Adenovirus-uteroglobin suppresses COX-2 expression via inhibition of NF-κB activity in lung cancer cells

Summary

Uteroglobin (UG, Clara cell secretory protein) is a steroid inducible, multifunctional protein that is secreted by the mucosal epithelia. UG has anti-proliferative and anti-metastatic effects in cancer cells. COX-2, which catalyzes the first step in the synthesis of prostanoids, has been shown to be overexpressed in tumors. This study investigated the effect of UG on the inhibition of COX-2 expression in lung cancer cells. The level of the COX-2 protein and its mRNA were decreased by UG, as demonstrated by Western blot and the RT-PCR, respectively. The EIA shows that UG suppressed PGE2 synthesis. Western blot showed that the NF-κB nuclear translocation was inhibited by the transduction of UG. In addition, an EMSA demonstrated the inhibition of the NF-κB-DNA binding by UG. The luciferase assay showed that UG also inhibited the NF-κB-mediated transcription activity. Furthermore, transfection of the lung cancer cell lines with the COX-2 reporter gene constructs demonstrated that the transcription of COX-2 gene was suppressed by UG. These results show that the inhibition of COX-2 expression by UG transduction correlated with the suppression of NF-κB activity in the lung cancer cells. This suggests that UG have the possibility for the treatment of lung cancer.

Introduction

Uteroglobin (UG, Clara cell secretory protein) is a steroid-inducible and low molecular mass (15.8 kDa) protein, which is synthesized by the mucosal epithelia from many organs, including those of the thymus, pituitary gland, respiratory and gastrointestinal tracts, and prostate and mammary gland [1]. UG was first discovered in the rabbit uterine fluids, as a critical element that has a stimulatory effect on the growth of blastocysts in early pregnancy, and was named blastokinin [2]. Since then, many different names based on the tissue or body fluid in which UG was detected, or molecules it interacted with, such as retinol and progesterone, has been ascribed to this protein. For this reason, UG is also known as the Clara cell secretory protein, lacryglobin, urine protein-1, retinol-binding protein and progesterone-binding protein [3]. This confusing nomenclature has led to a consensus for a new generic family name. Secretoglobin has been officially approved as the new generic name referring to the uteroglobin-like molecules with common characteristics, including secretion, small size, alpha-helical and dimeric structure [4]. Secretoglobin exhibits potent anti-inflammatory and immunomodulatory activity by its ability to inhibit the activity of phospholipase A2 [5], [6]. Normal bronchial epithelia express the UG gene at a high level. However, neither an adenocarcinoma nor cell lines express it at a markedly reduced level [7]. In addition, the incidence of cancer in UG-knockout mice is known to be extremely high. The induced expression of UG in some cancer cells appears to result in the loss of their transformed phenotype and suppression of metastasis [3], [8]. Overall, these results suggest that the loss of UG gene expression may be a common characteristic of cancer cells, and UG may act as a tumor suppressor.

Cyclooxygenase (COX), which is also known as prostaglandin (PG) H synthase (EC 1.14.99.1), catalyzes the rate-limiting steps in the formation of the PG endoperoxides [9]. Three isoforms of COX have been described. COX-3 is two smaller COX-1-derived proteins (partial COX-1 or PCOX-1 proteins). Cyclooxygenase-1 (COX-1) is expressed constitutively in most tissues, whereas cyclooxygenase-2 (COX-2), the other isoform, is not expressed under physiological conditions in most organs but is induced by cytokines and mitogens during the inflammatory process [10], [11]. The overexpression of COX-2 has been observed in many tumors [12], increasing the invasiveness of the malignant cells and inducing the formation of new blood vessels within the tumors [13], [14]. The COX-2 products, prostaglandins also contribute to tumor growth by inhibiting apoptosis [15]. Recent studies have demonstrated that an increased COX-2 expression level is observed frequently in human non-small cell lung cancer (NSCLC), and the elevated biosynthesis of the PGs has been identified in the NSCLC cell lines [16], [17]. Recent works also indicated that aspirin, a nonsteroidal anti-inflammatory drug (NSAID) and a nonselective COX-2 inhibitor, may inhibit the proliferation of the NSCLC cell lines and might reduce the number of lung adenoma induced by the tobacco-specific nitrosamine 4-(methynitro-samino)-1-(3-pyridyl)-1-butanone [18]. The COX-2 gene has been cloned, and there are two transcription nuclear factor-κB (NF-κB) consensus sites on the promoter region [19]. NF-κB exists in a latent form in the cytoplasm of non-stimulated cells. Following stimulation with a cytokine, the NF-κB is activated and then translocated into the nucleus [20]. The NF-κB pathway is involved in the important transcriptional regulation of immunity, inflammation and carcinogenesis [21].

The UG and COX-2 inhibitors have common features that inhibit proliferation, angiogenesis and metastasis. The aim of this study was to investigate the effect of adenovirus–uteroglobin (ad-UG) on the inhibition of COX-2 expression in lung cancer cells. The NF-κB pathway was assumed to be the mechanism of this effect.