IL-6 induces neuroendocrine dedifferentiation and cell proliferation in non-small cell lung cancer cells

Kuo-Ting Chang,1,2 Chun-Ming Tsai,3 Yih-Chy Chiou,5 Chao-Hua Chiu,3 King-Song Jeng,4 and Chi-Ying F. Huang1,2,5,6

1Graduate Institute of Life Sciences, National Defense Medical Center, Taipei; 2Division of Molecular and Genomic Medicine, National Health Research Institutes, Miaoli County; 3Section of Thoracic Oncology, Chest Department, Taipei Veterans General Hospital, and Department of Medicine, School of Medicine, National Yang-Ming University, Taipei; 4Institute of Molecular Biology, Academia Sinica, Nankang, Taipei; 5Institute of Biotechnology in Medicine, National Yang-Ming University, Taipei; and 6Department of Computer Science and Information Engineering, National Taiwan University, Taipei, Taiwan

Submitted 28 February 2005 ; accepted in final form 9 May 2005


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Interleukin-6 (IL-6) has been identified as an important growth regulator of lung cancer cells. Elevation of serum levels of IL-6 has been found in a subpopulation of lung cancer patients, but rarely in patients with benign lung diseases. Approximately 15% of non-small cell lung cancer (NSCLC) tumors exhibit neuroendocrine (NE) properties (NSCLC-NE) and have been suggested to have the biological characteristics similar to small cell lung cancer (SCLC) with early metastasis and initial responsiveness to chemotherapy. We recently showed that IL-6 promotes cell proliferation and downregulates the expression of neuron-specific enolase (NSE, one of the major NE markers) in NSCLC-NE cells. In this study, we show that IL-6 stimulates a transient increase of tyrosine phosphorylation of STAT3 in a dose-dependent fashion. Inhibition of STAT3 signaling pathway by either AG-490 (JAK2-specific inhibitor) or overexpression of STAT3Y705F (a dominant-negative STAT3) reverses NSE expression in IL-6- treated NSCLC-NE cells. In addition, IL-6 induces phosphorylation and activation of p38 MAPK. SB-203580, a p38 MAPK-specific inhibitor, inhibits IL-6-induced p38 MAPK phosphorylating activity and suppresses IL-6-stimulated cell proliferation. Together, our results indicate that STAT3 signaling pathway is involved in IL-6-induced NE differentiation and that p38 MAPK is associated with IL-6-stimulated growth regulation in NSCLC-NE cells. These data suggest that both kinase pathways play critical roles in the pathogenesis of NSCLC-NE malignancies, providing new molecular targets for future therapeutic approaches.

neuron-specific enolase; signal transducer and activator of transcription; p38 mitogen-activated protein kinase


LUNG CANCER PATIENTS who have coexisting inflammatory lung disease (e.g., lung abscess, empyema, tuberculosis, etc.) may have a worse prognosis than those without (36). Whether inflammatory cytokines play a role in modulating malignant phenotype of lung cancer cells is not clear. Interleukin-6 (IL-6), a cytokine initially recognized as a regulator of immune and inflammatory responses, is also a growth modulator of many tumor cells (11, 18). 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 (16, 22, 25, 45). It has been suggested that IL-6 may play a significant role in the pathophysiology of cancer and that IL-6 is a potential mediator in the development of cancer cachexia (16, 45). 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 (2, 14, 23, 48). Circulating IL-6 level is correlated with the extent of disease and disease recurrence and is associated with decreased survival. In some of these tumors, elevated levels of IL-6 were found in patients who were unresponsive to hormone therapy, radiotherapy, or chemotherapy (5, 14, 32). 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 or patients with benign lung diseases (48). Intriguingly, IL-6 may be required in the control of cell proliferation in a subset of non-small cell lung cancer (NSCLC) cell lines in an IL-6-dependent or -independent manner (4). Paradoxically, antitumor effects of IL-6 have been demonstrated in vitro and in vivo in patients with NSCLC and breast cancer (46).

Neuroendocrine (NE) differentiation has been found in a subgroup of a variety of carcinomas including prostate (1), breast (51), gastric, and colorectal cancers (3, 23), and NSCLC (15, 33) and has been suggested as a marker of poor prognosis of various carcinomas. Clinically and biologically, lung cancer is generally divided into small cell lung cancer (SCLC) and NSCLC. 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) (27). NSCLC-NE tumors express multiple NE cell markers, including neuron-specific enolase (NSE), L-dopa decarboxylase, chromogranin A, synaptophysin, and cytoplasmic dense core granules (8). 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 (30). NSE was the marker most frequently detected in NSCLC (38%), and, most importantly, it is probably a prognostic marker of clinical relevance (17).

The IL-6/IL-6 receptor reaction activates several signal transduction pathways that have been separately implicated in regulating cancer cell growth and differentiation. Currently, there are at least three signal transduction pathways associated with IL-6 receptor signaling. The first one is the Janus kinase-signal transducer and activator of transcription (JAK/STAT) pathway. STAT proteins are a family of latent cytoplasmic transcription factors that are activated by cytokines and growth factors. STATs are activated after tyrosine phosphorylation by JAKs associated with cytokine receptors. Subsequently, activated STATs form dimers and translocate to the nucleus where they bind specific promoter elements of target genes and regulate their transcription (12, 13, 20, 21). Some members of the STAT family play roles in cellular differentiation, survival, and proliferation. Among STAT family members, STAT3 is the most frequently associated with deregulated cell growth and neoplasia (19). At present, the effect of IL-6 on tumors is controversial. Most notably, IL-6 has been shown to mediate growth arrest and NSE upregulation in LNCaP (a human prostate cancer cell line) and murine M1 leukemia cells by STAT3 (34, 42, 43). On the other hand, other investigators have suggested that ectopic expression of IL-6 stimulates prostate cancer cell growth accompanied by activation of the STAT3 signaling pathway (29) or by activating extracellular signal-regulated kinase (ERK) through cross talk with the growth factor receptor kinase erbB2 (39) in LNCaP cells. It is not clear what causes such discrepancies even for the same cell line.

The second one is the phosphatidylinositol 3-kinase (PI3-K)/Akt pathway. The PI3-K pathway regulates various cellular processes, such as proliferation, growth, apoptosis, and cytoskeletal rearrangement. Akt, a serine-threonine kinase, is activated by recruitment to the plasma membrane through direct contact of its pleckstrin-homology domain with phosphatidyl inositol-3,4,5-triphosphate, and phosphorylation at threonine 308 and serine 473 (47). In LNCaP cells, IL-6 may induce a remarkable NE-like differentiation phenotype, with neurite extension and enhanced expression of NSE by the activation of PI3K/Etk (a member of Btk tyrosine kinase family) (40). Recent growing evidence suggests the involvement of PI3-K/Akt in IL-6-dependent survival and proliferative responses in several types of cells. In IL-6-dependent 7TD1 B-cell hybridoma cells, IL-6 induced PI3-K/Akt activation is essential for the optimal growth through upregulation of antiapoptosis protein XIAP (52).

The third one is the mitogen-activated protein kinase (MAPK) pathway. The superfamily of MAPKs (38) include three major groups: the ERKs, the Jun NH2-terminal kinases (JNKs), and the p38 MAPK (37). The MAPK signaling cascade is activated by a wide variety of different cellular stimuli. It has been suggested that JAK and STAT proteins are activated by IL-6 in multiple myeloma (MM) cells independently of cell proliferation, whereas ERK was activated only in cells showing a proliferative response to IL-6 (35). However, the signal cascades mediating IL-6-induced cell growth are not fully defined. Accumulating evidence indicates that MAPK pathways can mediate signals that either promote or suppress the growth of malignant cells. For example, members of the p38 MAPK are primarily activated by stress stimuli but are also activated during engagement of various cytokine receptors by their ligands. The functions of p38 MAPK include the regulation of apoptosis, induction of cell differentiation, cell growth, and inflammation (10). Moreover, p38 MAPK has been reported to be associated with acute-phase protein secretion from HepG2 hepatoma cells and proliferation of B9 hybridoma cells in the presence of IL-6 (50). These findings support the idea that p38 MAPK may play a role in the process of cell proliferation.

The aim of this study is to identify the possible IL-6-mediated signaling pathway(s) involved in controlling NE differentiation and cell proliferation in NCI-H820. In the present study, we demonstrate that STAT3 phosphorylation is involved in NSE downregulation and p38 MAPK activation is associated with cell proliferation upon IL-6 stimulation. These results suggest that two major signaling pathways, STAT3 and p38 MAPK, are involved in the biological functions of regulating NE dedifferentiation and cell growth in NSCLC-NE cells, respectively.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell line and reagents. The NCI-H820 human adenocarcinoma cell line, which has NE properties, was obtained from the National Cancer Institute (NCI)-Navy Medical Oncology Branch (Division of Cancer Treatment, NCI, Bethesda, MD) from tumor specimens obtained from previously untreated patients (27). Recombinant human IL-6 (rhIL-6) and heregulin {beta}-1 were purchased from R & D Systems (Minneapolis, MN). Antibodies against STAT1, STAT3, Akt, ERK, and p38 (including phosphorylated forms) were purchased from Cell Signaling Technology (Beverly, MA). Anti-STAT5 (including phosphorylated forms) antibody was from Upstate Biotechnology (Lake Placid, NY). Anti-NSE antibody was obtained from Neomarkers (Fremont, CA). Anti-{beta}-actin antibody and AG-490 were obtained from Calbiochem (La Jolla, CA). SB-203580 was from Promega (Madison, WI). All other reagents were of analytical grade.

Cell culture, treatment of rhIL-6, and transfection. NCI-H820 cells were cultured and maintained in serum-free ACL-4 medium (Invitrogen, Grand Island, NY) supplemented with penicillin (100 U/ml) and streptomycin (100 µg/ml). In 6 days of treatments, ACL-4 medium was renewed with the indicated concentration of IL-6 every 2 days. For short-term stimulation experiments, cells were starved in RPMI-1640 for 16 h followed by stimulation with IL-6 (10–200 ng) in RPMI-1640. When inhibitors were used, AG-490 was added to the medium and incubated for 16 h before rhIL-6 treatment (26, 31). For SB-203580 treatment (28), cells were pretreated with SB-203580 for 90 min before rhIL-6 stimulation. The rhIL-6 constitutive expression construct pCMV-IL-6 was transfected into NCI-H820 cells with the TransFast transfection reagent (Promega) according to the manufacturer's instructions. Stable transfectants were isolated and cultured in the selective culture medium with 200 µg/ml of the antibiotic Geneticin (G418, Invitrogen) for 3–4 wk. G418-resistant clones were isolated and expanded. Expression of soluble rhIL-6 was confirmed by ELISA analysis (Diaclone Research, Besançon, France) of supernatants from transfectant, control vector, and parental cell populations.

Cell growth. NCI-H820 cells were seeded in a T-25 culture flask at a density of 1 x 105 cells per 4 ml of ACL-4 medium with or without IL-6 (20 ng/ml). The medium was replaced every 2 days. Cells were disaggregated with a trypsin-EDTA solution, neutralized, and resuspended in culture medium. Viable cell counts were determined by trypan blue exclusion with a hemocytometer. The mean of the hemocytometer counts of cells from triplicate flasks was determined every other day for 6 days for growth-curve and doubling-time determinations. The reported results were the means of three independent 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.

Construction of the pStat3Y705F Adeno-X expression vector and infection. The dominant form of STAT3, Stat3Y705F, was generated by PCR-based mutagenesis (QuikChange Site-Directed Mutagenesis Kit; Stratagene, La Jolla, CA) employing STAT3 as the template. Stat3Y705F was subcloned into pIRES2/EGFP shuttle vector, which was flanked by PI-Sce I and I-Ceu I. The resulting construct was then subcloned into Adeno-X Viral DNA vector, which was predigested with PI-Sce and I-Ceu I, according to the manufacturer's instructions (BD Biosciences Clontech, Palo Alto, CA) to produce recombinant adenovirus. The adenovirus construct of Stat3Y705F, designated Ad-DNSTAT3, was transfected into a low-passage HEK-293 cell line with PacI-digested recombinant Adeno-X DNA, and recombinant adenovirus was harvested 10–14 days later. Virus titers were determined by with the Adeno-X Rapid Titer Kit (BD Biosciences Clontech). NCI-H820 cells were infected with 50–100 multiplicities of infection (MOI) of either Ad-DNSTAT3 or vector control (Ad-CTL) for 48 h. Viral infection was confirmed by visual observation of EGFP expression in infected cells.

Preparation of cell lysates and Western blot analysis. To investigate the cell signaling pathway, NCI-H820 cells were first cultured in ACL-4 medium for 16 h. Cells then either were left untreated or were treated with rhIL-6 (50, 100, and 200 ng/ml) or Neu differentiating factor (heregulin; 1.5 nM, as the positive control) for 15 min. The harvested cells were lysed in radioimmunoprecipitation assay buffer [20 mM Tris (pH 8.0), 150 mM NaCl, 10% glycerol, 1% Nonidet P-40, and 0.42% NaF] and protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 1 mM Na3VO4, 5 µg/ml aprotinin, and 5 µg/ml leupeptin). Total cell lysates (50 µg) were resuspended in SDS sample buffer and resolved by SDS-PAGE. Proteins were transferred to polyvinylidene difluoride (PVDF) membrane (NEN Life Science, Boston, MA) and detected with various antibodies. The PVDF membrane was then blocked with 3% BSA/PBST (PBS plus 0.1% Tween 20) for 1 h at room temperature. Antibodies were incubated with the membrane for 1 h at room temperature. The membranes were washed with PBST at room temperature for 30 min, and the wash was repeated three times. The resulting IgGs were detected by incubation with secondary antibodies conjugated to horseradish peroxidase and developed using an enhanced chemiluminescence system (LumiGLO; KPL, Gaithersburg, MD) and exposed to BioMax film (Kodak, Rochester, NY). All experiments were performed in triplicate.

p38 MAPK kinase activity assay. Cell lysates (600 µg) were incubated overnight with 20 µl of immobilized phospho-p38 MAPK (threonine 180/tyrosine 182) monoclonal antibody beads (50% slurry) (Cell Signaling Technology, Beverly, MA) according to the manufacturer’s instructions. After extensive washing, the kinase reaction was performed in the presence of 100 µM cold ATP and 2 µg of activating transcription factor (ATF)-2 fusion protein at 30°C for 30 min. Phosphorylation of ATF-2 at threonine 71 was measured by Western blotting using phospho-ATF-2 (threonine 71) antibody.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Activation of STAT3 in lung adenocarcinoma cells is mediated by IL-6. In this study, we investigated whether the IL-6-elicited NE differentiation in lung adenocarcinoma cells might use signaling pathways similar to those in prostate cancer cells. A panel of phosphorylated antibodies, which represent the activation state of the signaling pathways, was used to rapidly screen the possible involvement of the above-mentioned pathways. Of the 16 lung cancer cell lines surveyed, NCI-H820 was selected for further analysis because it contains a high gene expression of IL-6 receptor and exhibits NE markers, such as NSE (9). NCI-H820 treated with the indicated concentrations of rhIL-6 resulted in a dramatic elevation of STAT3 tyrosine 705 phosphorylation (Fig. 1A) but not in phosphorylation of STAT1 (Fig. 1A), STAT5a/b (Fig. 1A), STAT6 (data not shown), Akt serine 473 (Fig. 1B), and Akt threonine 308 (Fig. 1B). These initial screenings suggest that IL-6-elicited NE differentiation might be preferentially mediated by STAT3 phosphorylation, but not by PI3-kinase activation.



View larger version (37K):
[in this window]
[in a new window]
 
Fig. 1. STAT3 activation is induced by IL-6 in NCI-H820. A: IL-6 activated STAT3 by tyrosine 705 phosphorylation but did not activate other STATs involved in the JAK-STAT pathways. NCI-H820 cells (5 x 105 in 4 ml of ACL-4 culture medium) were seeded overnight, and ACL-4 culture medium was changed to RPMI basal medium for starvation. After 16 h, cells were either left untreated or treated with recombinant human (rh) IL-6 (50–200 ng/ml) for 10 min. Cells were harvested and lysed. Equal amounts of protein were analyzed by Western blotting using antibodies specific to the phosphorylated (p) or nonphosphorylated antibodies as indicated. Positive controls (+) were HeLa and NIH/3T3 cell lysates provided by Cell Signaling Technology. B: neither the serine (473) nor threonine (308) of Akt was phosphorylated after rhIL-6 stimulation. Positive control (+) indicates that NCI-H820 cells were treated with 1.5 nM heregulin {beta}-1 for 15 min. The results shown are representative of 3 independent experiments.

 
STAT3 activation is required for NE differentiation in NCI-H820 cells. To provide additional evidence that STAT3 activation is responsible for IL-6-induced NE differentiation, two approaches were taken. First, a JAK2-specific inhibitor, tyrphostin AG-490, was used to inhibit the rhIL-6-induced JAK2 activation, resulting in the blockage of downstream STAT3 phosphorylation of tyrosine 705. NCI-H820 cells were first serum and cytokine starved, treated with increasing concentrations of AG-490 for 16 h, and finally stimulated with rhIL-6 (50 ng/ml, 10 min). Phosphorylation of STAT3 was progressively blocked by 50 and 100 µM AG-490 (68 and 99%, respectively). All P values were <0.001 (Fig. 2A). Moreover, NE differentiation can be characterized by the NSE downregulation. In agreement with a previous observation, long-term stimulation of NCI-H820 with rhIL-6 (4 and 6 days) resulted in NSE downregulation. Compared with NSE expression in control cells with no rhIL-6 treatment, NSE expression in cells treated with 50 ng/ml of rhIL-6 was downregulated for 4 days to ~0.35-fold and for 6 days to ~0.32-fold (Fig. 2B). To evaluate the effect of AG-490 on NE differentiation, NCI-H820 was preincubated with 50 µM AG-490 and then treated with rhIL-6 (50 ng/ml) for 4 and 6 days. As shown in Fig. 2B, AG-490 could reverse the downregulation of NSE expression in the rhIL-6-treated NCI-H820.



View larger version (55K):
[in this window]
[in a new window]
 
Fig. 2. AG-490, a JAK2 inhibitor, attenuates IL-6-elicited STAT3 phosphorylation and neuron-specific enolase (NSE) downregulation. A: AG-490 abolished IL-6-elicited STAT3 phosphorylation. NCI-H820 cells were preincubated with either DMSO or increasing amounts of AG-490 (50 µM and 100 µM) for 16 h and then rhIL-6 (20 ng/ml) for 10 min. CTL, NCI-H820 cells without any treatment. B: AG-490 reversed IL-6-mediated NSE downregulation. NCI-H820 cells were preincubated with 50 µM AG-490 for 16 h, and then rhIL-6 (20 ng/ml) was added for different time intervals (0–6 days). Cell lysates were prepared and analyzed by Western blotting with NSE or {beta}-actin antibodies. Similar results were obtained in 3 independent experiments. The numbers in parentheses are the relative expression fold compared with the first lane (1.00).

 
Although AG-490 has been broadly used for JAK/STAT signaling pathway identification, it should be noted that this tyrphostin inhibitor specifically blocks JAK2 activation. Therefore, the second approach was to employ the dominant-negative form of STAT3 (STAT3Y705F), in which tyrosine 705 has been replaced by phenylalanine, to ascertain the involvement of STAT3 in rhIL-6 induced NSE downregulation. Because of the low transfection efficiency of NCI-H820, we prepared an adenovirus delivery system to increase the sensitivity of our assay. The adenovirus constructs [i.e., dominant-negative STAT3 and control vector (vehicle)] were designated Ad-DNSTAT3 and Ad-CTL, respectively. NCI-H820 was infected with 100 MOI of Ad-DNSTAT3 and Ad-CTL, respectively. After 48 h, infected cells were treated with increasing concentrations of rhIL-6 (5 and 20 ng/ml) for 10 min. The results showed that overexpression of Ad-DNSTAT3 could completely block IL-6-induced tyrosine phosphorylation of STAT3 but did not affect the phosphotyrosine status of JAK2 (Fig. 3A). For long-term (6 days) treatment, NCI-H820 was infected with 50 MOI of Ad-DNSTAT3 and Ad-CTL, respectively, and then assayed for NSE expression. Figure 3B shows that Ad-DNSTAT3, but not Ad-CTL, could reverse the downregulation of NSE expression when cells were stimulated with rhIL-6. Together, these findings raise the possibility that activation of STAT3 is an important mediator for IL-6-mediated NE differentiation in NCI-H820.



View larger version (49K):
[in this window]
[in a new window]
 
Fig. 3. Overexpression of dominant-negative STAT3 blocks IL-6-elicited STAT3 phosphorylation and NSE downregulation. A: overexpression of STAT3Y705F, the dominant-negative form of STAT3, inhibited IL-6-induced tyrosine phosphorylation of STAT3 but did not affect the phosphotyrosine status of JAK2. NCI-H820 cells (5 x 105) were seeded overnight and infected with 100 multiplicities of infection (MOI) of STAT3Y705F (Ad-DNSTAT3) and control vector (Ad-CTL), respectively. After 48 h, cells were starved for 16 h and exposed to IL-6 for 10 min. Cell lysates were prepared and analyzed by Western blotting with antibodies specific for phosphorylated STAT3 or JAK2. Membrane was reprobed with antibodies specific for nonphosphorylated STAT3 to show the amount of STAT3Y705F. B: overexpression of STAT3Y705F blocked IL-6-induced NSE downregulation. NCI-H820 cells (1 x 105) were seeded overnight and infected with 50 MOI of Ad-DNSTAT3 or Ad-CTL. After 48 h, cells were treated with rhIL-6 (20 ng/ml) for different time intervals (0–6 days). Cell lysates were prepared and analyzed by Western blotting with NSE or {beta}-actin antibodies. Membrane was reprobed with antibodies specific for nonphosphorylated STAT3 to show the amount of STAT3Y705F. The results shown are representative of 3 independent experiments.

 
IL-6 activates p38 MAPK in NCI-H820 cells. We next investigated whether the three well-known MAPK pathways were involved in the modulation of IL-6 in NCI-H820. Again, three phosphorylated antibodies, which represent the activation state of the p38 MAPK, ERK, and JNK signaling pathways, were used to rapidly screen the possible involvement of IL-6-elicited signaling pathways. NCI-H820 was treated with rhIL-6 (20 ng/ml) for 10 min. Cell lysates were examined by Western blotting with antibodies to the phosphorylated forms of p38 MAPK, ERK, and JNK. As shown in Fig. 4A, p38 phosphorylation was elevated in IL-6-treated cells. In contrast, ERK and JNK were not phosphorylated in response to rhIL-6 stimulation (results not shown). To provide additional evidence for the participation of p38 MAPK in rhIL-6 stimulation, we performed an immunoprecipitation/kinase assay using antibody against phosphorylated p38 MAPK. ATF-2 served as an in vitro substrate for p38 MAPK in the kinase assay. The results showed that p38 MAPK activity was enhanced 8.35-fold upon induction with rhIL-6 (Fig. 4B).



View larger version (45K):
[in this window]
[in a new window]
 
Fig. 4. IL-6 mediates p38 MAPK activation. A: IL-6 mediates p38 MAPK phosphorylation at threonine 180 and tyrosine 182. NCI-H820 cells were either left untreated or treated with rhIL-6 (20 ng/ml) for 10 min as described in the legend of Fig. 1. The positive control (+) was NCI-H820 cells treated with anisomycin (20 ng/ml) for 30 min. B: inhibition of p38 MAPK activity by SB-203580. NCI-H820 cells were either left untreated or treated with 10 µM SB-203580 and then stimulated or not with rhIL-6. Cell lysates were prepared and equal amounts of lysates were immunoprecipitated with phospho-p38 antibody. Phosphorylation of activating transcription factor (ATF)-2 at threonine 69/71 was measured by Western blotting using phospho-ATF-2 (threonine 69/71) antibody (bottom). Membrane was reprobed with antibody to phosphorylated p38 MAPK to determine the phosphorylation status of p38 MAPK (middle). Numbers in parentheses are the levels of phosphorylated ATF-2 and phospho-p38 MAPK quantified by densitometry, which are expressed in arbitrary units relative to the value of control cells, to which a value of 1 has been assigned. Similar results were obtained in 3 independent experiments. The loading control of p38 MAPK was added to show that equal amounts of lysates were used for the immunoprecipitation (top).

 
A pyridinyl imidazole compound, SB-203580, which has been shown to inhibit the p38 MAPK signaling pathway by binding to the ATP pocket of p38 MAPK (49), was used to examine the activity of p38 MAPK. In this assay NCI-H820 cells were exposed to 10 µM SB-203580 for 90 min before stimulation with rhIL-6 (20 ng/ml). p38 MAPK phosphorylation was slightly inhibited by SB-203580 (Fig. 4B), whereas 10 µM SB-203580 was sufficient to reduce p38 MAPK activity to the basal level (Fig. 4B). A similar effect was seen in another NSCLC-NE cell, NCI-H1355 (data not shown). These data support the idea that IL-6 activates p38 MAPK in NCI-H820 cells.

IL-6-elicited cell proliferation is mediated by p38 MAPK in NCI-H820 cells. In agreement with previous observation, treatment with rhIL-6 promoted cell growth as determined by direct cell counting (Fig. 5A). The finding prompted us to investigate whether the JAK/STAT and/or p38 MAPK signaling pathways were involved in IL-6-elicited cell proliferation. Treatment with AG-490 or overexpression of Ad-DNSTAT3 failed to demonstrate a dependency of cell growth on STAT3 activation in NCI-H820 (data not shown). In contrast, pretreatment of NCI-H820 with SB-203580 (5 µM) effectively blocked rhIL-6 (20 ng/ml)-induced cell proliferation (P < 0.05, n = 3, compared with the IL-6-stimulated cell growth) (Fig. 5A). To further corroborate these findings and elucidate the biological role of IL-6 in the NCI-H820 cells, an rhIL-6 constitutive expression construct was transfected into NCI-H820 and generated a stable rhIL-6-overexpressing subclone, which was designated H820.ILSC. Subsequently, we examined the H820.ILSC cell growth in the presence or absence of SB-203580. As shown in Fig. 5B, SB-203580 significantly suppressed H820.ILSC cell growth ~2.24-fold at concentrations of 5 µM (P < 0.05, n = 3) and 4.46-fold at 10 µM (P < 0.001, n = 3). Together, these data support the idea that IL-6-elicited cell proliferation is mediated through p38 MAPK.



View larger version (13K):
[in this window]
[in a new window]
 
Fig. 5. Effect of p38 MAPK inhibitor on IL-6-stimulated cell growth. A: SB-203580 inhibited cell growth induced by rhIL-6 treatment. NCI-H820 cells (1 x 105) were seeded overnight in 4 ml of ACL-4 culture medium. The next day, the medium was replaced with fresh medium. Cells were preincubated with or without 5 µM SB-203580 (SB5) for 90 min and treated with or without rhIL-6 (20 ng/ml) for 6 days. Culture medium containing either rhIL-6 or SB-203580 was changed every 2 days and cells were harvested at different time intervals (0–6 days). rhIL-6-induced cell proliferation was inhibited in the presence of 5 µM SB-203580 (statistical significance: *P < 0.05 compared with the cells with rhIL-6 treatment). B: SB-203580 suppressed cell proliferation of the rhIL-6 stable transfectant H820.ILSC. H820.ILSC cells were preincubated with 2, 5, and 10 µM SB-203580 (SB2, SB5, SB10), respectively, for 6 days. Culture medium with or without SB-203580 was changed every 2 days, and cells were harvested at different time intervals (0–6 days). The cell growth of H820.ILSC was markedly suppressed by 5 and 10 µM SB-203580 (statistical significance: **P < 0.001 compared with the cells without SB-203580 treatment). Viable cells were counted by trypan blue dye exclusion test every other day. Mean growth curves of the test cell lines are shown as the mean (data points) ± SE (bars) from 3 independent experiments. DMSO and CTL indicate AG-490 negative and blank control, respectively.

 

    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
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, involved in regulation of the immune response, acute phase reaction, cell differentiation, and cell proliferation (18, 33). This pleiotropism implies a complex signaling cascade that enables the development of different pathways and distinct biological functions. It is well established that the binding of IL-6 to IL-6 receptor activates the JAK/STAT signal transduction pathway, where STAT3 plays a central role in transmitting the signals from the membrane to the nucleus (Fig. 6). The biological readout of STAT3 can be proliferation, survival, apoptosis, and/or differentiation, depending on the target tissues. STAT3 was first described as the acute-phase response factor, a transcription factor activated by IL-6/IL-6 receptor in human hepatoma cells (50). Recent studies have demonstrated the activation of STAT3 in human cancers, e.g., multiple myelanoma, leukemia, and lymphoma (6). In addition, STAT3 has been suggested to be a key player in the pathogenesis of diverse human cancers, making this molecule a prime target for novel therapies.



View larger version (21K):
[in this window]
[in a new window]
 
Fig. 6. Proposed signaling pathways of IL-6-elicited JAK/STAT and p38 MAPK activation in NSCLC-NE. Binding of IL-6 to its cognate receptor leads to the recruitment and phosphorylation of JAK2. Activated JAK2 then phosphorylates STAT3 at tyrosine 705 (Y705), which promotes the homodimerization of STAT3 through reciprocal phosphotyrosine-SH2 domain interactions. Activated STAT3 dimers then translocate to the nucleus where they bind to consensus DNA sequences and transactivate target genes. Activation of p38 MAPK occurs through phosphorylation at threonine 180/tyrosine 182 (T180/Y182). Activated p38 MAPK then regulates downstream transcription factors and potently activates ATF-2 by phosphorylation at threonine 69/71 (T69/71). The activated transcription factors bind to specific promoter elements of the target genes and regulate their transcription. Sites of action of AG-490, AdDNSTAT3, and SB-203580 are depicted. RTK, receptor tyrosine kinase; ISRE, interferon-stimulated regulatory factor; GAS, gamma-activated site; CRE, cAMP response element; TRE, TPA response element.

 
The importance of STAT3 activation via IL-6 in prostate cancer development has been suggested. In LNCaP cells, IL-6 treatment could increase the growth of the cells concomitant with activation of STAT3 (19, 29). In contrast, other reports have observed that treatment of LNCaP cells with IL-6 results in terminal differentiation (NSE upregulation) and growth inhibition associated with STAT3 activation (42, 43). However, the molecular basis for the apparent contradiction remains unknown. On the other hand, IL-6-mediated proliferation of BAF3 cells is not dependent on activation of STAT3 (44). Although few direct studies into the role of STAT3 in human lung cancer have been undertaken, one recent study has demonstrated the role of STAT3 activation in transducing survival signals downstream of tyrosine kinases such as Src, EGF-R, and c-Met, as well as cytokines such as IL-6 in human NSCLCs (41). In the present study, the aim is to delineate the potential role of STAT3 in human non-small lung carcinomas by studying cell lines of non-small cell origin. We show that IL-6-elicited NSE downregulation in NCI-H820 is mediated by STAT3 activation using treatment with the JAK2-selective inhibitor (AG-490) (Fig. 2) and ectopic expression of a dominant-negative form of STAT3 (Fig. 3). Moreover, our results suggest that in IL-6-dependent NSCLC-NE cells, the JAK/STAT pathway appears to be more important in the regulation of NE differentiation than in cell growth promotion. To the best of our knowledge, this is the first report to directly demonstrate a role for STAT3 of the STAT family in NE differentiation in the established human NSCLC-NE cell line NCI-H820.

In addition to the classic JAK/STAT pathway, other signaling cascades may play important roles in signal transduction via the IL-6 receptor. It has been reported that ERK activation is essential for IL-6-dependent MM cell proliferation, whereas the phosphorylation of STAT1 and STAT3 is unrelated to melanoma cell growth (35). In contrast, the p38 MAPK pathway has been shown to be involved in cell proliferation in IL-6 signaling in B9 hybridoma cells (50). Therefore, different intracellular signaling pathways may contribute to the regulation of cell proliferation. In the present study, we show that activation of the p38 MAPK pathway, but not ERK or JNK pathway, is necessary for the induction of IL-6 (Fig. 4). Moreover, inhibition of p38 MAPK activity by SB-203580 resulted in suppression of the growth of cells induced by IL-6 and growth of an IL-6-expressing transfectant (Fig. 5), suggesting that p38 MAPK activation may play a critical role in IL-6-induced cell growth of NSCLC-NE cells. Together, the proposed signaling modules (Fig. 6), which depict IL-6-dependent NE dedifferentiation and cell proliferation in NSCLC-NE cells, exert their effect through the STAT3 and p38 MAPK signaling pathways.

Various new pharmacological agents are currently under clinical development for treatment of malignant tumors, including agents that either block growth-promoting signaling cascades or trigger apoptotic signals in neoplastic cells. The realization of signaling cascades that play important roles in the regulation of apoptosis and growth of cancer cells has led to extensive studies. Growing evidence points to a number of important STAT3 target genes that have been implicated in the formation of tumors (7, 12, 19). Inhibition of STAT3 through pharmacological blockage of upstream molecules such as Src and JAK may reduce the tumor formation. Our findings strongly suggest that the STAT3 and p38 MAPK signaling systems may play important roles in the development of lung cancers by either autocrine or paracrine mechanisms. This raises the possibility that, in addition to targeting STAT3, targeting the p38 MAPK pathway may have therapeutic implications in the treatment of NSCLC-NE tumors. Our results demonstrate that a thorough understanding of how specific signaling pathways regulate downstream gene expression may provide rationale for using targeted therapy to enhance cell killing.


    GRANTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This work was supported by grants from the National Health Research Institutes and National Science Council (National Research Program for Genomic Medicine NHRI93A1-NSCMM-11-5 and DOH94-TD-G-111-009 to C-Y. F. Huang) and Taipei Veterans General Hospital (2001#314, 2004#302 to C. M. Tsai).


    ACKNOWLEDGMENTS
 
We thank Dr. Chuang at the Division of Cancer Research, National Health Research Institutes, for generously providing the recombinant human IL-6 expression construct pCMV-IL-6.


    FOOTNOTES
 

Address for reprint requests and other correspondence: C.-Y. F. Huang, Natl. Health Research Inst., Div. of Molecular & Genomic Medicine, 35, Keyan Rd., Zhunan Town, Miaoli County 350, Taiwan, R. O. C. (e-mail: chiying{at}nhri.org.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.


    REFERENCES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Abrahamsson PA. Neuroendocrine differentiation in prostatic carcinoma. Prostate 39: 135–148, 1999.[CrossRef][ISI][Medline]
  2. Adler HL, McCurdy MA, Kattan MW, Timme TL, Scardino PT, and Thompson TC. Elevated levels of circulating interleukin-6 and transforming growth factor-beta1 in patients with metastatic prostatic carcinoma. J Urol 161: 182–187, 1999.[ISI][Medline]
  3. Bernick PE, Klimstra DS, Shia J, Minsky B, Saltz L, Shi W, Thaler H, Guillem J, Paty P, Cohen AM, and Wong WD. Neuroendocrine carcinomas of the colon and rectum. Dis Colon Rectum 47: 163–169, 2004.[CrossRef][ISI][Medline]
  4. Bihl M, Tamm M, Nauck M, Wieland H, Perruchoud AP, and Roth M. Proliferation of human non-small-cell lung cancer cell lines: role of interleukin-6. Am J Respir Cell Mol Biol 19: 606–612, 1998.[Abstract/Free Full Text]
  5. Borsellino N, Bonavida B, Ciliberto G, Toniatti C, Travali S, and D'Alessandro N. Blocking signaling through the Gp130 receptor chain by interleukin-6 and oncostatin M inhibits PC-3 cell growth and sensitizes the tumor cells to etoposide and cisplatin-mediated cytotoxicity. Cancer 85: 134–144, 1999.[CrossRef][ISI][Medline]
  6. Bowman T, Garcia R, Turkson J, and Jove R. STATs in oncogenesis. Oncogene 19: 2474–2488, 2000.[CrossRef][ISI][Medline]
  7. Bromberg J. Stat proteins and oncogenesis. J Clin Invest 109: 1139–1142, 2002.[Free Full Text]
  8. Carles J, Rosell R, Ariza A, Pellicer I, Sanchez JJ, Fernandez-Vasalo G, Abad A, and Barnadas A. Neuroendocrine differentiation as a prognostic factor in non-small cell lung cancer. Lung Cancer 10: 209–219, 1993.[CrossRef][ISI][Medline]
  9. Chang KT, Huang CYF, Tsai CM, Chiu CH, and Lok YY. Role of IL-6 in neuroendocrine differentiation and chemosensitivity of non-small cell lung cancer. Am J Physiol Lung Cell Mol Physiol 289: L438–L445, 2005.[Abstract/Free Full Text]
  10. Chang L and Karin M. Mammalian MAP kinase signalling cascades. Nature 410: 37–40, 2001.[CrossRef][ISI][Medline]
  11. Chen-Kiang S. Regulation of terminal differentiation of human B-cells by IL-6. Curr Top Microbiol Immunol 194: 189–198, 1995.[Medline]
  12. Darnell JE Jr. STATs and gene regulation. Science 277: 1630–1635, 1997.[Abstract/Free Full Text]
  13. Darnell JE Jr, Kerr IM, and Stark GR. Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins. Science 264: 1415–1421, 1994.[ISI][Medline]
  14. De Vita F, Orditura M, Auriemma A, Infusino S, Roscigno A, and Catalano G. Serum levels of interleukin-6 as a prognostic factor in advanced non-small cell lung cancer. Oncol Rep 5: 649–652, 1998.[ISI][Medline]
  15. Ferrigno D, Buccheri G, and Giordano C. Neuron-specific enolase is an effective tumour marker in non-small cell lung cancer (NSCLC). Lung Cancer 41: 311–320, 2003.[CrossRef][ISI][Medline]
  16. Giri D, Ozen M, and Ittmann M. Interleukin-6 is an autocrine growth factor in human prostate cancer. Am J Pathol 159: 2159–2165, 2001.[Abstract/Free Full Text]
  17. Graziano SL, Tatum A, Herndon JE II, Box J, Memoli V, Green MR, and Kern JA. Use of neuroendocrine markers, p53, and HER2 to predict response to chemotherapy in patients with stage III non-small cell lung cancer: a Cancer and Leukemia Group B study. Lung Cancer 33: 115–123, 2001.[CrossRef][ISI][Medline]
  18. Heinrich PC, Behrmann I, Haan S, Hermanns HM, Muller-Newen G, and Schaper F. Principles of interleukin (IL)-6-type cytokine signalling and its regulation. Biochem J 374: 1–20, 2003.[CrossRef][ISI][Medline]
  19. Hirano T, Ishihara K, and Hibi M. Roles of STAT3 in mediating the cell growth, differentiation and survival signals relayed through the IL-6 family of cytokine receptors. Oncogene 19: 2548–2556, 2000.[CrossRef][ISI][Medline]
  20. Ihle JN. STATs: signal transducers and activators of transcription. Cell 84: 331–334, 1996.[CrossRef][ISI][Medline]
  21. Ihle JN, Witthuhn BA, Quelle FW, Yamamoto K, Thierfelder WE, Kreider B, and Silvennoinen O. Signaling by the cytokine receptor superfamily: JAKs and STATs. Trends Biochem Sci 19: 222–227, 1994.[CrossRef][ISI][Medline]
  22. Jee SH, Shen SC, Chiu HC, Tsai WL, and Kuo ML. Overexpression of interleukin-6 in human basal cell carcinoma cell lines increases anti-apoptotic activity and tumorigenic potency. Oncogene 20: 198–208, 2001.[CrossRef][ISI][Medline]
  23. Kinoshita T, Ito H, and Miki C. Serum interleukin-6 level reflects the tumor proliferative activity in patients with colorectal carcinoma. Cancer 85: 2526–2531, 1999.[CrossRef][ISI][Medline]
  24. Kloppel G. [Neuroendocrine tumors of the gastrointestinal tract]. Pathologe 24: 287–296, 2003.[CrossRef][ISI][Medline]
  25. Lee JD, Sievers TM, Skotzko M, Chandler CF, Morton DL, McBride WH, and Economou JS. Interleukin-6 production by human melanoma cell lines. Lymphokine Cytokine Res 11: 161–166, 1992.[ISI][Medline]
  26. Levitzki A. Tyrphostins–potential antiproliferative agents and novel molecular tools. Biochem Pharmacol 40: 913–918, 1990.[CrossRef][ISI][Medline]
  27. Linnoila RI. Spectrum of neuroendocrine differentiation in lung cancer cell lines featured by cytomorphology, markers, and their corresponding tumors. J Cell Biochem Suppl 24: 92–106, 1996.[Medline]
  28. LoGrasso PV, Frantz B, Rolando AM, O'Keefe SJ, Hermes JD, and O'Neill EA. Kinetic mechanism for p38 MAP kinase. Biochemistry 36: 10422–10427, 1997.[CrossRef][ISI][Medline]
  29. Lou W, Ni Z, Dyer K, Tweardy DJ, and Gao AC. Interleukin-6 induces prostate cancer cell growth accompanied by activation of stat3 signaling pathway. Prostate 42: 239–242, 2000.[CrossRef][ISI][Medline]
  30. Marangos PJ and Schmechel DE. Neuron specific enolase, a clinically useful marker for neurons and neuroendocrine cells. Annu Rev Neurosci 10: 269–295, 1987.[CrossRef][ISI][Medline]
  31. Meydan N, Grunberger T, Dadi H, Shahar M, Arpaia E, Lapidot Z, Leeder JS, Freedman M, Cohen A, Gazit A, Levitzki A, and Roifman CM. Inhibition of acute lymphoblastic leukaemia by a Jak-2 inhibitor. Nature 379: 645–648, 1996.[CrossRef][ISI][Medline]
  32. Miyamoto Y, Hosotani R, Doi R, Wada M, Ida J, Tsuji S, Kawaguchi M, Nakajima S, Kobayashi H, Masui T, and Imamura M. Interleukin-6 inhibits radiation induced apoptosis in pancreatic cancer cells. Anticancer Res 21: 2449–2456, 2001.[ISI][Medline]
  33. Naka T, Nishimoto N, and Kishimoto T. The paradigm of IL-6: from basic science to medicine. Arthritis Res 4, Suppl 3: S233–242, 2002.[CrossRef][Medline]
  34. Nakajima K, Yamanaka Y, Nakae K, Kojima H, Ichiba M, Kiuchi N, Kitaoka T, Fukada T, Hibi M, and Hirano T. A central role for Stat3 in IL-6-induced regulation of growth and differentiation in M1 leukemia cells. EMBO J 15: 3651–3658, 1996.[Abstract]
  35. Ogata A, Chauhan D, Teoh G, Treon SP, Urashima M, Schlossman RL, and Anderson KC. IL-6 triggers cell growth via the Ras-dependent mitogen-activated protein kinase cascade. J Immunol 159: 2212–2221, 1997.[Abstract]
  36. Pass HI, Mitchell JB, Johnson DH, Turrisi AT, and Minna JD. Lung Cancer: Principles and Practice. Philadelphia, PA: Lippincott Williams & Wilkins, 2000.
  37. Platanias LC. The p38 mitogen-activated protein kinase pathway and its role in interferon signaling. Pharmacol Ther 98: 129–142, 2003.[CrossRef][ISI][Medline]
  38. Platanias LC. Map kinase signaling pathways and hematologic malignancies. Blood 101: 4667–4679, 2003.[Abstract/Free Full Text]
  39. Qiu Y, Ravi L, and Kung HJ. Requirement of ErbB2 for signalling by interleukin-6 in prostate carcinoma cells. Nature 393: 83–85, 1998.[CrossRef][ISI][Medline]
  40. Qiu Y, Robinson D, Pretlow TG, and Kung HJ. Etk/Bmx, a tyrosine kinase with a pleckstrin-homology domain, is an effector of phosphatidylinositol 3'-kinase and is involved in interleukin 6-induced neuroendocrine differentiation of prostate cancer cells. Proc Natl Acad Sci USA 95: 3644–3649, 1998.[Abstract/Free Full Text]
  41. Song L, Turkson J, Karras JG, Jove R, and Haura EB. Activation of Stat3 by receptor tyrosine kinases and cytokines regulates survival in human non-small cell carcinoma cells. Oncogene 22: 4150–4165, 2003.[CrossRef][ISI][Medline]
  42. Spiotto MT and Chung TD. STAT3 mediates IL-6-induced neuroendocrine differentiation in prostate cancer cells. Prostate 42: 186–195, 2000.[CrossRef][ISI][Medline]
  43. Spiotto MT and Chung TD. STAT3 mediates IL-6-induced growth inhibition in the human prostate cancer cell line LNCaP. Prostate 42: 88–98, 2000.[CrossRef][ISI][Medline]
  44. Stahl N, Farruggella TJ, Boulton TG, Zhong Z, Darnell JE Jr, and Yancopoulos GD. Choice of STATs and other substrates specified by modular tyrosine-based motifs in cytokine receptors. Science 267: 1349–1353, 1995.[ISI][Medline]
  45. Strassmann G, Fong M, Kenney JS, and Jacob CO. Evidence for the involvement of interleukin 6 in experimental cancer cachexia. J Clin Invest 89: 1681–1684, 1992.[ISI][Medline]
  46. Veldhuis GJ, Willemse PH, Sleijfer DT, van der Graaf WT, Groen HJ, Limburg PC, Mulder NH, and de Vries EG. Toxicity and efficacy of escalating dosages of recombinant human interleukin-6 after chemotherapy in patients with breast cancer or non-small-cell lung cancer. J Clin Oncol 13: 2585–2593, 1995.[Abstract/Free Full Text]
  47. Vivanco I and Sawyers CL. The phosphatidylinositol 3-kinase AKT pathway in human cancer. Nat Rev Cancer 2: 489–501, 2002.[CrossRef][ISI][Medline]
  48. Yanagawa H, Sone S, Takahashi Y, Haku T, Yano S, Shinohara T, and Ogura T. Serum levels of interleukin 6 in patients with lung cancer. Br J Cancer 71: 1095–1098, 1995.[ISI][Medline]
  49. Young PR, McLaughlin MM, Kumar S, Kassis S, Doyle ML, McNulty D, Gallagher TF, Fisher S, McDonnell PC, Carr SA, Huddleston MJ, Seibel G, Porter TG, Livi GP, Adams JL, and Lee JC. Pyridinyl imidazole inhibitors of p38 mitogen-activated protein kinase bind in the ATP site. J Biol Chem 272: 12116–12121, 1997.[Abstract/Free Full Text]
  50. Zauberman A, Zipori D, Krupsky M, and Ben-Levy R. Stress activated protein kinase p38 is involved in IL-6 induced transcriptional activation of STAT3. Oncogene 18: 3886–3893, 1999.[CrossRef][ISI][Medline]
  51. Zekioglu O, Erhan Y, Ciris M, and Bayramoglu H. Neuroendocrine differentiated carcinomas of the breast: a distinct entity. Breast 12: 251–257, 2003.[CrossRef][ISI][Medline]
  52. Zhang J, Li Y, and Shen B. PI3-K/Akt pathway contributes to IL-6-dependent growth of 7TD1 cells. Cancer Cell Int 3: 1, 2003.[CrossRef][Medline]
  53. Zych J, Sakowicz A, Radzikowska E, Zaleska J, and Rowinska-Zakrzewska E. Neuron-specific enolase (NSE) serum level in non small cell lung cancer–can it be an indicator of tumour chemosensitivity? Rocz Akad Med Bialymst 42, Suppl 1: 173–178, 1997.[Medline]




This Article
Abstract
Full Text (PDF)
All Versions of this Article:
289/3/L446    most recent
00089.2005v1
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Search for citing articles in:
ISI Web of Science (1)
Google Scholar
Articles by Chang, K.-T.
Articles by Huang, C.-Y. F.
Articles citing this Article
PubMed
PubMed Citation
Articles by Chang, K.-T.
Articles by Huang, C.-Y. F.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2005 by the American Physiological Society.