Abstract
OBJECTIVE To study the expression levels of Twist and epithelial-mesenchymal transitions in multidrug-resistant MCF-7/ADR breast cancer cells, and to study the relationship between multidrug resistance (MDR) and metastatic potential of the cells.
METHODS RT-PCR, immunohistochemical and Western blotting methods were used to examine the changes of expression levels of the transcription factor Twist, E-cadherin and N-cadherin in the MCF-7 breast cancer cell line and its multidrug-resistant variant, MCF-7/ADR.
RESULTS In MCF-7 cells, the expression of E-cadherin can be detected, but there is no expression of Twist or N-cadherin. In MCF-7/ADR cells, E-cadherin expression is lost, but the expression of two other genes was significantly positive.
CONCLUSION Epithelial-mesenchymal transitions induced by Twist, may have a relationship with enhanced invasion and metastatic potential during the development of multidrug-resistant MCF-7/ADR breast cancer cells.
keywords
INTRODUCTION
Epithelial-mesenchymal transitions (EMT) is a process whereby epithelial cell layers lose polarity and cell-cell contacts, and thus undergo a dramatic remodeling of the cytoskeleton. This type of conversion is the basic process of embryogenesis, tissue remodeling and tissue repair in adults. It also applies to the steps that mediate tumor progression, including local invasion, spread through the circulation and, distant metastasis[1,2]. Recent studies have reported that Twist, a highly conserved basic helix–loop–helix (bHLH) transcription factor known to be essential for EMT, also plays a key role in breast cancer metastasis. A number of studies have demonstrated enhanced invasive and metastatic potential of multidrug-resistant cancer cells compared with their parental counterparts[3,4]. In order to study the mechanism of enhanced metastatic ability in multidrug-resistant MCF-7/ADR breast cancer cells and the relationship between MDR and metastatic behavior, we compared the changes of expression levels of Twist, E-cadherin and N-cadherin in the MCF-7 and MCF-7/ADR breast cancer cell lines.
MATERIALS AND METHODS
Cell lines and cell culture conditions
MCF-7 human breast cancer cells (Adriamycin-sensitive) and their MCF-7/ADR multidrug-resistant variant (Adri-amycin-resistance) were obtained with the kind help of Mr. Zizheng Hou of the Detroit Hospital, Detroit USA. The cells were cultured in RPMI 1640 supplemented with 10% (v/v) fetal bovine serum, 100 U/ml penicillin and 100 U/ml streptomycin. The cultures were maintained in a humidified atmosphere at 37°C under 5% CO2, and were passaged every 2~3 days. The cells were digested with a mixture of 0.025% trypsin (GIBCOL BRL) and 0.01% EDTA (Sigma). The MCF-7/ADR cells were continuously exposed to 0.5 μΜ Adriamycin for the maintenance of the MDR phenotype, but cultured in drug-free medium for at least one month before use in any experiment.
RNA extraction and RT-PCR
The total RNA extraction reagent, Trizol and Reverse Transcription System, were obtained from Invitrogen Life Technologies, A Taq DNA polymerase kit for the PCR reactions was purchased from the Takara Co., Primers for PCR were synthesized by Augct Biotechnology Co., Beijing. Total RNA extraction and the reverse transcription reactions were performed in accordance with the manufacturer’s instructions. Twist was amplified using the forward and reverse primers as follows: 5′-GGG AGT CCG CAG TCT TAC GA-3′ and 5′-AGA CCG AGA AGG CGT AGC TG-3′ with generated fragments of 277 bp in length. E-cadherin was amplified with the following primers: 5′-CGG TGG TCA AAG AGC CCT TAC T-3′ and 5′-TGA GGG TTG GTG CAA CGT CGT TA-3′ with generated fragments of 171 bp in length. N-cadherin was amplified with the following primers: 5′-CCA CGC TGA GCC CCA GTA TC-3′ and 5′-CCC CCA GTC GTT CAG GTA ATC A-3′ with generated fragments of 232 bp in length. β-Actin was amplified with the following primers: 5′-CAC CCC ACT TCT CTC TAA GG-3′ and 5′-AAA AAG TAT TAA GGC GAA GAT-3′ with generated fragments of 126 bp in length. The PCR reaction conditions were as follows: initial DNA denaturization at 94°C for 5 min, followed by 35 cycles at 94°C for 1 min, 58°C(Twist for 60°C) for 30 s, 72°C for 30 s, and a final extension at 72°C for 7 min before stopping at 4°C. The amplified fragments were detected by 1% (w/v) agarose gel electrophoresis and staining with 0.3 mg/ml ethidium bromide. Each band was analyzed using a Kodak 2D image-analysis software (USA).
Immunocytochemical staining
Rabbit anti-human polyclonal antibodies and mouse anti-human monoclonal antibodies were obtained from Santa Cruz Co., America. A general type immunohistochemistry kit was purchased from the Zhongshan Co., Beijing. Cells grown on glass slides were washed three times with PBS for 3 min and then fixed with prechilled acetone for 10 min. After incubation in 3% H2O2 for 10 min to block endogenous peroxidase, the cells were washed three times with PBS for 3 min; then a blocking solution (normal goat serum) was added followed by incubation for 15 min at room temperature to reduce the background. After blotting off the excess serum, the sections were incubated overnight with the primary antibody at 4°C. The primary antibody was discarded and the cells were washed three times with PBS for 3 min. The biotinylated second antibody was added and incubation conducted at 37°C for 20 min, after which the antibody was discarded followed by three washes with PBS for 3 min. HRP and streptavidin conjugated third antibody were applied followed by incubation at 37°C for 30 min. After discarding the antibodies, the cells were washed three times with PBS for 3 min. DAB substrate-chromogen solution was added and incubated for 5 min. Then the cells were counterstained with hematoxylin and covered with a coverslip. For positive controls, breast cancer tissue slices known to be postive were used, and for negative controls, the primary antibody was omitted.
Western blot analysis
Total protein was extracted with RIPA cell lysis buffer. Protein quantification was performed by using the Bradford method. Total protein (100 μg) was separated by 10% SDS-PAGE and then the proteins was transfered to a PVDF membrane. The PVDF membrane was incubated in a blocking solution consisting of 5% milk in 10 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 0.1% Tween 20 at room temperature for 1 h. The primary antibodies were then added followed by incubation at 37°C for 2 h, washing with TBST three times for 10 min, adding enzyme-linked second antibody, and incubation at 37°C for 1 h and washing with TBST three times for 10 min. Detection by enzyme-linked chemiluminescence was performed according to the manufacturer’s protocol. β-Actin was used as a loading control.
RESULTS
Morphological changes of both cells types under light microscopy
When the morphology of the MCF-7 (Adriamycin-sensitive) and MCF-7/ADR (Adriamycin-resistant) cells routinely growing in culture was investigated by inverted microscopy, we observed that the MCF-7 cells, which are derived from breast adenocarcinoma cells, grew in clusters and still possessed epithelial properties. In contrast the MCF-7/ADR cells grew in a disperse manner, and had lost the majority of their cell-cell contacts. Notably, after they were digested by 0.25% trypsin and suspended in medium, they displayed a completely different morphological appearance. The MCF-7cells were clustered, compared to the MCF-7/ADR cells, which appeared to be single and totally dissociated.
Loss of E-cadherin expression and gain of Twist expression in the MCF-7/ADR cells
As Fig. 1. shows, the argrose electrophoretic analysis of the RT-PCR products indicated that the Twist and N-cadherin genes were positively expressed in the MCF-7/ADR cells, but E-cadherin expression was lost. In the MCF-7 cells, E-cadherin expression was positive, but Twist and N-cadherin expression could not be detected. The length of the specific RT–PCR products were 277 bp for Twist, 171 bp for E-cadherin, 232 bp for N-cadherin, and 126 bp for β-actin.
RT-PCR analysis of Twist, E-cadherin, N-cadherin gene mRNA expression in MCF-7 and MCF-7/ADR cells.
Results of immunocytochemical staining of MCF-7 and MCF-7/ADR cells
We analyzed the expression of E-cadherin, N-cadherin and Twist in MCF-7 and MCF-7/ADR cells. Strong membranous staining for E-cadherin was found in the MCF-7 cells and the circumsciption of the cells was clear, but with MCF-7/ADR cells there was no staining of E-cadherin and only the nuclei, counterstained by hematoxylin could be seen (Fig.2). However the MCF-7/ADR cells showed high levels of N-cadherin, which was also localized at the plasma membrane (Fig.3) and strong nuclear staining for Twist, but staining for N-cadherin and Twist in MCF-7 cells was negative (Fig.4).
Immunocytochemical staining of E-cadherin in MCF-7 and MCF-7/ADR cells.
Immunocytochemical staining of N-cadherin in MCF-7 and MCF-7/ADR cells.
Immunocytochemical staining of Twist in MCF-7 and MCF-7/ADR cells.
Results of Western blots
As Fig.5 shows, we observed positive expression of E-cadherin in the MCF-7 cells, but the MCF-7/ADR cells lost expression of E-cadherin and gained expression of N-cadherin and Twist.
Western blot analysis of twist, E-cadherin, N-cadherin expression in MCF-7 and MCF-7/ADR cells.
DISCUSSION
Recently, a number of studies have demonstrated that drug resistance and cancer metastasis are sometimes linked to each other. P-gp-overexpressing multidrug-resistant (MDR) MCF-7/ADR breast cancer cells display variations in invasive and metastatic behavior compared with parental MCF-7cells [5]. Although some reports have shown that the expression of adhesion factors and matrix metalloproteinases, that correlated with metastasis in the MCF-7/ADR cells, was different from the MCF-7parental cells, there is no clear mechanism for enhanced invasive or metastatic ability of drug-resistant cancer cells[5,6]. We hypothesize that enhancement of metastases during the acquisition of drug resistance in the MCF-7/ADR cells may be related to the activation of signaling pathways.
EMT applies to the steps that mediate tumor progression, including local invasion, spread through the circulation and distant metastasis[1]. Yang et al.[7] reported that Twist, a key transcription factor known was essential for EMT, and also played a key role in breast cancer metastasis. They found that Twist is only expressed in highly invasive breast cancer cell lines, and suppression of Twist expression by SiRNA could only reduce the number of metastatic nodules, but had little effect on formation of primary tumors. In our study, we found that the MCF-7/ADR cells demonstrated altered morphologic characteristics similar to mesenchymal cells, and gained Twist expression during acquisition of drug resistance. But, it is unclear as to how the MCF-7/ADR cells gained expression of Twist and EMT and as to whether EMT is a cause or result of Twist expression during development of drug resistance.
A hallmark of EMT is the loss of E-cadherin expression and appearance of N-cadherin[2]. E-cadherin is a central component of cell-cell adhesion junctions and is required for the formation of epithelia in the embryo and to maintain epithelial homeostasis in adults. The loss of E-cadherin–mediated adhesion has shown to play an important role in the transition of epithelial tumors from a benign to an invasive state[8,9]. Loss of E-cadherin can increase tumor cell invasiveness in vitro and contributes to the transition of adenoma to carcinoma in animal models[2]. Other studies have found that N-cadherin can promote tumor cell motility, invasion and metastasis[10,11]. In our study, we found that enhancement of metastasis in MCF-7/ADR cells maybe related to the loss of E-cadherin and gain of N-cadherin during acquisition of drug resistance. Loss of E-cadherin expression may be due to Twist, acting as a bHLH transcription factor, binding directly to the E-boxes in the E-cadherin promoter to suppress its transcription[7]. In addition to silencing E-cadherin transcription, Twist also can induce the expression of N-cadherin[12].
Several mechanisms have been suggested to contribute to the enhancement of metastasis during acquisition of drug resistance in MCF-7/ADR cells, such as altered expression of adhesion and matrix metalloproteinases. Our studies demonstrated that the appearance of Twist and EMT in MCF-7/ADR cells may play a key role in enhanced invasive or metastatic ability of this cell line. Mironchik et al.[13] and Raman[14] have generated a human breast cancer cell line with stable over-expression of Twist (MCF-7/Twist). The overexpression of Twist results in an epithelial to mesenchymal-like transition (EMLT) leading to increased invasiveness and cellular motility. In our study, we also found enhanced fibronectin and vimentin expression in the MCF-7/ADR cells, and protein markers representative of a mesenchymal transformation. Furthermore, increased expression of the CXCR4 chemokine receptor, was highly correlated with breast cancer metastasis (data not shown).
All these findings are consistent with two clinical phenomena: first, metastatic tumors are more resistant to chemotherapeutic drugs than those that are nonmetastatic; second, drug resistant tumors are more invasive/metastatic relative to non-resistant parental tumors. We hypothesize that drug resistance and tumor metastasis may be inseparable events during the progression of malignant tumors, as there is a functional linkage between the two phenotypes. Development of drug resistance and of metastatic potential are complex processes involving a possible common or cross signaling pathway. Therefore the activity and expression of drug resistance genes may simultaneously activate genes that lead to cancer metastasis.
Several studies have shown that transcriptional activation of MDR1 is regulated by the MAPK pathway[15-17], while others have reported that Snail, another transcription factor that mediats EMT, was also regulated by the MAPK pathway[1]. Interestingly, in Drosophila, Twist can induce the expression of Snail, a known repressor of E-cadherin transcription[18]. Since, the relationship between drug resistance and cellular metastatic ability remains unclear, more extensive studies will be necessary to further clarify this relationship.
Footnotes
This work was supported by the grants from National Natural Science Foundation (No.30370553) of China and Tianjin Medical University Natural Science Foundation (No.2005KY41).
- Received November 29, 2006.
- Accepted December 15, 2006.
- Copyright © 2007 by Tianjin Medical University Cancer Institute & Hospital and Springer
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