Experimental study on inhibition effects of the XAF1 gene against lung cancer cell proliferation.

Wen-Tao Yang,Fu-Quan Zhang,Ying-Chen Xia,Dong-Lai Chen,Duan-Shan Zhou,Rong-Ying Zhu,Yong-Bing Chen

doi:10.7314/apjcp.2014.15.18.7825

Abstract

To investigate the effect of high expression of XAF1 in vivo or in vitro on lung cancer cell growth and apoptosis. 1. The A549 human lung cancer cell line was transfected with Ad5/F35 - XAF1, or Ad5/ F35 - Null at the same multiplicity of infection (MOI); (hereinafter referred to as transient transfected cell strain); XAF1 gene mRNA and protein expression was detected by reverse transcription polymerase chain reaction (RT- PCR) and Western blotting respectively. 2. Methyl thiazolyl tetrazolium (MTT) and annexin V-FITC/PI double staining were used to detect cell proliferation and apoptosis before and after infection of Ad5/F35 - XAF1 with Western blotting for apoptosis related proteins, caspase 3, caspase - 8 and PARP. 3. After the XAF1 gene was transfected into lung cancer A549 cells by lentiviral vectors, and selected by screening with Blasticidin, reverse transcription polymerase chain reaction (RT-PCR) and Western blotting were applied to detect mRNA and protein expression, to establish a line with a stable high expression of XAF1 (hereinafter referred to as stable expression cell strain). Twenty nude mice were randomly divided into groups A and B, 10 in each group: A549/ XAF1 stable expression cell strain was subcutaneously injected in group A, and A549/Ctrl stable cell line stable expression cell strain in group B (control group), to observe transplanted tumor growth in nude mice. The mRNA and protein expression of XAF1 in A549 cells transfected by Ad5/F35 - XAF1 was significantly higher than in the control group. XAF1 mediated by adenovirus vector demonstrated a dose dependent inhibition of lung cancer cell proliferation and induction of apoptosis. This was accompanied by cleavage of caspase -3, -8, -9 and PARP, suggesting activation of intrinsic or extrinsic apoptotic pathways. A cell strain of lung cancer highly expressing XAF1 was established, and this demonstrated delayed tumor growth after transplantation in vivo. Adenovirus mediated XAF1 gene expression could inhibit proliferation and induce apoptosis in lung cancer cells in vitro; highly stable expression of XAF1 could also significantly inhibit the growth of transplanted tumors in nude mouse, with no obvious adverse reactions observed. Therefore, the XAF1 gene could become a new target for lung cancer treatment.

Highlights

Lung cancer is a common malignant tumor in China, and NSCLC patients take up 80%~85% of total cases in lung cancer (Aydiner et al, 2013; Cai et al, 2013; Kaya et al, 2013; Mutlu et al, 2013; Natukula et al, 2013; Oven et al, 2013; Unal et al, 2013; Wang et al, 2013)
The A549 human lung cancer cell line was transfected with Ad5/F35 - XAF1, or Ad5/ F35 – Null at the same multiplicity of infection (MOI);; XAF1 gene mRNA and protein expression was detected by reverse transcription polymerase chain reaction (RTPCR) and Western blotting respectively
After the XAF1 gene was transfected into lung cancer A549 cells by lentiviral vectors, and selected by screening with Blasticidin, reverse transcription polymerase chain reaction (RT-PCR) and Western blotting were applied to detect mRNA and protein expression, to establish a line with a stable high expression of XAF1

Summary

Introduction

Lung cancer is a common malignant tumor in China, and NSCLC patients take up 80%~85% of total cases in lung cancer (Aydiner et al, 2013; Cai et al, 2013; Kaya et al, 2013; Mutlu et al, 2013; Natukula et al, 2013; Oven et al, 2013; Unal et al, 2013; Wang et al, 2013). How to effectively treat patients with lung cancer is a focus in clinical settings (Yan et al, 2009). Apoptosis plays an important role in the development of lung cancer (Yan et al, 2009). This study investigated the effect of high expression of XAF1 with recombinant adenovirus as a carrier on inhibiting proliferation and inducing apoptosis in human lung cancer cell line A549 in vitro,, and used lentiviral vectors to build highly stable expression of XAF1 gene in lung cancer cell line, and injected lung cancer cell strains through the tail vein of nude mice to construct lung metastases model, provided experimental evidence for XAF1 gene therapy in vivo

Methods

Results

Conclusion