ONCOLOGY LETTERS 8: 657-662, 2014
Effects of damage‑regulated autophagy regulator gene on the SGC7901 human gastric cancer cell line BAO‑SONG ZHU*, KUI ZHAO*, XIN JIA, YONG‑YOU WU and CHUN‑GEN XING Department of General Surgery, The Second Affiliated Hospital, Soochow University, Suzhou, Jiangsu 215021, P.R. China Received September 1, 2013; Accepted March 4, 2014 DOI: 10.3892/ol.2014.2220 Abstract. The aim of this study was to investigate the effects of the adenoviral‑mediated autophagy gene, damage‑regulated autophagy regulator (DRAM), on the proliferation and autophagy of SGC7901 human gastric cancer cells in vitro. The recombinant adenovirus, AdMax‑pDC315‑DRAM‑EGFP, working as a virus vector of DRAM was constructed and infected into the SGC7901 human gastric cancer cell line. The MTT assay was used to determine the growth rate of the SGC7901 cells. Activation of autophagy was monitored with monodansylcadaverin (MDC) staining following AdMax‑pDC315‑DRAM‑EGFP treatment. Immunofluorescent staining was used to examine the expression of microtubule‑associated protein 1 light chain 3 (LC3), and western blotting was used to examine the expression of apoptosis‑ and autophagy‑associated proteins, including Beclin1, p53, p21 and B‑cell lymphoma 2 (Bcl‑2), in the culture supernatant. The viability of the SGC7901 cells was activated by AdMax‑pDC315‑DRAM‑EGFP treatment. The AdMax‑pDC315‑DRAM‑EGFP‑treated cells exhibited positive LC3 expression detected by immunoreactivity and MDC staining. Inductions in the expression of the apoptosis‑related proteins, p53 and p21, and the autophagic protein, Beclin1, were revealed by western blot analysis. By contrast, downregulation of the apoptosis‑related protein, Bcl‑2, following AdMax‑pDC315‑DRAM‑EGFP treatment was identified. In conclusion, the present study demonstrated that AdMax‑pDC315‑DRAM‑EGFP treatment resulted in upregulation of the level of autophagy and induction of cell proliferation in the SGC7901 human gastric cancer cell line in vitro.
Correspondence to: Professor Chun‑Gen Xing, Department of General Surgery, The Second Affiliated Hospital, Soochow University, 1055 Sanxiang Road, Suzhou, Jiangsu 215006, P.R. China E‑mail: [email protected] *
Contributed equally
Key words: damage‑regulated autophagy regulator, p53, autophagy, cell proliferation
Introduction Gastric cancer is the fourth most common type of cancer and the second leading cause of cancer‑related mortality worldwide (1), with approximately one million new cases diagnosed each year. One of the major factors that controls tumor cell death is the tumor suppressor, p53 (2). The importance of cell death to tumor suppression is exemplified by p53 (3). In response to various forms of cellular stress, including DNA damage, hypoxia and oncogene activation, p53 levels are elevated (2). p53 has also been linked to another cell process that controls cell death known as autophagy (4,5). Autophagy is a vesicular trafficking process that mediates the degradation of long‑lived proteins and is the only pathway within the cell for the degradation of organelles (6). In tumor development, autophagy is considered to act in either an oncogenic or tumor suppressive capacity and p53 has been reported to be an inducer of autophagy (4,5). Moreover, the discovery that damage‑regulated autophagy regulator (DRAM), a p53 target gene which is required for p53‑induced autophagy, is frequently downregulated in squamous cancers underscores the theory that autophagy is a component of tumor suppression downstream of p53 (5). DRAM has been identified as an effector molecule that is critical for p53‑mediated apoptosis, thus further supporting the tumor‑suppressive role of autophagy (5,7,8). The discovery of DRAM revealed a novel role for autophagy in p53‑induced apoptotic cell death (5), and DRAM is considered to be a crucial modulator in apoptosis and autophagy. The present study aimed to investigate the effects of AdMax‑pDC315‑DRAM‑EGFP on growth, apoptosis and autophagy of gastric cancer cells in vitro, and to compare the infection efficiency, biological and molecular mechanisms of AdMax‑pDC315‑DRAM‑EGFP. Materials and methods Reagents. The SGC7901 gastric cancer cell line was purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China). The RPMI‑1640 medium was purchased from Gibco‑BRL (Rockville, MD, USA). Fetal bovine serum (FBS) was obtained from Hangzhou Sijiqing Biological Engineering Material Co., Ltd. (Hangzhou, China), and L‑glutamine and MTT were provided by Sigma (St. Louis, MO, USA). Antibodies against p53 (1:500; Rabbit
658
ZHU et al: DRAM ACTIVATES AUTOPHAGY AND INDUCES CELL PROLIFERATION
monoclonal anti‑human), B cell lymphoma 2 (Bcl 2; 1:500; Rabbit monoclonal anti‑human), Beclin1 (1:700; Rabbit monoclonal anti‑human) and p21 (1;500; Rabbit monoclonal anti‑human) were supplied by Cell Signaling Technology, Inc. (Beverly, MA, USA). Adenoviral vectors and infections. The adenoviral vectors and NC‑RNAi‑GFP‑AD were purchased from Shanghai Jikai Biological Technology Co., Ltd. (Shanghai, China). Stocks of replication‑defective adenoviral vectors expressing green fluorescent protein (GFP) (AdMax‑pDC315‑DRAM‑EGFP) were stored at ‑80˚C. NC‑RNAi‑GFP‑AD was used as a control which was also stored at -80˚C. Infections were performed at 70‑75% confluence in Dulbecco's modified Eagle's medium supplemented with 2% fetal calf serum (FCS). The cells were subsequently incubated at 37˚C for at least 4 h, followed by the addition of fresh medium. Cells were then subjected to functional analyses at fixed time points following infection as described for individual experimental conditions (9). Determination of optimal multiplicity of infection (MOI). The SGC7901 cells (1x104 cells̸well) were seeded in 96‑well plates and reached 60‑70% confluence. Different MOI (MOI = 10, 20, 30, 50 and 100) values of the NC‑RNAi‑GFP‑AD 100‑µl diluted infected cells were added to the plates and, after 8 h, RPMI‑1640 medium containing 10% FBS was added. After 48 h of culture, the cells were counted under a fluorescence microscope (Leica DMI4000B; Leica Microsystems Wetzlar GmbH, Wetzlar, Germany) to calculate the number of cells expressing GFP. Cell culture and viability assay. The SGC7901 cells were maintained in RPMI‑1640 medium containing 10% heat‑inactivated FBS and 0.03% L‑glutamine, and incubated in an atmosphere of 5% CO2 at 37˚C. The cells in a mid‑log phase were used in the experiments. Cell viability was assessed by the MTT assay. To determine the effects of AdMax‑pDC315‑DRAM‑EGFP, the SGC7901 cells were plated into 96‑well microplates (7x10 4 cells̸well) and AdMax‑pDC315‑DRAM‑EGFP was added to the culture medium. Cell viability was assessed by the MTT assay 24 h after AdMax‑pDC315‑DRAM‑EGFP treatment. MTT (Sigma) solution was added to the culture medium (500 µg̸ml final concentration) for 4 h prior to the end of treatment and the reaction was inhibited by the addition of 10% acid sodium dodecyl sulfate (100 µl; Beijing Biosea Biotechnology Co., Ltd., Beijing, China). The absorbance value (A) at 570 nm was measured using an automatic multi‑well spectrophotometer (Bio‑Rad, Richmond, CA, USA). The percentage of cell proliferation was calculated as follows: Cell proliferation (%)= (1‑A of experiment well̸A of positive control well) x 100. Visualization of MDC‑labeled vacuoles. Exponentially growing cells were plated on 24‑chamber culture slides, cultured for 24 h and then incubated with the drug in 10% FCS̸RPMI‑1640 medium for 12 and 24 h. Autophagic vacuoles were labeled with MDC (Sigma) (10) by incubating cells with 0.001 mmol̸l MDC in RPMI‑1640 at 37˚C for 10 min. Following incubation, cells were washed three times with phosphate‑buffered saline (PBS) and immediately analyzed with a fluorescence Nikon Eclipse TE300 microscope (Nikon, Tokyo, Japan) equipped with a filter system (V‑2A excitation filter, 380‑420 nm; barrier filter,
Figure 1. Reduced cell viability following AdMax‑pDC315‑DRAM‑EGFP t reatment. SGC7901 cells (7x10 4 cells/m l) were cultured with AdMax‑pDC315‑DRAM‑EGFP (MOI, 60) for 24 h and cell viability was analyzed by the MTT assay. Values are expressed as the mean ± standard deviation of three independent experiments. #P
Effects of damage‑regulated autophagy regulator gene on the SGC7901 human gastric cancer cell line BAO‑SONG ZHU*, KUI ZHAO*, XIN JIA, YONG‑YOU WU and CHUN‑GEN XING Department of General Surgery, The Second Affiliated Hospital, Soochow University, Suzhou, Jiangsu 215021, P.R. China Received September 1, 2013; Accepted March 4, 2014 DOI: 10.3892/ol.2014.2220 Abstract. The aim of this study was to investigate the effects of the adenoviral‑mediated autophagy gene, damage‑regulated autophagy regulator (DRAM), on the proliferation and autophagy of SGC7901 human gastric cancer cells in vitro. The recombinant adenovirus, AdMax‑pDC315‑DRAM‑EGFP, working as a virus vector of DRAM was constructed and infected into the SGC7901 human gastric cancer cell line. The MTT assay was used to determine the growth rate of the SGC7901 cells. Activation of autophagy was monitored with monodansylcadaverin (MDC) staining following AdMax‑pDC315‑DRAM‑EGFP treatment. Immunofluorescent staining was used to examine the expression of microtubule‑associated protein 1 light chain 3 (LC3), and western blotting was used to examine the expression of apoptosis‑ and autophagy‑associated proteins, including Beclin1, p53, p21 and B‑cell lymphoma 2 (Bcl‑2), in the culture supernatant. The viability of the SGC7901 cells was activated by AdMax‑pDC315‑DRAM‑EGFP treatment. The AdMax‑pDC315‑DRAM‑EGFP‑treated cells exhibited positive LC3 expression detected by immunoreactivity and MDC staining. Inductions in the expression of the apoptosis‑related proteins, p53 and p21, and the autophagic protein, Beclin1, were revealed by western blot analysis. By contrast, downregulation of the apoptosis‑related protein, Bcl‑2, following AdMax‑pDC315‑DRAM‑EGFP treatment was identified. In conclusion, the present study demonstrated that AdMax‑pDC315‑DRAM‑EGFP treatment resulted in upregulation of the level of autophagy and induction of cell proliferation in the SGC7901 human gastric cancer cell line in vitro.
Correspondence to: Professor Chun‑Gen Xing, Department of General Surgery, The Second Affiliated Hospital, Soochow University, 1055 Sanxiang Road, Suzhou, Jiangsu 215006, P.R. China E‑mail: [email protected] *
Contributed equally
Key words: damage‑regulated autophagy regulator, p53, autophagy, cell proliferation
Introduction Gastric cancer is the fourth most common type of cancer and the second leading cause of cancer‑related mortality worldwide (1), with approximately one million new cases diagnosed each year. One of the major factors that controls tumor cell death is the tumor suppressor, p53 (2). The importance of cell death to tumor suppression is exemplified by p53 (3). In response to various forms of cellular stress, including DNA damage, hypoxia and oncogene activation, p53 levels are elevated (2). p53 has also been linked to another cell process that controls cell death known as autophagy (4,5). Autophagy is a vesicular trafficking process that mediates the degradation of long‑lived proteins and is the only pathway within the cell for the degradation of organelles (6). In tumor development, autophagy is considered to act in either an oncogenic or tumor suppressive capacity and p53 has been reported to be an inducer of autophagy (4,5). Moreover, the discovery that damage‑regulated autophagy regulator (DRAM), a p53 target gene which is required for p53‑induced autophagy, is frequently downregulated in squamous cancers underscores the theory that autophagy is a component of tumor suppression downstream of p53 (5). DRAM has been identified as an effector molecule that is critical for p53‑mediated apoptosis, thus further supporting the tumor‑suppressive role of autophagy (5,7,8). The discovery of DRAM revealed a novel role for autophagy in p53‑induced apoptotic cell death (5), and DRAM is considered to be a crucial modulator in apoptosis and autophagy. The present study aimed to investigate the effects of AdMax‑pDC315‑DRAM‑EGFP on growth, apoptosis and autophagy of gastric cancer cells in vitro, and to compare the infection efficiency, biological and molecular mechanisms of AdMax‑pDC315‑DRAM‑EGFP. Materials and methods Reagents. The SGC7901 gastric cancer cell line was purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China). The RPMI‑1640 medium was purchased from Gibco‑BRL (Rockville, MD, USA). Fetal bovine serum (FBS) was obtained from Hangzhou Sijiqing Biological Engineering Material Co., Ltd. (Hangzhou, China), and L‑glutamine and MTT were provided by Sigma (St. Louis, MO, USA). Antibodies against p53 (1:500; Rabbit
658
ZHU et al: DRAM ACTIVATES AUTOPHAGY AND INDUCES CELL PROLIFERATION
monoclonal anti‑human), B cell lymphoma 2 (Bcl 2; 1:500; Rabbit monoclonal anti‑human), Beclin1 (1:700; Rabbit monoclonal anti‑human) and p21 (1;500; Rabbit monoclonal anti‑human) were supplied by Cell Signaling Technology, Inc. (Beverly, MA, USA). Adenoviral vectors and infections. The adenoviral vectors and NC‑RNAi‑GFP‑AD were purchased from Shanghai Jikai Biological Technology Co., Ltd. (Shanghai, China). Stocks of replication‑defective adenoviral vectors expressing green fluorescent protein (GFP) (AdMax‑pDC315‑DRAM‑EGFP) were stored at ‑80˚C. NC‑RNAi‑GFP‑AD was used as a control which was also stored at -80˚C. Infections were performed at 70‑75% confluence in Dulbecco's modified Eagle's medium supplemented with 2% fetal calf serum (FCS). The cells were subsequently incubated at 37˚C for at least 4 h, followed by the addition of fresh medium. Cells were then subjected to functional analyses at fixed time points following infection as described for individual experimental conditions (9). Determination of optimal multiplicity of infection (MOI). The SGC7901 cells (1x104 cells̸well) were seeded in 96‑well plates and reached 60‑70% confluence. Different MOI (MOI = 10, 20, 30, 50 and 100) values of the NC‑RNAi‑GFP‑AD 100‑µl diluted infected cells were added to the plates and, after 8 h, RPMI‑1640 medium containing 10% FBS was added. After 48 h of culture, the cells were counted under a fluorescence microscope (Leica DMI4000B; Leica Microsystems Wetzlar GmbH, Wetzlar, Germany) to calculate the number of cells expressing GFP. Cell culture and viability assay. The SGC7901 cells were maintained in RPMI‑1640 medium containing 10% heat‑inactivated FBS and 0.03% L‑glutamine, and incubated in an atmosphere of 5% CO2 at 37˚C. The cells in a mid‑log phase were used in the experiments. Cell viability was assessed by the MTT assay. To determine the effects of AdMax‑pDC315‑DRAM‑EGFP, the SGC7901 cells were plated into 96‑well microplates (7x10 4 cells̸well) and AdMax‑pDC315‑DRAM‑EGFP was added to the culture medium. Cell viability was assessed by the MTT assay 24 h after AdMax‑pDC315‑DRAM‑EGFP treatment. MTT (Sigma) solution was added to the culture medium (500 µg̸ml final concentration) for 4 h prior to the end of treatment and the reaction was inhibited by the addition of 10% acid sodium dodecyl sulfate (100 µl; Beijing Biosea Biotechnology Co., Ltd., Beijing, China). The absorbance value (A) at 570 nm was measured using an automatic multi‑well spectrophotometer (Bio‑Rad, Richmond, CA, USA). The percentage of cell proliferation was calculated as follows: Cell proliferation (%)= (1‑A of experiment well̸A of positive control well) x 100. Visualization of MDC‑labeled vacuoles. Exponentially growing cells were plated on 24‑chamber culture slides, cultured for 24 h and then incubated with the drug in 10% FCS̸RPMI‑1640 medium for 12 and 24 h. Autophagic vacuoles were labeled with MDC (Sigma) (10) by incubating cells with 0.001 mmol̸l MDC in RPMI‑1640 at 37˚C for 10 min. Following incubation, cells were washed three times with phosphate‑buffered saline (PBS) and immediately analyzed with a fluorescence Nikon Eclipse TE300 microscope (Nikon, Tokyo, Japan) equipped with a filter system (V‑2A excitation filter, 380‑420 nm; barrier filter,
Figure 1. Reduced cell viability following AdMax‑pDC315‑DRAM‑EGFP t reatment. SGC7901 cells (7x10 4 cells/m l) were cultured with AdMax‑pDC315‑DRAM‑EGFP (MOI, 60) for 24 h and cell viability was analyzed by the MTT assay. Values are expressed as the mean ± standard deviation of three independent experiments. #P
