Biologically active MK-801 and SKF-10,047 binding sites distinct from those in rat brain are expressed on human lung cancer cells.

We have shown previously that cultured human lung cancer cells of different histologic types express multiple opioid receptors that can regulate their growth. In this report, we show that these cells also express specific, saturable, and high-affinity binding sites (Kd approximately 1 nM) for the non-opioid phencyclidine (PCP), [(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,b]cyclohepten-5,10-imine hydrogen maleate] (MK-801) and sigma N-allylnormetazocine (SKF-10,047) receptor ligands. Characterization of these binding sites showed them to be protein in nature and sensitive to the guanine nucleotide GTP. Pharmacological studies showed that (+) MK-801 and (+) SKF-10,047 competed with each other for their binding sites and also for the methadone binding site present in these cells. However, the mu and delta opioid ligands did not compete for (+) MK-801 and (+) SKF-10,047 binding sites. In addition, these binding sites on lung cancer cells appear to be distinct from the N-methyl D-aspartate/PCP receptor ionophore complex reported to be present in rat brain. MK-801 and SKF-10,047, at nM concentrations, were found to inhibit the growth of these cells in culture within a few hours of exposure, and this effect was irreversible after 24 h. The growth effects of these ligands could not be reversed by the opioid antagonist naloxone, suggesting involvement of nonopioid type receptors in the actions of these ligands. The abundant expression of biologically active MK-801 and SKF-10 047 binding sites in these cell lines, distinct from those in rat brain, suggests that these cell lines may prove to be a valuable source for further characterization and purification of these binding sites.     

Expression of glutamate receptor subunits in human cancers

Emerging evidence suggests a role for glutamate and its receptors in the biology of cancer. This study was designed to systematically analyze the expression of ionotropic and metabotropic glutamate receptor subunits in various human cancer cell lines, compare expression levels to those in human brain tissue and, using electrophysiological techniques, explore whether cancer cells respond to glutamate receptor agonists and antagonists. Expression analysis of glutamate receptor subunits NR1-NR3B, GluR1-GluR7, KA1, KA2 and mGluR1-mGluR8 was performed by means of RT-PCR in human rhabdomyosarcoma/medulloblastoma (TE671), neuroblastoma (SK-NA-S), thyroid carcinoma (FTC 238), lung carcinoma (SK-LU-1), astrocytoma (MOGGCCM), multiple myeloma (RPMI 8226), glioma (U87-MG and U343), lung carcinoma (A549), colon adenocarcinoma (HT 29), T cell leukemia cells (Jurkat E6.1), breast carcinoma (T47D) and colon adenocarcinoma (LS180). Analysis revealed that all glutamate receptor subunits were differentially expressed in the tumor cell lines. For the majority of tumors, expression levels of NR2B, GluR4, GluR6 and KA2 were lower compared to human brain tissue. Confocal imaging revealed that selected glutamate receptor subunit proteins were expressed in tumor cells. By means of patch-clamp analysis, it was shown that A549 and TE671 cells depolarized in response to application of glutamate agonists and that this effect was reversed by glutamate receptor antagonists. This study reveals that glutamate receptor subunits are differentially expressed in human tumor cell lines at the mRNA and the protein level, and that their expression is associated with the formation of functional channels. The potential role of glutamate receptor antagonists in cancer therapy is a feasible goal to be explored in clinical trials.     

Two distinct bombesin receptor subtypes are expressed and functional in human lung carcinoma cells.

Bombesin-like peptides have been implicated as autocrine growth factors influencing the pathogenesis and progression of some human lung carcinoma cells. To determine the pharmacologic and structural properties of the bombesin receptors expressed in human lung carcinoma cells, cDNA clones encoding a human gastrin-releasing peptide receptor (GRP-R) and a pharmacologically distinct neuromedin-B preferring bombesin-receptor (NMB-R) were isolated from a human small cell lung carcinoma cell line (NCI-H345). After expression in Xenopus oocytes, a GRP-R-specific antagonist was effective in blocking responses elicited from the cloned GRP-R, but not the NMB-R. Both GRP-R and NMB-R mRNA expression was detected at varying levels in a panel of human lung cancer cell lines. These results indicate heterogeneity of bombesin receptor subtypes exists in human lung carcinoma cells and should be considered in the design of bombesin receptor antagonists intended to inhibit tumor cell growth.     

Characterization of Opioid Receptor Subtypes in Solution

Stable opioid receptor binding activity that retains distinct subtype specificities (mu, delta, and kappa) has been obtained in high yields in digitonin extracts of rat brain membranes that had been preincubated with Mg2+ prior to solubilization. The dependence on Mg2+ ions for receptor activity is also expressed in the soluble state, where the presence of Mg2+ leads to high-affinity and high-capacity opioid peptide binding to the delta, mu, and kappa sites (the latter subtype measured by the binding of [3H]dynorphin1-8). Binding of opiate alkaloids to soluble receptor sites is less dependent on Mg2+ than is opioid peptide binding. Soluble opioid binding activity shows the same sensitivity to Na+ ions and guanine nucleotides as the membrane-bound receptor. The ligand-receptor interactions give evidence of strong positive cooperativity, which is interpreted in terms of association-dissociation of receptor subunits on ligand binding in solution. Binding of enkephalin peptides is associated with the large macromolecules present (apparent Stokes radii greater than 60 A), whereas both those and several small species present (less than 60 A) bind opiate alkaloids and dynorphin1-8.     

Human small cell lung cancer cells express high affinity naloxone-insensitive [125I]β-endorphin binding sites

Previous reports have demonstrated that β-endorphin stimulates the clonal growth of human small cell lung carcinoma (SCLC) cell lines. In this study, the human SCLC lines, NCI-H69, NCI-H345, and NCI-N417, were observed to be highly-enriched in saturable, high-affinity binding sites which are labeled by [¹²⁵I]β-endorphin. In contrast to conventional opioid receptors, [¹²⁵I]β-endorphin SCLC binding was insensitive to naloxone and other mu, delta, or kappa opioid ligands. Further analysis of the NCI-H69 cells demonstrated that specific (naloxone-insensitive) binding was dependent on receptor concentration, reversible, sensitive to sodium ion, but insensitive to the GTP analogue, Gpp(NH)p. These results suggest a role for naloxone-insensitive β-endorphin in modulating SCLC metabolism.     

Discoidin Domain Receptor 1 Contributes to Tumorigenesis through Modulation of TGFBI Expression

Discoidin domain receptor 1 (DDR1) is a member of the receptor tyrosine kinase family. The receptor is activated upon binding to its ligand, collagen, and plays a crucial role in many fundamental processes such as cell differentiation, adhesion, migration and invasion. Although DDR1 is expressed in many normal tissues, upregulated expression of DDR1 in a variety of human cancers such as lung, colon and brain cancers is known to be associated with poor prognosis. Using shRNA silencing, we assessed the oncogenic potential of DDR1. DDR1 knockdown impaired tumor cell proliferation and migration in vitro and tumor growth in vivo. Microarray analysis of tumor cells demonstrated upregulation of TGFBI expression upon DDR1 knockdown, which was subsequently confirmed at the protein level. TGFBI is a TGFβ-induced extracellular matrix protein secreted by the tumor cells and is known to act either as a tumor promoter or tumor suppressor, depending on the tumor environment. Here, we show that exogenous addition of recombinant TGFBI to BXPC3 tumor cells inhibited clonogenic growth and migration, thus recapitulating the phenotypic effect observed from DDR1 silencing. BXPC3 tumor xenografts demonstrated reduced growth with DDR1 knockdown, and the same xenograft tumors exhibited an increase in TGFBI expression level. Together, these data suggest that DDR1 expression level influences tumor growth in part via modulation of TGFBI expression. The reciprocal expression of DDR1 and TGFBI may help to elucidate the contribution of DDR1 in tumorigenesis and TGFBI may also be used as a biomarker for the therapeutic development of DDR1 specific inhibitors.     

Insulin receptors in the mammalian central nervous system: binding characteristics and subunit structure

Insulin receptors in rat and human central nervous system have been identified by binding of 125I-insulin on purified synaptic plasma membranes; affinity labelling of receptors by chemical cross-linking 125I-insulin; or phosphorylation of receptors with [gamma-32P]ATP. Brain insulin receptors showed significant differences in their binding characteristics and subunit structure when compared with receptors in other tissues like adipose and liver cells: absence of negatively cooperative interactions; a distinct binding specificity i.e. porcine proinsulin, coypu insulin and insulin-like growth factor I and II showed 2-5 times higher binding affinity in brain than in other cell types; a smaller molecular size of the brain receptor alpha-subunit than in other tissues (Mr approximately 115,000 instead of 130,000). In contrast, the size (Mr approximately 94,000) and function of the insulin receptor beta-subunit kinase was identical with that described in other cells. We conclude, that insulin receptors in mammalian brain represent a receptor subtype which may mediate growth rather than metabolic activity of insulin.     

Inhibition of the Growth Factor MDK/Midkine by a Novel Small Molecule Compound to Treat Non-Small Cell Lung Cancer

Midkine (MDK) is a heparin-binding growth factor that is highly expressed in many malignant tumors, including lung cancers. MDK activates the PI3K pathway and induces anti-apoptotic activity, in turn enhancing the survival of tumors. Therefore, the inhibition of MDK is considered a potential strategy for cancer therapy. In the present study, we demonstrate a novel small molecule compound (iMDK) that targets MDK. iMDK inhibited the cell growth of MDK-positive H441 lung adenocarcinoma cells that harbor an oncogenic KRAS mutation and H520 squamous cell lung cancer cells, both of which are types of untreatable lung cancer. However, iMDK did not reduce the cell viability of MDK-negative A549 lung adenocarcinoma cells or normal human lung fibroblast (NHLF) cells indicating its specificity. iMDK suppressed the endogenous expression of MDK but not that of other growth factors such as PTN or VEGF. iMDK suppressed the growth of H441 cells by inhibiting the PI3K pathway and inducing apoptosis. Systemic administration of iMDK significantly inhibited tumor growth in a xenograft mouse model in vivo. Inhibition of MDK with iMDK provides a potential therapeutic approach for the treatment of lung cancers that are driven by MDK.     

Opposite Role of Kindlin-1 and Kindlin-2 in Lung Cancers

Lung cancer is highly heterogenous and is composed of various subtypes that are in diverse differential stages. The newly identified integrin-interacting proteins Kindlin-1 and Kindlin-2 are the activators of transmembrane receptor integrins that play important roles in cancer progression. In this report we present the expression profiles of Kindlin-1 and Kindlin-2 in lung cancers using patient specimens and established their correlation with lung cancer progression. We found that Kindlin-1 was expressed in epithelia-derived non-small-cell lung cancer, especially in squamous cell lung cancer but expressed at low levels in poorly differentiated large cell lung cancer. However, Kindlin-2 was highly expressed in large cell lung cancer. Both Kindlin-1 and Kindlin-2 were found not expressed or expressed at very low levels in neuroendocrine-derived small cell lung cancer. Importantly, the Kindlin-1 expression level was positively correlated with the differentiation of squamous cell lung cancer. Surprisingly, we found that the very homologous Kindlin family proteins, Kindlin-1 and Kindlin-2, displayed counteracting functional roles in lung cancer cells. Ectopic expression of Kindlin-1 in non-small-cell lung cancer cells inhibited in vitro cell migration and in vivo tumor growth, while Kindlin-2 promoted these functions. Mechanistically, Kindlin-1 prohibited epithelail to mesenchymal transition in non-small-cell lung cancer cells, while Kindlin-2 enhanced epithelail to mesenchymal transition in these cells. Taken together, we demonstrated that Kindlin-1 and Kindlin-2 differentially regulate lung cancer cell progression. Further, the expression levels of Kindlin-1 might be potentially used as a marker for lung cancer differentiation and targeting Kindlin-2 might block the invasive growth of large cell lung cancer.     

Differential reactions of microglia to brain metastasis of lung cancer

The brain is a common metastatic site for various types of cancers, especially lung cancer. Patients with brain metastases have a poor prognosis in spite of radiotherapy and/or chemotherapy. It is postulated that immune cells in the brain may play a major role in cancer metastasis, dormancy, and relapse. Although microglia may serve as a major component in the brain immune system, the interaction between metastatic cancer cells and microglia is still largely unknown and remains to be elucidated. In this study, we have investigated microglial reactions in brain tissues with metastatic lung cancer cells and evaluated the cytotoxic effects of lipopolysaccharide (LPS)-activated microglia on metastatic lung cancer cells in vitro. In the vicinity of metastatic lung cancer mass in the brain, microglia showed signs of significant activation. There was an obvious increase in the number of microglia labeled with ionized calcium binding adaptor molecule 1 (Iba-1) antibody, a specific marker of microglia. The microglia were observed to form a clear boundary between the tumor mass and normal brain tissue. In the region where the tumor mass was situated, only a few microglia expressed inducible nitric oxide synthase (iNOS) and tumor necrosis factor-alpha (TNF-alpha), indicating differential activation in those microglia. The supernatant from LPS-activated microglia induced apoptosis of metastatic lung cancer cells in vitro in a dose- and time-dependent manner. However, at lower concentrations of activated microglial supernatant, trophic effects on cancer cells were observed, some lung cancer cells being insensitive to microglial cytotoxicity. Together with the observation that TNF-alpha alone induced proliferation of the tumor cells, the findings provide possible clues to the mechanism involved in metastasis of lung cancer cells to the brain.     

Characteristics of specific binding of epidermal growth factor (EGF) on human tumor cell lines

Using seventeen human tumor cell lines derived from a variety of tissues, specific binding sites for epidermal growth factor (EGF), a mouse submandibular gland-derived growth factor, has been characterized. A significant amount of membrane-bound EGF receptors, although considerably varied, was demonstrated in all the tumor cell lines studied. Epidermoid carcinoma appeared to have more EGF receptors than adenocarcinoma. One small cell carcinoma of the lung, one choriocarcinoma of the stomach and three bone tumors also possessed EGF receptors comparable to those of epidermoid carcinoma, while one adenoacanthoma of the stomach had less EGF receptors comparable to adenocarcinoma. Among a variety of phorbol esters tested, tetradecanoyl phorbol acetate, a potent tumor promotor, was shown to be the most effective compound in inhibiting 125I-labeled EGF binding to its receptors. Our results indicate that human tumor cells contain varying amounts of membrane-bound receptors for EGF and that phorbol esters interact with these EGF receptor sites. However, the relationship between EGF receptor sites on tumor cells and cellular proliferation and/or differentiation awaits further study.     

正常猕猴与树鼩神经系统和免疫系统组织中大麻素与阿片受体的表达

为应用猕猴和树鼩动物模型研究毒品成瘾对神经/免疫系统的影响提供基础数据,对大麻素及阿片受体在正常猕猴和树鼩神经系统和免疫系统的表达进行初步确定.采集正常猕猴和树鼩新鲜组织(皮质、小脑、脑干、海马、脊髓、脾脏),应用半定量逆转录PCR和实时定量PCR的方法检测大麻素与阿片受体mRNA在猕猴和树鼩各组织中的表达情况.猕猴脑部各区包括脾脏均表达大麻索受体1(CNR1),而大麻素受体2(CNR2)只表达于脾脏内.三类阿片受体中,mu(μ)受体表达最为广泛,在以上各组织中均有表达;delta(δ)受体表达的组织最少,只在海马表达;kappa(κ)受体表达介于两者之间,分别在皮质、小脑、脑干、脊髓中表达.在树鼩组织中,CNR1和CNR2表达于整个大脑重要脑区中,且CNR1表达量高于同一区域内CNR2表达的鼍:脾脏中CNR2的表达较高,而CNR1不表达.三类阿片受体只有检测到μ受体在脑部与脾脏表达,且在各个脑区的表达量明显高于脾脏的表达量;δ体和κ受体在被检各个组织中均无表达.总体而言,两种大麻素受体在猕猴和树鼩体内表达情况与人类和鼠的情况类似,而三类阿片受体在猕猴体内表达情况与人类吏为接近.猕猴和树鼩可能可用于人类毒品成瘾的研究;猕猴在某些神经受体的表达更接近人类,其在研究毒品成瘾的机理和对免疫系统的影响方面仍有不可替代的地位.     

Regulation of cell growth and motility by hepatocyte growth factor and receptor expression in various cell species.

Hepatocyte growth factor (HGF), a humoral mediator for regeneration of liver and kidney, possesses multiple biological activities. To investigate target cell specificity and to examine whether multiple actions of HGF are related to properties of the HGF receptor on target cells, we examined the effects of HGF on cell growth and motility and analyzed the HGF receptor in various species of cells. HGF stimulated growth and DNA synthesis of PAM212 (naturally immortalized mouse keratinocytes), Mv1Lu (mink lung epithelia), and A431 (human epidermoid carcinoma) cells, as well as mature hepatocytes, but inhibited those of IM-9 (human B-lymphoblasts). Conversely, HGF had a marked stimulatory effect on cell motility of MDCK (Mardin-Darby canine kidney epithelia) cells, but not on their growth. Also, HGF enhanced the motility of various species of cells, including A431, PAM212, HepG2 (human hepatoma), KB (human epidermoid carcinoma), and J-111 (human monocytes) cells. Scatchard analysis of 125I-HGF binding to hepatocytes indicated that the cells expressed both high- and low-affinity binding sites for HGF with Kd values of 23 and 260 pM, respectively. High-affinity HGF receptor with Kd values of 20-25 pM was detected at 40-720 sites/cell in MDCK, A431, PAM212, Lu99, and IM-9 cells, but not in fibroblasts and hematopoietic cells. In contrast, low-affinity binding sites were detected in all cell lines examined, even in those not responsive to HGF. Northern blots revealed that cells possessing a high-affinity HGF receptor expressed c-MET/HGF receptor mRNA. Therefore, HGF probably regulates both cell growth and motility of various types of epithelial cells and some types of mesenchymal cells. The multiple biological activities of HGF may be exerted through a high-affinity HGF receptor linked to multiple distinct intracellular signaling pathways.     

Angiotensin II type 1 receptor expression in human pancreatic cancer and growth inhibition by angiotensin II type 1 receptor antagonist

We investigated the expression of angiotensin II type 1 receptor (AT1) in pancreatic cancer. Both AT1 mRNA and protein were expressed in human pancreatic cancer tissues and cell lines. Binding assays showed that pancreatic cancer cells have specific binding sites for angiotensin II and that binding could be eliminated by treatment with a selective AT1 antagonist in a dose-dependent fashion. Surprisingly, the growth of cancer cells was significantly suppressed by treatment with antagonist, also in a dose-dependent manner. These observations suggest AT1 plays an important role in pancreatic cancer growth. Furthermore, ligand-induced inhibition of AT1 may be a useful therapeutic strategy.     

Lung tumor growth-promoting function of peroxiredoxin 6

This study compared lung tumor growth in PRDX6-overexpressing transgenic (Tg) mice and normal mice. These mice expressed elevated levels of PRDX6 mRNA and protein in multiple tissues. In vivo, Tg mice displayed a greater increase in the growth of lung tumor compared with normal mice. Glutathione peroxidase and calcium-independent phospholipase 2 (iPLA2) activities in tumor tissues of Tg mice were much higher than in tumor tissues of normal mice. Higher tumor growth in PRDX6-overexpressing Tg mice was associated with an increase in activating protein-1 (AP-1) DNA-binding activity. Moreover, expression of proliferating cell nuclear antigen, Ki67, vascular endothelial growth factor, c-Jun, c-Fos, metalloproteinase-9, cyclin-dependent kinases, and cyclins was much higher in the tumor tissues of PRDX6-overexpressing Tg mice than in tumor tissues of normal mice. However, the expression of apoptotic regulatory proteins including caspase-3 and Bax was slightly less in the tumor tissues of normal mice. In tumor tissues of PRDX6-overexpressing Tg mice, activation of mitogen-activated protein kinases (MAPKs) was much higher than in normal mice. In cultured lung cancer cells, PRDX6 siRNA suppressed glutathione peroxidase and iPLA2 activities and cancer cell growth, but the enforced overexpression of PRDX6 increased cancer cell growth associated with their increased activities. In vitro, among the tested MAPK inhibitors, c-Jun NH₂-terminal kinase (JNK) inhibitor clearly suppressed the growth of lung cancer cells and AP-1 DNA binding, glutathione peroxidase activity, and iPLA2 activity in normal and PRDX6-overexpressing lung cancer cells. These data indicate that overexpression of PRDX6 promotes lung tumor growth via increased glutathione peroxidase and iPLA2 activities through the upregulation of the AP-1 and JNK pathways.     

Role of toll-like receptors in lung cancer

Abstract

Lung cancer is a leading cause of death world-wide and the long-term survival rate for patients with lung cancer is one of the lowest for any cancer. Toll-like receptors (TLRs), evolutionarily conserved innate, are expressed in a wide variety of tissues and cell types, and they play key role in the innate immune system. TLRs have been found to be expressed by some kinds of tumor cells. However, what is the biological function of TLRs on tumor cells and whether human lung cancer cells can express TLRs remain to be fully understood. This review was performed to sum up the role of TLRs in lung cancer.     

Incidence of asthma and mortality in a cohort of young adults: a 7-year prospective study

Background

CpG-oligonucleotides (CpG-ODN), which induce signaling through Toll-like receptor 9 (TLR9), are currently under investigation as adjuvants in therapy against infections and cancer. CpG-ODN function as Th-1 adjuvants and are able to activate dendritic cells. In humans TLR9 has been described to be strongly expressed in B-lymphocytes, monocytes, plasmacytoid dendritic cells and at low levels in human respiratory cells. We determined whether a direct interaction of bacterial DNA with the tumor cells themselves is possible and investigated the expression and function of TLR9 in human malignant solid tumors and cell lines. TLR9 expression by malignant tumor cells, would affect treatment approaches using CpG-ODN on the one hand, and, on the other hand, provide additional novel information about the role of tumor cells in tumor-immunology.

Methods

The expression of TLR9 in HOPE-fixed non-small lung cancer, non-malignant tissue and tumor cell lines was assessed using immunohistochemistry, confocal microscopy, in situ hybridization, RT-PCR and DNA-sequencing. Apoptosis and chemokine expression was detected by FACS analysis and the Bio-Plex system.

Results

We found high TLR9 signal intensities in the cytoplasm of tumor cells in the majority of lung cancer specimens as well as in all tested tumor cell lines. In contrast to this non-malignant lung tissues showed only sporadically weak expression. Stimulation of HeLa and A549 cells with CpG-ODN induced secretion of monocyte chemoattractant protein-1 and reduction of spontaneous and tumor necrosis factor-alpha induced apoptosis.

Conclusions

Here we show that TLR9 is expressed in a selection of human lung cancer tissues and various tumor cell lines. The expression of functionally active TLR9 in human malignant tumors might affect treatment approaches using CpG-ODN and shows that malignant cells can be regarded as active players in tumor-immunology.