Elsevier

Lung Cancer

Volume 62, Issue 1, October 2008, Pages 8-14
Lung Cancer

Knockdown of Snail, a novel zinc finger transcription factor, via RNA interference increases A549 cell sensitivity to cisplatin via JNK/mitochondrial pathway

https://doi.org/10.1016/j.lungcan.2008.02.007Get rights and content

Summary

Previous reports have implicated epithelial-mesenchymal transition (EMT) as a major cause of cancer. Snail, a novel zinc finger transcription factor, was suggested to be an important inducer of EMT and therefore be involved in different phases of tumorigenicity. However, whether Snail could increase chemoresistance of cancer cells to chemotherapeutic agent remains unclear. To evaluate the roles and possible mechanisms of Snail in chemoresistance of lung cancer cells to cisplatin, we utilized RNA interference to knockdown Snail expression in A549 cells and further assessed the cell viability and apoptosis as well as possible signaling transduction pathways. The data showed that Snail depletion sensitized A549 cells to cisplatin possibly by inducing activation of JNK/mitochondrial pathway, suggesting critical roles of Snail in A549 cell chemoresistance to cisplatin and raising the possibility of Snail depletion as a promising approach to lung cancer therapy.

Introduction

Lung cancer is the leading cause of cancer-related death worldwide. Non-small-cell lung cancer (NSCLC) constitutes about 80% of all lung cancers. At the time of diagnosis, 40% of NSCLCs are often in an advanced stage. So far, chemotherapy has been the main method of treating NSCLCs for a period of time. In systematic chemotherapy, cisplatin, a DNA damaging agent, has been widely used for treatment of NSCLCs for many years. Nevertheless, the therapeutic effect of cisplatin has been weakened by chemoresistance of most NSCLCs. It is believed that clinical multidrug resistance to chemotherapeutic agents is a main obstacle to curative treatment for advanced NSCLCs. For this reason, to find new methods for enhancing the sensitivity of NSCLCs cells to chemotherapeutic agents is required. Gene therapy for NSCLCs might be a promising approach.

Recently, much attention has been focused on epithelial-mesenchymal transition (EMT), a key event of embryogenesis, by which tumor cells are prone to be much malignant [1]. EMT is essential for the formation of different tissues and organs during the process of embryonic development [2], whereas in adult tissue, it may be inhibited for maintaining epithelial integrity and homeostasis. Aberrant activation of EMT in epithelial tumor usually correlates with development and recurrence of neoplasm [3]. Some molecules such as Snail, Slug, TWIST, Zeb and bHLH factors have been regarded as the inducers of EMT and hence play important roles in it [4].

Snail, a newly discovered zinc finger transcription factor, has been detected in breast carcinomas [5], ovarian carcinomas [6], melanomas [7], oral squamous carcinomas [3], etc. In most cancers, Up-regulation of Snail is accompanied by a down-regulation of E-cadherin, suggesting a loss of adhesive function [8]. Therefore, Snail has been identified as one of E-cadherin repressors and a key factor triggering the EMT. Also, studies indicated that Snail may have a correlation with genesis and development of cancers. Espineda et al. [9] reported that Snail induces EMT by repressing Na, K-ATPase beta1-subunit in several carcinomas. Using in vitro and in vivo assays, Miyoshi et al. [10] demonstrated that Snail enhances invasive and metastasis abilities of hepatocellular cancer cells through up-regulation of MMP expression. In breast cancer, over-expression of Snail was shown to suppress the expression of Claudin-1, an integral membrane protein component of tight junctions in epithelial cells, thereby leading to progression of the tumor [11]. Therefore, Snail was suggested to be involved in both early events and advanced phases of tumors.

A few studies have been devoted to the roles of Snail in chemoresistance of cancer cells to anti-cancer agents. A recent report indicated the possibility by which Snail enhances chemoresistance of pancreatic cancer cells to 5-fluorouracil or gemcitabine [12]. However, to the best of our knowledge, the roles and possible mechanisms of Snail in chemoresistance of lung cancer cells to cisplatin have not virtually been reported in the open literature. In our preliminary experiments, over-expression of Snail expressed by A549 cells was observed, leading us to hypothesize that Snail might increase A549 cell chemoresistance to cisplatin, and, conversely, suppression of Snail may sensitize A549 cells to apoptosis induced by cisplatin.

The purpose of this study was to determine whether suppression of Snail could increase chemosensitivity of A549 cells to cisplatin. RNA interference was employed to knockdown Snail expression in A549 cells and the cell viabilities and apoptosis as well as possible involvement of signaling transduction pathways in response to cisplatin were further assessed.

Section snippets

Reagents

Cisplatin was purchased from Sigma Company (USA). Antibodies to Snail, Bcl-2, Bax, cytochrome C, caspase-3, p-JNK, JNK and GAPDH were from Santa Cruz Biotech Inc. (USA).

Cell culture

The human lung adenocarcinoma cell line, A549 cell line, was obtained from ATCC and conserved in the central laboratory of Xinqiao Hospital. Cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM, HyClone, Logan, UT) containing 5% fetal bovine serum in a humidified atmosphere containing 5% CO2 at 37 °C.

SiRNA construction and transfection

pSilencer™ 2.1-U6

Vector-mediated RNAi led to down-regulation of Snail

Previous studies have reported on some techniques for silencing Snail expression in cancer cells. Roy, et al. [13] used an antisense phosphorodiamidate morpholino oligomer (AS-PMO) to suppress Snail for treating colorectal cancer in mice, resulting in decreased number and reduced sizes of tumors. Using RNA interference, Olmeda et al. [14] successfully silenced Snail expression in MDCK-Snail cells, leading to a complete mesenchymal to epithelial transition.

In the present study, RNA interference

Conflict of interest

The authors promised there were not any possible conflicts of interest in this research.

Acknowledgements

This work was partially supported by the specific funds of Third Military Medical University for postgraduates (2005256) and National Natural Science Foundation of China (NSFC) grant (No. 30371586). We take this opportunity to specifically thank the reviewers and editors for their kind instructions that may be helpful for our further studies.

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  • Cited by (0)

    1

    These authors contributed equally to this work.

    2

    The author works at Guiyang Medical College.

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