Miyako Takata

Antibody-Dependent Cellular Cytotoxicity Mediated by Cetuximab against Lung Cancer Cell Lines

Purpose: Epidermal growth factor receptor (EGFR) is commonly overexpressed in lung cancer. Cetuximab is a chimeric mouse-human antibody targeted against EGFR. Compared with its inhibitory properties, its immunologic mechanisms have not been well studied. In this study, we investigated the antibody-dependent cellular cytotoxicity (ADCC) activity of cetuximab against lung cancer cell lines. Experimental Design: We studied the correlation between EGFR expression in lung cancer cell lines and the ADCC activity of cetuximab as well as the influence of interleukin-2 and chemotherapy on the ADCC activity. EGFR expression was measured by a quantitative flow cytometric analysis and immunohistochemistry. The ADCC activity was assessed by a 4-h 51Cr release assay. Peripheral blood mononuclear cells, purified T cells, natural killer (NK) cells, and monocytes from healthy donors or lung cancer patients were used as effector cells. Results: Fresh peripheral blood mononuclear cells exhibited cetuximab-mediated ADCC activity against lung cancer cell lines at a low concentration of cetuximab (0.25 μg/mL). A logarithmic correlation was observed between the number of EGFRs and ADCC activity. Even low EGFR expression, which was weakly detectable by immunohistochemistry, was sufficient for maximum ADCC activity, and further increases in EGFR expression on the target cells had no further effect on the ADCC activity. In addition, ADCC activity was enhanced by interleukin-2 mainly through activation of NK cells and was less susceptible to immunosuppression by chemotherapy than NK activity in lung cancer patients. Conclusions: These observations suggest the importance of ADCC activity as an immunologic mechanism of cetuximab in biological therapy for lung cancer patients.

Dexamethasone influences human clock gene expression in bronchial epithelium and peripheral blood mononuclear cells in vitro.

We determined whether human peripheral blood mononuclear cells (PBMCs) could be used to analyze clock genes by studying their mRNA expressions in human bronchial epithelium (BEAS‐2B) and PBMCs following stimulation by the glucocorticoid homologue dexamethasone (DEX) in vitro. PBMCs were obtained at 10:00 h from two diurnally active (∼07:00 to 23:00 h) healthy volunteers and were evaluated for hPer1 mRNA expression following DEX stimulation in vitro using real time‐PCR analysis. DEX stimulation of human BEAS‐2B cells and PBMCs in vitro led to a remarkable increase of hPer1 mRNA. The glucocorticoid rapidly affected the expression of hPer1 mRNA in PBMCs, suggesting that human PBMCs may be a useful surrogate marker for the investigation of drug effects on clock genes.

Clock gene dysfunction in patients with obstructive sleep apnoea syndrome.

Clock genes regulate mammalian circadian rhythms, and dysfunction of clock genes can contribute to various disorders. To investigate whether obstructive sleep apnoea syndrome (OSAS) influences clock gene function, the present authors examined Period1 (Per1) mRNA expression in vitro and in vivo. In eight healthy subjects and eight OSAS patients, plasma noradrenaline, serum interleukin (IL)-6, high-sensitivity C-reactive protein (hsCRP) and Per1 mRNA expression in peripheral whole blood were measured. Expression of Per1 mRNA in cultured cells was examined under IL-6 or noradrenaline stimulation in vitro. After noradrenaline was administered to mice in vivo, Per1 mRNA expression in the brain was examined. The concentrations of serum IL-6, hsCRP and plasma noradrenaline were elevated in OSAS patients, but improved by continuous positive airway pressure (CPAP) therapy. Per1 mRNA expression in the peripheral blood significantly decreased at 02:00 h by CPAP in OSAS patients. Stimulation with IL-6 did not directly induce Per1 mRNA in vitro. Administration of noradrenaline induced Per1 mRNA in the cerebral cortex of mice in vivo. The current study revealed that obstructive sleep apnoea syndrome caused clock gene dysfunction, and continuous positive airway pressure helped to improve it. Sympathetic activation and elevation of the plasma noradrenaline concentration in obstructive sleep apnoea syndrome may be one of the factors involved in disorders of Period1 mRNA expression.

Expression of functional leukotriene B4 receptors on human airway smooth muscle cells

BACKGROUND Leukotriene B4 (LTB4) increases in induced sputum and exhaled breath condensate in people with asthma. Furthermore, the T(H)2-type immune response and airway hyperresponsiveness induced by ovalbumin sensitization is markedly suppressed in LTB4 receptor (BLT) 1 null mice. These studies suggest that LTB4 may contribute to asthma pathophysiology. However, the direct effects of LTB4 on human airway smooth muscle (ASM) have not been studied. OBJECTIVES We sought to determine the expression of LTB4 receptors on human ASM and its functional role in mediating responses of human ASM cells, and the effect of LTB4 on these cells. METHODS Immunohistochemistry, RT-PCR, Western blotting, and flow cytometry were used to determine the expression of LTB4 receptors. To determine the effect of LTB4 on human ASM cells, cell proliferation was assessed by counting cells, and chemokinesis was assessed by gold particle phagokinesis assay. RESULTS We confirmed expression of both BLT1 and BLT2 in human ASM cells in bronchial tissue and in cell culture. LTB4 markedly induced cyclin D1 expression, proliferation, and chemokinesis of human ASM cells. LTB4 also induced phosphorylation of both p42/p44 mitogen-activated protein kinase (MAPK) and downstream PI3 kinase effector, Akt1. However, we observed no induction of c-Jun N-terminal kinase or p38 MAPK. Notably, LTB4-induced migration and proliferation of ASM cells were inhibited by the BLT1 specific antagonist, U75302, and by inhibitors of p42/p44 MAPK phosphorylation (U1026), and PI3 kinase (LY294002). CONCLUSIONS These observations are the first to suggest a role for a LTB4-BLT1 signaling axis in ASM responses that may contribute to the pathogenesis of airway remodeling in asthma.

Glucocorticoid administration increases hPer1 mRNA levels in human peripheral blood mononuclear cells in vitro or in vivo.

In mammals, biological clocks govern important physiological and behavioral circadian rhythms. Details of molecular mechanisms of the circadian oscillator have been studied in nocturnally active rodents (Okamura, 2004), but few reports have addressed clock gene expression in diurnally active humans (Bjarnason et al., 2001). Recently, human peripheral blood mononuclear cells (PBMCs) have been used as a surrogate marker for mRNAexpression in peripheral clock genes (Takata et al., 2002; Boivin et al., 2003; Kusanagi et al., 2004; Burioka et al., 2005). The greatest hPer1 transcription was observed in the morning (Takata et al., 2002; Kusanagi et al., 2004). However, these studies did not determine whether a drug could affect peripheral clock genes in human PBMCs in vivo. Here, we tested whether a glucocorticoid hormone analog, prednisolone, would induce the human clock gene hPer1 in human PBMCs in vitro and in vivo. According to reverse transcription–polymerase chain reaction (RT-PCR) analysis, both monocytes and lymphocytes increased hPer1 mRNA levels in response to prednisolone in vitro (Fig. 1). Figure 2 shows that 10 M prednisolone significantly increased hPer1 mRNA levels in both monocytes and lymphocytes, while hPer2 or hClock mRNA were not increased in vitro. We also evaluated the daily variation of clock gene mRNA expression in PBMCs in response to injection of prednisolone in vivo (see Appendix). On day 1, 3 healthy volunteers underwent blood sampling to determine clock gene expression by PBMCs at 0900, 1000, 1200, and 2100 h. In each subject, the value at 0900 h on the 1st day was taken as the baseline and was defined as 1. Relative hPer1, hPer2, and hClock mRNAlevels were measured at 0900, 1000, 1200, and 2100 h (Fig. 3 for hPer1, open circles). On day 2, the same subjects were injected with 20 mg of prednisolone at 0900 h to investigate the influence of the drug on expression (Fig. 3 for hPer1, filled circles). Baseline values (0900 h) were the same as those at 0900 h on day 1. Relative hPer1 mRNA expression was 0.89 ± 0.06 at 0900 h, with an increase at 1000 h (3.61 ± 0.66) that fell to 1.18 ± 0.78 at 1200 h and 0.42 ± 0.03 at 2100 h, apparently representing a prednisolone effect of limited duration. Relative hPer2 and hClock mRNA levels did not change relative to baseline (data not shown). Thus, the effect of prednisolone injection on clock gene expression was specific to hPer1. Three patients starting prednisolone therapy were also investigated. Each patient showed increased hPer1 mRNA expression after therapeutic prednisolone administration (Fig. 3B). Doses were higher, on average, than those used on healthy subjects (see Appendix). Expression of hPer2 and hClock mRNA were again not affected by prednisolone (data not shown). Multiple signaling pathways have been reported to elicit expression of mammalian clock genes (Balsalobre et al., 2000; Okamura, 2004). Glucocorticoids are a particularly potent stimulus for eliciting expression of clock genes in peripheral tissues. Le Minh et al. (2001) and Hida et al. (2000) suggested that the surge of Per1 mRNA accumulation was caused by glucocorticoid signaling via the glucocorticoid response element (GRE) consensus sequences on Per1. A previous study has reported that dexamethasone induced phase shifts in circadian gene expression in liver but not in the SCN, presumably because the glucocorticoid receptor is expressed in most peripheral cell types but not in SCN neurons (Balsalobre et al., 2000). We found that both in vitro and in vivo, a glucocorticoid hormone analog, prednisolone, could increase hPer1

β2-adrenoceptor agonists induce the mammalian clock gene, hPer1, mRNA in cultured human bronchial epithelium cells in vitro

The mammalian Per1 gene is one of the most important components of circadian clock function of the suprachiasmatic nucleus and peripheral tissues. We examined whether the β2‐adrenoceptor agonists, procaterol and fenoterol, induce human Per1 mRNA expression in human bronchial epithelium. The in vitro stimulation of β2‐adrenoceptor agonists in BEAS‐2B cells led to a remarkable increase in the level of hPer1 mRNA. Moreover, fenoterol or procaterol induced the phosphorylation of CREB in BEAS‐2B cells as verified by immunoblot analysis. β2‐adrenoceptor agonists induced human Per1 mRNA expression by the signaling pathways of cAMP‐CREB in BEAS‐2B cells.

Diagnostic and prognostic impact of serum‐soluble UL16‐binding protein 2 in lung cancer patients

UL16‐binding protein 2 (ULBP2) is one of the ligands for NKG2D (NKG2DL). ULBP2 expression is induced in transformed cells and is recognized by immune effector cells via the activating NKG2D immunoreceptor. Soluble forms of NKG2DL have been reported in the serum of patients with several types of cancer. The present study investigated the diagnostic and prognostic significance of serum‐soluble ULBP2 (sULBP2) in lung cancer patients. We used flow cytometry to evaluate the surface expression of NKG2DL by various lung cancer cells, while sULBP2 was measured using our original ELISA. In addition, the immunological effect of sULBP2 on peripheral blood mononuclear cells (PBMC) was examined by the 51Cr release assay. We found that ULBP2 was highly expressed and that the sULBP2 level was elevated in supernatants of cultured non‐small‐cell lung cancer (NSCLC) cells as well as in the serum of NSCLC patients. ULBP2 levels were especially high in squamous cell carcinoma (SQ) patients. Clinical stage IIIB and IV NSCLC patients with a sULBP2 level ≥8.7 pg/mL showed significantly shorter survival than patients with sULBP2 <8.7 pg/mL. In multivariate analysis, a sULBP2 level ≥8.7 pg/mL (hazard ratio [HR], 2.13; P = 0.038) and clinical stage IV (HR, 2.65; P = 0.019) were independent determinants of a poor outcome. As a possible mechanism, we demonstrated that sULBP2 directly suppresses the cytolytic activity of PBMC. In conclusion, ULBP2 is the most significant NKG2DL for lung cancer, and sULBP2 is useful in the diagnosis of SQ and as a prognostic indicator for patients with advanced NSCLC. (Cancer Sci, doi: 10.1111/j.1349‐7006.2012.02330.x, 2012)

Lack of AKT activation in lung cancer cells with EGFR mutation is a novel marker of cetuximab sensitivity.

Epidermal growth factor receptor (EGFR) mutation is the best marker of sensitivity to the EGFR tyrosine kinase inhibitor gefitinib, but a marker for the anti-EGFR antibody cetuximab has not been identified in lung cancer. The present study investigated markers for sensitivity to cetuximab. Sensitivity to cetuximab and gefitinib was compared with EGFR expression, EGFR and KRAS mutation, and EGFR gene copy numbers in lung cancer cell lines. We also studied the effect of these agents on the activation of EGFR, ERK, AKT, and STAT3 in cetuximab-sensitive and -resistant cell lines. We found one cetuximab-sensitive cell line with EGFR mutation among 19 lung cancer cell lines. Analysis of molecules downstream from EGFR revealed that AKT phosphorylation was suppressed in this cell line. Augmentation of AKT phosphorylation by transfection of a plasmid induced resistance to cetuximab. Acquisition of cetuximab resistance was associated with AKT activation in this cell line, while pharmacological inhibition of AKT markedly enhanced the growth inhibitory effect of cetuximab. Dephosphorylation of AKT in association with EGFR mutation is a candidate marker for sensitivity to cetuximab, and combined use of an AKT pathway inhibitor with cetuximab could be a novel therapeutic strategy for lung cancer.

Treatment with β2‐Adrenoceptor Agonist in Vivo Induces Human Clock Gene, Per1, mRNA Expression in Peripheral Blood

This study examined whether in vivo exposure to a β2‐adrenoceptor agonist, tulobuterol, induces human Period1 (hPer1) mRNA expression in cells from peripheral whole blood. In one experiment, oral tulobuterol was administered to five healthy volunteers at 22:00 h, while in another, a transdermally tulobuterol patch was applied to the same five subjects at 20:00 h. In each experiment, serum tulobuterol concentrations were measured at four time points, and total RNA was isolated from peripheral blood cells for determinations of hPer1 mRNA expression by real‐time polymerase chain reaction. Both the tulobuterol tablet and the transdermal patch increased hPer1 mRNA expression, suggesting that analyses of human peripheral blood cells could reliably represent peripheral clock gene mRNA expression in vivo.

Therapeutic antitumor efficacy of anti-epidermal growth factor receptor antibody, cetuximab, against malignant pleural mesothelioma

Epidermal growth factor receptor (EGFR) is commonly overexpressed in malignant pleural mesothelioma (MPM). Cetuximab is a chimeric mouse-human antibody targeted against EGFR and induces potent antibody-dependent cellular cytotoxicity (ADCC). The action of cetuximab against MPM cells has not been well studied. Therefore, in this study, we investigated the antitumor activity of cetuximab against MPM cell lines, particularly with respect to ADCC activity in vitro and in vivo. EGFR expression of MPM cells was measured by a quantitative flow cytometric analysis and immunohistochemistry. The effect of cetuximab on growth inhibition was assessed using a modified MTT assay. The ADCC activity was measured by a 4-h 51Cr release assay using fresh or IL-2-activated peripheral blood mononuclear cells. In vivo antitumor activity of cetuximab was evaluated using an orthotopic implantation mouse model. Cetuximab-mediated ADCC activity against MPM cells was observed at low concentration (0.25 mg/ml) and was enhanced by IL-2, whereas no direct effect on growth inhibition was detected. A logarithmic correlation was observed between the number of EGFRs on MPM cells and ADCC activity. Low EGFR expression on the MPM cells, which was weakly detectable by immunohistochemistry, was sufficient for maximum ADCC activity. In the mouse model, cetuximab treatment with or without IL-2 significantly inhibited intrathoracic tumor growth and prolonged their survival. Our study shows that cetuximab has potent anti-MPM activity both in vitro and in vivo, mainly through the immunologic mechanism of ADCC. Cetuximab has the potential to be used as a novel therapy for MPM patients.