E-cadherin decreased human breast cancer cells sensitivity to staurosporine by up-regulating Bcl-2 expression

doi:10.1016/j.abb.2008.10.021

Archives of Biochemistry and Biophysics

Volume 481, Issue 1, 1 January 2009, Pages 116-122

https://doi.org/10.1016/j.abb.2008.10.021 Get rights and content

Abstract

E-cadherin, a well-characterized cell–cell adhesion molecule, executes multifunction roles on cell behaviors. However, its effect on chemo-resistance remains controversial. In this study, we found that E-cadherin positive breast cell lines were less sensitive to staurosporine compared to E-cadherin negative ones. Next, we substantiated that the expression of E-cadherin in MDA-MB-435 cells could partly counteract the cytotoxic effect induced by staurosporine through a series of apoptosis assay. The resistance of E-cadherin over-expressing cells to staurosporine may due to the up-regulation of Bcl-2/Bax ratio. When E-cadherin interference plasmids were transfected into MCF-7 cells, Bcl-2 expression was down-regulated. Moreover, perturbation of E-cadherin function by blocking the cell–cell contact resulted in decreased cellular levels of Bcl-2 protein expression. All these results demonstrated the chemo-resistance function of E-cadherin in the condition of staurosporine treatment, therefore, might contribute effective therapeutic strategies in breast carcinoma.

Introduction

Apoptosis, a type of programmed cell death, is an evolutionarily conserved biochemical pathway in controlling cell suicide that plays an essential role in regulating normal development and homoeostasis in multicellular organisms [1]. Induction of apoptosis is considered to be the underlying mechanism that accounts for the efficiency of most chemotherapy drugs. Staurosporine (STS)², a potent protein kinase C inhibitor with a broad spectrum of activity, is used in vitro as an initiator of apoptosis in a wide variety of cell types. Nevertheless, STS has not been used as a clinical chemotherapy drugs until now. The potential factors that influence the STS-induced apoptosis remain largely unknown.

Generally, it is believed that a mitochondrial pathway plays a critical role in STS-induced apoptosis [2]. This involves release of mitochondrial apoptotic proteins such as cytochrome c, apoptosis-inducing factor (AIF) and second mitochondrial-derived activator of caspase (SMAC) [3], [4], [5]. On release, cytochrome c interacts with apoptotic proteinase-activating factor-1 and pro-caspase-9 to form apoptosomes. The latter activates caspase-9 and downstream effector caspases such as caspase-3 that are responsible for apoptotic destruction of the cells [6]. The anti-apoptotic Bcl-2 family members such as Bcl-2, Bcl-X_L and MCL1 appear to preserve the integrity of outer mitochondrial membrane [7], [8]. Over-expressions of Bcl-2 and Bcl-X_L inhibit mitochondria-dependent pathway to apoptosis in different kinds of cells, thus protecting those cells from killing by chemotherapeutic agents [9], [10]. And reduction of Bcl-2 levels increases the activity of chemotherapy against many tumor types in vitro and in vivo[10], [11], [12], [13], [14]. As such, the proto-oncogene Bcl-2 is thought to be directly associated with cellular transformation and resistance chemotherapy [9], [15].

E-cadherin, a well-characterized cell adhesion molecule, is essential for cell–cell adhesion, which in turn regulates various aspects of cell fate including developmental decisions, cellular differentiation and possibly cell survival [16], [17]. Just as integrins’ function to mediate cell–extracellular matrix interactions in anchorage-dependent survival, cadherins may also act in such a capacity, possessing a functional role in the regulation of intercellular adhesion-dependent survival. Several studies have reported the association between E-cadherin-mediated aggregation and survival of carcinoma cells [18]. However, the actual mechanism by which cadherins mediate these signals is not known. As a hallmark of tumor progression, E-cadherin expression varies in a larger range in different types and stages of tumor [19], [20]. It would be much helpful for the applications of chemotherapy if the relationship between the E-cadherin expression level and the susceptibility of tumor cells to chemotherapy drugs be clarified.

We investigated in this report that E-cadherin over-expressing breast cancer cells were less sensitive to STS, thereby reducing the effectiveness of chemotherapy. The mechanism by which E-cadherin protected cells from STS-induced apoptosis was due to the up-regulation of Bcl-2.

Section snippets

Cell culture

The human breast carcinoma cell lines MDA-MB-435, MDA-MB-231, T47D and MCF-7 were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% new-born bovine serum at 37 °C, at 5% CO₂.

MTT cell viability assay

Cell proliferation and viability were quantified using the methyl thiazol tetrazolium (MTT) cell proliferation reagent (Roche Diagnostics) according to the manufacturer’s protocol. Cells were seeded at a density of 2 × 10⁵ cells/ml into 96-well plates and incubated for 12 or 24 h in completed medium

Effect of STS on the growth inhibition of E-cadherin negative and positive breast cancer cells

To study the effect of STS on human breast cancer cells, E-cadherin negative cell lines (MDA-MB-435 and MDA-MB-231) and E-cadherin positive cell lines (MCF-7 and T47D) were treated with various doses of STS for 24 h, and assayed for growth inhibition. As shown in Fig. 1, STS treatment resulted in a loss of cell viability in a dose-dependent manner in each cell line. Among four breast carcinoma cell lines, MDA-MB-435 was the most sensitive cell line towards STS with IC₅₀ value of 0.56 μmol/L,

Discussion

The current work was done to evaluate that E-cadherin homophilic adhesion could initiate anti-apoptotic signaling by enhancing the Bcl-2 expression. Inhibition of homophilic binding of the E-cadherin extracellular domain correlated with decreased cellular levels of Bcl-2 protein expression. This work confirmed the chemo-resistance function of E-cadherin, therefore, might contribute effective therapeutic strategies in breast carcinoma.

As a critical epithelial cell adhesion molecule, E-cadherin

Acknowledgments

This investigation was supported by Shanghai Leading Academic Discipline Project (Project No. B110). We thank Dr. Cara J. Gottardi for providing the pcDNA3-E-cadherin constructs.

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^☆: This investigation was supported by Shanghai Leading Academic Discipline Project (Project No. B110).

¹: These authors are equally contributed to this work.

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E-cadherin decreased human breast cancer cells sensitivity to staurosporine by up-regulating Bcl-2 expression☆

Abstract

Introduction

Section snippets

Cell culture

MTT cell viability assay

Effect of STS on the growth inhibition of E-cadherin negative and positive breast cancer cells

Discussion

Acknowledgments

Trends Cell Biol.

Cell

Cell

Adv. Pharmacol.

Eur. J. Surg. Oncol.

Lancet

Blood

J. Biol. Chem.

Am. J. Pathol.

Biochem. Biophys. Res. Commun.

Curr. Opin. Cell Biol.

Exp. Cell Res.

Exp. Cell Res.

J. Biol. Chem.

Science