Enhancement of Antiproliferative Effects of Interleukin-1β and Tumor Necrosis Factor-α on Human Prostate Cancer LNCaP Cells by Coculture with Normal Fibroblasts through Secreted Interleukin-6

The cell-cell interactions between tumor cells and stromal cells are considered to be important in the regulation of tumor development at primary and metastatic secondary sites. We studied the effects of various cytokines on the cell-cell interactions between androgen-dependent LNCaP or androgen-independent PC-3 human prostate cancer cell lines and normal fibroblasts using a coculture system. Among the tested combinations of cytokines and fibroblasts, strong modulations of cytokine actions were seen in coculture with human normal fibroblasts WI-38. While interleukin (IL)-1β or tumor necrosis factor-α (TNF-α)) partially suppressed LNCaP cell growth in monoculture, each cytokine completely inhibited it in the case of coculture with WI-38 cells. On the other hand, they did not inhibit PC-3 cell growth significantly, regardless of monoculture or coculture. Conditioned medium prepared from WI-38 cells pretreated with IL-1β or TNF-α also strongly inhibited LNCaP cell growth. In the conditioned medium, marked IL-6 secretion was induced from WI-38 cells by IL-1β or TNF-α. Furthermore, neutralizing antibodies to IL-6 or IL-6 receptor abrogated the antiproliferative effects of IL-1β- and TNF-α-pretreated WI-38 conditioned medium. These results demonstrate that the antiproliferative effects of IL-1β and TNF-α on prostate cancer cells are enhanced by coculture with normal fibroblasts through some diffusible factor(s), such as IL-6, from the stimulated fibroblasts.     

Tumor-derived tumor necrosis factor-alpha promotes progression and epithelial-mesenchymal transition in renal cell carcinoma cells

Pro-inflammatory cytokines and chemokines are involved in promoting tumorigenesis by facilitating tumor proliferation and metastasis. The serum levels of interleukin (IL)-6, IL-1β, and tumor necrosis factor-alpha (TNF-α) are significantly elevated in patients with renal cell carcinoma (RCC). However, the mechanisms of how these cytokines participate in the progression of RCC remains unknown. In the present study, we investigated the effects of tumor-derived cytokines on invasion and the epithelial-mesenchymal transition (EMT) of RCC cells. We found that expression of IL-1β, IL-6, TNF-α, hypoxia-inducible factor-alpha (HIF-1α), and matrix metalloproteinase-2 (MMP2) were significantly elevated in high malignancy A498 cells compared to low malignancy 786-O cells. The invasion ability of A498 was three-fold higher than that of 786-O cells. The invasiveness of 786-O cells was markedly enhanced by adding conditioned medium derived from A498 cells. This phenomenon was significantly inhibited by immunodepletion of TNF-α followed by MMP2, IL-6, or IL-1β from A498 conditioned medium. Synergistic inhibition was also noted after simultaneous immunodepletion of TNF-α, IL-1β, and IL-6. RCC cell lines with higher malignancy produced more TNF-α, which was correlated with their stronger invasive ability. The invasiveness of 786-O cells was significantly promoted by TNF-α in a dose-dependent manner. Moreover, TNF-α induced the EMT of 786-O cells by repressing E-cadherin, promoting vimentin expression, and activating MMP9 activity. Our findings demonstrate that pro-inflammatory cytokines, especially TNF-α, can enhance invasion and the EMT of renal cancer cells, which provides a therapeutic target to prevent and treat advanced RCC. ( Cancer Sci 2008; 99: 905–913)     

Autonomous Expressions of Cytokine Genes by Human Lung Cancer Cells and Their Paracrine Regulation

Cell-to-cell interaction between tumors and host inflammatory cells is important for the subsequent cancer progression or regression. We examined the expressions of mRNAs for various proinflammatory cytokines by nine human lung cancer cell lines and the influences of cytokines on their gene expressions. The cytokines used were interleukin 1β (IL-1β), interleukin 6 (IL-6), tumor necrosis factor α (TNF-α), granulocyte-macrophage colony stimulating factor (GM-CSF) and monocyte chemotactic and activating factor. Gene expressions of cytokines were measured by Northern blot analysis. Substantial expressions of cytokine genes were detected in several lung cancer cell lines such as RERF-LC-MS, RERF-LC-OK and VMRC-LCD, although the levels of expression of each cytokine varied in different cell lines. Four lung cancer cell lines (RERF-LC-MS, RERF-LC-OK, A549 and YO-88) were used to examine the effects of exogenous cytokines (IL-1β, TNF-α and GM-CSF) on cytokine gene expressions by the cells. TNF-α and IL-1β caused significant changes in the levels of mRNA expressions of certain cytokines. Moreover, on stimulation with TNF-α, RERF-LC-OK cells produced IL-6 extracellularly. These extensive differences in the levels of gene expressions and productions of cytokines could have profound effects on the interactions between human lung cancer cells and the corresponding host cells.     

Transfection-induced Tumor Necrosis Factor-α Increases the Susceptibility of Human Glioma Cells to Lysis by Lymphokine-activated Killer Cells: Continuous Expression of Intercellular Adhesion Molecule-1 on the Glioma Cells

To develop more effective adoptive immunotherapy, we transfected the human tumor necrosis factor-α (TNF-α) gene into human glioma cells (U251-SP), which were used as target cells. TNF-α is known to increase both the expression of intercellular adhesion molecule-1 (ICAM-1) on the surface of glioma cells and the susceptibility of glioma cells to lymphokine-activated killer (LAK) cell cytolysis. We compared the expression of ICAM-1 induced by TNF-α generated by the TNF-α gene-transfected cells with that induced by exogenously added TNF-α. When the TNF-α gene was transfected into U251-SP cells, the expression of ICAM-1 was detected on the cell surface from 3 days after the transfection and continued until at least 9 days. In contrast, it was expressed only transiently in the case of exogenously added TNF-α. Also, the cytolytic activity of LAK cells induced by transfection-induced TNF-α was significantly stronger than that induced by exogenously added TNF-α. The increased susceptibility was quenched by anti-ICAM-1 monoclonal antibody. These data indicated that continuous expression of ICAM-1 induced by TNF-α gene transfection of glioma cells resulted in higher cytolytic activity of LAK cells.     

Effect of serum deprivation on constitutive production of granulocyte-colony stimulating factor and granulocyte macrophage-colony stimulating factor in lung cancer cells

We previously established 2 lung cancer cell lines, OKa-C-1 and MI-4, which constitutively produce an abundant dose of granulocyte-colony stimulating factor (G-CSF) and granulocyte macrophage-colony stimulating factor (GM-CSF). Many other cases with G-CSF or GM-CSF producing tumors have been reported up to the present. However, the biological properties of the overproduction of G-CSF and GM-CSF by tumor cells have not been well known. Several reports demonstrated the presence of an autocrine growth loop for G-CSF and GM-CSF in nonhematopoietic tumor cells. We showed that exogenous G-CSF and GM-CSF stimulated cell growth in a dose-dependent manner in OKa-C-1 and MI-4 cells. We could detect the presence of G-CSF and GM-CSF receptors in both cell lines by RT-PCR analysis. We have previously shown that inflammatory cytokines, tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta enhance the expression of G-CSF and GM-CSF in the cell lines. However, the factors that regulate constitutive production of G-CSF or GM-CSF by tumor cells are still unknown well. In our study, we first reported that serum deprivation stimulated constitutive production of G-CSF and GM-CSF by lung tumor cells through activation of nuclear factor (NF)-kappaB and p44/42 mitogen-activated protein kinase (MAPK) pathway signaling. We suggest that G-CSF and GM-CSF constitutively produced by tumor cells could grow tumor itself and rescue tumor cells from the cytotoxicity of serum deprivation.     

Gastric mucosal interleukin-17 and -18 mRNA expression in Helicobacter pylori-induced Mongolian gerbils

Helicobacter pylori infection causes characteristic mucosal infiltration of inflammatory cells, resulting in the development of peptic ulcers and gastric cancer in approximately 10% of cases. Different clinical expressions of the infection may reflect different patterns of cytokine expression. Interleukin (IL)-1ß, tumor necrosis factor (TNF)-α, IL-17, and IL-18 have been reported to be involved in H. pylori -induced gastric mucosal inflammation, but the details and relation to different patterns of inflammation remain unclear. Moreover, the proinflammatory virulence factor outer inflammatory protein (OipA) was reported to be associated with gastric mucosal inflammatory cytokine levels. To clarify these findings, Mongolian gerbils were infected for up to 12 months with wild-type H .  pylori 7.13 or with isogenic oipA mutants for 3 months, and mucosal cytokines (IL-1ß, IL-17, IL-18, and TNF-α) mRNA levels were then assessed using real-time RT-PCR. Antral mucosal IL-1β and IL-18 mRNA levels peaked 1 month after infection, whereas the peak of TNF-α mRNA was at 6–12 months; IL-17 levels peaked at 12 months. The inflammatory cell infiltration and mRNA levels of all cytokines studied were significantly lower in oipA mutants than in wild-type-infected gerbils. Mucosal IL-1ß, IL-17, and TNF-α expression, but not that of IL-18, were significantly associated with the grade of inflammatory cell infiltration. The pattern of increased inflammatory cytokines differed relative to the phase of the infection and pattern of inflammation. OipA appears to play a role in IL-1ß, IL-17, and TNF-α expression and the resulting inflammation. ( Cancer Sci 2009)     

Cyclooxygenase-2 inhibitor NS-398 suppresses cell growth and constitutive production of granulocyte-colony stimulating factor and granulocyte macrophage-colony stimulating factor in lung cancer cells

We previously established two lung cancer cell lines, OKa-C-1 and MI-4, which constitutively produce abundant granulocyte-colony stimulating factor (G-CSF) and granulocyte macrophage-colony stimulating factor (GM-CSF). Inflammatory cytokines, tumor necrosis factor-alpha (TNF-alpha) and interleukin (IL)-1beta stimulated the expression of G-CSF, GM-CSF, and cyclooxygenase (COX)-2 in the two cell lines. It is known that increased COX-2 activity promotes tumor growth and induces G-CSF and GM-CSF expression in non-malignant cells, and that selective COX-2 inhibitors inhibit the growth of some types of malignant cells. Therefore, we hypothesized that inhibition of COX-2 activity might suppress constitutive production of G-CSF or GM-CSF in addition to reducing the growth of malignant cells. We confirmed that the selective COX-2 inhibitor, NS-398 suppressed the constitutive production of G-CSF and GM-CSF, and the cell growth in both OKa-C-1 and MI-4 cell lines. Prostaglandin E2 (PGE2) reversed the inhibitions of G-CSF and GM-CSF expression, as well as cell growth, by NS-398. This result confirms that the effects of NS-398 are based on the inhibition of COX activity. Some studies have indicated that nuclear factor kappa B (NF-kappaB) or MAPK (mitogen-activated protein kinase) activation is related to upregulation of G-CSF, GM-CSF or COX-2 expression in some types of cells. Therefore, we examined if the actions of NS-398 might be mediated by the MAP kinase pathway or NF-kappaB activity in OKa-C-1 and MI-4 cells. We found that NS-398 inhibits G-CSF and GM-CSF production and cell growth through an extracellular signal-regulated kinase kinase (MEK) signaling pathway in these cell lines. The prognosis of non-small cell lung cancer showing G-CSF gene expression is significantly worse. G-CSF overproduction by tumor cells is observed at an advanced clinical stage. Our findings imply that a COX-2 inhibitor might improve the prognosis of patients with lung cancer through the reduction of G-CSF or GM-CSF.     

Hyperthermic Enhancement of the Antitumor Effect of Natural Human Tumor Necrosis Factor-α and -β: an in vitro and in vivo Study

A synergistic antitumor effect of natural human tumor necrosis factor-β (TNF-β) in combination with hyperthermia was found, in comparison with that of TNF-α, using an in vitro antiproliferative assay on a human colon cancer cell line (RPMI4788) and an in vivo tumor growth inhibition assay on Meth A sarcoma cells. In vitro combined treatment with TNF-β (10,000 U/ml) and hyperthermia (at 43° for 60 min) synergistically inhibited the proliferation of the cells. Combined effects of TNF-α or natural human interferon-α or -γ (IFN-α, -γ) and hyperthermia were also examined, and furthermore, the combinations of TNFs and IFNs were examined in combination with hyperthermia at 42°; their antiproliferative effects were further augmented by hyperthermia. In vivo growth of Meth A sarcoma cells (5 × 10⁵), transplanted subcutaneously into BALB/c mice, was inhibited significantly ( P <0.05) with the combination of TNF-α or -β (2 × 10⁵ U/mouse) and hyperthermia (at 43° for 60 min) as compared to either a single intravenous injection of TNF-α or -β alone or the hyperthermia alone. The influence of TNF-β and hyperthermia on the cell cycle was examined. Flow cytometric analysis showed that RPMI4788 cells treated with TNF-α or -β accumulated in the S phase of the cell cycle, and that hyperthermia (at 42° for 60 min) alone had no influence on the cell cycle and did not augment the S phase accumulation of the cells treated with TNF-α or -β.     

Activation of Protein Kinase C by Mycobacterial Cord Factor, Trehalose 6-Monomycolate, Resulting in Tumor Necrosis Factor-α Release in Mouse Lung Tissues

Cord factors are mycoloyl glycolipids in cell walls of bacteria belonging to Actinomycetales , such as Mycobacterium , Nocardia and Rhodococcus . They induce granuloma formation in the lung and interstitial pneumonitis, associated with production of macrophage-derived cytokines. We studied how cord factors induce biological activities in the cells. Cord factors isolated from M. tuberculosis , trehalose 6-monomycolate (mTMM) and trehalose 6,6'-dimycolate (mTDM), enhanced protein kinase C (PKC) activation in the presence of phosphatidylserine (PtdSer), diacylglycerol and Ca²⁺, and mTMM activated PKCα more strongly than PKCβ or γ under the same assay conditions. Kinetic studies of mTMM in response to PKC activation revealed that mTMM increased the apparent affinity of PKC to Ca²⁺ in the presence of both PtdSer and diolein. Although this is similar to observations with unsaturated fatty acids, such as arachidonic acid, mTMM was synergistic with PtdSer for PKC activation, but arachidonic acid was not. mTMM was also different as regards PKC activation, as phorbol ester was. A single i.p. administration of mTMM to mouse induced tumor necrosis factor-α (TNF-α) in serum and in the lung, which is a unique target tissue of cord factors. Based on our recent finding that TNF-α is an endogenous tumor promoter, the correlation between lung cancer and pulmonary tuberculosis is discussed.     

Production of Multiple Growth Factors by a Newly Established Human Thyroid Carcinoma Cell Line

A multiple growth factor-producing tumor cell line (NIM-1) was newly established from a patient with thyroid cancer and remarkable neutrophilia. NIM-1 cells also caused severe neutrophilia in nude mice bearing tumors. NIM-1-conditioned medium (NIM-1CM) contained activities that supported not only granulocyte, macrophage and eosinophil colony formation of human bone marrow cells but also the growth of colony-stimulating factor (CSF)-dependent cell lines, NFS60-KX and TF-1. Northern blot hybridization analysis revealed the constitutive expression of granulocyte-CSF (G-CSF), granulocyte/macrophage-CSF (GM-CSF) and interleukin(IL)-6 mRNAs in NIM-1 cells. Enzyme-linked immunosorbent assays (ELISA) using NIM-1CM also confirmed the production of IL-la and a small amount of IL-1β besides G-CSF, GM-CSF and IL-6 in NIM-1 cells. In addition, unexpected production of IL-11 in NIM-1 cells was detected by northern blot hybridization analysis and by bioassay using an IL-11-dependent cell line. Therefore, NIM-1 cell line is shown to produce multiple cytokines including potentially megakaryopoietic growth factors such as GM-CSF, IL-6 and IL-11.     

Parathyroid hormone-related protein induces interleukin 8 production by prostate cancer cells via a novel intracrine mechanism not mediated by its classical nuclear localization sequence

PTHrP (parathyroid hormone-related protein) overexpression by prostate carcinoma cells has been implicated in tumor progression. Although the biological effects of PTHrP can be mediated by the G-protein-coupled PTH/PTHrP receptor, PTHrP also has intracrine actions mediated by a nuclear localization sequence at residues 87-107. We investigated the effect of PTHrP transfection and treatment on production by prostate carcinoma cells of IL (interleukin)-8, which can regulate prostate cancer growth by angiogenic activity and growth-promoting effects. Six prostate cancer cell lines exhibited constitutive expression of PTHrP and IL-8 that were significantly correlated (r = 0.93; P < 0.01). We transfected wild-type and mutant PTHrP into these cells. Wild-type PTHrP1-173 and PTHrP33-173 lacking the PTH/PTHrP receptor-binding domain induced a 3-fold stimulation of IL-8 production but not production of another angiogenic factor, vascular endothelial growth factor. Transfection of the COOH-terminal truncation mutant PTHrP1-87 induced a 5-fold simulation of IL-8 and a 3-fold increase in IL-8 mRNA. Cells transfected with PTHrP1-87 and 1-173 also showed increased cell proliferation. In contrast, exogenous PTHrP1-34 and 1-86 peptides did not significantly affect IL-8 production; moreover, PTHrP-neutralizing antibodies did not inhibit the production of IL-8 by transfected PTHrP. Additional transfection studies with progressively COOH-terminally truncated PTHrP1-87 defined a 23-amino acid sequence, PTHrP65-87, required for PTHrP1-87 to robustly stimulate IL-8 in prostate cancer cells. Confocal microscopy and immunoassay demonstrated PTHrP1-87 nuclear localization. Our results demonstrate that PTHrP acts to induce IL-8 production in prostate cancer cells via an intracrine pathway independent of its classical nuclear localization sequence. This novel pathway could mediate the effects of PTHrP on the progression of prostate cancer.     

Enhancing Effect of Interleukin-2 on Production of Parathyroid Hormone-related Protein by Adult T-Cell Leukemia Cells

Leukemic cells from patients with adult T-cell leukemia (ATL) can produce a calcium-regulating protein, parathyroid hormone-related protein (PTHrP). Moreover, it has been reported that ATL cells produce some cytokines besides PTHrP and that these cells respond to the T-cell growth factors, interleukin-2 (IL-2) and interIenkin-4 (IL-4). To elucidate whether PTHrP produced by ATL cells is regulated by IL-2 or IL-4, we investigated the in vitro effects of IL-2 and IL-4 on the release of PTHrP. IL-2 increased the release of PTHrP into the conditioned medium from leukemic cells in some, but not all, ATL patients; however, IL-4 did not affect the PTHrP release. PTHrP messenger RNA (mRNA) levels were increased in ATL cells cultured in the presence of IL-2. These data suggest that IL-2 plays a role in the regulation of hypercalcemia by enhancing the production of PTHrP in ATL patients.     

Co-production of Interleukin-1 and Interleukin-6 in Tumor Cell Lines Elaborating Colony-stimulating Factors

Six carcinoma cell lines elaborating colony-stimulating factors (CSFs) were examined by enzyme-linked immtmosorbent assay and Northern blotting to determine whether or not they co-produced interleukin-lα (IL-lα), IL-lβ and IL-6. All 6 cell lines were co-producers; IL-lα was produced in all 6, IL-6 in 5 and IL-1β in 3 of them. These results indicate that IL-1 and IL-6 are commonly co-produced in CSF-producing tumors.     

Tumor Necrosis Factor-a Stimulates Colony Formation by a Megakaryoblastic Leukemia Cell Line, CMK

The CMK cell line is an acute megakaryoblastic leukemia cell line established from a patient with Down's syndrome, and is known to possess characteristics of normal megakaryocytes. Several cytokines with the ability to stimulate megakaryopoiesis, such as interleukin-3 (IL-3), interleukin-6 (IL-6) and granulocyte-macrophage colony-stimulating factor (GM-CSF), stimulated colony formation by CMK cells. The present study revealed that tumor necrosis factor-α (TNF-α) stimulated colony formation by CMK cells; the potency was almost equal to that of IL-3, IL-6 or GM-CSF. Scatchard plot analysis revealed that CMK cells possess two types of specific binding sites for TNF-α. The high-affinity binding sites had an affinity constant of 0.18 nM, and numbered 5,000. The low-affinity binding sites had an affinity constant of 1.8 nM/and numbered 19,000. These results raise the possibility that TNF-α can act as a growth-stimulating agent on megakaryocyte-Iineage cell line.     

Dipyridamole Combined with Tumor Necrosis Factor-α Enhances Inhibition of Proliferation in Human Tumor Cell Lines

In the search for cytokines whose antiproliferative action could be enhanced by combination with dipyridamole, 2,6-bis(diethanolamino)-4,8-dipiperidinopyrimido[5,4,d]pyrimidine, the combination of tumor necrosis factor-α (TNF-α) with this agent was evaluated in various human tumor cell lines. Inhibition of the proliferation of human melanoma cell lines MM-1CB and HMV-1 by TNF-α (1–10² U/ml) was enhanced in culture dishes by combination treatment with dipyridamole (0.1–10 μM). The enhancement effect was also detected in other tumor cell lines: T9S (glioma), SCC-1CB (squamous cell carcinoma), HAC-2 (ovarian clear-cell carcinoma), HLE (hepatoma), HEC-1 (endometrial adenocarcinoma) and HOC-21 (ovarian serous cystadenocarcinoma). The incorporation of [¹⁴C]amino acids and [³H]nridine into acid-insoluble cell materials in the combination-treated cells was not significantly different from that in cells treated with TNF-α or dipyridamole. However, the incorporation of [³H]thymidine was specifically inhibited in all cell lines examined after more than 12 h of the TNF-α and dipyridamole combination treatment, although neither agent alone inhibited this incorporation. On the other band, the growth of tumors induced by the injection of MM-1CB and HMV-1 cells into nude mice was more markedly inhibited by the subcutaneous administration of TNF-α in combination with orally administered dipyridamole than by either agent alone. The results presented suggested that dipyridamole is beneficial in assuring the effectiveness of anti-cancer cytokine therapy.