ONCOLOGY LETTERS 7: 927-932, 2014
Discrepancy between the effects of morronside on apoptosis in human embryonic lung fibroblast cells and lung cancer A549 cells JIAN‑PING CHEN1, DE‑GUO XU1, XIAO‑YI YU2, FENG‑MING ZHAO1, DONG‑QING XU1, XU ZHANG1, BAO‑CHANG CAI1 and MING‑YAN WANG1 1
Jiangsu Key Laboratory of Chinese Medicine Processing, Nanjing University of Chinese Medicine, Nanjing, Jiangsu 210029; 2China Pharmaceutical University, Nanjing, Jiangsu 210009, P.R. China Received July 2, 2013; Accepted December 13, 2013 DOI: 10.3892/ol.2014.1850
Abstract. Morroniside is a water‑soluble compound extracted from the fruit of Cornus officinalis and is used to protect lung activity against aging. In the present study, the manner in which morroniside regulates normal lung and cancer cells was examined. The human embryonic lung fibroblast (HELF) cell line and lung cancer A549 cell line, and their responses to morroniside treatment, were examined. Results showed that morroniside reverses the apoptotic effect of H 2O2 on HELF cell growth, protecting cell proliferation and normal cell morphology and inhibiting apoptosis. However, these effects were not present in A549 cells. Western blotting showed that morroniside also markedly downregulated retinoblastoma protein in HELF cells. These results suggest that morroniside treatment exhibits different effects on apoptosis in HELF and A549 cells, making it a viable compound for decreasing the side effects of anticancer medicines in normal cells. Introduction The fruit of Cornus officinalis is effective for treating liver and lung disease in traditional Chinese medicine (1). In recent decades, the pharmacology and phytochemistry of the fruit of Cornus officinalis have been extensively investigated. Clinical studies have demonstrated that this fruit has anticancer and antioxidative roles (2,3). Based on various clinical roles, several effective water‑soluble components of the fruit of Cornus officinalis have been identified. For instance, gallic acid has antitumor properties (4) and oleanolic acid has antiulcer properties and markedly reduces blood pressure and blood glucose
Correspondence
to: Professor Ming‑Yan Wang or Professor Bao‑Chang Cai, Jiangsu Key Laboratory of Chinese Medicine Processing, University of Chinese Medicine, 183 Xianling Road, Nanjing, Jiangsu 210049, China E‑mail: [email protected] E‑mail: [email protected] Key words: human embryonic lung fibroblasts cells, apoptosis, morroniside, retinoblastoma protein
levels (5). Morroniside is used to suppress the generation of superoxide anions in a concentration‑dependent manner, and protects nerve cells from H2O2‑induced oxidation injury (6,7). At the molecular level, morroniside can enhance the activity of alkaline phosphatase and decrease caspase‑3 mRNA levels (8). The protection of cell growth by morroniside is hypothesized to be involved in regulation of cell proliferation and apoptosis. The retinoblastoma (Rb)/pocket protein family is made up of tumor suppressor proteins involved in cell proliferation, apoptosis and cell differentiation (9,10). The Rb protein is one of the master regulators of the eukaryotic cell cycle, regulating the G1/G0 phase and also the S and G2 phases (11). Rb protein activity is regulated by phosphorylation. In normal cells during the M‑to‑G1 transition, the Rb protein is progressively dephosphorylated by protein phosphatase 1, enabling it to bind E2F, blocking transcription of multiple genes involved in cell cycle progression and elongating the G1/G0 phases. Following mitogenic stimulation of the cell, the Rb protein is phosphorylated by cyclin‑dependent kinase (CDK) 4/6-cyclin D complexes. Phosphorylation inactivates the Rb protein, resulting in release of E2F, enabling E2F to transcriptionally activate genes that facilitate the G1/S transition and S phase. The Rb protein remains phosphorylated/inactive throughout the S, G2 and M phases through hyperphosphorylation by cyclin E/CDK2 complexes (12-15). The present study showed that morroniside decreases cell apoptosis induced by H 2O2 in HELF cells but not in A549 cells. Additionally, the S phase of the cell cycle was returned to normal function and Rb protein levels were downregulated by morroniside. These results indicate that the effects of morroniside on HELF and A549 cells may correlate with the regulation of Rb protein levels and S phase length. Materials and methods Cells and reagents. HELF and A549 cells were purchased from Shanghai Institute of Biochemistry and Cell Biology (Shanghai, China). Morroniside was provided by Jiangsu ZhongKang New Drug Fingerprint R&D Co., Ltd (Nanjing, China) at a purity of >98%; MTT, acridine orange (AO), ethidium bromide (EB), RNase A, propidium iodide (PI) and trypsin were purchased from Sigma (St. Louis, MO, USA). RPMI 1640 medium was obtained from Gibco‑BRL (Carlsbad,
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CHEN et al: MORRONISIDE AND APOPTOSIS
CA, USA) and fetal bovine serum (FBS) was from Sijiqing Biotechnology (Hangzhou, China). Cell culture and drug incorporation. HELF and A549 cells were cultured in RPMI 1640 medium with 10% (v/v) FBS, 100 U/ml streptomycin and 100 U/ml penicillin, at 37˚C with 5% CO2. The cells digested with 0.25% trypsin were cultured in 96‑well plates (for proliferation analysis), 6‑well plates (for morphology observation) or flasks (for cell cycle analysis). In all experiments, HELF/A549 cells were cultured without treatment (normal control), with 300 µM H2O2/0.2 nM staurosporine (model) or with combined treatment of H2O2/staurosporine and morroniside at various doses (12.5, 25, 50, 100 and 200 µg/ml). Cell proliferation analysis. Cell proliferation was determined by MTT assay. HELF cells in exponential growth were seeded at a final concentration of 6x10 4 cells/ml in a 96‑well plate. At 50% confluency, the cells were pretreated with morroniside at various concentrations for 2 h, exposed to H2O2 or staurosporine (0.2 nM) and incubated for 48 h. Next, 50 ml MTT (1 mg/ml) was added to each well, followed by incubation for 4 h at 37˚C. The supernatants were discarded and the formazan product was dissolved in 100 µl dimethyl sulfoxide. The 96‑well plate was agitated on a micro‑vibrator for an additional 10 min. The optical density of each well was measured at λ 490 nm using an enzyme‑immunoassay instrument. Morphological examination. Cell groups were subjected to various designated treatments, as discussed. The cells were observed under a fluorescence microscope and photographed. Cells were seeded at a concentration of 1x105/well in a 6‑well culture plate. A cover slip was placed on the bottom of each well to allow cells to grow on the surface as a monolayer. At 40‑60% confluency, the cells were divided into three groups: Normal control group; H 2O2‑treated group, treated with 300 µM H2O2 and incubated for 48 h; morroniside plus H2O2‑treated group, pretreated with 100 µg/ml morroniside for 2 h and exposed to 300 µM H2O2. After a 48‑h incubation period, the cell culture was washed with phosphate‑buffered saline (PBS), stained with 10 µg/ml AO/EB for 2 min and observed under a fluorescence microscope (Olympus BX60; Olympus, Tokyo, Japan). Flow cytometry. Cells were treated as discussed. After 24‑h culture, cells were washed twice with ice‑cold PBS buffer (pH 7.4) prior to fixing with 75% ice‑cold ethanol and staining with PI (1 mg/ml) in the presence of 1% RNAase A for 30 min. The samples were subsequently analyzed by flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA). The Cell Quest software (BD Biosciences) was applied to analyze apoptosis rates and the cell cycle. Western blotting. HELF cells were lysed in cold lysis buffer [40 mM HEPES (pH 7.5), 150 mM NaCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM dithiothreitol and 1 mM fresh phenylmethylsulfonyl fluoride]. Lysates were centrifuged at 12,000 x g for 15 min. The supernatant was collected and the protein concentration was determined by Lowry protein assay. Next, 20 µg protein was loaded in each well, electrophoresed by SDS‑PAGE and transferred onto nitrocellulose membranes.
The membranes were blocked with 5% non‑fat dried milk in PBS with 0.5 ml/l Tween‑20 for 2 h at room temperature, and incubated overnight at 4˚C with primary antibodies against human P16, P27, P53 or Rb proteins, purchased from Beyotime (Nanjing, China). This was followed by incubation with a horseradish peroxidase‑conjugated secondary antibody (Bioworld Technology, St. Louis Park, MN, USA) at room temperature for 1 h. Enhanced chemiluminescence was used to detect the results. Statistical analysis. The statistical analysis among various groups was performed using one‑way analysis of variance with Scheffé's test. All data were processed with SPSS software (SPSS, Inc., Chicago, IL, USA). The results were expressed as the mean ± SD. P
Discrepancy between the effects of morronside on apoptosis in human embryonic lung fibroblast cells and lung cancer A549 cells JIAN‑PING CHEN1, DE‑GUO XU1, XIAO‑YI YU2, FENG‑MING ZHAO1, DONG‑QING XU1, XU ZHANG1, BAO‑CHANG CAI1 and MING‑YAN WANG1 1
Jiangsu Key Laboratory of Chinese Medicine Processing, Nanjing University of Chinese Medicine, Nanjing, Jiangsu 210029; 2China Pharmaceutical University, Nanjing, Jiangsu 210009, P.R. China Received July 2, 2013; Accepted December 13, 2013 DOI: 10.3892/ol.2014.1850
Abstract. Morroniside is a water‑soluble compound extracted from the fruit of Cornus officinalis and is used to protect lung activity against aging. In the present study, the manner in which morroniside regulates normal lung and cancer cells was examined. The human embryonic lung fibroblast (HELF) cell line and lung cancer A549 cell line, and their responses to morroniside treatment, were examined. Results showed that morroniside reverses the apoptotic effect of H 2O2 on HELF cell growth, protecting cell proliferation and normal cell morphology and inhibiting apoptosis. However, these effects were not present in A549 cells. Western blotting showed that morroniside also markedly downregulated retinoblastoma protein in HELF cells. These results suggest that morroniside treatment exhibits different effects on apoptosis in HELF and A549 cells, making it a viable compound for decreasing the side effects of anticancer medicines in normal cells. Introduction The fruit of Cornus officinalis is effective for treating liver and lung disease in traditional Chinese medicine (1). In recent decades, the pharmacology and phytochemistry of the fruit of Cornus officinalis have been extensively investigated. Clinical studies have demonstrated that this fruit has anticancer and antioxidative roles (2,3). Based on various clinical roles, several effective water‑soluble components of the fruit of Cornus officinalis have been identified. For instance, gallic acid has antitumor properties (4) and oleanolic acid has antiulcer properties and markedly reduces blood pressure and blood glucose
Correspondence
to: Professor Ming‑Yan Wang or Professor Bao‑Chang Cai, Jiangsu Key Laboratory of Chinese Medicine Processing, University of Chinese Medicine, 183 Xianling Road, Nanjing, Jiangsu 210049, China E‑mail: [email protected] E‑mail: [email protected] Key words: human embryonic lung fibroblasts cells, apoptosis, morroniside, retinoblastoma protein
levels (5). Morroniside is used to suppress the generation of superoxide anions in a concentration‑dependent manner, and protects nerve cells from H2O2‑induced oxidation injury (6,7). At the molecular level, morroniside can enhance the activity of alkaline phosphatase and decrease caspase‑3 mRNA levels (8). The protection of cell growth by morroniside is hypothesized to be involved in regulation of cell proliferation and apoptosis. The retinoblastoma (Rb)/pocket protein family is made up of tumor suppressor proteins involved in cell proliferation, apoptosis and cell differentiation (9,10). The Rb protein is one of the master regulators of the eukaryotic cell cycle, regulating the G1/G0 phase and also the S and G2 phases (11). Rb protein activity is regulated by phosphorylation. In normal cells during the M‑to‑G1 transition, the Rb protein is progressively dephosphorylated by protein phosphatase 1, enabling it to bind E2F, blocking transcription of multiple genes involved in cell cycle progression and elongating the G1/G0 phases. Following mitogenic stimulation of the cell, the Rb protein is phosphorylated by cyclin‑dependent kinase (CDK) 4/6-cyclin D complexes. Phosphorylation inactivates the Rb protein, resulting in release of E2F, enabling E2F to transcriptionally activate genes that facilitate the G1/S transition and S phase. The Rb protein remains phosphorylated/inactive throughout the S, G2 and M phases through hyperphosphorylation by cyclin E/CDK2 complexes (12-15). The present study showed that morroniside decreases cell apoptosis induced by H 2O2 in HELF cells but not in A549 cells. Additionally, the S phase of the cell cycle was returned to normal function and Rb protein levels were downregulated by morroniside. These results indicate that the effects of morroniside on HELF and A549 cells may correlate with the regulation of Rb protein levels and S phase length. Materials and methods Cells and reagents. HELF and A549 cells were purchased from Shanghai Institute of Biochemistry and Cell Biology (Shanghai, China). Morroniside was provided by Jiangsu ZhongKang New Drug Fingerprint R&D Co., Ltd (Nanjing, China) at a purity of >98%; MTT, acridine orange (AO), ethidium bromide (EB), RNase A, propidium iodide (PI) and trypsin were purchased from Sigma (St. Louis, MO, USA). RPMI 1640 medium was obtained from Gibco‑BRL (Carlsbad,
928
CHEN et al: MORRONISIDE AND APOPTOSIS
CA, USA) and fetal bovine serum (FBS) was from Sijiqing Biotechnology (Hangzhou, China). Cell culture and drug incorporation. HELF and A549 cells were cultured in RPMI 1640 medium with 10% (v/v) FBS, 100 U/ml streptomycin and 100 U/ml penicillin, at 37˚C with 5% CO2. The cells digested with 0.25% trypsin were cultured in 96‑well plates (for proliferation analysis), 6‑well plates (for morphology observation) or flasks (for cell cycle analysis). In all experiments, HELF/A549 cells were cultured without treatment (normal control), with 300 µM H2O2/0.2 nM staurosporine (model) or with combined treatment of H2O2/staurosporine and morroniside at various doses (12.5, 25, 50, 100 and 200 µg/ml). Cell proliferation analysis. Cell proliferation was determined by MTT assay. HELF cells in exponential growth were seeded at a final concentration of 6x10 4 cells/ml in a 96‑well plate. At 50% confluency, the cells were pretreated with morroniside at various concentrations for 2 h, exposed to H2O2 or staurosporine (0.2 nM) and incubated for 48 h. Next, 50 ml MTT (1 mg/ml) was added to each well, followed by incubation for 4 h at 37˚C. The supernatants were discarded and the formazan product was dissolved in 100 µl dimethyl sulfoxide. The 96‑well plate was agitated on a micro‑vibrator for an additional 10 min. The optical density of each well was measured at λ 490 nm using an enzyme‑immunoassay instrument. Morphological examination. Cell groups were subjected to various designated treatments, as discussed. The cells were observed under a fluorescence microscope and photographed. Cells were seeded at a concentration of 1x105/well in a 6‑well culture plate. A cover slip was placed on the bottom of each well to allow cells to grow on the surface as a monolayer. At 40‑60% confluency, the cells were divided into three groups: Normal control group; H 2O2‑treated group, treated with 300 µM H2O2 and incubated for 48 h; morroniside plus H2O2‑treated group, pretreated with 100 µg/ml morroniside for 2 h and exposed to 300 µM H2O2. After a 48‑h incubation period, the cell culture was washed with phosphate‑buffered saline (PBS), stained with 10 µg/ml AO/EB for 2 min and observed under a fluorescence microscope (Olympus BX60; Olympus, Tokyo, Japan). Flow cytometry. Cells were treated as discussed. After 24‑h culture, cells were washed twice with ice‑cold PBS buffer (pH 7.4) prior to fixing with 75% ice‑cold ethanol and staining with PI (1 mg/ml) in the presence of 1% RNAase A for 30 min. The samples were subsequently analyzed by flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA). The Cell Quest software (BD Biosciences) was applied to analyze apoptosis rates and the cell cycle. Western blotting. HELF cells were lysed in cold lysis buffer [40 mM HEPES (pH 7.5), 150 mM NaCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM dithiothreitol and 1 mM fresh phenylmethylsulfonyl fluoride]. Lysates were centrifuged at 12,000 x g for 15 min. The supernatant was collected and the protein concentration was determined by Lowry protein assay. Next, 20 µg protein was loaded in each well, electrophoresed by SDS‑PAGE and transferred onto nitrocellulose membranes.
The membranes were blocked with 5% non‑fat dried milk in PBS with 0.5 ml/l Tween‑20 for 2 h at room temperature, and incubated overnight at 4˚C with primary antibodies against human P16, P27, P53 or Rb proteins, purchased from Beyotime (Nanjing, China). This was followed by incubation with a horseradish peroxidase‑conjugated secondary antibody (Bioworld Technology, St. Louis Park, MN, USA) at room temperature for 1 h. Enhanced chemiluminescence was used to detect the results. Statistical analysis. The statistical analysis among various groups was performed using one‑way analysis of variance with Scheffé's test. All data were processed with SPSS software (SPSS, Inc., Chicago, IL, USA). The results were expressed as the mean ± SD. P
