Differentiation-dependent expression and mitogenic action of interleukin-6 in human colon carcinoma cells: relevance for tumour progression

Interleukin (IL)-6 mRNA expression in general is low in normal, adenomatous and cancerous human colon mucosa, except in rather undifferentiated lesions, in which IL-6 is overexpressed. Cytokeratin (CK) 8-positive carcinoma cells were identified by double immunostaining as almost exclusive source of IL-6. Likewise, in five (sub)clones of primary culture COGA-1 and COGA-13 human colon carcinoma cells, and in three established cell lines (Caco-2/AQ, Caco-2/15 and HT-29), efficient translation of IL-6 mRNA into protein was observed only in the least differentiated COGA-13 cells. Notably, IL-1beta (5 ng/ml) enhanced IL-6 release in COGA-13 cultures by three orders of magnitude. Although all cell clones studied expressed the IL-6 receptor (IL-6R), rhIL-6 (1-100 ng/ml) had a significant effect on cellular proliferation only in highly differentiated Caco-2 cells. Our data imply that IL-6, when released from rather undifferentiated carcinoma cells, particularly in response to IL-1beta, can advance tumour progression through paracrine growth stimulation of normal or highly differentiated colon tumour cells with intact STAT-3-mediated IL-6 signalling.     

Differential expression of S19 ribosomal protein, laminin-binding protein, and human lymphocyte antigen class I messenger RNAs associated with colon carcinoma progression and differentiation.

Three complementary DNA encoding S19 ribosomal protein (S19), laminin-binding protein (LBP), and HLA class I (HLA-I) genes were isolated from a colon tumor-enriched subtraction library. To evaluate this mRNA expression, surgically removed colon tumors as well as matched normal tissue and human colon carcinoma cell lines showing various differentiation states, anchorage dependence, and proliferation states were examined by Northern blot analysis. The mRNA level of S19 mRNA (0.6 kilobase) was higher in primary colon carcinoma tissue than in matched normal colon tissue in 5 of 6 cases. In 2 of 4 cases, the expression of LBP mRNA (1.2 kilobases) was higher in carcinoma than in normal tissue. In 12 human colon cell lines, the level of LBP mRNA was higher in poorly differentiated cells. On the other hand, HLA-I mRNA (1.7 kilobases) was higher in well-differentiated cells. Although the S19 mRNA was expressed in both well- and poorly differentiated cells, a concomitant increase with tumor progression was observed in two pairs of cell lines derived from the same patients (SW480 and SW620; COLO201 and COLO205). Anchorage dependence of butyrate-treated HT29 colon carcinoma cells was correlated with lower levels of S19 and LBP mRNAs and higher levels of HLA-I mRNA expression compared with untreated cells. While the expression of S19 and LBP mRNAs was not changed due to cell growth states, HLA-I mRNA levels were found to be low in proliferating HT29 cells but highly induced in contact-inhibited cells. In summary, therefore, high expression of S19 and LBP combined with low expression of HLA-I were well correlated with colon carcinoma cells of higher malignant potential.     

Serum factors attenuating the anti-tumor activity of 5-fluorouracil

In sera of cachectic patients bearing advanced cancers, the concentration of interleukin-1 (IL-1), interleukin-6 (IL-6), tumor necrotizing factor-alpha (TNF-alpha), and interferon-gamma (IFN-gamma) have been reported to elevate. In this study, we investigated whether those cytokines influenced in vitro anti-tumor effect of 5-fluorouracil (5-FU) on a human colon tumor cell line, HCT-15. Pretreatment of HCT-15 cells with IL-1 beta, IL-6 or TNF-alpha did not affect the anti-tumor effect of 5-FU at various concentrations. However, IFN-gamma attenuated the anti-tumor effect of 5-FU at the concentrations of 0.1-10 IU/ml. An experiment with tritium thymidine showed that 0.1 IU/ml of IFN-gamma did not suppress the growth of HCT-15 cells. As low as 0.1 IU/ml of IFN-gamma attenuated the anti-tumor effect of 5-FU in another experimental system where HCT-15 cells were exposed to 0.1 IU/ml of IFN-gamma before and during the treatment with 5-FU. This system mimicked the clinical condition around in situ cancer cells. Treatment of HCT-15 cells with 0.1-10 IU/ml of IFN-gamma did not change their DNA histogram pattern. An immunoblotting with the antibodies to thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD) in HCT-15 cells revealed that 0.1-10 IU/ml of IFN-gamma enhanced their TS and DPD expressions. Results of the immunoblotting gave some explanation to attenuation in the sensitivity of HCT-15 cells to 5-FU.     

Influence of low dose rIL-2 treatment on endogenous cytokine production, expression of surface IL-2R and the level of soluble IL-2R in patients with minimal residual disease

This study was designed to investigate the immunomodulatory effect of low-dose IL-2 therapy (100 microg/day for 3 weeks) on interferon (IFN), tumor necrosis factor (TNF) production in vivo and in vitro and on the expression of IL-2Ralpha/beta and soluble form of IL-2Ralpha. Patients enrolled in the study suffered from multiple myeloma (MM), Hodgkin's disease (HD) and non-Hodgkin's lymphoma (NHL) All of them were in remission after chemotherapy or radiotherapy. Our results indicated that IL-2 given subcutaneously at a low dose of 100 microg/day for 3 weeks induced IFN-gamma and TNF-alpha in plasma (measured 24 hrs after the last dose of IL-2) and affected the ability of blood leukocytes to produce cytokines. Production of IFN-gamma induced in vitro with PHA was enhanced, but TNF-alpha production induced by lipopolysaccharide (LPS) and virus (Newcastle Disease Virus) was depressed. The expression of both: surface IL-2R, especially beta subunit on total population of lymphocytes and NK cells, and soluble form of IL-2R, of chain were significantly enhanced after low-dose IL-2 therapy. Low dose IL-2 therapy was well tolerated by all patients, and side effects not exceeding II grade of toxicity according to WHO scale were observed. Five patients with MM relapsed 3-10 month after cessation of IL-2 therapy, but all patients with Hodgkin's and non-Hodgkin's lymphomas are still in remission (20 months of observation).     

Regulation of TNF-alpha, IL-1 and IL-6 synthesis in differentiating human monoblastoid leukemic U937 cells

The human monoblastoid tumor cell line U937 was induced to differentiate along the monocyte/macrophage lineage by treatment with 5 x 10(-9) M 12-O-tetradecanoyl phorbol-13-acetate (TPA). Between 2 h and 4 h following TPA-treatment U937 cells started to release significant amounts of TNF-alpha which remained detectable until 8-10 days. A significant IL-1 beta release was measured 24 h-48 h post stimulation and increased levels of IL-1 beta persisted until 20-22 days of culture. In contrast no release of either IL-1 alpha or IL-6 could be detected with 5 x 10(-9) M TPA during the whole time course of the experiments. The sequential induction of TNF-alpha and IL-1 beta appeared to be independently regulated since TNF-alpha release was not required for the onset of IL-1 beta production. Northern-blot analysis confirmed the sequential induction and the long term expression of TNF-alpha and IL-1 beta mRNAs. Western-blot analysis predominantly showed a high molecular weight IL-1 beta protein of about 35 kD. Further investigations on the regulation of cytokine production and release by TPA-differentiated U937 cells revealed that TNF-alpha and IL-1 beta synthesis was not influenced by exogenously added rhTNF-alpha or PGE2, whereas rh gamma-IFN specifically enhanced the IL-1 beta production. Thus, the regulation and intracellular processing of cytokines generated by differentiating U937 cells shows some differences when compared to mature monocytes/macrophages which may be related to the tumorigenic origin of U937 cells or to an incomplete differentiation.     

TNF-alpha modulates cytokine and cytokine receptors in C2C12 myotubes.

Incubation of C2C12 differentiated myotubes in the presence of 1000 units/ml (100 ng/ml) of recombinant murine tumor necrosis factor-alpha (TNF-alpha) for up 6 h caused an important increase in mRNA content of the cytokine (10-fold at 6 h). The levels of expression of the two cytokine receptors were not changed by the cytokine at short-time intervals, but 48 h of treatment resulted in a decreased expression of TNFR1 (33%). Interestingly, the presence of the cytokine resulted in significant increases in mRNA content for the catabolic pro-inflammatory cytokines IL-6 (17-fold) and IFN-gamma (8-fold). Similarly, TNF-alpha also caused a moderate increased expression of different anti-inflammatory cytokines such as IL-9 (104%), IL-10 (24%) and IL-15 (47%). The results suggest that other cytokines may be involved in mediating TNF-alpha action in skeletal muscle and that anti-inflammatory cytokines may be released as a counter-regulatory mechanism.     

Fibroblast-tumor interaction changes constitutive expression of IL-6 and IL-8 in human carcinoma-cells

To examine whether expression of interleukin-6 (IL-6) and interleukin-8 (IL-8) in human carcinoma cells can be influenced by host fibroblasts, we investigated the expression of IL-6 and IL-8 mRNA in carcinoma cells cocultured with fibroblasts. Four human cancer cell lines, NUGC3 and GaCa gastric carcinoma cells, RERF-LCOK lung carcinoma cells, and GBK-1 gallbladder carcinoma cells, which constitutively express large amounts of both IL-6 and IL-8, were cocultured with murine 3T3 fibroblasts, in which IL-6 and IL-8 were not detected, under the same conditions. By Northern blot analysis, the expression of IL-6 mRNA was significantly decreased in NUGC3, GBK-1, and RERF-LCOK cells but was increased in GaCa cells. The expression of IL-8 mRNA was significantly increased in GaCa and GBK-1 cells but decreased in RERF-LCOK cells. Using cell-free conditioned medium, only the NUGC3-3T3 culture supernatant showed little effect on IL-6 and IL-8 mRNA expression in the NUGC3 cells, thereby suggesting that changes in IL-6 and IL-8 mRNA expression observed in the coculture experiment depended mainly on 3T3-NUGC3 contact and not on soluble factors. Similar changes in IL-6 and IL-8 mRNA expression were noted when NUGC3 cells were cultured with paraformaldehyde-fixed 3T3 cells or the 3T3 membrane fraction, thereby supporting this notion. Northern blot analysis of transplanted NUGC3 tumors in nude mice showed a decrease in IL-6 mRNA expression and augmentation of IL-8 mRNA expression. Sera from the NUGC3-bearing mice showed only small differences in IL-6 and IL-8. The downregulation of IL-6 mRNA was reversed 20 hours after NUGC3 cancer cells were separated from the in vivo grown NUGC3 tumors and cultured in vitro. These results suggest that tumors producing IL-6 or IL-8 can be differently modulated by the host cell-mediated pathways, such as contact between fibroblast and tumor cells.     

Nitric oxide synthase is induced in tumor promoter-sensitive, but not tumor promoter-resistant, JB6 mouse epidermal cells cocultured with interferon-gamma-stimulated RAW 264.7 cells: the role of tumor necrosis factor-alpha

Expression of inducible nitric oxide synthase (iNOS) has been reported to be involved in certain organs of potential tumorigenesis, including the stomach and colon. The mechanisms for iNOS expression in epithelial cells, however, are not fully understood. In the present study, we investigated the role of macrophages in epithelial iNOS expression by coculturing a stimulated murine macrophage-like cell line, RAW 264.7, with either tumor promoter-sensitive (P+) or promoter-resistant (P-) JB6 murine epidermal cells. After monoculture, treatment of RAW 264.7 cells with IFN-gamma for 24 h generated a large amount of nitrite (NO2-), as reported previously, whereas no increase in NO2- concentration was observed in the IFN-gamma-treated P+ or P-subclones. Interestingly, when IFN-gamma-treated RAW 264.7 cells were cocultured with P+ but not P- cells, we observed a marked increase in NO2- concentration (30.8+/-3.6 microM), which significantly exceeded (P < 0.01) the sum of the concentrations (20.0+/-2.3 microM) added from each cell line monoculture. Western blotting analysis revealed that, after coculture, iNOS protein was up-regulated 55-fold more than the control in JB6 P+ but not in P- cells. IFN-gamma-treated RAW 264.7 cells secreted proinflammatory cytokines, including tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta. The addition of IFN-gamma-treated RAW 264.7 cell-conditioned media to P+ subclones led to a significant enhancement of NO2- formation that was diminished by the TNF-alpha-specific but not IL-1beta-specific antibody. When combined with IFN-gamma, the recombinant TNF-alpha (1-100 ng/ml) enhanced NO2- formation in JB6 P+ cells, whereas IL-1beta (1-100 ng/ml) did not. These results led us to conclude that IFN-gamma-treated RAW 264.7 cells release TNF-alpha to induce iNOS expression in promoter-sensitive JB6 cells. Thus, we propose the hypothesis that macrophages stimulate neoplastic cells with TNF-alpha via a paracrine loop to induce epithelial iNOS protein expression.     

ONO-4007, a synthetic lipid A analog,induces Th1-type immune response in tumor eradication and restores nitric oxide production by peritoneal macrophages

We have reported that ONO-4007, a novel synthetic lipid A derivative with low toxic activities, produced large amount of tumor necrosis factor (TNF)-alpha selectively in the tumor tissues and brought about complete cures in about 60% of rats bearing TNF-alpha sensitive KDH-8 cells, which also secreted a large amount of transforming growth factor (TGF)-beta. In our present study, to explore ONO-4007 induced Th1-type immune response, we investigated the mRNA expression of interferon (IFN)-gamma, Interleukin (IL)-1beta, IL-6, IL-10, IL-12 in KDH-8 bearing rats. We next examined the nitric oxide (NO) production. We found that IFN-gamma, IL-1beta, IL-6 and IL-12 mRNA expression of the tumor tissue were higher in the ONO-4007 treated rats than in phosphate buffer saline (PBS) treated rats. Western blotting also revealed that IL-12 protein production was increased. NO production from peritoneal macrophages were suppressed in tumor-bearing rats, but ONO-4007 restored it up to the normal level. These results suggest that ONO-4007 induces and restores Th1-type immune response through cytokine production cascade, followed by initial TNF-alpha production, eventually leading to tumor eradication.     

Radiation, 5-FU and OK-432: inhibitory effect of IL-10 and TGF-beta

We investigated the effect of 5-FU and radiation in OK-432-induced cytokine production. Stimulation of peripheral blood mononuclear cells (PBMCs) with OK-432 (1 micro/ml) for 24 h induced Th1-type cytokines (IFN-gamma, TNF-alpha, IL-12, IL-18) as well as IL-10 and TGF-beta. When the PBMCs were stimulated by 5-FU (5 microg/ml) or X-ray (2 Gy) simultaneously with OK-432, production of IL-10 and TGF-beta was significantly inhibited, while no significant change in Th1 cytokine production was observed. Although OK-432 also enhanced the expression of the genes encoding SOCS-1 and SOCS-3, which are negative regulators for cytokine signaling, this was reduced by 5-FU or X-irradiation. Induction of IL-10 and TGF-beta by OK-432 was significantly decreased by adding antisense ODN for SOCS-1 or that for SOCS-3. Radiation and 5-FU induce Th1-dominant state by inhibiting the OK-432-induced production of IL-10 and TGF-beta mediated by regulation of SOCS-1 and SOCS-3 expression, and are suggested to increase anti-cancer immunity.     

Inhibition of CD40 expression and CD40-mediated dendritic cell function by tumor-derived IL-10

As CD40 plays a key role in both antitumor immunity and DC maturation, we have studied the regulation of its expression during DC hematopoiesis (dendropoiesis) in vitro and in vivo in the tumor microenvironment. Using MC38 colon adenocarcinoma tumor models, we have demonstrated that DCs generated in vitro from bone marrow precursors obtained from tumor-bearers have significantly lower expression of CD40 molecules compared to DCs generated from tumor-free mice. Furthermore, CD40 expression on DCs isolated from the spleens of tumor-bearing mice was also significantly reduced, suggesting that tumor-derived factors inhibit CD40 expression on DCs during dendropoiesis both in vitro and in vivo. Interestingly, CD40 ligation on DCs generated from tumor-bearers did not result in inducible expression of IL-12 protein or IL-12 p40 mRNA. However, Staphylococcus aureus-induced IL-12 production by DCs was not altered in tumor-bearers, confirming that inhibition of IL-12 production by DCs generated in vitro from tumor-bearing mice was due to reduced expression of CD40 on DCs. We have also shown that MC38 tumor cells produce IL-10 and that exogenous IL-10 causes downregulation of CD40 expression on DCs. In addition, endogenous IL-10 produced by colon carcinoma cells inhibited CD40-dependent IL-12 production by DCs since tumor-induced inhibition of IL-12 production was abrogated by neutralizing anti-IL-10 antibody. Finally, systemic administration of FLT3L and/or CD40L reversed CD40 and IL-12 (p40) deficiency of DCs in tumor-bearing mice in vivo. These findings thus demonstrate that tumor-derived factors, including IL-10, inhibit CD40 expression on DCs and DC precursors and suppress their maturation and function.     

T helper 1 inhibitor TAK-603 inhibits IFN-gamma and IL-12 production with no effect on IL-18: an observation in sarcoidosis patients

BACKGROUND AND AIM: Sarcoidosis is immunologically characterized by highly enhanced Th1 responses in active stage. TAK-603 is a new quinoline derivative, which selectively suppresses Th1 cytokine production. Thus, the present study was designed to investigate whether TAK-603 ameliorates excess IFN-gamma production in active sarcoidosis. METHODS: We evaluated inhibitory effects of TAK-603 on IFN-gamma, IL-12 and IL-18 production in stimulated bronchoalveolar lavage (BAL) fluid cells and peripheral blood mononuclear cells (PBMCs) of sarcoidosis patients and healthy subjects. RESULTS: TAK-603 inhibited IFN-gamma production in stimulated BAL fluid cells and PBMCs of sarcoidosis patients and healthy subjects. TAK-603 inhibited IL-12 production in stimulated BAL fluid cells of sarcoidosis patients and healthy subjects. TAK-603 tended to inhibit IL-12 production in stimulated PBMCs of sarcoidosis patients and healthy subjects. However, TAK-603 did not affect IL-18 production in stimulated BAL fluid cells and PBMCs. TAK-603 did not affect TNF-alpha production in stimulated BAL fluid cells of sarcoidosis patients. TAK-603 inhibited IL-12 production in stimulated blood monocytes of healthy subjects, whereas IL-18 was increased by treatment with TAK-603. TAK-603 inhibited IFN-gamma production in PHA-stimulated blood T lymphocytes of healthy subjects with stimulation of IL-12 or a combination of IL-12 and IL-18. TAK-603 did not increase IL-10 or TGF-beta1 production in LPS-stimulated BAL fluid cells of sarcoidosis patients. CONCLUSIONS: TAK-603 inhibits IFN-gamma production in activated T lymphocytes and IL-12 production in activated monocytes/macrophages independently of increased production of IL-10 and TGF-beta1. TAK-603 may ameliorate excess IFN-gamma production and be a therapeutic tool for refractory sarcoidosis.     

Suppression of apoptosis in COLO 205 cells by the phorbol ester TPA may be mediated by the PKC isoenzyme alpha

Apoptosis induced by an antibody to CD95/APO-1/FAS in the colon carcinoma cells COLO 205 and HT-29 is suppressed by the phorbol ester TPA. Inhibition is much more effective in COLO 205 than in HT-29 cells. The TPA effect is abrogated by the protein kinase C (PKC)-specific inhibitor Go6983 indicating a role of PKC in this process. Bryostatin 1, unlike TPA, is unable to suppress apoptosis, but inhibits the TPA-induced suppression of apoptosis. TPA also inhibits indomethacin-induced apoptosis in COLO 205 cells. COLO 205 and HT-29 cells contain the PKC isoenzymes alpha, beta(II) delta, epsilon, eta, mu and zeta. Expression and activity of PKC alpha are at least 5 times higher in COLO 205 than in HT-29 cells. This correlates with the fact that inhibition of CD95-mediated apoptosis by TPA is more prominent in COLO 205 than in HT-29 cells. Thus, these findings suggest that PKC alpha has an important role in the TPA-induced inhibition of apoptosis.     

Overexpression of IL-1ra gene up-regulates interleukin-1beta converting enzyme (ICE) gene expression: possible mechanism underlying IL-1beta-resistance of cancer cells.

We investigated the interaction of endogenous interleukin (IL)-1beta, IL-1ra, and interleukin-1beta converting enzyme (ICE) in four human urological cancer cell lines, KU-19-19, KU-1, KU-2 and KU-19-20. Northern blot analysis showed that IL-1beta gene was expressed in all cell lines. On the other hand, in KU-19-19 and KU-19-20, the gene expressions of both IL-1ra and ICE were suppressed. MTT assay revealed that IL-1beta (10 ng ml(-1)) promoted cell growth in KU-19-19 and KU-19-20, while it inhibited in KU-1 and KU-2. An ICE inhibitor, Acetyl-Tyr-Val-Ala-Asp-CHO (YVAD-CHO) blocked IL-1beta-induced growth inhibition in KU-1 and KU-2. Overexpression of the secretory type IL-1ra with adenovirus vector (AxlL-1ra) enhanced ICE gene expression, while exogenous IL-1ra (100 ng ml(-1)) did not enhance it. Furthermore, AxIL-1ra treatment promoted endogenous IL-1beta secretion and induced significant growth inhibition and apoptotic cell death on KU-19-19 and KU-19-20. Treatment with either IL-1ra (100 ng ml(-1)), IL-1beta antibody (100 microg ml(-1)), or YVAD-CHO blocked AxlL-1ra-induced cell death in KU-19-19 and KU-19-20. These results suggest that IL-1beta-sensitivity depends on the level of ICE gene expression, which is regulated by the level of endogenous slL-1ra expression. This is a first report on the intracellular function of slL-1ra and these findings may provide key insights into the mechanism underlying the viability of cancer cells.