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Cancer Therapy: Preclinical |
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Authors' Affiliations: 1 Department of Pharmacology, College of Medicine, National Taiwan University; and 2 Department of Forensic Medicine and Pathology and 3 Division of Gastroenterology, Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan
Requests for reprints: Ching-Chow Chen, Department of Pharmacology, College of Medicine, National Taiwan University, No. 1, Jen-Ai Road, Section 1, Taipei 10018, Taiwan. Phone: 886-2-23123456, ext. 8321; Fax: 886-2-23947833; E-mail: ccchen{at}ha.mc.ntu.edu.tw.
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Abstract |
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Experimental Design: Expression of ICAM-1 protein in human lung cancer specimens was assessed by immunohistochemistry. ICAM-1 overexpressing A549 cells (A549/ICAM-1) were established to investigate the direct effect of ICAM-1 on in vitro cell invasion and in vivo tumor metastasis. Transient transfection and luciferase assay, electrophoretic mobility shift assay, and chromatin immunoprecipitation were done to assess the activity and binding of nuclear factor-
B to the ICAM-1 promoter. A xenograft model in nude mice was conducted to evaluate the anticancer effect of thalidomide.
Results: High expression of ICAM-1 in human lung cancer specimens was correlated with a greater risk of advanced cancers (stages III and IV). A549/ICAM-1 cells were shown to induce in vitro cell invasion and in vivo tumor metastasis. Anti-ICAM-1 antibody and thalidomide had inhibitory effect on these events. Thalidomide also suppressed tumor necrosis factor-
–induced ICAM-1 expression through inhibition of nuclear factor-B binding to the ICAM-1 promoter. The in vivo xenograft model showed the effectiveness of thalidomide on tumor formation.
Conclusion: These studies provide a framework for targeting ICAM-1 as a biologically based therapy for cancer, and thalidomide might be effective in human lung cancer.
ICAM-1 is expressed constitutively at low levels on endothelial cells and some lymphocytes and monocytes. Stimulation with inflammatory cytokines, such as interleukin-1, tumor necrosis factor (TNF-), and IFN-
, or with lipopolysaccharide has been documented to increase ICAM-1 expression on multiple cell types (12–14). The promoter region of human ICAM-1 gene has been shown to contain putative recognition sequences for a variety of transcription factors, including nuclear factor-B (NF-B), activator protein-1, activator protein-2, and the IFN-stimulated response (15). NF-B family proteins are essential for enhanced ICAM-1 expression, and inhibition of NF-B suppresses the expression of ICAM-1, resulting in the reduction of lung cancer cell invasion (9).
Thalidomide was originally used to treat morning sickness, but was banned in the 1960s due to serious congenital birth defects. Despite its history as human teratogen, thalidomide is emerging as a treatment for inflammatory disease and cancers because of its antiangiogenic and anti-inflammatory effects (16). Different stages of clinical trials have been conducted to treat malignant diseases, such as hematologic cancers and solid tumors, including renal cell carcinoma; ovarian, breast, and androgen-independent prostate cancer (17–19). Rational drug design of specific small-molecule inhibitors with better pharmacologic profiles leads to the identification of a specific molecular target for thalidomide (16). In this study, ICAM-1 was shown to be a molecular target for lung cancer. We investigated its role in lung cancer metastasis and the effect of thalidomide. High expression of ICAM-1 was detected in advanced human lung cancers (stages III and IV), and ICAM-1–overexpressing A549 cells induced in vitro cell invasion and in vivo tumor metastasis. Thalidomide showed anti-ICAM-1 and antitumor effects on the in vivo xenograft tumor model. Its molecular mechanism was shown to act through attenuation of NF-B binding to the ICAM-1 promoter. These studies provide a framework for targeting ICAM-1–dependent metastasis as a biologically based therapy for cancer.
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Materials and Methods |
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B-luciferase expression plasmid was purchased from Stratagene (La Jolla, CA). Human TNF- recombinant and mouse monoclonal anti-human ICAM-1 antibody were purchased from R&D Systems (Minneapolis, MN). The rabbit polyclonal antibodies specific for p65 and IKK/ß were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-phospho-IKK (Ser180)/ß (Ser181) and anti-phospho-p65 (Ser536) antibodies were obtained from Cell Signaling (Danvers, MA). Reagents for SDS-PAGE were from Bio-Rad (Hercules, CA). [-32P]ATP (3,000 Ci/mmol) was from Dupont-New England Nuclear (Beverly, MA). Thalidomide, a gift from TTY Biopharm (Taipei, Taiwan), was dissolved in DMSO. Cell culture. A549 cells, an alveolar epithelial carcinoma cell line, were obtained from American Type Culture Collection (Manassas, VA), and cultured in DMEM supplemented with 10% fetal bovine serum, 100 units/mL penicillin, and 100 µg/mL streptomycin at 37°C in a humidified atmosphere of 5% CO2/95% air.
Patients and specimens. Lung cancer specimens were obtained from a total of 40 patients who underwent surgical resection at the National Taiwan University Hospital from 2003 to 2005. Patients who had previous history of cancers or had been treated with neoadjuvant chemotherapy and radiation therapy were not included. Paraffin-embedded, formalin-fixed surgical specimens were collected for ICAM-1 immunohistochemical staining. Tumor size, local invasion, lymph node metastasis, and differentiation status were determined at pathologic examination. The final disease stage was determined by a combination of surgical and pathologic findings according to the current tumor-node-metastasis staging system for lung cancer. Follow-up data were obtained from the patients' medical charts and from our tumor registry service.
Immunohistochemistry. The 4-µm sections of paraffin-embedded tissue on glass slides were rehydrated and incubated in 3% hydrogen peroxide to block the endogenous peroxidase activity. After trypsinization, sections were blocked by incubation in 3% bovine serum albumin in PBS. The primary antibody monoclonal mouse anti-human ICAM-1 antibody (Novocastra, Newcastle, United Kingdom) was applied to the slides at a dilution of 1:25 (diluted in 3% bovine serum albumin) and incubated at 4°C overnight. After three washes in PBS, the samples were treated with goat anti-mouse IgG biotin-labeled secondary antibodies at a dilution of 1:50 (diluted in 0.05% PBS-Tween 20). Bound antibodies were detected with an ABC kit (Vector Laboratories). The slides were stained with chromogen diaminobenzidine, washed, counterstained with Delafield's hematoxylin, dehydrated, treated with xylene, and mounted. A score system from level 0 (no expression) to level 2 (highest expression) was established based on a positive staining area for ICAM-1 expression in tumors. Low and high ICAM-1 expression were defined by the levels 0, 1, and 2, respectively. Level 0 represents <5% of cells showing ICAM-1 expression, level 1 represents 6% to 50%, and level 2 represents 51% to 100%.
Stable transfected clone selection. Purified plasmid of human full-length ICAM-1 cDNA (3 µg) was transfected into A549 cells with TransFast transfection reagent. Twenty-four hours after transfection, stable transfectants were selected in gentamicin (G418; Invitrogen, Carlsbad, CA) at a concentration of 600 µg/mL. Thereafter, the selection medium was replaced every 3 days. After 2 weeks of selection in G418, clones of resistant cells were isolated and allowed to proliferate in medium containing G418 at 100 µg/mL.
Flow cytometry detection of ICAM-1 expression. ICAM-1 expression was determined by flow cytometry using an anti-ICAM-1 antibody and analyzed on a FACScalibur with Cellquest software.
Cell growth assays. A549/ICAM-1 cells were grown under 0.1% serum DMEM, and cell growth was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Sigma, St. Louis, MO) colorimetric method.
In vitro invasion assay. The invasion assay was carried out using Transwell cell culture chambers (Corning Costar 3422, Corning, Cambridge, MA) as previously described (9). Briefly, polyvinylpyrrolidone-free polycarbonate filters (8.0-µm pore size, Nuclepore, Pleasanton, CA) were precoated with 5 µg Matrigel (BD Biosciences, Bedford, MA) on the upper surface. A549 cells were harvested with 1 mmol/L EDTA and then resuspended in DMEM supplemented with 0.1% fetal bovine serum. Cell suspensions (104 cells) were added to the upper compartment of the chamber. Cells were challenged with TNF following pretreatment with thalidomide for 30 minutes. After 24-hour incubation, the top side of the insert membrane was scrubbed free of cells with a cotton swab and the bottom side was fixed with 3.7% paraformaldehyde, stained with 0.5% crystal violet in 20% methanol. The crystal violet dye retained on the filters was extracted with DMSO and colorimetrically assessed by measuring its absorbance at 590 nm on an ELISA reader (Bio-Tek, Birmingham, United Kingdom).
In vivo metastasis assay. A549/Mock and A549/ICAM-1 cells were resuspended in PBS. Subsequently, 5 x 106 cells in 0.1 mL of PBS were injected into the lateral tail vein of 6-week-old nude mice. In anti-ICAM-1 antibody experiment, mice were randomly divided into two groups: The control group was i.v. injected with PBS, whereas the other group was i.v. injected with anti-ICAM-1 antibody (0.8 mg/g) on the 1st and 8th day. In thalidomide experiment, mice were randomly divided and orally received either vehicle (soy oil) or thalidomide (200 mg/kg/d) suspended in soy oil everyday. Mice were killed after 2 weeks, and all organs were examined for metastasis formation. The lungs were removed and fixed in 10% formalin. The number of lung tumor colonies was counted. Representative lung tumors were removed, fixed, and embedded in paraffin. Embedded tissue was sectioned into 4-µm sections, and the sections were stained with H&E for histologic analysis. All animal work was done under protocols approved by the Institutional Animal Care and Use Committee of the College of Medicine, National Taiwan University.
Quantification of ICAM-1 expression. The level of cell surface ICAM-1 expression was determined by ELISA as described previously (20).
Reverse transcription-PCR. Total RNA was isolated from A549 cell using Trizol reagent (Life Technologies). Two micrograms of total RNA were reverse transcribed into cDNA using oligo(dT) primer, then amplified for 30 cycles using two oligonucleotide primers derived from published ICAM-1 or ß-actin sequence, including 5'-TGCGGCTGCTACCACAGTGATGAT-3' and 5'-CCATCTACAGCTTTCGGCGCCCA-3' (ICAM-1), or 5'-TGACGGGGTCACCCACACTGTGCCCATCTA-3' and 5'-CTAGAAGCATTTGCGGG GACGATGGAGGG-3' (ß-actin). The PCR products were subjected to 1.5% agarose gel electrophoresis.
Transient transfection. The vectors (pIC339, B, and ß-galactosidase reporters) were transiently transfected into A549 cells with Tfx-50 transfection reagent (Promega, Madison, WI). Briefly, 2 µg of plasmid DNA and 1 µg of ß-gal and 3 µL transfection reagents were mixed, and the transfection protocol was carried out according to the manufacturer's instructions (Promega). Six hours after transfection, the cells were cultured in normal complete medium for another 16 hours. The transfected cells were subjected to luciferase assay.
Electrophoretic mobility shift assay. Oligonucleotides corresponding to the consensus sequences of NF-B (5'-AGCTTGGAAATTCCGGA-3') and of the human ICAM-1 promoter (5'-TTCCGGAATTTCCAAGCT-3') were synthesized, annealed, and end labeled with [-32P]ATP using T4 polynucleotide kinase. Electrophoretic mobility shift assay was done as previously described (20).
Chromatin immunoprecipitation assay. Chromatin immunoprecipitation assay analysis was done as described (21). Immunoprecipitated DNA was purified, resuspended in H2O, and subjected to PCR. To amplify the regions of ICAM-1 promoter, PCR was done with the following pair of primers: 5'-AGACCTTAGCGCGGTGTAGA-3' and 5'-GCGACTCGAGGAGACGATGA-3'. PCR products were then resolved by 1.5% agarose-ethidium bromide gel electrophoresis and visualized by UV.
Western blot analysis. Following treatment with thalidomide, total cell lysates or nuclear extracts were prepared and subjected to SDS-PAGE using 10% polyacrylamide gels as described previously (12).
Immunofluorescence staining. A549 cells, grown on coverslips, were pretreated with 1 µmol/L thalidomide for 30 minutes before incubation with 10 ng/mL TNF- for 60 minutes. The immunofluorescence staining was done as previously described (12).
Animal xenograft assay. Four- to 6-week-old female BALB/c nude mice were injected with 107 A549 cells (suspended in 0.1 mL PBS and mixed with 0.1 mL Matrigel) in the rear left flank. Two weeks after administration, 100 to 200 mm3 tumors were apparent on all mice. At this time, animals were divided into two groups (n = 6), and orally received either control vehicle (soy oil) or thalidomide (200 mg/kg/d) suspended in soy oil. Mice were treated everyday for 30 days with thalidomide or control vehicle, and tumor growth was measured twice per week with calipers. Tumor volume was calculated using the formula V (mm3) = 0.52 x [ab2], where a is the length and b is the width of the tumor.
Statistical analysis. All results were presented as mean ± SE. The two-tailed Student's t test was used to calculate the statistical significance between groups. Pearson 
2 test was used to compare the clinicopathologic characteristics of patients with high ICAM-1 and low ICAM-1 expression. ANOVA test was used to compare the growth rate and tumor growth.
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Results |
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Inhibitory effect of thalidomide on TNF-–induced cell invasion and ICAM-1 expression. Previous study had found that ICAM-1 expression induced by TNF- elicited cell invasion (9); therefore, the effect of thalidomide on TNF-–induced ICAM-1–dependent cell invasion was also examined. As shown in Fig. 3A
, thalidomide inhibited TNF-–induced cell invasion in a dose-dependent manner. It also inhibited TNF-–induced ICAM-1 protein and mRNA expressions (Fig. 3B and C). To elucidate the molecular mechanism, transient transfection was done using a human ICAM-1 promoter-luciferase construct, pIC339. Thalidomide inhibited TNF-–induced ICAM-1 promoter activity in a dose-dependent manner (Fig. 3D).
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–stimulated NF-B activity and binding to the ICAM-1 promoter. Because NF-B is critical for TNF-–induced ICAM-1 expression (13), the effect of thalidomide on NF-B activation was examined. The NF-B luciferase activity and NF-B–specific DNA-protein binding induced by TNF- were inhibited by thalidomide in a dose-dependent manner (Fig. 4A and B
). Chromatin immunoprecipitation assay showed that the in vivo binding of p65 to the ICAM-1 promoter was also inhibited by thalidomide (Fig. 4C).
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–stimulated NF-B activity was inhibited by thalidomide, its effect on the IKK/IB/NF-B pathway was further examined. When cells were treated with TNF- for 5, 10, 30, or 60 minutes, phosphorylation of IKK was seen after a 5-minute treatment and was sustained for 10 minutes (Fig. 5A, lanes 2-5
). These events were inhibited by thalidomide (Fig. 5A, lanes 6-9).
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B. Therefore, Ser536 phosphorylation was examined using a specific anti-p65 phospho-Ser536 antibody. TNF-–induced p65 phosphorylation was seen at a 5-minute treatment and was sustained for 60 minutes (Fig. 5A, lanes 2-5); this effect was also inhibited by thalidomide (Fig. 5A, lanes 7-9). The dose-response effect of thalidomide on the phosphorylation of IKK and p65 was further examined. As shown in Fig. 5B, TNF-–induced IKK and p65 phosphorylations were both inhibited by thalidomide (Fig. 5B, lanes 3-5).
The translocation of NF-B to the nucleus is tightly regulated by its interaction with the inhibitory IB; we further examined the nuclear translocation of p65 using Western blot and immunofluorescence. As shown in Fig. 5C, translocation of p65 to the nucleus was seen after treatment with TNF- for 60 minutes (Fig. 5C, lane 2), but was not inhibited by 1 to 40 µmol/L thalidomide (Fig. 5C, lanes 3-5). Similar results were also shown by immunofluorescence (Fig. 5D).
Inhibitory effect of thalidomide on tumor growth of lung cancer xenograft and on metastasis. To further investigate the effect of thalidomide on the progression of lung cancer and metastasis, A549 cells were introduced into nude mice via s.c. administration. Fifteen days after injection, tumor formation of 
100 mm3 was seen in all mice. Mice were then randomly selected for oral administration of thalidomide (200 mg/kg/d). As shown in Fig. 6A and B
, a dramatic increase in tumor size and weight in nude mice on the 45th day after A549 cells injection was seen. The volume of tumor approached 2,000 mm3 and the weight of tumor reached 1.85 g. After oral administration of thalidomide qd for 30 days, the tumor growth was inhibited by 63% and the tumor weight decreased to <1 g. No toxicity was found in mice treated with thalidomide.
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To evaluate the inhibition of thalidomide on angiogenesis and tumor formation, the expression of vascular endothelial growth factor and ICAM-1 protein in the excised tumors was examined by Western blotting. As shown in Fig. 6E, the expression of vascular endothelial growth factor and ICAM-1 was elevated in excised tumors and was abolished after thalidomide treatment, further demonstrating the involvement of ICAM-1 in carcinogenesis.
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Discussion |
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B activation. ICAM-1 is up-regulated in response to a variety of cytokines and is associated with inflammatory and immune responses (23). In addition to its role in leukocyte adhesion and cancer cell invasion (9, 12), several lines of evidence in this study show that ICAM-1 plays an important role in tumorigenesis and metastasis. First, ICAM-1 protein expression was higher in the specimens with advanced lung cancer, especially those in stages III and IV. Second, tumors excised from nude mice s.c. injected with A549 lung cancer cells showed high expression of ICAM-1. Third, i.v. injection of A549/ICAM-1 cells into the nude mice induced more metastatic lung nodules than A549/Mock cells. Fourth, A549/ICAM-1 cells induced more invasion than A549/Mock. Cell invasion and tumor metastasis induced by A549/ICAM-1 cells were inhibited by the specific anti-ICAM-1 antibody. However, the inhibition of the in vitro cell invasion was greater than that of the in vivo metastasis. It is probable that more factors, such as stability and distribution, might be involved in the effect of the in vivo application of anti-ICAM-1 antibody. Although ICAM-1 has been reported to be associated with myeloma and breast cancer cell invasion, we provide the direct evidence that ICAM-1 is required for lung cancer formation and metastasis. Adhesion molecules expressed in cancer cells can attract inflammatory cells such as macrophages or lymphocytes, which release trophic factors to enhance cancer cell survival and facilitate instability of tumor environment (24, 25). For example, tumor-associated macrophage, the major component of the infiltrating inflammatory cells, had been reported to release growth and angiogenic factors that stimulate tumor cell proliferation, promote angiogenesis, and even favor invasion and metastasis (26, 27). In this study, human lung cancer with higher ICAM-1 expression aggregated more leukocytes and macrophages,4 suggesting the dependence on ICAM-1 for the recruitment of inflammatory cells by tumor cells. This ICAM-1–mediated leukocyte accumulation may be a potential trigger for tumorigenesis.
Cancer cells use adhesion mechanisms to aid its migration and homing to distant metastatic sites (25). A549 cells overexpressing ICAM-1 were established to show the direct role of ICAM-1 in the in vitro cell invasion and in vivo tumor metastasis. These results were further strengthened by the clinical characteristics of human lung carcinoma—high ICAM-1 expression was associated with more metastatic nodules, lymph node metastasis, and poor differentiation (Table 1). According to the logic regression model to analyze multiple outcomes in 40 lung adenocarcinoma patients, ICAM-1 might indirectly affect tumor status and could predict lung cancer patients' tumor status and prognosis. Hence, ICAM-1 might be a potential marker for the evaluation of the outcome of lung cancer patients. Metastasis is a highly organized process (28). The first phase consists of changes in tumor cell adhesion, induction of cell motility, and invasion to local sites, followed by dissemination to regional lymph nodes or circulation and homing to secondary organs (29, 30). Some metastatic cells may eventually become precursors of secondary tumors that arise years after resection of the primary tumor (31). In the present study, we first showed that in vivo metastasis was induced by ICAM-1–overexpressing A549 cells. ICAM-1 can be an important target for metastasis and might develop as a new biomarker for metastatic lung cancer. The mechanism of ICAM-1–mediated metastasis was probably linked to its outside-in signaling. It has been shown that ICAM-1 can initiate a calcium-mediated signal in breast cancer cells (32). ICAM-1 engagement has also been documented to activate the oncogenic mitogen-activated protein kinase/extracellular signal-regulated kinase cascade through phosphorylation, resulting in activator protein-1 activation and production of cytokine, such as interleukin-8 and RANTES; adhesion molecules (ICAM-1 and vascular cell adhesion molecule-1); matrix metalloproteases; and reactive oxygen species (33–36).
Thalidomide has been shown to be a first-line or salvage therapy in patients with multiple myeloma (16). Chemotherapy (cisplatin, cyclophosphamide, epirubicin, and etoposide) with or without thalidomide for lung cancer on clinical trails has been reported (37). Our study on preclinical trial addresses the efficiency of thalidomide in lung adenocarcinoma, a subtype of non–small cell lung cancer. Several studies have reported that thalidomide reduces vascular endothelial growth factor expression in tumor tissues, leading to a dramatic decrease in vascular density and permeability, and an increase in tumor cell apoptosis (38). Our results also showed that thalidomide inhibits the overexpression of vascular endothelial growth factor in tumors excised from nude mice. However, two lines of evidence showed that the anticancer effect of thalidomide was associated with the inhibition of ICAM-1. First, A549/ICAM-1–induced in vitro cell invasion and in vivo metastasis were inhibited by thalidomide. Second, high expression of ICAM-1 in tumors excised from nude mice s.c. injected with A549 cells disappeared when treated with thalidomide. To our knowledge, this is the first report linking thalidomide and ICAM-1 in tumorigenesis. The inhibition of tumor growth and metastasis by thalidomide in a preclinical lung cancer xenograft model supports the finding that thalidomide may be a potential chemotherapeutic agent for lung cancer. NF-B is crucial for TNF-–induced ICAM-1 expression (13). Thalidomide was shown to attenuate the phosphorylation of IKK and p65, and to inhibit the binding of NF-B to the ICAM-1 promoter. CYL-19s and CYL-26z, two structurally related -methylene--lactone compounds, were also identified by this laboratory to show inhibition of TNF-–induced ICAM-1 expression and ICAM-1–elicited cell invasion (9). Their mechanism of action was shown to be inhibition of the TNF-–induced IB phosphorylation and degradation, and NF-B activation via direct targeting of the IKK complex. Although both thalidomide and -methylene--lactone compounds have inhibitory effect on NF-B activation and ICAM-1 expression, their chemical structures are different. Moreover, -methylene--lactone compounds are IKK inhibitors that inhibit IB degradation (9); however, thalidomide did not inhibit IB degradation (data not shown) and p65 translocation (Fig. 5C and D).
In summary, ICAM-1 is a molecular target for lung cancer. Its role in tumorigenesis, including cancer formation and metastasis, was shown. New insight on how thalidomide suppression of ICAM-1–dependent metastasis and of NF-B activation is effective in cancer therapy was also shown. ICAM-1 might be developed as new biological marker for the diagnosis of lung cancer. These studies provide a framework for targeting ICAM-1 as a biologically based therapy for cancer.
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Footnotes |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 6/ 8/06; revised 8/14/06; accepted 9/12/06.
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pathway. Carcinogenesis 2004;25:1805–12.-methylene--butyrolactone derivatives. Carcinogenesis 2004;25:1925–34.(v)ß3 integrin and intercellular adhesion molecule-1 expression in cutaneous malignant melanoma lesions. Cancer Res 1997;57:1554–60.-induced epithelial ICAM-1 expression and monocyte adhesion. Involvement of protein kinase C-dependent c-Src tyrosine kinase activation pathway. J Biol Chem 2002;277:7118–26.-induced activation of downstream NF-B site of the promoter mediates epithelial ICAM-1 expression and monocyte adhesion. Involvement of PKC, tyrosine kinase, and IKK2, but not MAPKs, pathway. Cell Signal 2001;13:543–53.[CrossRef][Medline] but not p44/42 mitogen-activated protein kinase, p38, or c-Jun NH(2)-terminal kinase is required for intercellular adhesion molecule-1 expression mediated by interleukin-1ß: involvement of sequential activation of tyrosine kinase, nuclear factor-B-inducing kinase, and IB kinase 2. Mol Pharmacol 2000;58:1479–89.-induced up-regulation of intercellular adhesion molecule-1 (ICAM-1) in respiratory epithelial cells through activator protein-1 and nuclear factor-B: structure-activity relationships. Mol Pharmacol 2004;66:683–93.B-independent mechanism. J Immunol 1999;162:2990–6.This article has been cited by other articles:
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