Contributed equally
Although Beclin 1 has been demonstrated to exert an important role in cell autophagy during carcinogenesis, its biological function in lung cancer has yet to be fully elucidated. A previous study by our laboratory identified that knockdown of Beclin 1 promoted cell growth and inhibited apoptosis in the A549 lung cancer cell line. In the present study, a Beclin 1 lentiviral expression vector was constructed, and an A549 cell line was established with a steady expression of Beclin 1. Furthermore, the effect of Beclin 1 overexpression on cell invasion and apoptosis, changes in the activities of the apoptosis-associated caspases-3 and -9, and the overexpression of esophageal cancer-related gene 4 (ECRG4) were examined. The results demonstrated that the overexpression of Beclin 1 in A549 cells reduced cell invasion by Matrigel invasion assay and promoted apoptosis by flow cytometric analysis (P<0.01) compared with Lenex-packaged lentiviral particles and non-transfected control groups. Furthermore, the overexpression of Beclin 1 in A549 cells increased the activities of caspases-3 and -9 and the expression of ECRG4 (P<0.01) compared with Lenex-packaged lentiviral particles and non-transfected control groups. In conclusion, the overexpression of Beclin 1 promoted apoptosis and decreased invasion by upregulating the expression of ECRG4 in A549 lung adenocarcinoma cells. Therefore, the selection of Beclin l as a target for gene therapy represents a more effective method for the treatment of lung cancer.
Beclin l (the mammalian counterpart of the yeast Atg6 gene) is an essential player in autophagy. Allelic loss or deficiency of the Beclin 1 gene has been demonstrated in human breast cancer, ovarian cancer and prostate cancer; in lung cancer, hepatocellular carcinoma, cervical cancer and lymphoma, the expression of Beclin 1 is very low/almost undetectable (
Although Beclin 1 has been demonstrated to exert an important role in cell autophagy during carcinogenesis, its biological function in lung cancer has yet to be fully elucidated. A previous study by our laboratory identified that knockdown of Beclin 1 promoted cell growth and inhibited apoptosis in the A549 lung cancer cell line (
Human lung adenocarcinoma cell line A549 was obtained from the Chinese Center for Type Culture Collection (Wuhan, China). An A549 cell suspension was added into a centrifuge tube containing 5 ml RPMI-1640 culture medium (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and centrifuged at 1,200 × g for 5 min. The supernatant was subsequently discarded, and fresh RPMI-1640 medium was added. Following the second centrifugation, cells were resuspended in 10 ml RPMI-1640 culture medium containing 10% fetal bovine serum (FBS) and cultured overnight in a 5% CO2 incubator at 37°C. A549 cells at a density of 5×105 cells/ml were seeded into a 6-well culture plate (2 ml per well). When cells had grown to 70–80% confluence, 2 ml of RPMI-1640 medium containing 10% FBS and different concentrations of G418 solution (Invitrogen; Thermo Fisher Scientific, Inc.) were added. A G418 concentration (800
A549 cells were divided into three groups: Cells infected with lentiviral particles packaged with the recombinant vector, pLenex-Beclin 1; those infected with lentiviral particles packaged with the empty vector, pLenex; and non-transfected A549 cells. Recombinant lentiviruses were generated from our previously constructed pLenex-Beclin 1 and pLenex vectors (
Non-infected A549 cells and two groups of infected cells were added into tubes containing RLT buffer. After the cells had been harvested and lysed, the protein concentration was determined using a bicinchoninic acid protein assay kit. Samples were dissolved in loading buffer, boiled for 5 min, and subsequently loaded onto 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels. Protein bands were subsequently transferred onto a polyvinylidene difluoride membrane. The membrane was blocked overnight at 4°C in Tris-buffered saline-Tween 20 (TBST) containing 5% defatted milk, incubated with a rat anti-human Beclin 1 or ECRG4 primary antibody (dilution, 1:150; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) for 2 h, washed three times and incubated with horseradish peroxidase-conjugated rabbit anti-rat secondary antibody (dilution, 1:2,000) for 1 h at room temperature. The bands were visualized using 3,3′-diaminobenzidine (Pierce Biotechnology, Appleton, WI, USA).
Infected and non-infected cells were cultured for 48 h. Following trypsin digestion, cells were adjusted to a density of 5×105-1×106 cells/ml and fixed with 75% cold ethanol overnight. Following washing, cells were fully centrifuged at 1,200 g, mixed with 100
A total of ~5×105 cells in each group were centrifuged at 1,200 rpm for 5 min. The harvested cells were fixed in an ice-cold cell lysate buffer for 10 min at a density of 2×105 cells/50
The Boyden chamber assay was performed. Matrigel previously stored at −20°C was kept on ice overnight. With pre-cooled tips, 100
Statistical analyses were performed using SPSS v.16 software (SPSS, Inc., Chicago, IL, USA). Numerical data were expressed as the mean ± standard deviation. Analysis of variance was used to compare means of multiple groups, and the t-test was used for comparison of means between two groups. P<0.05 was considered to indicate a statistically significant difference.
The Beclin 1 lentivirus expression vector was initially constructed, and the A549 cell line with Beclin 1 lentivirus steady infection was successfully established. After 2 weeks of G418 selection, anti-G418 resistant cells survived and formed a stable clone. As shown in
No significant differences were identified in terms of the average number of cells passing through the Transwell membrane between non-transfected A549 cells and A549 cells infected with pLenex-packaged lentiviral particles (15.8±2.7 vs. 18.2±3.1; P>0.05). However, the average number of cells passing through the Transwell membrane was significantly lower in A549 cells infected with pLenex-Beclin 1-packaged lentiviral particles (6.4±2.1) compared with non-transfected A549 cells and A549 cells infected with pLenex-packaged lentiviral particles (P<0.05) (
As determined from the flow cytometric analysis, no statistical differences were identified in the apoptotic rate between non-transfected A549 cells and A549 cells infected with pLenex-packaged lentiviral particles (9.12±1.21% vs. 6.87±1.17%; P>0.05). However, the apoptotic rate was significantly higher in A549 cells infected with pLenex-Beclin 1-packaged lentiviral particles (17.12±2.12%) compared with non-transfected A549 cells and A549 cells infected with pLenex-packaged lentiviral particles (P<0.05), indicating that the overexpression of Beclin 1 promoted apoptosis of the A549 cells (
No significant differences were identified in the activities of caspases-3 and caspase-9 between non-transfected A549 cells and A549 cells infected with pLenex-packaged lentiviral particles (1.28±0.15 vs. 1.23±0.14, and 0.67±0.11 vs. 0.63±0.08, respectively; P>0.05 for both). However, the activities of caspase-3 and caspase-9 were significantly higher in A549 cells infected with pLenex-Beclin 1-packaged lentiviral particles (1.81±0.26 vs. 1.16±0.18) compared with the non-transfected A549 cells and A549 cells infected with pLenex-packaged lentiviral particles (P<0.05 for both) (
The results of the western blot analysis showed that no significant differences were identified in the expression of ECRG4 between non-transfected A549 cells and A549 cells infected with pLenex-packaged lentiviral particles. However, the expression of ECRG4 was significantly higher in A549 cells infected with pLenex-Beclin 1-packaged lentiviral particles compared with non-transfected A549 cells and A549 cells infected with pLenex-packaged lentiviral particles (P<0.05 for both) (
Autophagy is a form of programmed cell death that has an important role in biological growth and development, self-renewal, as well as disease development and tumor formation (
Invasion and metastasis are the most significant characteristics of cancer cells. The Matrigel invasion assay is a commonly used
Although there are significant differences between autophagy and apoptosis in biochemical pathways, their functions may be linked. Autophagy does not depend on the involvement of caspases, and its most notable feature is the presence of autophagosomes, which are eventually removed via the lysosomal system (
In conclusion, the overexpression of Beclin 1 promoted apoptosis and decreased invasion by upregulating the expression of ECRG4 in A549 lung adenocarcinoma cells. Therefore, the selection of Beclin 1 as a target for gene therapy represents a more effective method for the treatment of lung cancer.
This study was supported by the Chinese National Natural Science Foundation (no. U1304817) and the Zhengzhou City Science Research Project (no. 141PPTGHG298)
Fluorescent micrographs of cells transfected with a lentiviral vector expressing green fluorescent protein for 48 h. The micrographs illustrate (A) A549 cells infected with pLenex-Beclin 1-packaged lentiviral particles; (B) A549 cells infected with pLenex-packaged lentiviral particles; and (C) non-transfected A549 cells (original magnification, ×40).
Western blot analysis, revealing the increased protein expression of Beclin 1 in A549 cells infected with pLenex-Beclin 1-packaged lentiviral particles. Lane 1: A549 cells infected with pLenex-Beclin 1-packaged lentiviral particles; lanes 2 and 3: A549 cells infected with pLenex-packaged lentiviral particles; lane 4: non-transfected A549 cells. β-actin was used as a loading control.
Flow cytometry analysis. (A-C) Apoptosis of the different groups of cells, as determined by flow cytometry. (D) Comparison of the apoptotic rates among the different groups of cells. *P<0.05 vs. the control groups.
Caspase-3 and caspase-9 activities among the different cell groups. *P<0.05 compared with the empty plasmid and blank control groups.
The protein expression of ECRG4 determined by Western blotting. Lane 1, non-transfected A549 cells; lane 2, A549 cells infected with pLenex-packaged lentiviral particles; lane 3, A549 cells infected with pLenex-Beclin 1-packaged lentiviral particles. ECGR4, esophageal cancer-related gene 4.