Cancer Letters

Cancer Letters

Volume 253, Issue 2, 18 August 2007, Pages 249-257
Cancer Letters

Negative growth control of osteosarcoma cell by Bowman–Birk protease inhibitor from soybean; involvement of connexin 43

https://doi.org/10.1016/j.canlet.2007.01.021Get rights and content

Abstract

Bowman–Birk protease inhibitor (BBI) from soybean acts as a potential chemopreventive agent in several types of tumors. However, the mechanism is still unclear. The present study was undertaken to estimate a mechanism of BBI-dependent negative growth control of human osteosarcoma cell (U2OS cell). BBI had negative growth control of the cells via induction of connexin (Cx) 43, a tumor suppressor gene in U2OS cells. This negative growth control by BBI was abrogated under down-regulation of Cx43 induced by a Cx43 antisense nucleotide treatment. It was also found that the BBI-dependent induction of Cx43 was due to elevation of Cx43 mRNA and stabilization of Cx43 protein. Especially, BBI-dependent inhibition of chymotrypsin-like activity in proteasome contributed to stabilization of Cx43 protein. These results suggest that a major negative growth effect of BBI is based on the restoration of Cx43 expression in U2OS cells.

Introduction

Epidemiological studies indicate that diets containing high amounts of soybean products contribute to low cancer incidence and low mortality rates [1], [2]. Several components isolated from soybeans such as isoflavones and protease inhibitors are intensively studied as cancer chemopreventive agents [3]. Recently, protease inhibitors have been developed as a class of well-established cancer chemopreventive agents because of their strong anticarcinogenic activity in vivo and in vitro in cancer model systems [4], [5]. The most predominant protease inhibitor in soybean is the Bowman–Birk inhibitor (BBI) [6]. BBI, a 71-amino acid protein (∼8 kDa) and a serine protease inhibitor with both trypsin and chymotrypsin inhibitory activities, has been shown to be a valid suppressor of carcinogenesis in a human phase IIa clinical trial [7], [8], [9]. Interestingly, BBI is the only protease inhibitor derived from soybeans that possesses a chymotrypsin inhibitory activity which might contribute to its anticarcinogenic activity, whereas trypsin inhibitory activity is not essential [10], [11], [12]. Although BBI has a broad spectrum of cancer-protective activities [13], [14], [15], knowledge of the exact mechanism(s) by which BBI exerts its anticarcinogenic effects remains limited.

Among the different types of cell-cell interaction in mammalian cells, gap junctional intercellular communication (GJIC) is considered to be the only route allowing freely direct transfer of ions and hydrophilic molecules of up to 1000–1500 Da in size between cells, thereby maintaining the electrical and metabolic cell homeostasis [16], [17], [18]. The gap junction is made up of juxtaposed transmembrane hemichannels (connexons) provided by adjacent cells, and each connexon consists of six individual transmembrane proteins called connexin (Cx) [19], [20]. In general, it is well known that the Cx gene acts as a tumor suppressor gene by maintaining homeostatic control in multicellular organisms via GJIC. Additionally, it has been reported that an important chemopreventive effect of several components depends on maintaining and/or restoring expression and function of Cx gene during carcinogenic process [21]. Thus, it is reasonable that Cx gene is an effective target for cancer prevention [22].

In a recent study, we have reported that BBI suppresses the development of M5076 ovarian sarcoma in mice and induces elevation of Cx43 protein in the same sarcoma [23]. However, we have not confirmed that the increase of Cx43 is directly associated with the suppression of sarcoma development recorded in the study [23]. In general, it has been well known that Cx43 acts as a tumor suppressor gene in non-epithelial tumors such as sarcoma [24]. In this context, the present study was undertaken to estimate if the restoration of Cx43 expression by BBI could contribute to negative growth control of sarcoma cells. Since it is clearly demonstrated that Cx43 acts as a tumor suppressor gene in a human osteosarcoma cell (U2OS cell) [25], we utilized this cell line in this study.

Section snippets

Chemicals

BBI, Lucifer yellow CH, MG132 and cycloheximide were obtained from Sigma (St Louis, MO, USA). Protein A-Sepharose was from Amersham Biosciences (Piscataway, NJ, USA). Anti-Cx43 antibody was from Zymed Laboratories (San Francisco, CA, USA). Anti-ubiquitin antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Other chemicals and other antibodies were purchased from Wako Pure Chemicals (Osaka, Japan) and BD Transduction Laboratories (Lexington, KY, USA), respectively.

Cell culture and treatment

Human

Negative growth control of U2OS cells by BBI

To investigate whether BBI has negative growth effect on U2OS cells, we examined several markers related to cell growth and tumorigeneic phenotype. As shown in Fig. 1a, cell growth measured as cell viability was significantly inhibited by BBI in a dose-dependent manner. Similarly, BBI suppressed saturation density in U2OS cells, and the suppressive effect was the same as that in Cx43-expressed U2OS cells (Fig. 1b). This result suggested that BBI restored contact inhibition in U2OS cells. In

Discussion

From a recent output in phase IIa clinical trial, BBI shows promise of becoming an effective nontoxic chemopreventive agent against human cancers [8], but the exact mechanism by which BBI exerts its anticarcinogenic effects are still unclear at present. In this study, we have indicated that restoration of Cx43 expression contributes to negative growth control of human osteosarcoma cells by BBI.

It has been widely known that BBI has negative growth control for various types of tumor cells in vitro

Acknowledgements

We thank Miss K. Fukumoto for her excellent technical assistance. Also, we thank Dr. Ikuo Morita for supplying U2OS cells and Cx43-expressed U2OS cells. This study was supported by Japan Health Sciences Foundation (KH21012) and Fuji Foundation for Protein Research.

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