Role of IL-6 in neuroendocrine differentiation and chemosensitivity of non-small cell lung cancer

Kuo-Ting Chang,1,2 Chi-Ying F. Huang,1,2 Chun-Ming Tsai,3 Chao-Hua Chiu,3 and Ying-Yung Lok3

1Graduate Institute of Life Sciences, National Defense Medical Center, Taipei; 2Division of Molecular and Genomic Medicine, National Health Research Institutes, Miaoli; and 3Section of Thoracic Oncology, Chest Department, Taipei Veterans General Hospital, and Department of Medicine, School of Medicine, National Yang-Ming University, Taipei, Taiwan, Republic of China

Submitted 19 January 2005 ; accepted in final form 5 May 2005


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Interleukin-6 (IL-6) has been shown to regulate both growth and neuroendocrine (NE) differentiation in some types of human cancer cells, and erbB2 may be a critical component of IL-6 signaling. Non-small cell lung cancer (NSCLC) tumors that demonstrate NE properties have been suggested to have biological characteristics similar to small cell lung cancers with initial responsiveness to chemotherapy. We investigated whether IL-6 is implicated in the cell growth, NE differentiation, and chemosensitivity of NSCLC-NE cells. NSCLC-NE cells were treated with exogenous IL-6, and a subclone of an IL-6-transfected NSCLC cell line that constitutively expressed IL-6 receptor was also generated. These cells were assessed for cell proliferation by cell counting and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assays, chemosensitivity to cisplatin and etoposide by MTT assays, and NE differentiation by observing morphological changes and immunoblotting for neuron-specific enolase (NSE). The IL-6-treated cells and the IL-6-transfected cells showed enhanced cell proliferation and downregulated NSE expression, but little change in chemosensitivity. In the culture medium, IL-6-transfected cells grew as looser aggregates than the parental cells. IL-6 could not activate the erbB genes. In conclusion, IL-6 can induce cell proliferation and NE dedifferentiation but has little effect on chemosensitivity in IL-6 receptor-expressing NSCLC-NE cells. The status of NSE expression is unlikely to be a crucial factor for chemosensitivity in NSCLC cells.

interleukin-6


INTERLEUKIN-6 (IL-6), a pleiotropic cytokine with a wide range of biological activities in immune regulation, hematopoiesis, inflammation, and oncogenesis, is produced by various types of human normal and transformed tumor cells and is involved in regulation of the immune response, acute phase reaction, and cell proliferation (17, 27, 30).

Consistent with this prominent role in cell proliferation, IL-6 has been detected in primary squamous cell carcinomas, adenocarcinomas, and sarcomas, as well as in tumor cell lines (3, 9, 13, 18, 25). It has been suggested that IL-6 may play a significant role in the pathophysiology of cancer, and it is a potential mediator in the development of cancer cachexia (13, 39). Several clinical reports have highlighted the prognostic importance of IL-6 in a variety of human solid tumors, including prostate, breast, colon, renal, bladder, ovary, and lung cancers (3, 10, 22, 44), suggesting that circulating IL-6 level is correlated with the extent of disease and disease recurrence and is associated with worse survival. In some of these tumors, higher levels of IL-6 were found in patients who were unresponsive to hormone therapy, radiotherapy, or chemotherapy (7, 10, 29). However, whether IL-6 is directly involved in the development of treatment resistance is as yet unclear.

Neuroendocrine (NE) differentiation has been found in a subgroup of a variety of carcinomas, including prostate, breast, gastric, and colorectal cancers, and non-small cell lung cancer (NSCLC) (2, 5, 8, 23, 45). In some of these tumors, NE differentiation may suggest an adverse influence on patient survival (2, 5).

Several recent studies with prostate cancer cells have demonstrated that IL-6 can promote NE differentiation through different intracellular signal transduction pathways, including the phosphatidylinositol 3-kinase/epithelial and endothelial tyrosine kinase (PI 3-kinase/Etk) pathway (33), signal transducers and activators of transcription 3 (STAT3) pathway (38), and mitogen-activated protein kinase (MAPK) pathway (21, 34). The protein of erbB2 gene, one member of the erbB gene family, is shown to be a critical component of IL-6 signaling through the MAPK pathway (34). Data suggest that IL-6 acts as an autocrine and paracrine growth factor in hormone refractory human prostate cancer cell lines, and, in contrast, as a paracrine growth inhibitor in hormone-dependent cell lines (7).

Clinically and biologically, lung cancer is generally divided into small cell lung cancer (SCLC) and NSCLC. Increased serum level of IL-6 was found in 39% of lung cancer patients, whereas IL-6 was not detected in the serum of healthy people as well as patients with benign lung diseases (44). Bihl et al. (6) have demonstrated that IL-6 may be required in the control of cell proliferation in a subset of NSCLC cell lines, and there are two subgroups of NSCLC, IL-6 dependent and independent. Paradoxically, anti-tumor effects of IL-6 have been demonstrated in vitro and in vivo in patients with NSCLC and breast cancer (42). The real role of IL-6 in lung cancer still needs further study.

Whereas SCLC is responsive to chemotherapy, NSCLC is only moderately responsive to such therapy at the time of diagnosis. SCLC has distinct NE properties and is recognized as one of the NE neoplasms. In contrast, only ~10–20% of NSCLC tumors have NE properties (NSCLC-NE) (26). These express multiple NE cell markers, including neuron-specific enolase (NSE), L-dopa decarboxylase, chromogranin A, synaptophysin, and cytoplasmic dense core granules. Among these markers, NSE, a glycolytic enzyme enolase, has been detected in tissue extracts derived from either brain or various NE tissues. NSE is a specific biochemical marker for both neurons and peptide-secreting NE cells as well as a useful index of neuronal differentiation. NSE is the only marker that can be utilized to identify all the NE cells in the lung (28), is the NE marker most frequently detected in NSCLC (38%), and, most importantly, is probably a prognostic marker of clinical relevance (16).

Previously, Gazdar et al. (12) have shown that NSCLC-NE cell lines are as chemosensitive as SCLC cell lines, whereas NSCLC lines lacking NE markers are relatively chemoresistant. They suggested that NE cell differentiation may delineate a relatively chemoresponsive subset of NSCLC and could be used as a predictor of better prognosis. However, the association of the presence of NE markers with an increased likelihood of response to chemotherapy and a better prognosis in patients with NSCLC is less consistent. Some have found that NE differentiation predicts chemoresponsiveness (8, 15), but others have failed to demonstrate any correlation with chemosensitivity (11, 14, 16, 36). Similar discrepancies have also been observed in terms of patient survival (1, 5, 8, 11, 14, 19, 24, 32, 36, 37). Moreover, the evidence of direct correlation of NE differentiation with tumor chemosensitivity at the molecular level in NSCLC is still lacking.

Data demonstrated survival was poorer in patients with NSCLC whose tumor expressed a high level of erbB2 than in those with tumors expressing lower levels (20, 40), suggesting that erbB2 is a prognostic factor for patients with NSCLC. Data also suggested that erbB2 may be a predictor of response to chemotherapy (4, 41).

Thus far, the association between IL-6 and NE differentiation in NSCLC has not been identified. We are interested in the relationships between erbB2 expression, NE differentiation, and chemosensitivity in NSCLC cells. We therefore conducted this study to investigate the effects of IL-6 on the expression of erbB genes and biological features, including cell growth, NE differentiation, and chemosensitivity, in NSCLC-NE cells. We demonstrated that IL-6 could stimulate cell growth and downregulate the expression of NSE, whereas exhibiting very little change in chemosensitivity in IL-6 receptor expressing NSCLC-NE cells. The biological effects of IL-6 are not associated with the erbB signaling.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
NSCLC-NE cells and cell culture. Three lung adenocarcinoma cell lines, National Cancer Institute (NCI)-H1155, NCI-H1355, and NCI-H820, were selected for this study. These three cell lines exhibited NE properties (26) and were established and characterized at the NCI-Navy Medical Oncology Branch (Division of Cancer Treatment, NCI, Bethesda, MD) from tumor specimens obtained from previously untreated patients. All these cell lines were cultured and maintained in serum-free ACL-4 medium (Life Technologies, Grand Island, NY) unless otherwise indicated. Cells were cultured in a humidified atmosphere of 5% CO2 at 37°C. Cultured cells were observed, and cells in log-phase growth were photographed in phase contrast with a Nikon ECLIPSE TE-300 inverted microscope.

Establishment of IL-6 expression-stable transfectants. The recombinant human IL-6 (rhIL-6) constitutive expression construct pCMV-IL-6, which carries 0.64-kb full-length IL-6 cDNA (43), was generously provided by Dr. Chuang at the Division of Cancer Research, National Health Research Institute. Transfections were performed using TransFast transfection reagent (Promega, Madison, WI). Stable transfectants were selected and grown in media containing 200 µg/ml of the antibiotic Geneticin (G418; Roche Molecular Biochemicals, Indianapolis, IN) for 3–4 wk. G418-resistant clones were picked and expanded. Expression of soluble rhIL-6 was measured on the culture supernatants from transfectants, control vector, and parental cell populations by ELISA according to the manufacturer's instructions (DIACLONE Research, Besançon, France). Briefly, a flat-bottomed immunoassay plate was precoated with mouse anti-human IL-6 monoclonal antibody and then incubated with nondiluted supernatants or standard human IL-6. After incubating the plate with a biotinylated anti-IL-6 conjugate for 2 h at room temperature, we washed it, and a substrate solution was added. The reaction was stopped with 1 M sulfuric acid, and the resulting color was read at 450 nm with a microplate reader. Data of eight independently performed assays were obtained; each assay was done in duplicate.

Cell growth. Cell growth in the presence and absence of IL-6 was determined using the tetrazolium dye colorimetric {3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT)} assay for cells in log phase. The assay is well suited for precise analysis of cell growth and growth factor effects because the cell number is directly proportional to MTT reduction under defined conditions, and a doubling of optical density indicates a doubling of cell number (31). Cells were harvested and washed three times in PBS and then were plated at the appropriate seeding density in 180 µl of medium into 96-well microtiter plates. After ~16 h of incubation at 37°C in a 5% CO2 atmosphere, 20 µl of the test solutions, which consisted of culture medium plus IL-6 at a final concentration of 0, 5, or 50 ng/ml, were added to each well. Cell density was determined by preliminary experiments, and assay conditions were established so that growth of the tumor cells remained constant through the analysis period.

There were five replicate plates prepared for each cell line per test. After addition of IL-6, the microtiter plates were incubated. For each cell line, the MTT assay was performed immediately after adding IL-6 (day 0) and one plate per day for four more succeeding days as described previously (41). Each data point, representing the mean value from eight culture wells, was used for the construction of growth curves. Each experiment was done three times.

For NCI-H820, cell growth was also evaluated by direct counting of viable cells. Cells were seeded in a T-25 culture flask at a density of 1 x 105 cells per 4 ml of ACL-4 culture medium with and without IL-6 (50 ng/ml). Culture medium with an assigned concentration of rhIL-6 was changed every 2 days, and cells were harvested after incubation for 0, 2, 4, and 6 days. Cells were disaggregated with a trypsin-EDTA solution, neutralized, and resuspended in culture medium. Viable cell counts were determined by trypan blue exclusion using a hemocytometer. The mean of the hemocytometer counts of cells from triplicate flasks was determined every other day for 6 days for the construction of growth curves and to determine doubling times. The reported results were the means of three independently performed experiments.

Paired t-tests were performed to evaluate whether significant differences in cell proliferation occurred between the tested conditions. All tests were two sided, and significance was assumed if P < 0.05.

Immunoblottings of rhIL-6-treated cells. After being plated at the appropriate seeding density for 24 h, cells were incubated with rhIL-6 (0, 50, and 100 ng/ml) for 6 days. Culture medium with the assigned concentration of rhIL-6 was changed every 2 days, and cells were harvested after incubation for 0, 2, 4, and 6 days for evaluating NSE expression. To ascertain erbB expression, cells were cultured in RPMI 1640 for 16 h, incubated without or with rhIL-6 (50, 100, and 200 ng/ml) or neu differentiation factor (NDF; 15 nM, as the positive control) for 15 min, and then harvested. The harvested cells were lysed in RIPA buffer [20 mM Tris (pH 8.0), 150 mM NaCl, 10% glycerol, 1% Nonidet P-40, and 0.42% NaF] containing inhibitors (1 mM phenylmethylsulfonyl fluoride, 1 mM Na3VO4, 5 µg/ml aprotinin, and 5 µg/ml leupeptin). For immunoblotting, cell lysates containing 50 µg of protein were resolved on an SDS-polyacrylamide gel. Proteins were transferred to polyvinylidene fluoride membranes (NEN Life Science, Boston, MA) using an electroblotting apparatus (Bio-Rad, Hercules, CA). Filters were blocked in blocking solution (BSA/PBS-T; PBS, 0.1% Tween 20, 3% BSA) for 1 h at room temperature. Blots were incubated with primary antibody (anti-erbB1, anti-erbB2, anti-erbB3, anti-NSE, or anti-{beta}-actin) for 1 h at room temperature and washed three times with PBS-T. The blots were then incubated with horseradish peroxidase-conjugated secondary antibody for 1 h, washed three times with PBS-T, and then developed using an enhanced chemiluminescence technique (LumiGLO; KPL, Gaithersburg, MD) and exposed to BioMax Film (Kodak, Rochester, NY). The same blot was stripped and reprobed for {beta}-actin. The experiments were performed in triplicate.

RT-PCR of IL-6 and IL-6 receptor. Total RNA was isolated using Tri-Reagent (Sigma Chemical, St. Louis, MO) following the manufacturer's instructions. Five micrograms of total RNA were used for each RT reaction using the Superscript II reagent (Invitrogen, Carlsbad, CA). PCRs were performed using Taq DNA polymerase (BioTools, Edmonton, AB, Canada) with 30 amplifying cycles. The primer sequences were as follows: IL-6, forward: 5'-CTG GAT TCA ATG AGG AGA CTT GC-3'; reverse: 5'-GGA CAG GTT TCT GAC CAG AAG-3'; IL-6R, forward: 5'-AAG GAC CTC CAG CAT CAC TGT GTC A-3'; reverse: 5'-CCT TCA GAG CCC GCA GCT TCC ACG T-3'; NSE, forward: 5'-TGA CAG TGA CCA ACC CAA AA-3', reverse: 5'-CAC CAG GTC AGC AAT GAA TG-3'; and the housekeeping gene GAPDH, forward: 5'-ATC AAG AAG GTG GTG AAG CAG G-3'; reverse: 5'-GCA ACT GTG AGG AGG GGA GAT T-3', which was used as the mRNA internal control. The experiments were performed in triplicate.

Drug testing with or without IL-6. In vitro drug sensitivity testing was performed using the MTT assay. Cells were washed three times with PBS, disaggregated, resuspended in the ACL-4 medium, and seeded in 160 µl at the appropriate seeding density into 96-well microtiter plates. After ~16 h of incubation, 20 µl of rhIL-6 (a final concentration of 0, 5, or 50 ng/ml) plus 20 µl of cisplatin or etoposide (final concentration of 0, 0.031, 0.1, 0.31, 1, 3.1, or 10 µM) were added to the control and test wells. After exposure to the drug with or without rhIL-6 for 4 days, the remaining steps of the assay were performed as previously described (41). Each assay was done in eight replicate wells, and the experiment was performed in triplicate on each of the cell lines. The results were reported as IC50 values that were the means of three independently performed assays and were defined as the drug combination required to inhibit cell growth by 50%.

Reagents. rhIL-6 and NDF (her-reguline) were purchased from R&D Systems (Minneapolis, MN). Anti-NSE antibody was obtained from Neomarkers (Fremont, CA). Anti-erbB1 was from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-erbB2 and anti-{beta}-actin were obtained from Calbiochem (La Jolla, CA). Anti-erbB3 and anti-phosphotyrosine (4G10) were from Upstate Biotechnology (Lake Placid, NY). Cisplatin and etoposide were provided by Farmitalia Carlo Erba (Milan, Italy) and Bristol-Myers (Troisdorf, Germany), respectively. MTT was purchased from Sigma.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Expression of NSE in NSCLC-NE cells. Although the NE properties of the three tested cell lines had been well characterized (26), the expression of NSE, the major NE marker, was confirmed by immunoblotting with specific antibody. As shown in Fig. 1A, the NCI-H1155 cell line expressed the highest and the NCI-H1355 cell line expressed the lowest levels of NSE.



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Fig. 1. Expression of neuron-specific enolase (NSE; A) was determined by immunoblotting, and interleukin-6 (IL-6) and IL-6 receptor (IL-6R; B) expression was determined by RT-PCR analyses in the 3 non-small cell lung cancer-neuroendocrine (NSCLC-NE) cell lines. The experiments were performed in triplicate, and 1 example of the experiments is shown. Numbers in parentheses are the levels of NSE (A) and IL-6R (B), expressed in arbitrary units relative to the value of control cells, to which a value of 1 has been assigned. NSE and IL-6R levels were also normalized by densitometry of the {beta}-actin and GAPDH signals, respectively.

 
Expression of IL-6 and IL-6 receptor and effects of rhIL-6 on cell proliferation. We determined the expression of IL-6 and IL-6 receptor of the tested cell lines by RT-PCR. The results are shown in Fig. 1B. The NCI-H1155 cell line expressed almost no detectable IL-6 and IL-6 receptor (lane 2). The NCI-H1355 cell line (lane 3) expressed a higher level of IL-6 but a lower level of IL-6 receptor than the cell line NCI-H820 (lane 4). To observe the influence of IL-6 on NSCLC-NE cell proliferation, cells were treated with exogenous rhIL-6 at either 5 or 50 ng/ml for 4 or 6 days. The growth curves with and without rhIL-6 treatment determined by MTT assays are shown in Fig. 2. Exogenous rhIL-6 had very little effect on cell proliferation of NCI-H1155 (Fig. 2A, left), which expressed undetectable IL-6 and IL-6 receptor. Exogenous rhIL-6 produced a minimal but statistically significant growth stimulation (P < 0.05) of the cell line NCI-H1355 (Fig. 2A, middle), which expressed a high level of IL-6 but a relatively lower level IL-6 receptor (Fig. 1B). In contrast, rhIL-6 markedly enhanced NCI-H820 cell growth (P < 0.001) in a dose-dependent manner (Fig. 2A, right). The NCI-H820 cell line expressed a minimal amount of IL-6 but a relatively higher level of IL-6 receptor (Fig. 1B). These findings suggested that the growth effect of rhIL-6 was correlated with the expression level of IL-6 receptor. Growth curves that were determined by direct cell counting confirmed the growth stimulation of rhIL-6 on the NCI-H820 cells (Fig. 2B). Compared with the growth of control cells with no IL-6 treatment, cell growth of H820 was increased to ~150% after rhIL-6 (50 ng/ml) treatment for 4 days (by MTT assay, Fig. 2A, right) or 6 days (by direct counting, Fig. 2B). All the P values were <0.001.



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Fig. 2. Growth-stimulating effects of IL-6 are shown. Mean growth curves of the test cell lines are shown as the means (data points) ± SE (bars) from 3 independent experiments. A: mean growth curves of the NCI-H1155 (left), NCI-H1355 (middle), and NCI-H820 (right) cell lines in the absence or presence of recombinant human (rh)IL-6. Cells were seeded in 96-well cell culture plates with ACL-4 medium supplemented with a final concentration of either 5 or 50 ng/ml of rhIL-6. 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assays were performed immediately after adding rhIL-6 and then every 24 h as described in MATERIALS AND METHODS. B: mean growth curves of the NCI-H820 cells cultured in ACL-4 with or without 50 ng/ml of rhIL-6. Viable cells were counted by the trypan blue dye exclusion test every other day. **P < 0.001 compared with the control cells with no rhIL-6 treatment. Exogenous rhIL-6 markedly induced cell proliferation in the 2 IL-6R expressors H820 and H1355, but not in the receptor lacking H1155 (A, statistical significance: *P < 0.05 and **P < 0.001 compared with the control cells with no rhIL-6 treatment). H820 expressed a higher level of IL-6R and exhibited more rhIL-6-induced growth stimulation than H1355. In H820, rhIL-6 stimulated cell growth in a dose-dependent manner (A, right, 50 ng/ml of rhIL-6 induced more cell growth than 5 ng/ml, P = 0.017).

 
Downregulation of NSE expression by IL-6 in NSCLC-NE cells. It has been reported that IL-6 upregulates the expression of NSE in the human prostate cancer cell line LNCaP (23, 40, 45). To evaluate the long-term effect of IL-6 on NE marker expression, we treated the NCI-H820 cell line (a high IL-6 receptor and NSE expressor) with 50 and 100 ng/ml of rhIL-6 for 6 days. The results showed that NSE expression was downregulated by rhIL-6 after treatment for longer than 2 days in the NCI-H820 cell line, and there were very similar effects at concentrations of 50 and 100 ng/ml (Fig. 3, top). In the NCI-H1355 cells, treatment with rhIL-6 at 50 ng/ml for 4 and 6 days also downregulated NSE (Fig. 3, bottom left), but to a lesser degree when compared with its effect on the H820 cells. In contrast, rhIL-6 showed no detectable effects on NSE expression in the IL-6 receptor lacking NCI-H1155 cells (Fig. 3, bottom right). These findings demonstrated a trend suggesting that the effect of rhIL-6 on NSE expression was dependent on the expression status of IL-6 receptor in this panel of NSCLC-NE cell lines.



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Fig. 3. Effects of exogenous rhIL-6 on NSE expression are shown. NSE expression was downregulated obviously after rhIL-6 treatment for 4 and 6 days in H820, and there was little difference between the effects of 50 and 100 ng/ml (top). In the NCI-H1355 cells, rhIL-6 (50 ng/ml) treatment for 4 and 6 days also downregulated NSE, but to a lesser degree when compared with the effects on H820 (bottom left). In contrast, rhIL-6 showed no detectable effects on NSE expression in the IL-6R lacking H1155 (bottom right). Numbers in parentheses are the levels of NSE, expressed in arbitrary units relative to the value of control cells, to which a value of 1 has been assigned. NSE levels were also normalized by densitometry of the {beta}-actin signal.

 
Crucial role of IL-6 in cell proliferation and NSE regulation in H820. We have demonstrated that IL-6 exhibited profound effects on cell proliferation and NSE expression in the NCI-H820 cell line. To corroborate these findings and elucidate the biological role of IL-6 in the H820 cells, we transfected a rhIL-6 constitutive expression construct into this cell line and generated a stably rhIL-6-overexpressing subclone, which was designated "H820.ILSC." As shown in Fig. 1B, the endogenous IL-6 was very low in the parental H820 cell line; therefore, the transfected form would be the major source of IL-6 produced by the cells in these experiments.

To examine whether IL-6 was increased in the H820.ILSC, we detected the level of IL-6 mRNA and the amount of secret protein using RT-PCR and ELISA, respectively. As shown in Fig. 4A, IL-6 expression at the mRNA level and production of protein product in the culture medium of the H820.ILSC were much higher than those of the parental and the vector control lines (30- and 250-fold, respectively). Expression of NSE was also examined by RT-PCR and Western blot analysis and found to be markedly downregulated at the mRNA and the protein levels in the H820.ILSC cells (Fig. 4B). The findings were consistent with the results found in the rhIL-6-treated H820 cells (Fig. 3, top).



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Fig. 4. Biological characteristics of the rhIL-6 stable transfectant clone H820.ILSC. A: level of soluble IL-6 was expressed as the means (columns) ± SE (bars) of 8 independently performed assays; each assay was done in duplicate experiments. Compared with the parental H820 cells, the vector control H820.SC cell line did not show elevated IL-6 expression and production, whereas the H820.ILSC cells demonstrated robustly increased levels of IL-6 expression (30-fold of control by RT-PCR, top) and production (250-fold of control by ELISA, bottom). B: compared with the parental H820 cells, the vector control H820.SC cells showed similar level of NSE expression, whereas the H820.ILSC cells showed downregulated NSE expression at the mRNA (43% of control, by RT-PCR, top) and the protein (57% of control, by immunoblotting, bottom) levels. C: mean growth curves constructed by direct cell counting are shown as the means (data points) ± SE (bars) from 3 independent experiments. Compared with the parental H820 cells, the vector control H820.SC cell line showed no detectable change, whereas the H820.ILSC cells demonstrated marked enhancement (2.7-fold, P < 0.001) in cell proliferation. Numbers in parentheses are the levels of IL-6 (A) and NSE (B).

 
To determine whether IL-6 produced by the H820.ILSC cells might affect growth in an autocrine manner, we examined the cell proliferation of the H820.ILSC cells. Notably, the H820.ILSC cells exhibited a significantly increased growth rate (2.7-fold, P < 0.001) compared with the parental and the vector control lines (Fig. 4C). Together, our findings identified IL-6 as a crucial factor capable of triggering cell proliferation and modulating NE differentiation in NSCLC-NE cells.

Morphological change in the IL-6-transfected NCI-H820 cells. To further assess the effect of IL-6 on cell growth, we continuously observed and recorded the morphological difference between IL-6 constitutively expressing stable clone H820.ILSC and the parental cell line. In contrast with compact globelets of the parental and H820.SC vector control cells, the aggregates of the H820.ILSC cells grew loosely and unglobally after incubation for longer than 2 wk (Fig. 5).



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Fig. 5. Shown is the in vitro growth appearance of the NCI-H820 parental cells (left), the vector control H820.SC (middle), and the rhIL-6-transfected cells H820.ILSC (right) under microscopy. Compared with H820 and H820.SC, which grew in tight spherical aggregates, the aggregates of the H820.ILSC cells grew loosely and unglobally after incubation for 2 wk. Bar = 20 µm.

 
No activation of erbB signaling in IL-6-treated H820 cells. A previous report demonstrated that IL-6 might induce NE differentiation through erbB2 signaling in a prostate cancer NE cell line LNCaP (8). Presently, Western blots revealed detectable levels of erbB1, erbB2, and erbB3 in the NCI-H820 cell line (Fig. 6). To study the possibility of involvement of the erbB2 signaling in the IL-6-induced biological alterations, we examined the expression of erbB genes in the H820 cells with or without rhIL-6 treatment by Western blotting. We found that NDF but not rhIL-6 (from 50 to 200 ng/ml) could induce tyrosine phosphorylation at ~185 kDa, indicating that IL-6 was not able to activate erbB genes (Fig. 6). Although the antibody used for this blot was nonspecific and could detect all of the activated tyrosine kinases, the IL-6-treated cells showed no evidence of tyrosine kinase activation at ~185 kDa compared with the NDF-treated cells, indicating that neither erbB2 nor erbB3 was activated by IL-6.



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Fig. 6. A: expression of the erbB1, erbB2, and erbB3 genes in the H820 cells by immunoblotting. The same blot was immunoblotted after stripping of the previous test signals. Immunoblot analysis of erbB2 was performed first, followed by erbB3 and erbB1. Immunoblotting for {beta}-actin was performed last. B: cells were incubated without or with rhIL-6 or neu differentiation factor (NDF; 15 nM, used as control) for 15 min after being cultured in RPMI 1640 for 16 h, and phosphotyrosine-containing proteins were detected by immunoblotting. NDF (right lane) but not rhIL-6 (at 50, 100, and 200 ng/ml) could induce tyrosine phosphorylation at ~185 kDa, indicating that IL-6 is not able to activate erbB genes. A and B: 1 example of each of 3 independently performed experiments is shown.

 
Little effect on chemosensitivity by IL-6 in H820. Thus far, whether NE differentiation confers drug sensitivity to NSCLC-NE cells has not been clarified. In this study, we took advantage of the finding that IL-6 downregulated the expression of NSE to examine the influence of NE differentiation on drug sensitivity of the tested cells. The chemosensitivities (expressed as the IC50 value) to two commonly used anti-cancer agents (cisplatin and etoposide) were determined in the H820, H820.SC, and H820.ILSC cell lines as well as in the H820 cell line in the absence or presence of 5 or 50 ng/ml of exogenous rhIL-6. As shown by the dose-effect curves and the mean values of IC50 (Fig. 7), the response of the test cell lines was very similar to the same anti-cancer agent. These results indicated that NSE expression modulated by adding exogenous IL-6 or by endogenous IL-6 production was not related to chemosensitivity in NSCLC-NE cells.



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Fig. 7. Dose-effect curves of 2 anti-cancer agents, cisplatin (top) and etoposide (bottom), of the IL-6 (0, 5, 50 ng/ml)-treated H820 cells (left) and the rhIL-6-transfected H820.ILSC cells (right). In vitro chemosensitivities were determined by the tetrazolium assays, with drug exposure for 96 h. The percentage of control absorbance was considered to be the surviving fraction of cells. The mean surviving fractions of 3 separate experiments are used to construct the curves; bars, SE. The results, reported as IC50 values, are the means (±SE) of 3 independently performed assays and are defined as the drug concentrations required to inhibit cell growth by 50%. H820.SC: vector control.

 

    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
IL-6 is produced by some types of cancer cells and by normal stromal cells, such as fibroblasts and endothelial cells. By acting as an autocrine and paracrine growth factor, IL-6 is able to promote tumor cell proliferation through upregulation of anti-apoptotic and angiogenic proteins in tumor cells (17). Thus IL-6 is one of the mediators responsible for communications and interactions between tumor cells and the surrounding environment.

There are two types of NSCLC cells. IL-6-independent NSCLC cells, such as NCI-H1155, express neither IL-6 receptor nor IL-6 and have no response to exogenous rhIL-6. IL-6-dependent NSCLC cells express IL-6 receptor and may either highly express IL-6, such as NCI-H1355, or minimally or not express IL-6, such as H820. IL-6 acts as an autocrine growth stimulator in the H1355 cells while being a paracrine growth stimulator in the H820 cells. Exogenous IL-6 stimulated cell growth and suppressed NSE expression to a minor degree in H1355 cells but markedly enhanced cell proliferation and downregulated NSE expression in H820 cells.

Transfection of the IL-6 gene into the H820 cell line increased IL-6 production, which was accompanied by enhanced cell proliferation and decreased NSE expression. More interestingly, compared with the parental H820 cells that grew as floating aggregates of tightly packed cells, cells of the IL-6-transfected subclone H820.ILSC grew as loose floating aggregates in the culture medium (Fig. 5). The differences in the biological characteristics of in vitro culture morphology, rate of cell growth, and NE differentiation between the H820 parental cell line and its IL-6-transfected subclone were very similar to those differences observed between the two major in vitro subtypes of SCLC, namely, classic and variant types. Classic SCLC cell lines are characterized by a long doubling time, typical SCLC morphology (floating tight spherical aggregates), and high expression of NE markers. By contrast, variant lines replicate faster, have altered morphology (floating loose cell aggregates), and display the selective loss or lower levels of NE properties. It is well documented that classic SCLC is a moderately differentiated cell type. In contrast, variant SCLC is defined as a subset of morphologically poorly differentiated cells (26). Together, our results in the present study provide solid evidence that IL-6 stimulates cell growth, induces morphological alteration, and leads to the process of NE dedifferentiation in IL-6-expressing NSCLC-NE cells. Our results also suggest that the effects of IL-6 on NSE expression and cell proliferation are correlated with the expression level of IL-6 receptor. Accordingly, IL-6 may induce cell dedifferentiation in the H820 cells. The relationship between IL-6 and cell-cell adhesion molecules in NSCLC cells is currently under investigation.

The possible mechanisms by which IL-6 can regulate NE differentiation could involve the MAPK, PI 3-kinase/Etk, or STAT signal transduction pathway. Recently, we clearly demonstrated that IL-6 is involved in modulation of NSE expression through STAT3 signaling in IL-6 receptor expressing NSCLC cells (9).

Previously, several reports suggested that NE properties may have clinical correlates. SCLC is highly responsive to chemotherapy, whereas NSCLC is only moderately responsive to such therapy. SCLC has distinct NE properties. By contrast, NE properties exist in only 10–20% of NSCLCs, including 5 of 35 (14%) established cell lines (26). In four of the five NSCLC-NE cell lines, the in vitro drug sensitivity was similar to the pattern seen in SCLC (12).

Although a number of studies have suggested that NSCLC patients whose tumors have NE features may be more responsive to chemotherapy, and possibly have better survival, the clinical significance of NE differentiation in NSCLC still remains controversial. A study of the prognostic impact of NSE and chromogranin A in lung adenocarcinoma showed that patients with NSE-positive tumors responded significantly better to chemotherapy than patients with NSE-negative tumors (37). The Eastern Cooperative Oncology Group reported a retrospective study looking at NE markers in advanced lung cancer in a large patient population and found that NSE correlates with response to chemotherapy and that the expression of Leu7 correlates with longer survival (35). However, the Cancer and Leukemia Group B (CALGB) conducted a prospective study to determine whether there was a correlation between NE expression and response or survival in stage III NSCLC. The results did not support the hypothesis that NE-positive tumor cells are preferentially killed by chemotherapy in patients with NSCLC (16). Our finding that IL-6-induced downregulation of NSE expression does not influence chemosensitivity of NSCLC-NE cells is in accordance with the results of the CALGB study, suggesting that the level of NSE (the most commonly studied NE marker in the reports) expression is not directly correlated with responsiveness to chemotherapeutic agents in NSCLC.

De Vita et al. (10) have evaluated serum levels of IL-6 in a group of advanced NSCLC patients and found that patients who respond to cisplatin-based chemotherapy have lower serum IL-6 levels compared with unresponsive patients. Their data suggested that NSCLC patients with high levels of IL-6 have a worse clinical outcome and may manifest resistance to cisplatin chemotherapy. In the present study, however, we failed to demonstrate that exogenous or endogenous IL-6 could influence cisplatin or etoposide sensitivity of the tested NSCLC cells at the cellular level.

We previously reported that overexpression of erbB2 in NSCLC cells correlated with intrinsic resistance to chemotherapy (41). Studies suggested that differentiation of prostate NE cells might be associated with activation of the erbB protein (34), and erbB2 might be a critical component of IL-6 signaling through the MAPK pathway. In the present study, however, our data demonstrated that erbB genes are unlikely to be involved in the signaling of the IL-6-induced biological alterations in NSCLC-NE cells.

In conclusion, IL-6 stimulates cell proliferation and induces NE dedifferentiation in IL-6 receptor expressing NSCLC-NE cells. Our finding that no obvious changes in the chemosensitivities to etoposide and cisplatin occur in vitro in NSCLC-NE cells after cellular modulation of NSE expression by IL-6 suggests that IL-6 production or expression of NE features is unlikely to be a crucial determinant for chemosensitivity in NSCLC cells.


    GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 
This work was supported in part by grants from the National Health Research Institutes and National Science Council (National Research Program for Genomic Medicine, NHRI93A1-NSCMM11–5 to C.-Y. F. Huang) and Taipei Veterans General Hospital (2001#314, 2002#321, 2003#290 to C.-M. Tsai).


    ACKNOWLEDGMENTS
 
We thank Dr. Hsing-Jien Kung for valuable suggestions.


    FOOTNOTES
 

Address for reprint requests and other correspondence: C.-M. Tsai, Section of Thoracic Oncology, Chest Dept., Taipei Veterans General Hospital, Taipei 11217, Taiwan (e-mail: cmtsai{at}vghtpe.gov.tw)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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 DISCUSSION
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