Estrogens and insulin-like growth factor 1 modulate neoplastic cell growth in human cholangiocarcinoma.

We investigated the expression of estrogen receptors (ERs), insulin-like growth factor 1 (IGF-1), and IGF-1R (receptor) in human cholangiocarcinoma and cholangiocarcinoma cell lines (HuH-28, TFK-1, Mz-ChA-1), evaluating the role of estrogens and IGF-1 in the modulation of neoplastic cell growth. ER-alpha, ER-beta, IGF-1, and IGF-1R were expressed (immunohistochemistry) in all biopsies (18 of 18) of intrahepatic cholangiocarcinoma. ER-alpha was expressed (Western blot) only by the HuH-28 cell line (intrahepatic cholangiocarcinoma), whereas ER-beta, IGF-1, and IGF-1R were expressed in the three cell lines examined. In serum-deprived HuH-28 cells, serum readmission induced stimulation of cell proliferation that was inhibited by ER and IGF-1R antagonists. 17beta-Estradiol and IGF-1 stimulated proliferation of HuH-28 cells to a similar extent to that of MCF7 (breast cancer) but greater than that of TFK-1 and Mz-ChA-1, inhibiting apoptosis and exerting additive effects. These effects of 17beta-estradiol and IGF-1 were associated with enhanced protein expression of ER-alpha, phosphorylated (p)-ERK1/2 and pAKT but with decreased expression of ER-beta. Finally, transfection of IGF-1R anti-sense oligonucleotides in HuH-28 cells markedly decreased cell proliferation. In conclusion, human intrahepatic cholangiocarcinomas express receptors for estrogens and IGF-1, which cooperate in the modulation of cell growth and apoptosis. Modulation of ER and IGF-1R could represent a strategy for the management of cholangiocarcinoma.

Cell proliferation and drug resistance in hepatocellular carcinoma are modulated by Rho GTPase signals.

Hepatocellular carcinoma is highly resistant to chemotherapeutic agents, thus the need to discover effective therapeutic molecules to suppress cancer cell growth and to overcome drug resistance is urgent. The Rho GTPase is implicated in cancer and metastasis and is directly activated by the Lymphoid blast crisis (Lbc) protooncogene, a Rho guanine-nucleotide exchange factor. The aim of the study was to analyze the expression of Lbc in hepatocarcinoma and to determine the effect of Lbc-induced Rho signaling on expression, growth rate and resistance to genotoxic stress. We found, by immunohistochemical analysis of biopsy samples and Northern and Western blot analyses of cell lines, that Lbc is absent in normal adult liver but is abundantly expressed in hepatocarcinoma, implying an increased Rho pathway signaling. Lbc stably transfected hepatocarcinoma cells exhibit increased proliferation and levels of ERK and cyclin D1 activation, which are blocked by a Rho inhibitor. In contrast, AKT activation was not altered. Moreover, Lbc expression confers increased resistance to genotoxic stress induced by doxorubicin, which is associated with upregulation of Bcl-2 and BAD phosphorylation, and this is reversed by a Rho inhibitor. In conclusion, these data support a role for Rho in liver cancer progression and resistance to therapy and may provide a basis for developing effective treatment for hepatocarcinoma.

Effect of pirfenidone on rat hepatic stellate cell proliferation and collagen production.

BACKGROUND/AIMS: Pirfenidone has been recently shown to reduce dimethynitrosamine-induced liver fibrosis in the rat, but no information are available on the effect of this drug on cultured hepatic stellate cells (HSC). METHODS: HSC proliferation was evaluated by measuring bromodeoxyuridine incorporation; PDGF-receptor autophosphorylation, extracellular signal-regulated kinase (ERK1/2) and pp70(S6K) activation were evaluated by western blot; protein kinase C activation was evaluated by western blot and by ELISA; type I collagen accumulation and alpha1(I) procollagen mRNA expression were evaluated by ELISA and northern blot, respectively. RESULTS: Pirfenidone significantly inhibited PDGF-induced HSC proliferation, starting at a concentration of 1 microM, with a maximal effect at 1000 microM, without affecting HSC viability and without inducing apoptosis. The inhibition of PDGF-induced HSC proliferation was associated neither with variations in PDGF-receptor autophosphorylation, or with ERK1/2 and pp70(S6K) activation. On the other hand, pirfenidone was able to inhibit PDGF-induced activation of the Na(+)/H(+) exchanger, which is involved in PDGF-induced HSC proliferation in HSC, with a maximal effect at 1000 microM and inhibited PDGF-induced protein kinase C activation. Pirfenidone 100 and 1000 microM inhibited type I collagen accumulation in the culture medium induced by transforming growth factor(beta1) by 54% and 92%, respectively, as well as TGF(beta1)-induced alpha1(I) procollagen mRNA expression. RESULTS: Pirfenidone could be a new candidate for antifibrotic therapy in chronic liver diseases.

Association of Lbc Rho guanine nucleotide exchange factor with alpha-catenin-related protein, alpha-catulin/CTNNAL1, supports serum response factor activation.

The Rho GTPase signaling pathway is required for actin cytoskeletal organization and serum response factor-dependent gene transcription. Lbc is a Rho-specific guanine nucleotide exchange factor that contains a modulatory C-terminal region. To elucidate Lbc regulatory mechanism(s), a yeast two-hybrid screen for proteins that interact with the Lbc C-terminal region was carried out, resulting in multiple isolation of cDNAs encoding the same 734-amino acid Lbc interacting protein. The Lbc interacting protein has homology with the alpha-catenin cell adhesion component and is identical to the alpha-catenin-like alpha-catulin protein of unknown function. The human alpha-catulin gene (CTNNAL1) maps to 9q31-32. Here we identify the predicted endogenous alpha-catulin product, document alpha-catulin and Lbc co-expression in multiple human cell lines, and show alpha-catulin and Lbc subcellular co-fractionation and intracellular localization. The required regions for Lbc and alpha-catulin interaction were mapped, and complex formation between Lbc and alpha-catulin in mammalian cells was detected. Functionally, alpha-catulin co-expression leads to increased Lbc-induced serum response factor activation in vivo as measured by a transcriptional reporter assay. Furthermore, alpha-catulin co-expression enhances Lbc-induced GTP-Rho formation in vivo. These results support the concept that the recently identified alpha-catulin protein may modulate Rho pathway signaling in vivo by providing a scaffold for the Lbc Rho guanine nucleotide exchange factor.