposure to AαC and that the relative contribution of each pathway to bioactivation or detoxification of N-OH-AαC differs significantly from other N-hydroxy heterocyclic or aromatic amines

Direct oacetylation of N-hydroxy arylamines by acetylsalicylic acid to form carcinogen-DNA adducts

Acetylsalicylic acid (aspirin) has been shown to acetylate a number of drugs and biological macromolecules. Since enzymatic O-acetyiation of N-hydroxy arylamines is regarded as an important activation step for DNA adduct formation, we initially examined the ability of aspirin to serve as an acetyl donor for this reaction, using rabbit liver cytosol. Instead, a direct non-enzymatic reaction was observed. Arylamine-DNA binding was enhanced from 3- to 25-fold at pH 7 by addition of aspirin to reactions containing the N-hydroxy derivatives of 2-aminofluorene (AF), 4-aminobiphenyl, 2-amino-3, 8-dimethylimidazo[4, 5-f]quinoxaline and 2-amino-1-methyl-6-phenylimidazo[4, 5-b]pyridine, but not for 2-amino-3-methylimidazo[4, 5-f]quinoline or 2-amino-6-methyldipyrido[l, 2-a: 3', 2'-d]imidazole. Further studies with N-hydroxy-AF showed that reaction rates were first order with respect to both aspirin (0.1-10 mM) and N-hydroxy-AF (0.01-0.1 mM) concentrations. In contrast, aspirin had no effect on reactions conducted at pH 5 where N-hydroxy-AF is known to undergo protonation and react with DNA to form high levels ofN-(deoxyguanosin-8-yI)-AF. N-Acetylation of AF by aspirin under these conditions was also negligible. However, the formation of the adduct from N-hydroxy-AF occurred at high yield (64-82%) at pH 7 with either DNA or 2'-deoxyguanosine. HPLC analyses showed only an aspirin-dependent loss of N-hydroxy-AF and concomitant adduct formation, with no detectable formation of solvolysis products. This indicated that the reaction proceeds to a significant extent only upon addition of the nucleophile, and suggests the formation of an O-tetrahedral intermediate that is in equilibrium with both the N-hydroxy derivative and the reactive N-acetoxy arylamine. Thus, the apparent O-acetylation of certain N-hydroxy arylamines selectively by aspirin offers a convenient route for the synthesis of arylamine - DNA adducts. The potential biological significance of this reaction in vivo is also discussed

Metabolic activation pathway for the formation of DNA adducts of the carcinogen 2-amino-l-methyl-6-phenyumidazo[4,5-b]pyridine (PhIP) in rat extrahepatic tissues

The food-borne mutagen 2-amino-l-methyl-6-phenylimidazo[ 4,5-b]pyridine (PhIP) induces tumors in colon of male rats and has been implicated in the etiology of human cancers, particularly colorectal cancer. This study was conducted to examine: (1) the biliary and/or circulatory transport of N-hydroxy- PhIP and its N-glucuronides, N-sulfonyloxy-PhIP and N-acetoxy-PhIP; (2) their role as proximate and ultimate carcinogenic metabolites of PhIP; (3) the potential role of glutathione in modulating PhIP-DNA adduct formation. PhIP-DNA adducts, measured by the P-postlabeling method, were highest in the pancreas (361 adducts/10 nucleotides or 100%), followed by colon (56%), lung (28%), heart (27%) and liver (2%), at 24 h after a single oral dose of PhIP (220 μmol/kg) to male rats. In each tissue examined, we observed two major adducts, each of which accounted for 35-45% of the total, and one minor adduct, which represented about 10-20% of the total. One of the major adducts was identified as N-(deoxyguanosin-8-yl)-2-amino-l-methyl- 6-phenylimidazo[4,5-b]pyridine by chromatographic comparisons with an authentic standard. The major urinary metabolites of PhIP in these rats were 4'-hydroxy-PhIP and its glucuronide and sulfate conjugates, followed by N-hydroxy-PhIP N3-glucuronide, N-hydroxy-PhIP N-glucuronide and unchanged PhIP. In bile duct-ligated rats, the urinary excretion of the N-OH-PhIP N3-glucuronide was increased two-fold, but there was no effect on PhIP-DNA adduct formation in the colon, heart, lung, pancreas or liver. 2,6-Dichloro-4-nitrophenol, which strongly inhibits arylsulfo-transferase-mediated DNA binding in vivo, had no effect on PhIP-DNA adduct levels in liver or in extrahepatic tissues. Pretreatment of rats with buthionine sulfoximine, which results in hepatic glutathione depletion, caused a five-fold increase in adduct formation in the liver. Intravenous administration (10 μmol/kg) of N-hydroxy-PhIP and N-acetoxy-PhIP each led to high levels of PhIP-DNA adducts in each of the extrahepatic tissues examined. Adduct levels ranged from two- to six-fold higher (for N-hydroxy-PhIP) and four- to 28-fold higher (for N-acetoxy-PhIP) as compared to that after an i.v. dose of the parent compound, indicating that these two bioactivated derivatives of PhIP are sufficiently stable to be transported through the circulation to extrahepatic tissues. Analyses of whole blood obtained at 2-8 h after oral administration of [H]PhIP failed to detect N-hydroxy-PhIP

Expression of monomorphic and polymorphic N-acetyltransferases in human colon

The metabolism of sulfamethazine (SMZ) and p-aminobenzoic acid (PABA) by N-acetyltransferase (NAT) was measured in human colorectal cytosols from 12 slow and 11 rapid acetylators whose genotype was determined independently by a specific polymerase chain reaction. SMZ metabolism was significantly greater in the rapid than in the slow phenotype (192±22 versus 94±11 pmol N-acetylsulfamethazine/min/mg protein), while PABA metabolism was similar in both phenotypes (23.7±4.4 versus 23.0±3.9 nmol N-acetyl-p-aminobenzoic acid/min/mg protein). Both monomorphic and polymorphic NAT mRNAs were detected by the polymerase chain reaction in the colorectal mucosa of most samples. The finding that polymorphic NAT is expressed in a phenotype-dependent manner in colorectal mucosa indicates that this tissue has the capacity to participate in local bioactivation of dietary and environmental aryl- or heterocyclic amine carcinogens and may explain, in part, the phenotype-dependent occurrence of colorectal cancer