Maria Gabriella Caruso

EGF, TGF-a, and EGF-R in Human Colorectal Adenocarcinoma

: The over-expression of epidermal growth factor receptor (EGF-R), EGF and transforming growth factor alpha (TGFalpha) could be a mechanism for colorectal tumor cells to escape normal growth controls. Our aims were: (i) to evaluate EGF, TGFalpha, and EGF-R concentrations in neoplastic tissue and surrounding mucosa from 40 patients with colorectal adenocarcinoma, and (ii) to assess the expression of these growth factors and their receptor in relation to the tumor site. EGF, TGFalpha, and EGF-R were detected in either colorectal neoplastic tissue or surrounding mucosa. Significantly increased levels of EGF and EGF-R were present in neoplastic samples compared to surrounding mucosa. Furthermore, a significant increase in TGFalpha and in EGF levels was observed in the left-sided surrounding mucosa and left-sided neoplastic tissue, respectively. EGF-R stimulation by its ligands may play an important role in colorectal neoplastic tissue. Moreover, the higher content of growth factors in the left-side than the right-side colon suggests different growth properties in the proximal and distal colon.

Effects of olive oil polyphenols on fatty acid synthase gene expression and activity in human colorectal cancer cells

Oleuropein (OL) and hydroxytyrosol (HT), the main olive oil polyphenols, possess anti-proliferative effects in vitro. Fatty acid synthase, a key anabolic enzyme of biosynthesis of fatty acids, plays an important role in colon carcinoma development. Our aim was to investigate whether gene expression of FAS, as well as its enzymatic activity, is regulated by HT and OL in two human colon cancer cell lines, as HT-29 and SW620. In addition, we investigated the effects of these polyphenols on growth and apoptosis in these cells. FAS gene expression and activity in treated HT-29 and SW620 cells were evaluated by real-time PCR and radiochemical assay, respectively. Cell growth and apoptosis, after polyphenols treatment, were measured by MTT test and flow cytometry, respectively. The inhibition of proliferation, detected after HT treatment, was mediated by an inhibition of FAS expression and its enzymatic activity in SW620 cells, while the anti-proliferative effect in HT-29 cells seems to be independent from FAS. OL exerted an anti-proliferative effect only on SW620 cells with a mechanism which excluded FAS. Olive oil polyphenols used were able to induce apoptosis in both cell lines studied. The increase of apoptosis in these cells was accompanied by the block of cell cycle in the S phase. This study demonstrates that HT and OL may induce anti-proliferative and pro-apoptotic effects only in certain human colorectal cancer cell types. These effects are FAS mediated only in SW620 cells after treatment with HT.

Polyunsaturated fatty acids reduce Fatty Acid Synthase and Hydroxy-Methyl-Glutaryl CoA-Reductase gene expression and promote apoptosis in HepG2 cell line

Backgroundn-3 and n-6 polyunsaturated fatty acids (PUFAs) are the two major classes of PUFAs encountered in the diet, and both classes of fatty acids are required for normal human health. Moreover, PUFAs have effects on diverse pathological processes impacting chronic disease, such as cardiovascular and immune disease, neurological disease, and cancer.AimTo investigate the effects of eicosapentaenoic acid (EPA) and arachidonic acid (ARA) on the proliferation and apoptosis of human hepatoma cell line HepG2 after exposure to increasing concentrations of EPA or ARA for 48 h. Moreover, in the same cells the gene expression of Fatty Acid Synthase (FAS) and 3-Hydroxy-3-Methyl-Glutaryl Coenzyme A Reductase (HMG-CoAR) was also investigated.MethodCell growth and apoptosis were assayed by MTT and ELISA test, respectively after cell exposure to increasing concentrations of EPA and ARA. Reverse-transcription and real-time PCR was used to detect FAS and HMG-CoAR mRNA levels in treated cells.ResultsOur findings show that EPA inhibits HepG2 cell growth in a dose-dependent manner, starting from 25 μM (P < 0.01, one-way ANOVA test and Dunnetts post test) and exerts a statistically significant pro-apoptotic effect already at 1 μM of EPA. Higher doses of ARA were need to obtain a statistically significant inhibition of cell proliferation and a pro-apoptotic effect in these cells (100 μM, P < 0.01, one-way ANOVA test and Dunnetts post test). Moreover, a down-regulation of FAS and HMG-CoAR gene expression was observed after EPA and ARA treatment in HepG2 cells, starting at 10 μM (P < 0.05, one-way ANOVA test and Dunnetts post test).ConclusionOur results demonstrate that EPA and ARA inhibit HepG2 cell proliferation and induce apoptosis. The down-regulation of FAS and HMG-CoAR gene expression by EPA and ARA might be one of the mechanisms for the anti-proliferative properties of PUFAs in an in vitro model of hepatocellular carcinoma.

N6-isopentenyladenosine arrests tumor cell proliferation by inhibiting farnesyl diphosphate synthase and protein prenylation

The physiological effects of a variety of N6‐substituted adenine and adenosine derivatives called cytokinins have been documented in plants, but information on their occurrence and function in other biological system is limited. Here we investigated the anti‐proliferative effect of N6‐isopentenyladenosine (i6A), an adenosine and isoprenoid derivative, in a thyroid cell system, FRTL‐5 wild‐type, and K‐ras transformed KiMol cells. Addition of i6A to FRTL‐5 cells caused a dose‐dependent arrest of the G0‐G1 cell phase transition associated with a reduction of cells in the S phase that was much more evident in KiMol cells. I6A arrested tumor cell proliferation by inhibiting farnesyl diphosphate synthase (FPPS) and protein prenylation. Indeed the addition of farnesol reversed these effects and i6A affected protein prenylation, in particular lamin B processing. I6A effect was not mediated by the adenosine receptor but was due to a direct modulation of FPPS enzyme activity as a result of its uptake inside the cells. I6A inhibited FPPS activity more efficaciously in KiMol cells than in normal FRTL‐5. Moreover, the i6A anti‐proliferative effect was evaluated in vivo in a nude mouse xenograft model, where KiMol cells were implanted subcutaneously. Mice treated with i6A showed a drastic reduction in tumor volume. Our findings indicate that this isoprenoid end product might be used for antineoplastic therapy, an application emulating that of the lovastatin and/or farnesyltransferase inhibitors.—Laezza, C., Notarnicola, M., Caruso, M. G., Messa, C., Macchia, M., Bertini, S., Minutolo, F., Portella, G., Fiorentino, L., Stingo, S., Bifulco, M. N6‐isopentenyladenosine arrests tumor cell proliferation by inhibiting farnesyl diphosphate synthase and protein prenylation. FASEB J. 20, 412–418 (2006)

Higher Farnesyl diphosphate synthase activity in human colorectal cancer inhibition of cellular apoptosis

Objective: Farnesyl diphosphate synthase (FPPs) produces FPP which is considered a branch-point intermediate in the synthesis of sterols and isoprenylated cellular metabolites. In this study we investigated whether detectable FPPs activity was present in human colorectal cancer (CRC), also evaluating in vitro the role of this enzyme in the growth and apoptosis of CRC cells by using Pamidronate (PAM), a FPPs activity inhibitor. Methods: The activity level of FPPs was determined in CRC and the normal surrounding mucosa of 50 patients by radiochemical assay. The FPPs mRNA expression was investigated in 15 of 50 patients by quantitative reverse transcriptase polymerase chain reaction (RT-PCR). K-ras mutation was evaluated using PCR and restriction enzyme analysis. Cell growth and apoptosis, after PAM treatment, in human CRC cell line DLD-1 were measured by MTT test and DNA fragmentation, respectively. Results: FPPs activity was detectable in human CRC. FPPs activity and its mRNA were significantly more abundant in cancer samples than in normal mucosa. In vitro PAM resulted in a significant reduction of cell growth and also gave rise to a marked proapoptotic effect. Conclusions: This study provides the first evidence of the presence of FPPs activity in human CRC. Moreover, FPPs enzyme was found to play a significant role in colon cancer proliferation.

Serum levels of galectin-3 and its ligand 90k/mac-2bp in colorectal cancer patients

Galectin-3 is an endogenous lectin that binds glycan epitopes of cell membrane and some extracellular glycoproteins such as integrins and laminin. Galectin-3 is involved in several biological activities including regulation of cellular cycle, modulation of adhesion and tumor progression and metastasis. 90K/Mac-2BP glycoprotein is also a serum galectin-3 ligand. 90K is able to modulate the immune reaction against tumors and viruses and its level increases in sera of several neoplastic diseases. In our study, we have evaluated levels of both glycoproteins in sera of non metastatic colon cancer patients. Interestingly, galectin-3 ranged higher in cancer patients than in controls (p<0.0001), particularly in more differentiated tumors (p<0.04). Moreover, 90K mean values ranged higher in right-side than in left-side colon cancer. In conclusion, serum galectin3 might represent a useful biomarker to evaluate colon cancer transformation and, together with its ligand 90K, could contribute to the characterization of colon cancer.

Estrogenic induction of cannabinoid CB1 receptor in human colon cancer cell lines

Objective. Cannabinoids are a class of compounds that have the ability to activate two specific receptor subtypes, the cannabinoid CB1 and CB2 receptors. CB1 receptor is a G-protein-coupled receptor that is linked to the signal transduction pathways. The cumulative effects of this receptor have important implications in the control of cell survival and cell death having the potential to regulate tumor cell growth. In this connection, interest has been focused on factors such as sex steroid hormones, which regulate CB1 receptor expression. The aim of this study was to investigate the effects of 17β-estradiol exposure on the CB1 receptor gene and its protein expression in human primary tumor colon cancer cell lines, such as DLD-1, HT-29 and one lymph node metastatic cell line, SW620. Material and methods. CB1 gene expression was determined using quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) in DLD-1, HT-29 and SW620 cells treated at different times and doses of 17β-estradiol exposure. CB1 protein expression was detected by Western immunoblot. Results. 17β-estradiol induced CB1 gene expression in all the human colon cancer cells studied. The early induction of CB1 receptor mRNA in DLD-1 and SW620 cells was mediated by the estrogen receptor because the pure estrogen antagonist, ICI 182,780, was able to counteract this effect. Estrogenic induction of the CB1 receptor was also detectable at protein level in all cell types tested. Conclusions. The CB1 receptor can be considered an estrogen-responsive gene in DLD-1, HT-29 and SW620 cells. Up-regulation of CB1 expression by 17β-estradiol is a further mechanism of estrogens to control colon cancer proliferation.

N6-isopentenyladenosine inhibits cell proliferation and induces apoptosis in a human colon cancer cell line DLD1.

N6‐isopentenyladenosine (i6A) is a modified nucleoside with a pentaatomic isopentenyl derived from mevalonate that induces inhibition of tumor cell proliferation and apoptosis in several tumor cell lines. In this study, we reported that N6‐isopentenyladenosine inhibited the proliferation and promotes apoptosis in DLD1 human colon cancer cells. It suppressed the proliferation of cells through inhibition of DNA synthesis, causing a cell cycle arrest that correlated with a decrease in the levels of cyclin E, cyclin A and cyclin D1 and with a concomitant increase in the levels of cyclin‐dependent kinase inhibitor p21waf and p27kip1. Moreover, it induced apoptosis through an increase in the number of annexin V‐positive cells, a downregulation of antiapoptotic products and caspase‐3 activation. The apoptotic effects of N6‐isopentenyladenosine were accompanied by sustained phosphorylation and activation of c‐jun N‐terminal kinase (JNK) that induced phosphorylation of c‐jun. Overall, our data show that JNK, could play an important role in i6A‐mediated apoptosis in DLD1 human colon cancer cells

Modified HMG-CoA reductase and LDLr regulation is deeply involved in age-related hypercholesterolemia.

During the ageing process in rats hypercholesterolemia occurs in concert with full activation, lowered degradation rate and an unchanged level of the rate limiting cholesterol biosynthesis enzyme, 3‐hydroxy‐3‐methylglutaryl coenzyme A reductase (HMG‐CoAR). The molecular bases of the HMG‐CoAR unchanged level and lowered degradation rate in aged rats is not clear. In fact no data are available during ageing, on transcription and degradation of HMG‐CoAR, so well defined in adult animal. So, aim of this work was to measure mRNA levels of the enzyme and the level of the proteins of the regulatory complex responsible of the cholesterol metabolism. To complete the picture, the level of sterol regulatory element binding proteins (SREBPs), SREBP cleavage activating protein, and insulin‐induced gene has been measured. The levels of other related proteins, whose transcription is SREBP dependent, that is low density lipoprotein receptor (LDLr) and Caveolin 1, have been also measured. The age‐related reduced Insigs levels, joined to a reduced insulin sensitivity, could explain the decreased degradation rate of the HMG‐CoAR and the increased active SREBP‐2. The SREBP‐2 in particular seems to be committed in multiple way to gene transcription. The obtained data represent a good contribution to explain the age‐related hypercholesterolemia. J. Cell. Biochem. 98: 1044–1053, 2006.

Estrogenic regulation of cholesterol biosynthesis and cell growth in DLD-1 human colon cancer cells

Objective. The main molecules of cholesterol homeostasis are 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoAR), the key enzyme of the biosynthetic pathway, and the low-density lipoprotein receptor (LDL-R), which is responsible for the uptake of plasma lipoproteins. The increase in the endogenous cholesterol biosynthesis results in stimulation of DNA synthesis, while the inhibition of cholesterogenesis suppresses cell growth. Estrogens have been reported to regulate hepatic LDL-R expression and modulate cell proliferation in different tissues. In order to clarify the mechanisms of estrogenic growth control in colorectal carcinoma, we have investigated the effects of 17β-estradiol exposure on LDL-R gene expression and its protein, as well as on HMG-CoAR gene expression, its protein as well as enzyme activity in the DLD-1 human colon cancer cell line. The effect of 17β-estradiol on both cell growth and apoptosis in this cell line was also investigated. Material and methods. LDL-R and HMG-CoAR gene expressions were determined using quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) in DLD-1 cells at different times and doses of 17β-estradiol exposure. LDL-R and HMG-CoAR protein expression was detected by Western immunoblotting. HMG-CoAR activity was evaluated using a radiometric assay. Cell proliferation was measured by colorimetric MTT test and incorporation of [3H]-thymidine in DNA. Apoptotic death was estimated by DNA fragmentation analysis. Results. Estrogens induced an early increase of LDL-R, at both mRNA and protein level, and later decreased HMG-CoAR activity and protein expression. DLD-1 cells treated with 17β-estradiol exhibited inhibition of DNA synthesis and apoptosis. Conclusions. The results of this study suggest that estrogens regulate LDL-R and HMG-CoAR and influence cell growth and apoptosis in colon cancer cells.