Activation of paracrine growth factors by heparan sulphate induced by glucocorticoid in A549 lung carcinoma cells.

Alkaline phosphatase, a marker of differentiation in the human alveolar adenocarcinoma cell line A549, is inducible by conditioned medium from lung fibroblasts and by cytokines including oncostatin M and interleukin 6, but only in the presence of a glucocorticoid, dexamethasone. Dexamethasone was shown to induce incorporation of [3H]glucosamine into three fractions of medium and cell trypsinate from subconfluent A549 cells, eluting from DEAE ion-exchange chromatography. The first peak did not correspond to any of the unlabelled glycosaminoglycans and was not characterized further. Induction was seen in two other peaks, corresponding to hyaluronic acid and heparan sulphate. Of these, heparan sulphate, eluting as one well-defined peak (referred to as HS1) and another of lower activity and less well defined (HS2), was selected as the most likely to interact with growth factors and cytokines and was isolated from the eluate, concentrated and desalted, and used in alkaline phosphatase induction experiments in place of dexamethasone. HS1 isolated from the medium (HS1m) of subconfluent A549 cells was shown to replace dexamethasone in induction experiments with fibroblast-conditioned medium, oncostatin M and interleukin 6. HS1 from the cell trypsinate and HS2 from the medium and trypsinate were inactive. As the activity of HS1m could be abolished by heparinase and heparitinase but not by chondroitinase ABC, it was concluded that HS1m was a fraction of heparan sulphate involved in the regulation of paracrine growth factor activity in lung fibroblast-conditioned medium, and in the regulation of other growth factors with potential roles in the paracrine control of cell differentiation.     

Activity of interferon alpha, interleukin 6 and insulin in the regulation of differentiation in A549 alveolar carcinoma cells.

The differentiation of A549, a human tumour cell line from type II pneumocytes, can be induced by a crude fibroblast-derived factor (FDF) isolated from the conditioned medium of glucocorticoid-treated lung fibroblasts. In the present report, we have used alkaline phosphatase as a differentiation marker to investigate the activity of a number of growth factors as potential candidates for this paracrine activity. This showed that insulin, interleukin 6 (IL-6), and interferon alpha (IFN-alpha) could simulate the activity of conditioned medium. Their effects were dexamethasone (DX) dependent, additive and reversible with a half-life of 1 week. Transforming growth factor alpha and beta, IL-1 alpha and epidermal growth factor, were all inhibitory, and inhibition was opposed, partially or completely, by DX. The most potent inducer was IL-6, but as DX was shown to decrease the concentration of IL-6 in lung fibroblast-conditioned medium it seems an unlikely candidate for FDF. Unlike FDF, all of the positive-acting factors were shown to induce plasminogen activator. FDF has also been shown to be active in the absence of DX. This suggests that differentiation-inducing activity may be present in several paracrine factors, but that so far a candidate for FDF has not been identified.     

Interaction between non-small-cell lung cancer cells and fibroblasts via enhancement of TGF-β signaling by IL-6

Introduction

Fibroblasts are key components of the tumor microenvironment. We clarified the role of transforming growth factor (TGF)-β and interleukin (IL)-6 in the interaction between fibroblasts and non-small-cell lung cancer (NSCLC) cells.

Methods

We used NSCLC cells (A549, NCI-H358) and normal human lung fibroblast (NHLF) cells to evaluate phenotypic changes in the presence of human IL-6, TGF-β1, and conditioned media (CM) from these cells. Possible pathways were evaluated with SB431542, a TGF-β receptor inhibitor, or an anti-human IL-6 receptor neutralizing antibody (IL-6R-Ab).

Results

A549 and NCI-H358 cells incubated with IL-6 (50 ng/mL) and TGF-β1 (2 ng/mL) showed significantly increased epithelial–mesenchymal transition (EMT) signaling compared to those treated with TGF-β1 alone. Furthermore, NHLF cells were synergistically activated by IL-6 and TGF-β1. IL-6 increased the expression of TGF-β type I receptors on the surface of A549, NCI-H358 and NHLF cells and enhanced TGF-β signaling. TGF-β1 induced phenotypic changes were attenuated by IL-6R-Ab. NHLF cells were activated and A549 cells showed induction of EMT in response to CM from the other cell type. These activities were attenuated by SB431542 or IL-6R-Ab, suggesting that interplay between NSCLC cells and NHLF may lead to increased EMT signaling in NSCLC cells and activation of NHLF cells through TGF-β and IL-6 signaling. Subcutaneous co-injection of A549 and NHLF cells into mice resulted in a high rate of tumor formation compared with injection of A549 cells without NHLF cells. SB431542 or IL-6R-Ab also attenuated the tumor formation enhanced by co-injection of the two cell types.

Conclusion

IL-6 enhanced epithelial cell EMT and stimulated tumor progression by enhancing TGF-β signaling. IL-6 and TGF-β may play a contributing role in maintenance of the paracrine loop between these two cytokines in the communication between fibroblasts and NSCLC cells for tumor progression.     

Autophagy in fibroblasts induced by cigarette smoke extract promotes invasion in lung cancer cells

We investigated the effects of cigarette smoke extract (CSE) on lung fibroblasts and found that the invasiveness of lung cancer cells was facilitated by the conditioned medium from CSE-treated fibroblasts. CSE induced autophagy in fibroblasts and increased the expression of autophagy-related proteins, including optineurin and Ras-related protein Rab1B. Afterward, the fibroblasts produced high levels of interleukin-8 (IL-8), which promoted cancer cell invasion. The inhibition of either optineurin or Rab1B abrogated a rise in microtubule-associated protein 1 light chain 3 β and a decrease in p62 protein, as well as the production of IL-8, in CSE-treated fibroblasts. A three-dimensional invasion assay using cancer cell spheroids revealed that the invasion of cancer cells alone and the fibroblast-led cancer cell invasion were both enhanced by the conditioned media from CSE-treated fibroblasts. These results suggest that cigarette smoke may induce autophagy and IL-8 secretion in lung fibroblasts and modify the microenvironment to favor invasion of lung cancer cells.     

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)     

Low-dose gamma-irradiation inhibits IL-6 secretion from human lung fibroblasts that promotes bronchial epithelial cell transformation by cigarette-smoke carcinogen

Despite decades of research in defining the health effects of low-dose (<100 mGy) ionizing photon radiation (LDR), the relationship between LDR and human cancer risk remains elusive. Because chemical carcinogens modify the tumor microenvironment, which is critical for cancer development, we investigated the role and mechanism of LDR in modulating the response of stromal cells to chemical carcinogen-induced lung cancer development. Secretion of proinflammatory cytokines such as interleukin-6 (IL-6), CXCL1 and CXCL5 from human lung fibroblasts was induced by cigarette-smoke carcinogen benzo[a]pyrene diol epoxide (BPDE), which was inhibited by a single dose of LDR. The activation of NF-κB, which is important for BPDE-induced IL-6 secretion, was also effectively suppressed by LDR. In addition, conditioned media from BPDE-treated fibroblasts activated STAT3 in the immortalized normal human bronchial epithelial cell line Beas-2B, which was blocked with an IL-6 neutralizing antibody. Conditioned medium from LDR-primed and BPDE-treated fibroblast showed diminished capacity in activating STAT3. Furthermore, IL-6 enhanced BPDE-induced Beas-2B cell transformation in vitro. These results suggest that LDR inhibits cigarette smoke-induced lung carcinogenesis by suppressing secretion of cytokines such as IL-6 from fibroblasts in lung tumor-prone microenvironment.     

Cytokine production of lung cancer cell lines: Correlation between their production and the inflammatory/immunological responses both in vivo and in vitro

Cytokines produced by tumor cells may have various effects on antitumor immune responses and tumor growth. In the present study, the cytokine production of 31 lung cancer cell lines was evaluated, while any correlation with the histological type, the induction of tumor-specific cytotoxic T lymphocytes (CTL) in vitro, and angiogenesis and the infiltration of inflammatory cells in tumor tissues were also examined. Production of interleukin (IL)-1alpha, IL-1beta, IL-4, IL-6, IL-8, IL-10, tumor necrosis factor (TNF)-alpha, granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor, transforming growth factor (TGF)-beta and vascular endothelial growth factor (VEGF) in the culture supernatant was measured using enzyme-linked immunosorbent assay. Each cytokine was produced in a substantial number of the tumor cell lines. In particular, IL-6, IL-8, TGF-beta and VEGF were produced in 18 (55%), 29 (94%), 31 (100%) and 28 (90%) of 31 cell lines, respectively. However, neither IL-4 nor TNF-alpha was produced at all by any tumor cell line. TGF-beta production was significantly higher in adenocarcinoma than in squamous cell carcinoma (P = 0.03). Immunohistochemical staining revealed the magnitude of macrophage infiltration, and angiogenesis in surgically resected tumor tissue specimens correlated well with GM-CSF and IL-8 production from the corresponding cell lines. Among six lung cancer cell lines, CTL were induced in the three lung cancer cell lines that produced a lower amount of TGF-beta (<100 pg/mL). These findings suggested that TGF-beta produced by tumor cells could inhibit the induction of CTL in vitro. The present results suggest that the production of various cytokines from tumor cells might exert various paracrine effects both in vivo and in vitro.     

Tumor-derived transforming growth factor-β1 and interleukin-6 are chemotactic for lymphokine-activated killer cells

Adherent lymphokine-activated killer (A-LAK) cells are purified IL-2 activated natural killer (NK) cells with potent anti-tumor cytotoxic activity. They have been used in the adoptive immunotherapy of metastatic canters. However, it has been shown that intravenously transferred LAK cells have a poor horning capacity to tumor sites. For the present study, the effects of tumor-derived factors on the in vitro migratory capacity of A-LAK cells was investigated. In a micropore migration assay the conditioned medium from 3LL Lewis lung carcinoma cell cultures was found to exert a strong chemotactic, but not chemokinetic effect on A-LAK cells. This effect was partially inhibited by neutralizing antibodies against the cytokines TGF-β1 and IL-6. A combination of the 2 antibodies completely suppressed the chemotactic activity of tumor-cell-conditioned medium. Purified TGF-β1 and recombinant IL-6 were chemotactic for A-LAK cells. Biological activities of both cytokines were detectable in the tumor-cell-conditioned medium. The in vivo relevance of these findings, with respect to tissue infiltration of NK cells and LAK cells in inflammation or cancer, remains to be elucidated.     

阻断巨噬细胞NFKB-p65通路对香烟促进非小细胞肺癌NCI—H520细胞株增殖的影响

研究香烟对非小细胞性肺癌（NSCLC）NCI-H520细胞株增殖的影响以及巨噬细胞在此过程中的作用。方法 采用“Transwell？ Inserts”细胞培养室培养系统共培养人原代巨噬细胞或分化为巨噬细胞的人单核巨噬细胞株U937细胞和NCI-H520细胞。应用免疫印迹法和凝胶电泳迁移实验检测NF？B通路的激活;应用NF？B -p65 shRNA干扰质粒抑制U937细胞p65 表达;利用BrdU ELISA分析香烟抽提物（CSE）对NCI-H520细胞的促增殖效应;ELISA分析炎症细胞因子TNF？和IL-6的释放。结果 共培养及香烟刺激增强了人巨噬细胞的NF？B -p65的入核,以及同靶基因结合的活力。与对照组相比, NCI-H520细胞经不同浓度CSE单独处理,并没有发现肺癌细胞增殖明显加快（p〉0.05）;而经过与人原代巨噬细胞或分化为巨噬细胞的U937细胞共培养4天,不仅发现巨噬细胞明显促进NCI-H520细胞的增殖（p〈0.01）,而且观察到CSE的刺激显著增强这一效应（p〈0.01）。NF？B -p65shRNA干扰质粒明显抑制了U937细胞的NF？B -p65表达,抑制了NF？B通路的激活,减弱了CSE对NCI-H520细胞增殖的影响（p〈0.01）,同时也抑制了炎性因子IL-6和TNF？释放（p〈0.01）。结论 香烟通过巨噬细胞的中介促进NCI-H520细胞的增殖。采用RNA干扰技术阻断巨噬细胞的NF？B通路显著地抑制香烟促进的肺癌细胞的增殖,削弱香烟引起的巨噬细胞的炎症因子释放可能是其作用机制。     

Expression of neuroendocrine and epithelial markers in an adherent subline derived from a classic small cell lung cancer cell line.

Small cell lung cancer (SCLC) cell lines usually grow as floating aggregates, in contrast to the adherent monolayers formed by non small cell lung cancer (NSCLC). Induction of an adherent phenotype by a variety of methods has been the subject of a number of recent publications. In this study, cultivation of the classic SCLC cell line, NCI-H69, on a substratum provided by the pretreatment of tissue culture dishes with medium conditioned by the growth of a well differentiated squamous carcinoma cell line, HN5, induced an adherent phenotype with a variety of epithelioid morphologies, commencing within 24 hr of plating. From such cultures an adherent subline, H69A, has been established, which differs in its growth, morphological characteristics, and immunocytochemical marker expression from the parent NCI-H69 cells, and in its marker expression from other adherent SCLC cell lines. H69A retained expression of neural cell adhesion molecule (NCAM) and the neuroendocrine markers neuron specific enoclase, chromogranin A, and synaptophysin, but showed diminished expression of the epithelial cell surface markers AUA1, Ber-EP4, epithelial membrane antigen (EMA), and desmosomal protein. Compared to NCI-H69 cells, the amounts of cytokeratin 18 detected were elevated, while those of cytokeratin 19 were diminished in H69A cells. Focal expression of cytokeratin 4 was found in some H69A cells, indicative of a capacity for partial squamous differentiation. The expression of the cell surface glycoproteins detected by AUA1 and Ber-EP4 was reduced throughout cultivation of the H69A subline, while that of EMA and desmosomal protein was further diminished with continued passage. Changes in the expression of these markers and NCAM were evident in NCI-H69 cells grown on an HN5-derived substratum.     

The effect of recombinant cytokines on the proliferative potential and phenotype of cells of the human myelomonocytic leukaemia line, RC-2A

Cells of the human myelomonocytic line RC-2A can be induced to differentiate towards mature monocytes by culture in the presence of phytohaemagglutinin-treated lymphocyte conditioned medium (Lyons and Ashman, Leukemia Res. 11, 797, 1987). We have now examined the effect on RC-2A cells of some (recombinant) cytokines which might be present in conditioned medium. Gamma interferon most closely mimicked the effect of conditioned medium in inducing clonogenic suppression and the induction of monocytic maturation over 7 days of culture. Granulocyte colony stimulating factor induced enhancement of proliferation followed by clonogenic suppression, while granulocyte-macrophage colony stimulating factor had a purely stimulatory effect on proliferation over a 7-day period. Tumour necrosis factor alpha failed to affect cell proliferation or to induce characteristic monocytic differentiation, but did increase the expression of C3bi receptors. We conclude that RC-2A cells have receptors for all four cytokines studied, and that gamma interferon is a major differentiation-inducing stimulus for these cells.     

Monocyte Chemotactic Protein 1 Promotes Lung Cancer-Induced Bone Resorptive Lesions In Vivo

Lung cancer is the leading cause of cancer-related deaths. The morbidity and mortality of lung cancer have markedly increased in the past decade with at least 75% of patients with lung cancer having evidence of metastases at the time of diagnosis. It frequently metastasizes to bone resulting in osteolytic lesions with unknown mechanisms. The aim of this study was to identify factors that mediate lung cancer-induced osteoclast activity in vivo. Using a human cytokine antibody array, we first determined cytokine levels in a conditioned medium collected from non-small cell lung cancer A549 and H1299 cells and the non-neoplastic human bronchial epithelial BEAS2B cells. Both A549 and H1229 cells produced significantly higher amount of several cytokines including monocyte chemotactic protein 1 (MCP-1) and interleukin 8 (IL-8) compared with BEAS2B cells. These findings were confirmed by ELISA. From clinical serum specimens, we also observed that MCP-1 and IL-8 levels were increased in lung cancer patients with bone metastases compared with the patients with localized tumor. Next, we investigated the effects of MCP-1 on osteoclast formation in vitro using murine bone marrow-derived monocytes. A549 conditioned medium induced osteoclast formation that was inhibited by neutralizing antibodies against MCP-1. Finally, A549 cells were stably transfected with MCP-1 short hairpin RNA. The MCP-1 knockdown A549 cells were implanted into the tibia of severe combined immunodeficient mice for 4 weeks. The MCP-1 knockdown significantly diminished A549 cell growth. We conclude that MCP-1 promotes lung cancer-induced osteoclast activity and thus bone resorptive lesions in vivo.     

Combined Effects of Differentiation-inducing Factor and Other Cytokines on Induction of Differentiation of Mouse Myeloid Leukemic Cells

Mouse myeloid leukemic M1 cells are induced to differentiate into macrophage-like cells by differentiation-inducing factors (D-factors) and granulocyte colony-stimulating factor. We examined the effects of recombinant human tumor necrosis factor (rTNF), lymphotoxin (rLT) and interleukin 1 (rIL-1) on the induction of differentiation of M1 cells, compared with the effects of D-factor purified from the conditioned medium of mouse Ehrlich as cites tumor cells and recombinant human granulocyte colony-stimulating factor (rG-CSF). rIL-1 induced phagocytic activity, a typical marker of cell differentiation, in at most 30% of M1 cells at concentrations ranging from 10^-10 M to 10^-7 M . The differentiation-inducing activity of rIL-1 was similar to that of rG-CSF and less than that of D-factor. rTNF induced phagocytic activity in 14% of M1 cells only at a high concentration (10^-7 M ). rLT did not induce differentiation of the cells even at 10^-7 M . rTNF stimulated induction of differentiation of M1 cells by D-factor, rG-CSF or rIL-1 by two or three fold. The combination of any two of the cytokines D-factor, rG-CSF and rIL-1 induced differentiation of M1 cells more efficiently than any of these cytokines alone. Moreover, the combination of three cytokines rG-CSF, rIL-1 and rTNF, all of which are known to be produced by macrophages, was more effective than the combination of any two of these cytokines in induction of differentiation of M1 cells.     

Interference of Frizzled 1 (FZD1) reverses multidrug resistance in breast cancer cells through the Wnt/β-catenin pathway

Skeletal metastases are a frequent complication of prostate, breast and lung cancer, and the interactions of tumor cells with bone-forming osteoblasts and bone-resorbing osteoclasts have been suggested to play critical roles in disease progression. We have previously shown that treatment of primary murine osteoblasts with conditioned medium of the human osteolytic prostate cancer cell line PC-3 results in a rapid induction of chemokine expression, thereby providing further evidence for a molecular crosstalk between bone and tumor cells. The aim of our current study was to identify PC-3-derived molecules mediating this effect. Using Affymetrix Gene Chip hybridization followed by qRT-PCR we were able to confirm that the expression of chemokine-encoding genes is markedly induced in human primary osteoblasts following incubation with PC-3-conditioned medium. Since this induction was significantly affected upon alteration of p65-levels in PC-3 cells, we performed a second genome-wide expression analysis to identify p65-regulated cytokines, which were then tested for their ability to induce chemokine expression. Here we observed that interleukin-1β (IL-1B) did not only increase the expression of chemokines in osteoblasts, but also the phosphorylation of p65 and thereby its own expression. Since immunohistochemistry on bone biopsy sections from prostate cancer metastases demonstrated IL-1B expression in both, tumor cells and osteoblasts, our data suggest that IL-1B is one of the relevant cytokines involved in the skeletal complications of cancer metastases.     

Combined effects of differentiation-inducing factor and other cytokines on induction of differentiation of mouse myeloid leukemic cells

Mouse myeloid leukemic M1 cells are induced to differentiate into macrophage-like cells by differentiation-inducing factors (D-factors) and granulocyte colony-stimulating factor. We examined the effects of recombinant human tumor necrosis factor (rTNF), lymphotoxin (rLT) and interleukin 1 (rIL-1) on the induction of differentiation of M1 cells, compared with the effects of D-factor purified from the conditioned medium of mouse Ehrlich ascites tumor cells and recombinant human granulocyte colony-stimulating factor (rG-CSF). rIL-1 induced phagocytic activity, a typical marker of cell differentiation, in at most 30% of M1 cells at concentrations ranging from 10(-10) M to 10(-7) M. The differentiation-inducing activity of rIL-1 was similar to that of rG-CSF and less than that of D-factor. rTNF induced phagocytic activity in 14% of M1 cells only at a high concentration (10(-7) M). rLT did not induce differentiation of the cells even at 10(-7) M. rTNF stimulated induction of differentiation of M1 cells by D-factor, rG-CSF or rIL-1 by two or three fold. The combination of any two of the cytokines D-factor, rG-CSF and rIL-1 induced differentiation of M1 cells more efficiently than any of these cytokines alone. Moreover, the combination of three cytokines rG-CSF, rIL-1 and rTNF, all of which are known to be produced by macrophages, was more effective than the combination of any two of these cytokines in induction of differentiation of M1 cells.