Journal List > Tuberc Respir Dis > v.69(2) > 1001577

Jang, Jo, Hwhang, Kim, Lee, Moon, Shin, Cho, Lee, Lee, Park, Park, Kim, and Yang: The Effect of Autophagy to Cell Death in Nutrient-Deprived H460 Cells

Abstract

Background

Autophagy is an important adaptive mechanism in normal development and in response to changing environmental stimuli in cancer. Previous papers have reported that different types of cancer underwent autophagy to obtain amino acids as energy source of dying cells in nutrient-deprived conditions. However, whether or not autophagy in the process of lung cancer causes death or survival is controversial. Therefore in this study, we investigated whether nutrient deprivation induces autophagy in human H460 lung cancer cells.

Methods

H460, lung cancer cells were incubated in RPMI 1640 medium, and the starved media, which are BME and RPMI media without serum, including 2-deoxyl-D-glucose according to time dependence. To evaluate the viability and find out the mechanism of cell death under nutrient-deprived conditions, the MTT assay and flow cytometry were done and analyzed the apoptotic and autophagic related proteins. It is also measured the development of acidic vascular organelles by acridine orange.

Results

The nutrient-deprived cancer cell is relatively sensitive to cell death rather than normal nutrition. Massive cytoplasmic vacuolization was seen under nutrient-deprived conditions. Autophagic vacuoles were visible at approximately 12 h and as time ran out, vacuoles became larger and denser with the increasing number of vacuoles. In addition, the proportion of acridine orange stain-positive cells increased according to time dependence. Localization of GFP-LC3 in cytoplasm and expression of LC-3II and Beclin 1 were increased according to time dependence on nutrient-deprived cells.

Conclusion

Nutrient deprivation induces cell death through autophagy in H460 lung cancer cells.

Figures and Tables

Figure 1
Effect of nutrient deprivation on cell survival in H460 cell line. Cells were seeded at a density of 1x105 cells/mL in 48 well plates and incubated in completely fresh medium for 20 hours. The cells were washed with PBS and the medium was then changed to a medium without the nutrient factors. Basal medium eagle (BME) was deprived of each nutrient and prepared by adding 10 mM 2-deoxyl-D-glucose (2DG) or 10% of dialyzed fetal bovine serum to the basal medium. Cell survival in the presence (A) and absence (B) of serum were shown. Cell numbers at the start of starvation were considered to be 100%. The viability of cell was measured by MTT assay. Means±SD (n=4). *p≤0.05, p≤0.01.
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Figure 2
Apoptosis is not related with cell death induced by nutrient deprivation in NCI-H460 cells. Cells were incubated in RPMI 1640 medium, basal medium eagle (BME) medium or 10 mM 2-deoxyl-D-glucose (2DG) with BME medium (12 hours) assayed for apoptosis by FACS following annexin V-FITC staining. Control cells were contained RPMI 1640 Medium. 20 µM cisplatin was used as a positive control. The data shown are representative of three independent experiment.
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Figure 3
No apoptotic nuclear change occurred in nutrient-deprived H460 cells. H460 cells were plated on glass coverslips. Cells were incubated in RPMI 1640 medium, basal medium eagle (BME) medium (A) or 10 mM 2-deoxyl-D-glucose (2DG) with BME medium (B) for various periods. Hoechst staining was used to identify morphological features of cell death such as nuclear condensation and fragmentation. Scale bar, ×200. Samples were examined under a fluorescence microscope.
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Figure 4
No apoptosis and necrosis is not associated with the nutrient-deprived H460 cell death. Cells probed with Western immunoblots were incubated in RPMI 1640 medium (CTR), basal medium eagle (BME) medium (A) or 10 mM 2-deoxyl-D-glucose (2DG) with BME medium (B) for various periods. The equal amounts of protein from cell lysate were subjected on 12% and 15% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto polyvinylidene fluoride (PVDF) membrane and immunoblotted with antibodies against Caspase 3 and cleaved caspase 3, PARP, cleaved PARP, HMGB1 and Actin. The immunoreactive signals were visualized by enhanced chemiluminescent kit (ECL). Actin was used as a roading control.
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Figure 5
Cytoplasmic Vacuoles were increased according to time dependence in nutrient-deprived H460 cells. H460 cells were incubated in RPMI 1640 medium (CTR), basal medium eagle (BME) medium or 10 mM 2-deoxyl-D-glucose (2DG) with BME medium for 12 hours and 24 hours. Scale bar, ×200. Samples were examined under a light microscope.
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Figure 6
Red acidic autophagic vacuoles were detected in cytoplasm of nutrient-deprived H460 cells according to time dependence. H460 cells were incubated in RPMI 1640 medium (CTR), basal medium eagle (BME) medium or 10 mM 2-deoxyl-D-glucose (2DG) with BME medium for 12 hours and 24 hours as shown. And then stained by 1 µg/mL acridine orange in medium for 15 minutes. The acidic autolysosomes induced by nutrition deprivation were stained orange-red.
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Figure 7
Development of acidic vesicular organelles (AVO) in nutrient-deprived H460 cells and its quantification using FACS analysis. H460 cells were incubated in RPMI 1640 medium, basal medium eagle (BME) medium or 10 mM 2-deoxyl-D-glucose (2DG) with BME medidum for 6 hours, 12 hours, and 24 hours. AVOs were counted in cytoplasm of H460 cells. *p≤0.05, p≤0.01.
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Figure 8
Fluorescence staining of GFP-LC3 in response to nutrient deprivation. H460 cells were plated on glass coverslips. Following transfection with GFP-LC3 plasmid, cells were incubated in RPMI 1640 medium, basal medium eagle (BME) medium, 10 mM 2-deoxyl-D-glucose (2DG) with BME medium for 12 hours and 24 hours as indicated. The transfected cells were observed using a single-photon fluorescence microscope.
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Figure 9
Induction of autophagic protein LC3-I and formation of LC3-II and Beclin 1 in nutrient deprived condition. Lung cancer cells were incubated in basal medium eagle (BME) medium (A) and 10 mM 2-deoxyl-D-glucose (2DG) With BME medium (B) for various time periods as indicated. Beclin 1, the product of autophagy-promoting gene Beclin 1 (ATG6) expression was detected. LC3-I induction and conversion of LC3-I to LC3-II were determined by western blot analysis. GAPDH protein levels served as loading control.
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Figure 10
Effect of 3-Methyladenine (3-MA) on nutrient deprived cell death in H460 cells. Relative cell viability was determined 12 hours and 24 hours after nutrient-deprived medium cultured in the presence and absence of 10 mM 3-MA, respectively. Cell numbers at the start of starvation were considered to be 100%. The viability of cell was measured by MTT assay. Means±SD (n=4). *p≤0.01.
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Table 1
Media formulation
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Table 2
Induction of S-phase arrest in Nutrient-deprived condition
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BME: basal medium eagle; 2DG: 2-deoxyl-D-glucose.

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