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Meccanismi estrinseci ed intrinseci del processo apoptotico nell'epatocarcinogenesi
Full text linkThe mechanisms underlying the strict correlation between viral infection, cirrhosis and HCC development have been investigated in deep, but, despite this several aspects are still largely unknown or understood only in part. Hepatitis B and C viruses seem to be involved in neoplastic transformation through both direct and indirect mechanisms, due to inflammation process and the capacity of viral proteins in modulating the proliferation and the death of infected hepatocytes. Moreover it has been hypothesized that dysregulation of apoptotic process in infected cells could be a crucial step for the cronicity of infection, in the failure to antiviral treatment and in the neoplastic transformation.
Although apoptosis can be triggered by several different stimuli, apoptotic signalling within the cell is transduced mainly via two defined molecular pathways: the death receptor pathways, also called extrinsic pathway (Fas/FasL, TNF-?/TNF-R1-R2) and the mitochondrial pathway also called the intrinsic pathway (Bcl-2, Bax, Bad). Other plasma membrane receptors such as IL-1b and TGF-b1 and intrinsic mediators as BI-1 and NF-kB are involved in the apoptotic pathways. In this study mRNA expression of mediators of the extrinsic and intrinsic apoptotic process was evaluated. The liver biopsies and the surgical hepatic tissues obtained from patients with different degree of liver disease were used. mRNA transcripts were detected by gene amplifications techniques. on the basis of our results human hepatocytes cultures were performed. Primary hepatocytes were treated with TNF-a, Staurosporine and LY294002, protein-kinase inhibitors, in order to evaluate the activation and/or the inhibition of these mediators in the apoptotic process.
Aims
In the first part the study was performed in order to:
1) evaluate the mRNA expression of Fas/FasL, TNF-?, IL-1?, TGFß-1, Bcl-2, Bax, Bad, BI-1 and NF-kB in liver tissues from patients with different degrees of liver damage, from chronic hepatitis, to cirrhosis and hepatocellular carcinoma.
2) determine whether HCV and HBV infection modulates their expression
3) analyze the protein expression of the mediators identified by Western Blot, in liver tissues from patients with HCC
the second part included an "in vitro" study in order to:
4) culture primary human hepatocytes
5) evaluate the different apoptotic pathways in human hepatocytes after stimulation with:
a) TNF-?, mainly proinflammatory cytokine found in abundance in the inflammatory process relating to liver injury
b) Staurosporine (STS), a potent protein tyrosine/serine kinase inhibitor with an antiproliferative and anti-apoptotic activity in many tumoral cells
c) LY294002 synthetic inhibitor of PI3K (phosphoinositide 3-kinase)
Material and methods
We examined 62 patients: 39 with chronic hepatitis (CH), 7 with cirrhosis (CIRR), 13 with hepatocellular carcinoma HCC) and 3 controls (Contr). The Fas/FasL, IL-ß and TNF-? mRNAs were quantified by Semiquantitative RT-PCR, while the Bcl-2, Bax, Bad, BI-1, NF-kB and TGFß-1 mRNAs were quantified by quantitative Real Time PCR, using ß-actin as the housekeeping gene. The protein expression of NF-kB in tissues with HCC was analyzed by Western Blot.
Results
Tissues:
Fas/FasL mRNA expression:
Fas mRNA levels were significantly increased in CIRR when compared with HCC (p=0.026) while FasL mRNAs showed a statistically significant differences between CIRR and cirrhotic tissues surrounding tumours (PHCC) (p=0.018) or HCC (p=0.005).
IL-1ß mRNA expression:
PHCC tissues had higher IL-1b levels than either CH tissues (p<0,0001) or CIRR tissues (p=0,005).
TNF-? mRNA expression:
TNF-ß mRNAs were increased in CIRR tissues compared with PHCC (p=0,038) and HCC tissues (p=0,05).
TGFß-1 mRNA expression:
In CH tissues, TGFß-1 mRNA expression was significantly lower than in HCC (p=0,014) or PHCC tissues (p=0,028).
Bcl-2 mRNA expression:
Bcl-2 mRNA levels were statistically higher in PHCC when compared with normal liver tissues (p=0,026), with CH (p=0,004), and with CIRR (p=0,05).
BI-1 mRNA expression:
CH tissues expressed the highest BI-1 mRNA amount that was significantly higher when compared to cirrhotic tissues (p=0,033), cirrhotic tissues surrounding tumours (p<0,001) and HCC (p<0,001).
Bad mRNA expression:
Contr tissues showed the highest Bad transcript levels, which were statistically higher than in CH (p=0,006), PHCC (p=0,005), or HCC (p=0,002). CH coincided with higher Bad levels than in PHCC or HCC tissues (p=0,00006 and p=0,0007, respectively)
Bax mRNA expression:
No differences in Bax expression were observed.
NF-kB mRNA expression:
CONTR expressed the lowest NF-kB mRNA amount, significantly lower when compared with CH (p=0.05) and PHCC (p=0.05).
HBV HCV infection:
In CH, only TNF-?, IL-1ß and Bad mRNA expression were significantly different in HBV-related infection versus HCV-related infection.
Human hepatocytes:
TNF-? treatment:
Human hepatocytes incubated with TNF-? showed the highest levels of FasL (solo a 12h), IL-1ß(until 24h), TGFß-1 (24 e 48h), Bcl-2 (4h, 24h), Bad (24 and 48h) and NF-kB (8h) respect to unstimulated cells.
No differences in Fas, Bax, Bad (until 12h) , NF-kB (until 4h) e TGFß-1 (until 12h) mRNA levels were observed.
Staurosporine (STS) treatment:
In cells treated with STS, Bcl-2 mRNA expression until 48h of culture, was significantly lower than in unstimulated cells. No difference in TGFß-1, Bax, and NF-kB mRNAs levels were observed at the different time of incubation. While an increase in Bad mRNA at 12h and in BI-1 mRNA at 8 and 24h were observed.
LY294002 treatment:
LY294002 induce a strong inhibition of Bcl-2 mRNA (until 24h) in hepatocyte culture when compared with hepatocyte culture with medium alone. No difference in TGFß-1, Bax, and NF-kB mRNAs levels were observed while high levels of Bad mRNA at 12, 24 and 48h and of BI-1 from 8 to 24h of incubation were observed.
Pre-treatment with STS and LY294002 :
The pre-treatment of human hepatocytes for 1h with LY294002 and the following treatment with TNF-? inhibits the Bcl-2 mRNA expression until 48h when compared with cell treated with TNF-? without the pre-treatment. This inhibition was completely resolved after 48h of incubation.
In cells pretreated with LY294002 respect to the cells treated with TNF-? alone, were observed the highest levels of Bax after 4h, Bad after 24h e BI-1 after 24h of incubation.
No difference in TGFß-1 and NF-kB mRNAs expression were observed
In the cells pre-treated with STS only the BI-1 transcript levels were increased until 24h.
TISSUES
Fas/FasL system expression is upregulated in chronically-damaged livers while the onset of cancer is associated with a shut-down of this system in HCC, as a defense mechanisms adopted by HCC against the immune system.
High levels of IL-1ß mRNA transcripts observed in cirrhotic tissues surrounding tumours, may be the result of an increased activation of lymphocytes o monocytes in the liver.
Contrary, the low expression of TNF-? mRNA transcripts in cirrhotic tissues surrounding tumours and HCC may be the result of loss function of Kupffer cells, one of the main sources of production of TNF-? in the liver.
The Bcl-2 hyperexpression observed in cirrhotic peritumoral tissues seems to play an important part in regulating cell survival in damaged liver cells, which may contribute to the persistence of HBV or HCV and thus activate cell proliferation and cell transformation.
The upregulation of the Bad-induced pro-apoptotic pathway in CH seems to be more relevant in controlling apoptosis in liver tissues with HBV infection suggesting an important role of the proteins of HBV particularly in early liver diseases.
Moreover the BI-1 expression decrease correlates closely with the progression of liver damage from CH to cirrhosis and HCC and indicate a role of BI-1 as regulator of apoptosis in early diseases. The high BI-1 mRNA levels observed in CH may provide protection against apoptosis of liver cells virus infected, on the other hand the down regulation of BI-1 observed in HCC tissues seems to favour the hapatocellular carcinogenesis.
The persistently elevated levels of NF-kB in all stage of liver diseases, seems to depends on a chronic inflammatory with an consequently increased risk of hepatic fibrosis and of an HCC milieu.
The different pattern of IL-1ß, TNF-? and Bad expression in HCV- and HBV-related liver disease points to a different modulation of the immune response on the one hand, and to a different apoptotic activation pathways on the other.
HUMAN HEPATOCYTES
IL-1ß and TNF-? are proinflammatory cytokines always secreted during inflammation.
The high IL-1ß mRNA transcripts induced by TNF-? stimulation in primary human hepatocytes suggest that the presence of TNF-? in the hepatic microenvironment contributes to the increase in IL-1ß with a consequent intensification of inflammatory, proliferative and regenerative processes.
No difference in Fas/FasL mRNAs expression was observed after TNF-? treatment, suggesting that the expression of these mediators was not modulated by the extracellular TNF-? factor in our experimental conditions.
Bcl-2 is an antiapoptotic member of the intrinsic Bcl-2 family and its activation by TNF-? might explain the resistance to apoptotic process induced by TNF-? also observed in mouse and rat hepatocyte cultures.
Pre-treatment and treatment with STS, LY924002 and TNF-? reduce the Bcl-2 transcript levels by comparison with the levels obtained from cells treated with TNF-? alone. These effects were particularly evident using LY924002. STS is considered a scarcely-selective kinase inhibitor known for its toxicity, while LY924002 represents a highly specific PI3K inhibitor.
Bax and Bad are pro-apoptotic members of the BCl-2 family.
TNF-?, STS and LY294002 had no effect on Bax expression in human hepatocytes compared with hepatocytes incubated with medium alone, whereas Bad mRNA levels increased after stimulation with TNF-? STS and LY294002, particularly after 12 to 48 hours of incubation.
Costimulation with LY294002 and TNF-? increases the Bad mRNAs after 24h and 48h of incubation. So, when the antiapoptotic pathway (Bcl-2) was inhibited in primary human hepatocytes, members of the apoptotic pathway (Bad and Bax) were activated
The Unfolded Protein Response and Autophagy in Chronic Liver Diseases, HCC and Metastases
No full textOxidative damage in the progression of chronic liver disease to hepatocellular carcinoma: An intricate pathway.
Full text linkThe histo-pathologic and molecular mechanisms leading to initiation and progression of hepatocellular carcinoma (HCC) are still ill-defined; however, there is increasing evidence that the gradual accumulation of mutations, genetic and epigenetic changes which occur in preneoplastic hepatocytes results in the development of dysplastic foci, nodules, and finally, overt HCC. As well as many other neoplasias, liver cancer is considered an "inflammatory cancer", arising from a context of inflammation, and characterized by inflammation-related mechanisms that favor tumor cell survival, proliferation, and invasion. Molecular mechanisms that link inflammation and neoplasia have been widely investigated, and it has been well established that inflammatory cells recruited at these sites with ongoing inflammatory activity release chemokines that enhance the production of reactive oxygen species. The latter, in turn, probably have a major pathogenic role in the continuum starting from hepatitis followed by chronic inflammation, and ultimately leading to cancer. The relationship amongst chronic liver injury, free radical production, and development of HCC is explored in the present review, particularly in the light of the complex network that involves oxidative DNA damage, cytokine synthesis, telomere dysfunction, and microRNA regulation
TGF-beta 1 and IGF-1 and Anastomotic Recurrence of Crohn's Disease After Ileo-Colonic Resection
No full textTGF-beta1 and IGF-1 and anastomotic recurrence of Crohn's disease after ileo-colonic resection.
Scarpa M, Bortolami M, Morgan SL, Kotsafti A, Ruffolo C, D'Incà R, Bertin E, Polese L, D'Amico DF, Sturniolo GC, Angriman I.
Source
Clinica Chirurgica I, Dipartimento di Scienze Chirurgiche e Gastroenterologiche, Policlinico Universitario, Università di Padova, via Giustiniani 2, 35128, Padova, Italy. [email protected]
Abstract
BACKGROUND:
After bowel resection, Crohn's disease (CD) recurs frequently in the site of the anastomosis. Alteration of normal healing processes may play a role in this phenomenon. Transforming growth factor beta (TGF-beta) and insulin-like growth factor (IGF-1) are involved in wound healing mechanisms with pro-fibrogenic properties. The aim of this study was to assess the expression of TGF-beta1 and insulin-like growth factor 1 (IGF-1) in the different zones of the bowel wall to understand why side-to-side anastomosis are associated to a lower recurrence rate compared to end-to-end ones.
PATIENTS AND METHODS:
Seventeen patients affected by CD who underwent ileo-colonic resection from 2004 to 2005 were enrolled in this study. Full-thickness tissue samples were obtained from the mesenteric, the lateral, and the anti-mesenteric sides of the macroscopically diseased and healthy ileum for each patient. TGF-beta1 and IGF-1 messenger RNAs (mRNAs) were quantified by real-time polymerase chain reaction. Myeloperoxidase activity and histological disease activity were assessed to quantify the ileal inflammation. Vimentin, desmin, and alpha-smooth muscle actin were stained with immunohistochemistry to assess the fibroblast, smooth muscle cell, and myofibroblasts populations. Comparisons and correlations were carried out with nonparametric tests.
RESULTS:
In diseased ileum, TGF-beta1 mRNA transcripts in the antimesenteric side were significantly lower than those of the mesenteric side (p = 0.05), and a significant correlation between TGFbeta-1 levels in diseased bowel and the sampling site was observed (tau = 0.36, p = 0.03). On the contrary, neither the IGF-1 mRNA transcripts nor the distribution of fibroblast, smooth muscle cell, and myofibroblasts populations showed any relation with the sampling site.
CONCLUSION:
TGF-beta1 mRNA expression was lower in the anti-mesenteric side of the diseased ileum, and this was consistent with the success of side-to-side anastomosis in preventing CD recurrence. Since high expression of TGF-beta1 was associated to early recurrence, it seems rationale to construct the anastomosis on the anti-mesenteric side of the bowel
Aberrant gene methylation in non-neoplastic mucosa as a predictive marker of ulcerative colitis-associated CRC
Full text linkBackground Promoter: hypermethylation plays a major role in cancer through transcriptional silencing of critical genes. The aim of our study is to evaluate the methylation status of these genes in the colonic mucosa without dysplasia or adenocarcinoma at the different steps of sporadic and UC-related carcinogenesis and to investigate the possible role of genomic methylation as a marker of CRC.
Results: The expression of Dnmts 1 and 3A was significantly increased in UC-related carcinogenesis compared to non inflammatory colorectal carcinogenesis. In non-neoplastic colonic mucosa, the number of methylated genes resulted significantly higher in patients with CRC and in those with UC-related CRC compared to the HC and UC patients and patients with dysplastic lesion of the colon. The number of methylated genes in non-neoplastic colonic mucosa predicted the presence of CRC with good accuracy either in non inflammatory and inflammatory related CRC.
Methods: Colonic mucosal samples were collected from healthy subjects (HC) (n = 30) and from patients with ulcerative colitis (UC) (n = 29), UC and dysplasia (n = 14), UC and cancer (n = 10), dysplastic adenoma (n = 14), and colon adenocarcinoma (n = 10). DNA methyltransferases-1, -3a, -3b, mRNA expression were quantified by real time qRT-PCR. The methylation status of CDH13, APC, MLH1, MGMT1 and RUNX3 gene promoters was assessed by methylation-specific PCR.
Conclusions: Methylation status of APC, CDH13, MGMT, MLH1 and RUNX3 in the non-neoplastic mucosa may be used as a marker of CRC: these preliminary results could allow for the adjustment of a patient's surveillance interval and to select UC patients who should undergo intensive surveillance
TGF-beta1 and IGF-1production and recurrence of Crohn's disease after ileo-colonic resection.
Full text linkAbstract: Background. Recurrence after surgery is a major problem in the treatment of Crohn's disease (CD). Alteration of healing processes may play a role in this phenomenon. Transforming growth factor beta (TGF-beta) and insulin-like growth factor (IGF-1) have pro-fibrogenic properties and are involved in wound-healing mechanisms. The aim of this study was to assess their role in the CD recurrence after ileo-colonic resection.
Patients and methods. Twenty patients with CD, who underwent ileo-colonic resection in the period between 1999 and 2005, were enrolled in this study. Tissue samples were obtained from macroscopically diseased and healthy ileum. The TGF-beta 1 and IGF-1 mRNAs were quantified by real-time polymerase chain reaction using glyceraldehyde 3-phosphate dehydrogenase as the housekeeping gene. Histological severity of the disease was assessed to quantify the ileal inflammation. Patients' follow-up was investigated. Comparisons and correlations were carried out with nonparametric tests and survival analysis was performed.
Results. Histological inflammation was moderately severe in the diseased bowel, while it was absent in healthy segments (P < 0.01). TGF-beta 1 production in healthy bowels showed a direct correlation with clinical CD recurrence (tau = 0.43, P = 0.04) and survival analysis showed that patients who expressed high TGF-beta 1 mRNA transcripts in healthy intestines had higher cumulative recurrence rates than those who expressed low TGF-beta 1 mRNA levels (P = 0.02).
Conclusion. Our study suggests that the high levels of TGF-beta 1 in healthy bowels of patients who undergo ileo-colonic resection for CD are associated with early clinical disease recurrence, while there seems to be no association between IGF-1 and CD recurrence
Carcinogenesis in Ulcerative Colitis: Immunosurveillance Mechanisms
No full textBackground The inconsistency between dysplasia rate and incidence of cancer in ulcerative colitis (UC)suggests the presence of an immunosurveillance mechanism. The aim of our study was to analyse the expression of costimulatory molecules CD80 and CD86, the lamina propria mononuclear cells populations and the cytokines network at the different stages of carcinogenesis in UC compared to the non inflammatory omologues. Patients and methods Five groups of patients affected by UC (19 pts), UC with colonic dysplasia (10 pts), UC and cancer (7 pts), colonic adenoma (11 pts), sporadic colonic cancer (15 pts) and a group of healthy control (11 pts) were enrolled. Mucosal CD80 and CD86 mRNA levels were quantified with Real Time PCR. Mucosal expression of CD80, CD86, CD3, CD20, CD68 and p53 was analysed by immunohistochemistry. Mucosal levels of IL-1β, IL-2 and TNF- α were measured with immunometric assays. Kruskal-Wallis ANOVA, Mann-Whitney U test and Kendall's tau correlation test were used. Results In UC, CD80 expression was higher in patients with dysplasia (p=0.017). This difference was not evident in the non inflammatory carcinogenesis (fig 1). CD80 expression directly correlated with IL-2 expression and with lamina propria T and B lymphocytes and monocytes populations. In UC, CD86 expression resulted significantly higher than in non inflammatory carcinogenesis without any differences between the steps of the carcinogenesis. CD86 expression correlated with inflammation severity and with IFNgamma expression. Conclusion In UC, CD80 is significantly more expressed in the dysplastic colonic tissue and it seems to be down regulated in cancer. CD80 mucosal levels correlate with T cell population and with IL-2 expression suggesting its involvement in the immunosurveillance mechanism in UC that prevent that all the dysplasia progress to cancer
Mucosal immune environment in colonic carcinogenesis: CD80 up-regulation in colonic dysplasia in ulcerative colitis
Full text linkAbstract: Background: In patients with ulcerative colitis (UC) the inconsistency between the rate of dysplasia and actual cancer incidence suggests the presence of an immunosurveillance mechanism. The aim of our study was to analyse the expression of CD80 and CD86 during the different stages of UC-associated and in non-inflammatory carcinogenesis.
Patients and methods: Sixty-two patients affected with UC, UC with colonic dysplasia, UC and cancer, colonic adenoma, or colonic cancer and 11 healthy subjects were enroled in our study. Tissue samples were taken from surgical specimens during colonic resection or during colonoscopy. Mucosal mRNA expression of CD80 and CD86 was quantified with real time polymerase chain reaction (RT-PCR). CD80, CD86 and p53 expressions and lamina propria mononuclear cell populations (CD3, CD20 and CD68) were analysed by immunohistochemistry. Mucosal levels of IL-1 beta, IL-2 and IFN-gamma were measured with immunometric assays.
Results: Among UC patients, CD80 protein expression was higher in those with dysplasia (p = 0.017). In non-inflammatory carcinogenesis pathway CD80 protein and mRNA expressions were lower compared to the corresponding steps in the UC pathway. CD80 expression was directly correlated with the lamina propria mononuclear cell populations (T and B lymphocytes and monocytes). CD80 protein, but not CD80 mRNA, expression was significantly and directly correlated with IL-2 expression.
Conclusion: CD80 resulted to be up-regulated in UC with dysplasia, while it was down-regulated in cancer. CD80 mucosal levels correlate with lamina propria T-cell and with IL-2 expression suggesting that it may elicit an active role in the immunosurveillance mechanis
TGF-Beta1 and IGF-1 and Anastomotic Recurrence of Crohn's Disease After Ileo-Colonic Resection
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