Roberto Spisni

Genetics, Cytogenetics, and Epigenetics of Colorectal Cancer

Most of the colorectal cancer (CRC) cases are sporadic, only 25% of the patients have a family history of the disease, and major genes causing syndromes predisposing to CRC only account for 5-6% of the total cases. The following subtypes can be recognized: MIN (microsatellite instability), CIN (chromosomal instability), and CIMP (CpG island methylator phenotype). CIN occurs in 80–85% of CRC. Chromosomal instability proceeds through two major mechanisms, missegregation that results in aneuploidy through the gain or loss of whole chromosomes, and unbalanced structural rearrangements that lead to the loss and/or gain of chromosomal regions. The loss of heterozygosity that occur in the first phases of the CRC cancerogenesis (in particular for the genes on 18q) as well as the alteration of methylation pattern of multiple key genes can drive the development of colorectal cancer by facilitating the acquisition of multiple tumor-associated mutations and the instability phenotype.

Sulphation of resveratrol, a natural compound present in wine, and its inhibition by natural flavonoids

1. Resveratrol, a polyphenolic compound present in grape and wine, has beneficial effects against cancer and protective effects on the cardiovascular system. Resveratrol is sulphated, and the hepatic and duodenal sulphation might limit the bioavailability of this compound. The aim of this study was to see whether natural flavonoids present in wine, fruits and vegetables inhibit the sulphation of resveratrol in the human liver and duodenum. 2. In the liver, IC50 for the inhibition of resveratrol sulphation was 12 ± 2 pM (quercetin), 1.0 ± 0.04 μM (fisetin), 1.4 ± 0.1 μM (myricetin), 2.2 ± 0.1 μM (kaempferol) and 2.8 ± 0.2 μM (apigenin). Similarly, in the duodenum, IC50 was 15 ± 2 pM (quercetin), 1.3 ± 0.1 μM (apigenin), 1.3 ± 0.5 μM (fisetin), 2.3 ± 0.1 μM (kaempferol) and 2.5 ± 0.3 μM (myricetin). 3. The type of inhibition of quercetin on resveratrol sulphation was studied in three liver samples and was determined to be non-competitive and mixed in nature. Km (mean ± SD; μM) was 0.23 ± 0.07 (control), 0.40 ± 0.08 (5 pM quercetin) and 0.56 ± 0.09 (10 pM quercetin). Vmax (mean ± SD; pmol·min−1·mg−1) was 99 ± 11 (control), 73 ± 15 (5 pM quercetin) and 57 ± 10 (10 pM quercetin). K1 and K1es estimates (mean ± SD) were 3.7 ± 1.8 pM and 12.1 ± 1.7 pM respectively (p = 0.010). 4. Chrysin was a substrate for the sulphotransferase(s) and an assay was developed for measuring the chrysin sulphation rate in human liver. The enzyme followed Michaelis‐Menten kinetics and Km and Vmax (mean ± SD) measured in four livers were 0.29 ± 0.07 μM and 43.1 ± 1.9 pmol·min−1·mg−1 respectively. 5. Catechin was neither an inhibitor of resveratrol sulphation nor a substrate of sulphotransferase. 6. These results are consistent with the view that many, but not all, flavonoids inhibit the hepatic and duodenal sulphation of resveratrol, and such inhibition might improve the bioavailability of this compound.

Genetic and epigenetic biomarkers for diagnosis, prognosis and treatment of colorectal cancer

Colorectal cancer (CRC) is one of the most common cancer worldwide and results from the accumulation of mutations and epimutations in colonic mucosa cells ultimately leading to cell proliferation and metastasis. Unfortunately, CRC prognosis is still poor and the search of novel diagnostic and prognostic biomarkers is highly desired to prevent CRC-related deaths. The present article aims to summarize the most recent findings concerning the use of either genetic or epigenetic (mainly related to DNA methylation) biomarkers for CRC diagnosis, prognosis, and response to treatment. Recent large-scale DNA methylation studies suggest that CRC can be divided into several subtypes according to the frequency of DNA methylation and those of mutations in key CRC genes, and that this is reflected by different prognostic outcomes. Increasing evidence suggests that the analysis of DNA methylation in blood or fecal specimens could represent a valuable non-invasive diagnostic tool for CRC. Moreover, a broad spectrum of studies indicates that the inter-individual response to chemotherapeutic treatments depends on both epigenetic modifications and genetic mutations occurring in colorectal cancer cells, thereby opening the way for a personalized medicine. Overall, combining genetic and epigenetic data might represent the most promising tool for a proper diagnostic, prognostic and therapeutic approach.

Comparison Study of MS-HRM and Pyrosequencing Techniques for Quantification of APC and CDKN2A Gene Methylation

There is increasing interest in the development of cost-effective techniques for the quantification of DNA methylation biomarkers. We analyzed 90 samples of surgically resected colorectal cancer tissues for APC and CDKN2A promoter methylation using methylation sensitive-high resolution melting (MS-HRM) and pyrosequencing. MS-HRM is a less expensive technique compared with pyrosequencing but is usually more limited because it gives a range of methylation estimates rather than a single value. Here, we developed a method for deriving single estimates, rather than a range, of methylation using MS-HRM and compared the values obtained in this way with those obtained using the gold standard quantitative method of pyrosequencing. We derived an interpolation curve using standards of known methylated/unmethylated ratio (0%, 12.5%, 25%, 50%, 75%, and 100% of methylation) to obtain the best estimate of the extent of methylation for each of our samples. We observed similar profiles of methylation and a high correlation coefficient between the two techniques. Overall, our new approach allows MS-HRM to be used as a quantitative assay which provides results which are comparable with those obtained by pyrosequencing.

Role of cyclooxygenases 1 and 2 in the modulation of neuromuscular functions in the distal colon of humans and mice

Background: Cyclooxygenase isoforms (COX-1, COX-2) may exert differential regulatory actions on enteric motor functions under normal or pathological conditions. Aims: To examine the occurrence and functions of COX-1 and COX-2 in the neuromuscular compartment of normal distal colon using human and murine tissue. Methods: Gene expression (human, mouse), protein expression (human), gene deletion (mouse), and the effects of dual and isoform specific COX inhibitors on in vitro motility (human, mouse) were investigated. Results: Reverse transcription-polymerase chain reaction (RT-PCR) showed mRNA expression of COX-1 and COX-2 in human and wild-type mouse colonic muscle whereas only COX-2 or COX-1 was detected in COX-1 or COX-2 knockout animals. Immunohistochemistry localised both isoforms in neurones of myenteric ganglia, COX-1 in circular layer myocytes, and COX-2 in longitudinal muscle. Indomethacin (COX-1/COX-2 inhibitor), SC-560 (COX-1 inhibitor), or DFU (COX-2 inhibitor) enhanced atropine sensitive electrically induced contractions of human longitudinal muscle. The most prominent actions were recorded with indomethacin or SC-560 plus DFU. These results were confirmed under pharmacological blockade of non-cholinergic nerves. Atropine sensitive contractions evoked by carbachol in the presence of tetrodotoxin were enhanced by indomethacin or DFU but not by SC-560. In wild-type mice, contractile responses to electrical stimulation were enhanced by indomethacin, SC-560, or DFU. SC-560 potentiated electrically induced contractions in COX-2, but not COX-1, knockout mice. In contrast, DFU enhanced the contractions elicited by electrical stimuli in COX-1, but not in COX-2, knockout mice. Conclusions: These results indicate that COX-1 and COX-2 are expressed in the neuromuscular compartment of normal human colon where they modulate cholinergic excitatory control of colonic motility at prejunctional and postjunctional sites, respectively.

Differential inhibition of human liver and duodenum sulphotransferase activities by quercetin, a flavonoid present in vegetables, fruit and wine

1. Quercetin is a natural flavonoid present in vegetables, fruit and wine, and is known to inhibit sulphotransferase. Drugs are often taken orally and the intestinal mucosa is an early site of drug metabolism. The aims of this investigation were to study the inhibition of dopamine, (-)-salbutamol, minoxidil and paracetamol sulphation by quercetin in the duodenal mucosa and liver and to compare the IC 50 in these tissues. 2. The rates (pmol min -1 mg -1) of sulphation of 4-nitrophenol were 343 92 (liver) and 164 22 (duodenum; p = 0.031), of dopamine were 15 11 (liver) and 656 516 (duodenum; p = 0.049), of (-)-salbutamol were 153 31 (liver) and 654 277 (duodenum; p = 0.018), of minoxidil were 156 47 (liver) and 105 7 (duodenum; n.s.), and of paracetamol were 229 86 (liver) and 328 187 (duodenum; n.s.). 3. The IC 50 of quercetin for 4-nitrophenol was 48 11 nM (liver) and 56 1 nM (duodenum, n.s.), for dopamine was 5.7 0.7 µM (liver) and 170 12µM (duodenum, p < 0.0001), for (-)-salbutamol was 54 4 nM (liver) and 16 8µM (duodenum; p = 0.025), for minoxidil was 134 22 nM (liver) and 3 0.3 µM (duodenum, p = 0.013), and for paracetamol was 57 7 nM (liver) and 35 1 µM (duodenum; p = 0.0002). 4. Quercetin inhibited the sulphation of 4-nitrophenol, dopamine, (-)-salbutamol, minoxidil and paracetamol both in liver and duodenum. With dopamine, (-)-salbutamol, minoxidil and paracetamol as substrates, quercetin was a more potent inhibitor in the liver than the duodenum. Such a difference may reflect the different composition of sulphotransferase forms in the liver and duodenum.

Curcumin is a potent inhibitor of phenol sulfotransferase (SULT1A1) in human liver and extrahepatic tissues

1. Curcumin has anti-carcinogen effects and is under clinical evaluation as a potential colon cancer chemopreventive agent. The first aim was to see whether curcumin inhibited phenol sulfotransferase (SULT1A1) and, if so, to study the variability of the IC 50 of curcumin for SULT1A1 in 50 human liver samples. For comparative purposes, the inhibition of catechol sulfotransferase (SULT1A3) in five human liver specimens was studied. The second aim was to measure the IC 50 of curcumin against SULT1A1 in five samples of human duodenum, colon, kidney and lung. 2. Curcumin was a potent inhibitor of SULT1A1 in human liver; the mean ± SD and median of IC 50 were 14.1 ± 7.3 nM and 12.8 nM, respectively. The IC 50 ranged from 6.2 to 30.6 nM between the 5th and 95th percentiles and the fold of variation was 4.9. The distribution of IC 50 was positively skewed (skewness 1.2) and deviated from normality (p = 0.0004). 3. Curcumin inhibited human SULT1A3, and the inhibition was studied in five liver specimens with an IC 50 of 4324 ± 1026 nM. This inhibition was greater than the IC 50 of curcumin for SULT1A1 (p < 0.0001). 4. In the extrahepatic tissues, the IC 50 of curcumin for SULT1A1 was 25.9 ± 4.8 nM (duodenum), 25.4 ± 6.8 nM (colon), 23.4 ± 2.2 nM (kidney) and 25.6 ± 5.6 nM (lung). Inhibition in these tissues is greater than that of curcumin for SULT1A1 in human liver (p < 0.0001). 5. In conclusion, curcumin is a potent inhibitor of SULT1A1 in human liver, duodenum, colon, kidney and lung. The IC 50 of curcumin for SULT1A1 varied 4.9-fold in human liver. The comparison of the present data with those of the literature revealed that the IC 50 of curcumin in the liver and extrahepatic tissues is one order of magnitude lower that the peak serum concentration of curcumin after therapeutic doses of 4 g to humans.

Gene promoter methylation in colorectal cancer and healthy adjacent mucosa specimens: Correlation with physiological and pathological characteristics, and with biomarkers of one-carbon metabolism

We evaluated the promoter methylation levels of the APC, MGMT, hMLH1, RASSF1A and CDKN2A genes in 107 colorectal cancer (CRC) samples and 80 healthy adjacent tissues. We searched for correlation with both physical and pathological features, polymorphisms of folate metabolism pathway genes (MTHFR, MTRR, MTR, RFC1, TYMS, and DNMT3B), and data on circulating folate, vitamin B12 and homocysteine, which were available in a subgroup of the CRC patients. An increased number of methylated samples were found in CRC respect to adjacent healthy tissues, with the exception of APC, which was also frequently methylated in healthy colonic mucosa. Statistically significant associations were found between RASSF1A promoter methylation and tumor stage, and between hMLH1 promoter methylation and tumor location. Increasing age positively correlated with both hMLH1 and MGMT methylation levels in CRC tissues, and with APC methylation levels in the adjacent healthy mucosa. Concerning gender, females showed higher hMLH1 promoter methylation levels with respect to males. In CRC samples, the MTR 2756AG genotype correlated with higher methylation levels of RASSF1A, and the TYMS 1494 6bp ins/del polymorphism correlated with the methylation levels of both APC and hMLH1. In adjacent healthy tissues, MTR 2756AG and TYMS 1494 6bp del/del genotypes correlated with APC and MGMT promoter methylation, respectively. Low folate levels were associated with hMLH1 hypermethylation. Present results support the hypothesis that DNA methylation in CRC depends from both physiological and environmental factors, with one-carbon metabolism largely involved in this process.

(-)-Salbutamol sulphation in the human liver and duodenal mucosa : interindividual variability

1. Salbutamol as a beta 2-adrenergic agonist used in the treatment of lung obstructive disease and premature labour. It has a bioavailability of 50% and sulphation is the main route of its metabolism. (-)-Salbutamol retains most of the beta 2-adrenergic activity and, thereby, we describe the interindividual variability in the sulphation rate of (-)-salbutamol in 100 specimens of human liver and duodenal mucosa. 2. The mean rate (pmol/min/mg of salbutamol sulphation was 498 in the duodenum and 141 in the liver with 4-fold variation within +/-2 SD units in both tissues. 3. A modelling approach based on the comparison of the best fittings obtained using a gaussian and the sum of two gaussian curves revealed the presence of two subgroups in the hepatic rate of salbutamol sulphation and their means were 69.5 and 105 pmol/min/mg (p < 0.05). In the duodenum, the rate of salbutamol sulphation approached normality. 4. The rates of salbutamol and 4-nitrophenol sulphation correlated highly (r = 0.853; p < 0.001) in the liver whereas in duodenum the rates of salbutamol and dopamine correlated highly (r = 0.914; p < 0.001), 4-Nitrophenol and dopamine are the diagnostic substrates of phenol- and catechol-sulphotransferases respectively. These findings are consistent with the view that the rate of salbutamol sulphation is higher in the gut than in liver and it varies considerably in both tissues.

Effect of IL-6 overexpression on the metastatic potential of rat hepatocellular carcinoma cells

AbstractBackground: Previous studies demonstrated that excess IL-6 production correlated with the metastatic potential of rat hepatocellular carcinoma cells. In the work reported here a retroviral construct containing the gene for murine IL-6 was introduced into otherwise nonmetastatic tumor cells to directly determine the effect of IL-6 overexpression on tumor metastatic potential. Methods: The clonal cell lines 1682.C.2.9.L0 (L0, poorly metastatic) and 1682.C.2.9.L10 (L10, highly metastatic) were selected from a parental hepatocellular carcinoma induced in ACI rats by feeding an ethionine-containing diet. Viral supernatant was used to infect the PA317 amphotropic cell line, and retrovirus produced from these cells infected the poorly metastatic L0 hepatocellular carcinoma cell line. Neomycin-resistant cells were selected in G418 and designated L0-IL-6. Results: As determined by bioassay, L0 cells produce 10±1.2 U/mL IL-6 in culture, whereas L10 cells release 95±11 U/mL (P<0.01, Studentst-test). Retroviral-mediated IL-6 gene transfer resulted in the production of 1266±48 U/mL IL-6 by L0-IL-6 cells under identical culture conditions. When an inoculum of 5×106 cells is injected subcutaneously, both L0 and L10 cell lines result in primary tumors with equivalent rates of growth; only L10 cells metastasize to the lung, however. A similar inoculation of L0-IL-6 cells produced local tumors in all 24 animals tested. Interestingly, 15 of 24 (62%) animals presented with metastatic nodules in the abdominal cavity, whereas no such tumors were found in animals receiving L10 cells. Conclusion: Overexpression of IL-6 increases metastatic potential of tumor cells, with preferential metastases to the abdominal cavity when compared with tumor cells elaborating endogenous IL-6.