Constitutive activation of the gastrin-releasing peptide receptor expressed by the nonmalignant human colon epithelial cell line NCM460

Gastrin-releasing peptide (GRP) causes multiple effects in humans by activating a specific heptaspanning receptor. Within the gastrointestinal tract, GRP receptors (GRP-R) are not normally expressed by mucosal epithelial cells except for those lining the gastric antrum. In contrast, recent studies have shown that up to 40% of resected colon cancers aberrantly express this receptor. This is important because the GRP-R can cause the proliferation of many, but not all, tissues in which it is expressed. Since GRP and other agonists are not known to exist in the colonic lumen, it has not been clear how or even if GRP-R expression in colon cancer contributes to cell proliferation. To evaluate the functional consequence of GRP-R expression on colonic epithelium, we transfected the recently isolated nonmalignant human colon epithelial cell line NCM460 with the cDNA for this receptor. All NCM460 cell lines expressing varying numbers of GRP-R bound selected agonists and antagonists indistinguishably from receptors expressed by other human tissues. Furthermore GRP-R-expressing transfected cell lines, but not wild-type NCM460 cells, proliferated independently of serum or other growth factors. Further evaluation revealed that GRP-R in these cells tonically stimulated G alpha q/11, resulting in increased phospholipase C activation. Since transfected cells do not secrete GRP, nor is their growth influenced by exposure to receptor-specific antagonists, these data indicate that GRP-R ectopically expressed by NCM460 cells are constitutively active. This report provides the first evidence of mutation-independent heptaspanning receptor constitutive activation resulting in cell proliferation, and identifies a potential mechanism whereby the GRP-R may act as an oncogene in human colon cancer.     

Ligands for peroxisome proliferator-activated receptorgamma and retinoic acid receptor inhibit growth and induce apoptosis of human breast cancer cells in vitro and in BNX mice

Induction of differentiation and apoptosis in cancer cells through ligands of nuclear hormone receptors (NHRs) is a novel and promising approach to cancer therapy. All-trans-retinoic acid (ATRA), an RA receptor-specific NHR ligand, is now used for selective cancers. The NHR, peroxisome proliferator-activated receptor gamma (PPARgamma) is expressed in breast cancer cells. Activation of PPARgamma through a synthetic ligand, troglitazone (TGZ), and other PPARgamma-activators cause inhibition of proliferation and lipid accumulation in cultured breast cancer cells. TGZ (10(-5) M, 4 days) reversibly inhibits clonal growth of MCF7 breast cancer cells and the combination of TGZ (10(-5) M) and ATRA (10(-6) M, 4 days) synergistically and irreversibly inhibits growth and induces apoptosis of MCF7 cells, associated with a dramatic decrease of their bcl-2 protein levels. Similar effects are noted with in vitro cultured breast cancer tissues from patients, but not with normal breast epithelial cells. The observed apoptosis mediated by TGZ and ATRA may be related to the striking down-regulation of bcl-2, because forced over-expression of bcl-2 in MCF7 cells cultured with TGZ and ATRA blocks their cell death. TGZ significantly inhibits MCF7 tumor growth in triple immunodeficient mice. Combined administration of TGZ and ATRA causes prominent apoptosis and fibrosis of these tumors without toxic effects on the mice. Taken together, this combination may provide a novel, nontoxic and selective therapy for human breast cancers.     

Magnetic resonance imaging and invasive evaluation of development of heart failure in transgenic mice with myocardial expression of tumor necrosis factor-alpha

The intestine is a large endocrine organ, but the dependence of colon cancer on hormones remains unknown. We show here that neurotensin, a paracrine/endocrine peptide in the gut, and the neurotensin receptor antagonist SR 48692 control colon cancer cell growth in vitro and in vivo by interacting with receptors that are ectopically expressed in colon cancers. In cell culture, neurotensin stimulates the growth of human colon cancer cell lines (SW480, SW620, HT29, HCT116 and Cl.19A) expressing the neurotensin receptor NTR1 but does not change the growth of Caco2 cells, which do not express NTR1. In SW480 cells, neurotensin is active in the 10(-10) to 10(-6) M concentration range (ED50 = 0.47 nM) while the neurotensin fragment (I-II) is inactive. Neurotensin also enhances the cellular cloning efficiency of SW480 cells in soft agar by inducing a 50% increase of colony formation. This effect is blocked by SR 48692, which alone does not alter colony formation. Subcutaneous delivery of neurotensin (0.54 micromol/kg every 24 hr) by osmotic pumps to nude mice that have been xenografted with SW480 cells results in a significant increase of tumor volume, i.e., up to 255% of control at day 20 of treatment. SR 48692 administered alone (1.7 micromol/kg every 24 hr) by daily i.p. injections reduces the development of tumors formed by xenografting SW480 cells in nude mice. A significant mean reduction of tumor volume of 38% is observed during the 22-day period of treatment. SR 48692 alone is also active at reducing tumor volume after xenografting HCT116 cells in nude mice. Our results support the notion that colon cancer growth may be dependent on blood-borne neurotensin and suggest that non-peptide neurotensin antagonists, such as SR 48692, may be useful for the development of novel therapeutic strategies of colon cancer.     

The GDB Human Genome Database Anno 1997

MMP-9 (gelatinase B) and urokinase-type plasminogen activator receptor (u-PAR), which are involved in cancer cell invasion and metastasis, are reported to be predominantly expressed by immune/inflammatory cells in human colorectal cancers. To investigate their significance in cancer progression, we morphometrically analyzed the tissue expression of MMP-9 and u-PAR among different stages of colorectal cancer. The numbers of MMP-9- and u-PAR-positive cells along the invasive margin were significantly smaller in cases with liver metastasis than in cases without liver metastasis, and were also smaller in cases with an infiltrating margin than in cases with an expanding margin. Both variables were larger in colon cancer cases with conspicuous lymphocytic infiltration. These results indicated that the degree of tissue expression of MMP-9 and u-PAR by host cells is inversely associated with liver metastasis and an infiltrating growth pattern in human colorectal cancers. Essentially the same results were obtained for the number of macrophages distributed along the invasive margin. We also found that the expression pattern of MMP-9 was similar to that of MMP-8 (polymorphonuclear leukocyte collagenase). These data are consistent with clinicopathologic studies of host cells. Therefore, our data suggest a dual role of MMP-9 and u-PAR expression in colon cancer tissue; i.e., not only are these proteinases cancer-promoting factors, but also they are related to the host defensive mechanism when they are expressed by host cells.     

Enoxaparin increases the incidence of postoperative intracranial hemorrhage when initiated preoperatively for deep venous thrombosis prophylaxis in patients with brain tumors

Previous studies have shown that PKC-alpha protein expression is decreased in sporadic human colon cancers, as well as in colonic tumors of rats induced by chemical carcinogens. To elucidate the potential role of PKC-alpha on several phenotypic characteristics of colon cancer cells, we have transfected cDNAs for PKC-alpha in sense or antisense orientations into CaCo-2 cells, a human colonic adenocarcinoma cell line. Transfected clones were isolated that demonstrated approximately 3-fold increases (sense transfectants) and approximately 95% decreases (antisense transfectants) in PKC-alpha expression with no significant alterations in other PKC isoforms. Transfection of CaCo-2 cells with PKC-alpha in the antisense orientation resulted in enhanced proliferation and decreased differentiation, as well as in a more aggressive transformed phenotype compared with empty vector-transfected control cells. In contrast, cells transfected with PKC-alpha cDNA in the sense orientation demonstrated decreased proliferation, enhanced differentiation, and an attenuated tumor phenotype compared with these control cells. These data show that alterations in the expression of PKC-alpha induce changes in the proliferation, differentiation, and tumorigenicity of CaCo-2 cells. Furthermore, these findings indicate that loss of PKC-alpha expression in sporadic human and chemically induced colonic cancers may confer a relative growth advantage during colonic malignant transformation.     

Role of transforming growth factor beta 1 (TGF beta 1) in mediating androgen-induced growth inhibition in human adrenal cortex in vitro

We have previously found that the androgen receptor gene is expressed both in normal and adenomatous human adrenal cortex and in the NCI-H295 human adrenocortical cancer cell line. Furthermore, we have observed that dihydrotestosterone (DHT) at physiological concentrations (10(-11) M) inhibits human adrenocortical cell growth in vitro and slightly decreases c-myc RNA levels in NCI-H295 cells. As c-myc is probably not the main mechanism mediating DHT-induced inhibition of cell growth, other genes controlling cell proliferation may be involved. Transforming Growth Factor beta (TGF beta) is a regulatory peptide that acts by both autocrine and paracrine mechanisms to control proliferation and differentiation, and there is previous evidence that TGF beta may exert an antimitotic effect on human fetal adrenal cells in vitro. This study examines a possible role for TGF beta 1 in mediating the DHT-induced reduction of human adrenocortical cell growth. TGF beta 1 and its receptor (TGF beta RII) are expressed in DHT-treated and nontreated NCI-H295 cells; on Northern blot analysis 24-h treatment with DHT (10(-11) M) produced a small increase in TGF beta RII RNA, and quantitative RT-PCR showed a 1.5-fold increase in TGF beta 1 RNA levels. These findings suggest that TGF beta 1 and its receptor may be involved in DHT-induced inhibition of human adrenocortical cell growth.     

Comparative effects of overexpression of p27Kip1 and p21Cip1/Waf1 on growth and differentiation in human colon carcinoma cells

Recent studies have shown that decreased expression of p27Kip1 is associated with high grade tumors and an unfavorable prognosis in several types of human cancer. To clarify the role of p27Kip1 in colon cancer, we have overexpressed this protein in the HT29 colon cancer cell line. The derivatives displayed an increase in the p27Kip1 protein in cyclin E/CDK2 immunoprecipitates and a decrease in cyclin E-associated kinase activity when compared to vector control clones, providing evidence that the overexpressed protein was functional. Clones with a high level of p27Kip1 displayed partial growth inhibition in monolayer culture and a decrease in plating efficiency, even though they expressed increased levels of the cyclin D1 protein. Using alkaline phosphatase expression as a marker, we found that the p27Kip1 overexpressor clones displayed a 2-3-fold increase in sensitivity to induction of differentiation by 2 mM sodium butyrate. In contrast to these results, derivatives of HT29 cells that stably overexpressed p21Cip1/Waf1 displayed decreased sensitivity to the induction of differentiation. These findings may explain why decreased levels of p27Kip1 in certain human cancers is associated with high grade (poorly differentiated) tumors, and suggest that strategies that increase the level of p27Kip1 may be useful in cancer therapy.     

1,25-Dihydroxyvitamin D3 receptor as a marker of human colon carcinoma cell line differentiation and growth inhibition

The antiproliferative action of 1,25-dihydroxyvitamin D3 in osteosarcoma, breast carcinoma, and colon carcinoma cell lines has been described. In this study, the level of vitamin D receptor was analyzed in a panel of colon adenoma and adenocarcinoma cell lines and the receptor level was correlated with the response to treatment with 1,25-dihydroxyvitamin D3. Ribonuclease protection and ligand-binding assays quantitated the level of vitamin D receptor mRNA expression and the level of functional receptors, respectively. The more well-differentiated cell lines, such as VACO 330, showed higher levels of vitamin D receptor than less-differentiated cell lines, such as SW620. Proliferation assay, clonogenic assay, and growth curve study in HT29 and SW620 cell lines assessed the antiproliferative effect of 1,25-dihydroxyvitamin D3 at concentrations ranging from 10(-11) to 10(-6) M. HT29 showed significant (P < 0.05) growth inhibition at 10(-9) to 10(-6) M concentrations, but growth of SW620 remained unchanged. The amount of vitamin D receptor in 12 malignant colonic tumors was compared with that of adjacent normal tissue, and in 9 cases, the tumor expressed a lower vitamin D receptor level. Our results suggest that the level of vitamin D receptor correlates with the degree of differentiation in human colon cancer cell lines and may serve as a useful biological marker in predicting clinical outcome in patients.     

Location and characterization of the human GRP receptor expressed by gastrointestinal epithelial cells

The exact location of normal gastrin-releasing peptide (GRP) receptor expression by epithelial cells lining the human gastrointestinal (GI) tract is not known; yet this receptor is found on upwards of 50% of GI cancers. Furthermore, the pharmacology reported for GRP receptors expressed by GI cancers varies considerably. Therefore, the purpose of this study was to determine the normal distribution of GRP receptor expression by cells lining the human GI tract, and then determine the normal pharmacology of the human receptor when ectopically expressed by the nonmalignant human colon epithelial cell line NCM460. We obtained endoscopic pinch biopsies of, and extracted the RNA from, epithelial cells lining the esophagus, stomach, jejunum, ileum, and proximal and descending colon, RT-PCR demonstrated that GRP-R expression is limited to cells lining the gastric antrum, indicating that this receptor is aberrantly expressed by GI cancers. To determine the normal pharmacology of this receptor when expressed by nonmalignant human tissues for the first time, we transfected NCM460 cells with the cDNA for the human GRP receptor. By studying three stable NCM460 cell lines expressing varying numbers of receptors, we demonstrate that agonist and antagonist binding affinity, binding kinetics, and G-protein coupling are all independent of receptor number. Finally, by comparing GRP receptors expressed by GI cancers with those on NCM460-transfected cells, we show that the pharmacology of the aberrantly expressed receptors is significantly altered. Thus, these data demonstrate that GI cancers aberrantly express GRP receptors that then behave abnormally.     

Activation of c-Src by receptor tyrosine kinases in human colon cancer cells with high metastatic potential

Recent data suggest that signal transduction may have a critical role in the development and regulation of the metastatic phenotype. Here, we investigated the role of c-Src activation in the process of human colon cancer metastasis to the liver. Our data, derived from two different sets of human colon cancer cell line metastatic variants, suggest that not only do highly-metastatic cells display constitutively elevated c-Src protein kinase activity when compared to poorly metastatic cells, but also that receptor tyrosine kinases participate in the ligand-activation of c-Src above basal levels. Specifically, the epidermal growth factor receptor (EGFR), p185HER2/Neu and the hepatocyte growth factor receptor (c-Met) appear to be linked to the process because they preferentially activate c-Src in highly-metastatic cells. EGFR was found to associate with c-Src in colon cancer cells and specific inhibitors of the EGFR resulted in a reduction of c-Src activity to basal levels. In addition, c-Src transfectants displayed partially-activated EGFRs, suggesting a feedback role for c-Src in the regulation of the EGFR. p185HER2/Neu was also identified in immunocomplexes of c-Src following ligand activation of the EGFR, but only in highly-metastatic cells. Collectively, these observations suggest a paradigm whereby c-Src interacts with multiple cell-surface growth factors in a catalytic fashion for the development of tumor cells with metastatic potential.     

sst2 somatostatin receptor expression reverses tumorigenicity of human pancreatic cancer cells

Among the five cloned somatostatin receptor subtypes (sst1 to sst5), sst2 mediates the antiproliferative effect of somatostatin analogues in vitro. Somatostatin analogues have been shown to inhibit cell growth in vitro and in vivo in pancreatic cancer models that expressed sst2. We recently demonstrated the loss of sst2 gene expression in human pancreatic adenocarcinomas and most of the derived pancreatic cancer cell lines. In the present study, we corrected the sst2 defect in human pancreatic cancer BxPC-3 and Capan-1 cells by stable transfection with human sst2 cDNA. In the absence of exogenous ligand, both BxPC-3 and Capan-1 cells expressing sst2 showed a significant reduction in cell growth. This inhibitory effect was blocked by treatment with antiserum to somatostatin. sst2-expressing cells produced somatostatin-like immunoreactivity that mainly corresponded to somatostatin 14, indicating the induction of a negative autocrine loop. In other respects, sst2 expression in Capan-1 cells induced a significant reduction of clonogenicity in soft agar. Moreover, a significantly reduced (Capan-1 cells) or suppressed (BxPC-3 cells) tumor growth in athymic nude mice was observed. The reversal of tumorigenicity induced by the restoration of sst2 expression suggests that the loss of sst2 contributes to the malignancy of human pancreatic cancers.     

Activators of the nuclear receptor PPARgamma enhance colon polyp formation

A high-fat diet increases the risk of colon, breast and prostate cancer. The molecular mechanism by which dietary lipids promote tumorigenesis is unknown. Their effects may be mediated at least in part by the peroxisome proliferator-activated receptors (PPARs). These ligand-activated nuclear receptors modulate gene expression in response to fatty acids, lipid-derived metabolites and antidiabetic drugs. To explore the role of the PPARs in diet-induced carcinogenesis, we treated mice predisposed to intestinal neoplasia with a synthetic PPARgamma ligand. Reflecting the pattern of expression of PPARgamma in the gastrointestinal tract, treated mice developed a considerably greater number of polyps in the colon but not in the small intestine, indicating that PPARgamma activation may provide a molecular link between a high-fat diet and increased risk of colorectal cancer.     

Neutralizing capacity of ++antiophidic sera against the venom of Bothrops moojeni (lance-headed viper)

Human oncostatin M (OM) is a M(r) 28,000 glycoprotein that has been shown to regulate cell proliferation and differentiation. The biological activities of OM can be mediated by two different heterodimeric receptor complexes, the leukemia inhibitory factor (LIF)/OM shared receptor and the OM-specific receptor. In this study, we have examined the growth-regulatory effect of OM on 10 breast cancer cell lines derived from human tumors. The cellular proliferation of seven of these breast cancer cell lines was inhibited by OM. The three cell lines that did not respond to OM treatment lacked the expression of OM receptors. The growth-inhibitory activity of OM is examined further in the H3922 breast cancer cell line, which expresses the high-affinity OM receptor at a relatively higher level. We found that the cellular proliferation of H3922 cells was induced strongly by extrogenous epidermal growth factor (EGF), EGF-like factor, and basic fibroblast growth factor. The proliferative activities of these growth factors can be abolished totally by cotreatment of H3922 cells with OM. Treatment of H3922 cells with OM for 24 h did not block EGF binding or the induction of EGF receptor tyrosine phosphorylation. This finding suggests that OM interferes with the mitogenic signal at steps distal to the EGF receptor. Examination of proto-oncogene expression demonstrated that OM down-regulates the c-myc gene in H3922 cells. The biological effects reported herein are not shared by the OM-related cytokines interleukin 6 or LIF, as demonstrated by the inability of these proteins to inhibit cell growth or modulate c-myc gene expression in breast cancer cells. Additionally, the high-affinity binding of labeled OM cannot be displaced by LIF. Together, these data suggest that OM is a growth inhibitor for breast cancer cells. The inhibitory activity is mediated predominantly through the OM-specific receptor, and activation of this receptor abrogates growth factor stimulation and down-regulates the c-myc proto-oncogene.