The bifunctional autophagic flux by 2-deoxyglucose to control survival or growth of prostate cancer cells

Abstract

Background: Recent reports using metabolism regulating drugs showed that nutrient deprivation was an efficient tool to suppress cancer progression. In addition, autophagy control is emerging to prevent cancer cell survival. Autophagy breaks down the unnecessary cytoplasmic components into anabolic units and energy sources, which are the most important sources for making the ATP that maintains homeostasis in cancer cell growth and survival. Therefore, the glucose analog 2-deoxyglucose (2DG) has been used as an anticancer reagent due to its inhibition of glycolysis.

Methods: Prostate cancer cells (PC3) were treated with 2DG for 6 h or 48 h to analyze the changing of cell cycle and autophagic flux. Rapamycin and LC3B overexpressing vectors were administered to PC3 cells for autophagy induction and chloroquine and shBeclin1 plasmid were used to inhibit autophagy in PC3 cells to analyze PC3 cells growth and survival. The samples for western blotting were prepared in each culture condition to confirm the expression level of autophagy related and regulating proteins.

Results: We demonstrated that 2DG inhibits PC3 cells growth and had discriminating effects on autophagy regulation based on the different time period of 2DG treatment to control cell survival. Short-term treatment of 2DG induced autophagic flux, which increased microtubule associated protein 1 light chain 3B (LC3B) conversion rates and reduced p62 levels. However, 2DG induced autophagic flux is remarkably reduced over an extended time period of 2DG treatment for 48 h despite autophagy inducing internal signaling being maintained. The relationship between cell growth and autophagy was proved. Increased autophagic flux by rapamycin or LC3B overexpression powerfully reduced cell growth, while autophagy inhibition with shBeclin1 plasmid or chloroquine had no significant effect on regulating cell growth.

Conclusion: Given these results, maintaining increased autophagic flux was more effective at inhibiting cancer cell progression than inhibition of autophagic flux, which is necessary for the survival of PC3 cells. Autophagic flux should be tightly regulated to maintain metabolic homeostasis for cancer cell growth and survival in PC3 cells and is a suitable target for cancer therapy.

Fig. 1

2-deoxyglucose inhibits PC3 prostate cancer cell growth and inhibits autophagy. PC3 cells were incubated with different concentrations of 2-deoxyglucose (2DG). a Cell growth rate was measured by counting the cell numbers at the indicated time points for 3 days. Data are represented as means ± SD. ***P < 0.001, control versus 5 mM 2DG; ***P < 0.001, control versus 10 mM 2DG; ***P < 0.001, control versus 20 mM 2DG. (b) After 2DG treatment for 48 h, autophagy levels were detected using western blotting analysis. p62 was used as an autophagy substrate and LC3B showed autophagy levels. (c) The level of autophagy was confirmed with confocal microscopy. Blue, nucleus; Green, LC3B; Red, p62

Fig. 2

Long-term exposure to 2DG enhances cell cycle arrest. PC3 cells were treated with 2DG for a short time (6 h) or long time (48 h). CQ was used as an autophagy inhibitor at a final concentration of 20 μM for 2 h. Rapamycin was used as an autophagy inducer at 100 nM for 6 h. a After 2DG treatment, each cell was collected for FACS analysis. Cells were stained with iodine for 5 min and analyzed for the amount of DNA content. Each DNA content rate is expressed as G1, S, and G2 phase. Data are represented as the means ± SD. *P < 0.05, **P < 0.01, **P < 0.001. (b) Expression levels of cell cycle related genes were observed using western blotting analysis

Fig. 3

Autophagic flux is differentially regulated by exposure time to 2DG. 2DG was administered to PC3 cell, with CQ or alone, to confirm autophagic flux. a Protein levels of p62 and LC3B in 2DG containing culture medium for the indicated time followed by CQ or no further treatment were observed with western blotting. L.E. means long exposure. (b) Autophagic flux calculated by the accumulated amount of LC3B with treatment of CQ autophagy blocker for 2 h. Data are represented as the means ± SD. ***P < 0.001 for both culture conditions. (c) The changes in intracellular signaling pathway related autophagy regulation were verified for each condition. Phosphorylated levels of mTOR and AKT were checked for whether the inhibitory signal of autophagy was induced. The AMPK signaling pathway was confirmed as an autophagy activating condition

Fig. 4

Knockdown of Beclin1 is not effective at regulating the cell cycle. The samples for western blotting were prepared in each culture condition. Afterwards, a changing autophagy levels, (b) the kinetics of signaling proteins in autophagy regulation, and (c) the expression levels of cell cycle related genes were checked in both normal and Beclin1 knocked-down PC3 cells using shRNA introduction. shN.T., non-targeting shRNA; shBec1, shRNA for Beclin1

Fig. 5

Induction of autophagy suppresses cell growth and survival. Autophagic flux was regulated by chemical treatment or genetic modification to prove the relationship between autophagic flux and cell growth. Autophagy was blocked with a various concentrations of CQ treatment or (b) Beclin1-targeting shRNA. The level of Beclin1 expression was confirmed with western blotting for Beclin1 (Right panel). (c) Autophagy was induced with rapamycin at the indicated concentrations. The level of LC3B expression was confirmed with western blotting for LC3B (Right panel). Data are represented as the means ± SD, ***P < 0.001 versus 1 nM, ***P < 0.001 versus 10 nM, ***P < 0.001 versus 100 nM, ***P < 0.001 versus 1000 nM. (d) The LC3B plasmid to overexpress LC3B was introduced into PC3 cells. Data are represented as the means ± SD. ***P < 0.001 versus 2DG, ***P < 0.001 versus LC3B, ***P < 0.001 versus LC3B + 2DG. Cell growth rate was measured using an MTT assay (a and c) or cell counting (b and d). Autophagy blocking did not show a synergistic effect with 2DG to regulate cell growth. However, autophagy induction suppressed cell growth, not only with LC3B overexpression, but also with 2DG combination treatment, which showed a synergistic cell suppression effect