Inhibited Hepatocyte Growth Factor Research Articles

Transforming growth factor β receptor family ligands inhibit hepatocyte growth factor synthesis and secretion from astrocytoma cells

Transforming growth factor β (TGFβ) and hepatocyte growth factor (HGF) promote glioma progression. Using U87human astrocytoma cells, which express TGFβ receptors (TβRs), we show (1) mRNA expression of Smads (2, 3, 4), bone morphogenetic protein (BMP)- and activin-A receptors; (2) TGFβ1 inhibits and HGF induces proliferation; (3) TGFβ1 and activin-A equipotently inhibit HGF secretion more than BMP-2, but none alters c-Met expression. Because interfering with TβR signaling might nullify the beneficial inhibition of HGF secretion, activin-A should instead be considered for combination glioma therapy.

Induction of Apoptosis in Human Bladder Cancer Cells In Vitro and In Vivo Caused by FTY720 Treatment

FTY720 is a unique immunosuppressant that induces apoptosis in activated lymphocytes but not in other hematopoietic cells. We examined whether FTY720 has anticancer effects on human bladder cancer cells by inducing apoptosis and we investigated its molecular pathway. We used the 3 human bladder cancer cell lines T24, UMUC3 and HT1197, and the human fibroblast derived cell line CRL-2096 (American Type Tissue Collection, Rockville, Maryland) in this study. The difference in drug susceptibility to FTY720 in cancer cells and fibroblasts was examined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and cell growth assays. FTY720 induced apoptosis was determined by morphological analysis under light and electron microscopy, and DNA electrophoresis, and its molecular pathway was evaluated by Western blot analysis focusing on the p42/p44 mitogen activated protein kinase pathway. We then tested the in vivo effect of this agent using 2 mouse models of human bladder cancer xenograft. FTY720 treatment in vitro induced selective apoptosis in cancer cells at a concentration of less than 10 microM. Morphological analysis revealed features characteristic of apoptosis, including small cytoplasm with fragmented nuclei and condensed chromatin. DNA electrophoresis confirmed apoptosis, as evidenced by a distinct oligosomal ladder. Western blot analysis revealed that the agent significantly inhibited hepatocyte growth factor induced p42/p44 mitogen activated protein kinase activity. The in vivo anticancer effect was clearly confirmed by significantly decreased tumor growth without notable side effects in the 2 xenograft models. FTY720 treatment may induce selective apoptosis in vitro as well as in vivo in cancer cells. We suggest that FTY720 is a potent and clinically applicable anticancer agent for bladder cancer.

Aspirin and NS-398 inhibit hepatocyte growth factor–induced invasiveness of human hepatoma cells

Nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit cyclooxygenase (COX) activity and are considered to exert antitumor actions in a variety of cancer cells, although the effects are unlikely entirely due to COX inhibition. Because clinical observations suggest that hepatocyte growth factor (HGF) can promote metastasis of hepatoma cells while stimulating tumor invasiveness, we investigated the effect of aspirin and NS-398, a selective COX-2 inhibitor, on HGF-mediated invasiveness of HepG2 human hepatoma cells. HGF stimulated the invasiveness of HepG2 cells in Matrigel cell invasion assay, together with increased expression of matrix metalloproteinase (MMP) 9. Addition of aspirin or NS-398, similar to PD98059, which acts as a specific inhibitor of mitogen-activated protein kinase/extracellular signal–regulated kinase (MEK), an upstream kinase regulating extracellular signal–regulated kinase (ERK)1/2, abrogated such actions of HGF without affecting cell viability. Aspirin and NS-398, in contrast to PD98059, did not suppress ERK1/2 phosphorylation induced by HGF. However, both agents inhibited the kinase activity of ERK1/2 induced by HGF and repressed HGF-induced phosphorylation of 90-kd ribosomal S6 kinase (RSK) and Elk-1, key downstream substrates of ERK1/2, resulting in the suppression of transcriptional activity of Elk-1 as well as nuclear factor κB (NF-κB) and AP-1, which are involved in MMP-9 gene regulation. In conclusion, our results suggest that aspirin and NS-398 inhibit HGF-induced invasiveness of HepG2 human hepatoma cells through ERK1/2. (H EPATOLOGY 2002;35:1117-1124.)

Activins A, AB, and B inhibit hepatocyte growth factor synthesis by MRC-5 human lung fibroblasts.

The effect of activins A, AB, and B on hepatocyte growth factor (HGF) synthesis stimulated by 12-O-tetradecanoylphorbol beta-acetate (TPA) was studied in MRC-5 human lung fibroblasts. Activins A, AB, and B inhibited the increase in HGF secretion induced by TPA in different dose-dependent manners and potencies. At 5 ng/ml, activins A and AB inhibited the increase approximately 30% and 10%, respectively, and at 25 ng/ml both activins produced almost maximal inhibition, i.e., approximately 40%. Activin B caused 10% inhibition at 12 ng/ml, and at 25 ng/ml produced almost maximal inhibition, approximately 30%. Further analysis with activin A indicated that the inhibition was caused by decreased HGF mRNA levels, followed by decreased cellular HGF levels. At 25 ng/ml, activin A inhibited the increase in HGF in the cellular lysate and the increase in HGF mRNA level approximately 80% and 40%, respectively.

Endostatin Acts Through Glypicans

Endostatin (ES), a proteolytic fragment of collagen, inhibits endothelial cell migration and is a potent angiogenesis inhibitor in animal model systems. However, its mechanism of action is not well understood. For example, it has not been clear whether ES action requires binding to heparan sulfate proteoglycans (HSPGs). Karumanchi et al. report that ES binds with low affinity to glycosyl-phosphatidylinositol anchored HSPGs called glypicans that are present on the surface of both endothelial and nonendothelial cells. Binding in vivo specifically required the presence of a heparan sulfate-glycosaminoglyan moiety on the glypicans. When glypican-1 expression was blocked in endothelial cells by antisense-based inhibition, ES binding decreased. Consequently, the ability of ES to inhibit endothelial cell migration in response to a particular form of vascular endothelial growth factor (VEGF) also decreased. Although some forms of VEGF require glypican to bind to their own receptor, the authors used a form of VEGF that does not require cell-surface proteoglycan for signaling to show that ES inhibited VEGF actions by inhibiting intracellular VEGF-mediated signaling and not by competing for glypican binding. ES also inhibited hepatocyte growth factor (HGF)-induced branching of nonepithelial renal tubular cells by binding to glypican-4. The authors suggest that glypicans are low-affinity receptors that present ES to a high-affinity receptor that has yet to be identified. S. A. Karumanchi, V. Jha, R. Ramchandran, A. Karihaloo, L. Tsiokas, B. Chan, M. Dhanabal, J. Hanai, G. Venkataraman, Z. Shriver, N. Keiser, R. Kalluri, H. Zeng, D. Mukhopadhyay, R. L. Chen, A. D. Lander, K. Hagihara, Y. Yamaguchi, R. Sasisekharan, L. Cantley, V. P. Sukhatme, Cell surface glypicans are low-affinity endostatin receptors. Mol. Cell 7 , 811-822 (2001). [Online Journal]

Agonists of the retinoic acid- and retinoid X-receptors inhibit hepatocyte growth factor secretion and expression in U87 human astrocytoma cells

Retinoids participate in the onset of differentiation, apoptosis and the inhibition of growth in a wide variety of normal and cancerous cells. Several recent reports have shown that hepatocyte growth factor (HGF), and its receptor, c-Met, are expressed at abnormally high levels in various human malignant gliomas and exert a strong proliferative action in an autocrine fashion. These results, consequently, imply that HGF and its receptor may represent a major contributor to the progression of such malignancies. Since astrocytomas are the most frequently occurring glioma, we have shown here that U87 cells — a well-established, human astrocytoma cell line — express both HGF and c-Met, thereby providing a suitable astrocytic tumor model for studying the potential role of HGF, functioning in an autocrine mode, in astrocytic tumorigenesis. Furthermore, we demonstrated the expression of the retinoic acid receptor (RAR) isoforms, RARα, -β and -γ, as well as the retinoid x-receptor (RxR) isoforms, RxRα and -β, by RT-PCR and western blot analysis in these cells. Since ligands of the RARs and RxRs are known to exert growth inhibitory effects on various tumor cells which include some astrocytomas, we speculated that such effect of retinoids might be mediated via inhibition of HGF secretion in human astrocytoma cells. Indeed, we have shown that the RAR agonists, all- trans retinoic acid (ATRA) and ( E)-4-[2-(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthylenyl)-1-propenyl] benzoic acid (TTNPB), inhibited HGF secretion with half maximal inhibition occurring at 3.0 μM and 15 nM, respectively, as did the RxR agonists, 9- cis- and 13- cis retinoic acid (9cRA and 13cRA, respectively), which exerted half-maximal inhibitory effects at 40 and 25 nM, respectively. These actions of the RAR and RxR agonists appear to be exerted at the transcriptional level as assessed by Northern blot analysis. Taken together, our results show for the first time that retinoids, acting via the RAR and RxRs, significantly inhibit both the secretion and expression of HGF, thereby interrupting a potentially highly tumorigenic autocrine loop in astrocytoma cells.

Angiostatin Binds to Smooth Muscle Cells in the Coronary Artery and Inhibits Smooth Muscle Cell Proliferation and Migration In Vitro

Angiostatin is an inhibitor of angiogenesis that is known to reduce endothelial cell proliferation and consequently prevent the progression of tumor metastases. However, the modest effect of angiostatin on endothelial cell proliferation raises the possibility that angiostatin might exert its effects on other cells. To determine the cellular distribution of angiostatin binding in tissues with neovasculature (atherosclerotic coronary arteries), we developed a fusion protein consisting of placental alkaline phosphatase and the first 3 kringles of plasminogen. Angiostatin binding colocalized with smooth muscle cells and could be inhibited by a 50-fold molar excess of plasminogen and 10 mmol/L epsilon-amino-n-caproic acid. The fusion protein also bound to smooth muscle cells in culture. Angiostatin inhibited hepatocyte growth factor-induced proliferation and migration of smooth muscle cells, suggesting that they are a target for the antiangiogenic effect of angiostatin.

Nonsteroidal anti-inflammatory drugs may prevent colon cancer through suppression of hepatocyte growth factor expression

Nonsteroidal anti-inflammatory drugs which inhibit cyclooxygenase have been reported to suppress colon carcinogenesis. However the mechanism has not yet been elucidated. Growth factors such as hepatocyte growth factor, which are produced by fibroblasts, have been shown to be important in carcinogenesis and the progression of various human cancers. In the present study, we tested the hypothesis that nonsteroidal anti-inflammatory drugs inhibit hepatocyte growth factor expression through an endogenous prostaglandin-mediated pathway in cultured human colonic fibroblasts. Human colonic fibroblasts were obtained from a resected colon and cultured. Hepatocyte growth factor and prostaglandin E 2 were measured by enzyme-linked immunosorbent assay. Induction of cyclooxygenase-1 and cyclooxygenase-2 protein was estimated by immunoblotting. Prostaglandins increased hepatocyte growth factor production significantly in a dose- and time-dependent manner. Cholera toxin and 8-bromo cAMP also stimulated hepatocyte growth factor production. Further, prostaglandin E 1 significantly increased cellular cAMP. The prostaglandin EP 2 and EP 4 receptors were detected by reverse transcription-polymerase chain reaction. Interleukin-1β dramatically increased prostaglandin E 2 production and significantly stimulated hepatocyte growth factor synthesis. Interleukin-1β induced cyclooxygenase-2 but not cyclooxygenase-1 protein. Indomethacin significantly reduced interleukin-1β-induced prostaglandin E 2 release and hepatocyte growth factor production. These results suggest that prostaglandin is a factor for the production of hepatocyte growth factor by human colonic fibroblasts. Nonsteroidal anti-inflammatory drugs may suppress colon carcinogenesis, in part, through the suppression of hepatocyte growth factor expression by inhibiting endogenous prostaglandin production.