Endothelin Inhibits Cholangiocarcinoma Growth by VEGF Expression Decrease
Endothelin Inhibits Cholangiocarcinoma Growth by VEGF Expression Decrease
Background: Endothelins (ET-1, ET-2, ET-3) are peptides with vasoactive properties interacting with ETA and ETB receptors. ET-1 inhibits secretin-stimulated ductal secretion (hallmark of cholangiocyte growth) of cholestatic rats by interaction with ET receptors.
Aim: The aims of the studies were to evaluate (i) the effect of ET-1 on cholangiocarcinoma growth in Mz-ChA-1 cells and nude mice and (ii) whether ET-1 regulation of cholangiocarcinoma growth is associated with changes in the expression of vascular endothelial growth factor-A (VEGF-A), VEGF-C, VEGF receptor-2 (VEGFR-2) and VEGFR-3.
Methods: We determined the expression of ETA and ETB receptors on normal and malignant (Mz-ChA-1) cholangiocytes and human cholangiocarcinoma tissue and the effect of ET-1 on the proliferation and expression of VEGF-A, VEGF-C (regulators of tumour angiogenesis) and its receptors, VEGFR-2 and VEGFR-3, in Mz-ChA-1 cells. In vivo, Mz-ChA-1 cells were injected into the flanks of athymic mice and injections of ET-1 or saline into the tumours were performed daily. The effect of ET-1 on tumour size, cell proliferation, apoptosis, collagen quantity and the expression of VEGF-A and VEGF-C and VEGFR-2 and VEGFR-3 were measured after 73 days.
Results: Higher expression of ETA and ETB was observed in malignant compared with normal cholangiocytes. ET-1 inhibited proliferation and VEGF-A, VEGF-C, VEGFR-2 and VEGFR-3 expression of Mz-ChA-1 cells. Chronic ET-1 treatment decreased tumour volume, tumour cell proliferation and VEGF-A and VEGF-C expression but increased apoptosis and collagen tissue deposition compared with controls.
Conclusions: Modulation of VEGF-A and VEGF-C (by ET-1) may be important for managing cholangiocarcinoma growth.
Cholangiocarcinoma, the tumour originating from the biliary epithelium, accounts for about 3% of all gastrointestinal cancers and represents the second most common primary liver tumour after hepatocarcinoma. Biliary tumours are extremely aggressive and display a poor prognosis. The lack of therapeutic tools for such a devastating disease is due, at least in part, to the lack of knowledge regarding the mechanisms regulating cholangiocarcinoma growth. However, increasing evidence has shown that neuropeptides and neuroendocrine hormones are among those factors that are able to affect cholangiocarcinoma biology, either promoting or inhibiting its growth. While oestrogens and insulin-like growth factor 1 stimulate cholangiocarcinoma proliferation, gastrin inhibits cholangiocarcinoma growth by activation of protein kinase C-α. In Mz-ChA-1 cells, α2-adrenoreceptor stimulation causes upregulation of cyclic adenosine 3',5'-monophosphate, which leads to inhibition of cholangiocarcinoma growth by decreased extracellular-signal-regulated kinase1/2 (ERK1/2) activity. A recent study has shown that leptin increased the cholangiocarcinoma growth by phosphorylation of STAT-3 and ERK1/2. The two endocannabinoids, anandamide and 2-arachidonylglycerol, exert differential effects on cholangiocarcinoma growth via receptor-independent mechanisms, with anandamide inhibiting growth and 2-arachidonylglycerol stimulating cholangiocarcinoma cell growth.
Endothelin-1 (ET-1), a 21 amino acid vasoconstrictor peptide, regulates the pathogenesis of hypertension and heart failure. ET-1 exerts a multitude of effects in liver cells. ET receptors are expressed by rat hepatic stellate cells ET-1 has been shown in human gallbladder epithelial cells. and in bile ducts of human cirrhotic liver. The importance of ET-1 in participating to liver homeostasis is supported by the finding that cholangiocytes synthesize hepatic ET-1 during the development of hepatopulmonary syndrome after extrahepatic bile duct obstruction. ET-1 inhibits secretin-induced ductal secretion (a hallmark of cholangiocyte proliferation) in cholestatic rats by interacting with ETA receptors on cholangiocytes.
A number of angiogenic factors including vascular endothelial growth factor (VEGF) are overexpressed by human cholangiocarcinoma tissues and cell lines such as KMC-1, KMC-2, KMBC and KMG-C. Overexpression of VEGF has been suggested to contribute to the 'angiogenic switch' of the malignant phenotype in human cholangiocarcinoma. Increased expression of VEGF (by oestrogens) plays an important role in regulating human cholangiocarcinoma growth. A number of studies have shown that VEGF regulates the function of normal and hyperplastic cholangiocytes. No information exists regarding the in vitro and in vivo effects of ET-1 on (i) the growth and apoptosis of cholangiocarcinoma cells and (ii) the expression of the angiogenic factor, VEGF-A and VEGF-C, and its receptors VEGFR-2 and VEGFR-3 in cholangiocarcinoma. On the basis of these studies, we evaluated (i) the expression of ETA and ETB in the human cholangiocarcinoma cell line, Mz-ChA-1 and the normal cell line, H69, and tissue arrays containing cholangiocarcinoma and normal biopsy samples, (ii) the in vitro effects of ET-1 on the growth of H69 and Mz-ChA-1 cells, (iii) the in vivo effect of ET-1 on the growth of tumour cell lines implanted in nude mice and (iv) whether the in vitro and in vivo effects of ET-1 on Mz-ChA-1 cell growth are associated with changes in VEGF-A and VEGF-C expression.
Abstract and Introduction
Abstract
Background: Endothelins (ET-1, ET-2, ET-3) are peptides with vasoactive properties interacting with ETA and ETB receptors. ET-1 inhibits secretin-stimulated ductal secretion (hallmark of cholangiocyte growth) of cholestatic rats by interaction with ET receptors.
Aim: The aims of the studies were to evaluate (i) the effect of ET-1 on cholangiocarcinoma growth in Mz-ChA-1 cells and nude mice and (ii) whether ET-1 regulation of cholangiocarcinoma growth is associated with changes in the expression of vascular endothelial growth factor-A (VEGF-A), VEGF-C, VEGF receptor-2 (VEGFR-2) and VEGFR-3.
Methods: We determined the expression of ETA and ETB receptors on normal and malignant (Mz-ChA-1) cholangiocytes and human cholangiocarcinoma tissue and the effect of ET-1 on the proliferation and expression of VEGF-A, VEGF-C (regulators of tumour angiogenesis) and its receptors, VEGFR-2 and VEGFR-3, in Mz-ChA-1 cells. In vivo, Mz-ChA-1 cells were injected into the flanks of athymic mice and injections of ET-1 or saline into the tumours were performed daily. The effect of ET-1 on tumour size, cell proliferation, apoptosis, collagen quantity and the expression of VEGF-A and VEGF-C and VEGFR-2 and VEGFR-3 were measured after 73 days.
Results: Higher expression of ETA and ETB was observed in malignant compared with normal cholangiocytes. ET-1 inhibited proliferation and VEGF-A, VEGF-C, VEGFR-2 and VEGFR-3 expression of Mz-ChA-1 cells. Chronic ET-1 treatment decreased tumour volume, tumour cell proliferation and VEGF-A and VEGF-C expression but increased apoptosis and collagen tissue deposition compared with controls.
Conclusions: Modulation of VEGF-A and VEGF-C (by ET-1) may be important for managing cholangiocarcinoma growth.
Introduction
Cholangiocarcinoma, the tumour originating from the biliary epithelium, accounts for about 3% of all gastrointestinal cancers and represents the second most common primary liver tumour after hepatocarcinoma. Biliary tumours are extremely aggressive and display a poor prognosis. The lack of therapeutic tools for such a devastating disease is due, at least in part, to the lack of knowledge regarding the mechanisms regulating cholangiocarcinoma growth. However, increasing evidence has shown that neuropeptides and neuroendocrine hormones are among those factors that are able to affect cholangiocarcinoma biology, either promoting or inhibiting its growth. While oestrogens and insulin-like growth factor 1 stimulate cholangiocarcinoma proliferation, gastrin inhibits cholangiocarcinoma growth by activation of protein kinase C-α. In Mz-ChA-1 cells, α2-adrenoreceptor stimulation causes upregulation of cyclic adenosine 3',5'-monophosphate, which leads to inhibition of cholangiocarcinoma growth by decreased extracellular-signal-regulated kinase1/2 (ERK1/2) activity. A recent study has shown that leptin increased the cholangiocarcinoma growth by phosphorylation of STAT-3 and ERK1/2. The two endocannabinoids, anandamide and 2-arachidonylglycerol, exert differential effects on cholangiocarcinoma growth via receptor-independent mechanisms, with anandamide inhibiting growth and 2-arachidonylglycerol stimulating cholangiocarcinoma cell growth.
Endothelin-1 (ET-1), a 21 amino acid vasoconstrictor peptide, regulates the pathogenesis of hypertension and heart failure. ET-1 exerts a multitude of effects in liver cells. ET receptors are expressed by rat hepatic stellate cells ET-1 has been shown in human gallbladder epithelial cells. and in bile ducts of human cirrhotic liver. The importance of ET-1 in participating to liver homeostasis is supported by the finding that cholangiocytes synthesize hepatic ET-1 during the development of hepatopulmonary syndrome after extrahepatic bile duct obstruction. ET-1 inhibits secretin-induced ductal secretion (a hallmark of cholangiocyte proliferation) in cholestatic rats by interacting with ETA receptors on cholangiocytes.
A number of angiogenic factors including vascular endothelial growth factor (VEGF) are overexpressed by human cholangiocarcinoma tissues and cell lines such as KMC-1, KMC-2, KMBC and KMG-C. Overexpression of VEGF has been suggested to contribute to the 'angiogenic switch' of the malignant phenotype in human cholangiocarcinoma. Increased expression of VEGF (by oestrogens) plays an important role in regulating human cholangiocarcinoma growth. A number of studies have shown that VEGF regulates the function of normal and hyperplastic cholangiocytes. No information exists regarding the in vitro and in vivo effects of ET-1 on (i) the growth and apoptosis of cholangiocarcinoma cells and (ii) the expression of the angiogenic factor, VEGF-A and VEGF-C, and its receptors VEGFR-2 and VEGFR-3 in cholangiocarcinoma. On the basis of these studies, we evaluated (i) the expression of ETA and ETB in the human cholangiocarcinoma cell line, Mz-ChA-1 and the normal cell line, H69, and tissue arrays containing cholangiocarcinoma and normal biopsy samples, (ii) the in vitro effects of ET-1 on the growth of H69 and Mz-ChA-1 cells, (iii) the in vivo effect of ET-1 on the growth of tumour cell lines implanted in nude mice and (iv) whether the in vitro and in vivo effects of ET-1 on Mz-ChA-1 cell growth are associated with changes in VEGF-A and VEGF-C expression.