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. 2021 Nov;42(11):1875-1887.
doi: 10.1038/s41401-021-00612-9. Epub 2021 Feb 19.

Autophagy inhibitors increase the susceptibility of KRAS-mutant human colorectal cancer cells to a combined treatment of 2-deoxy-D-glucose and lovastatin

Xiao-Ming Huang  1 , Jia-Jun Huang  1 , Jing-Jing Du  1 , Na Zhang  1 , Ze Long  1 , You Yang  1 , Fang-Fang Zhong  1 , Bo-Wen Zheng  1 , Yun-Fu Shen  1 , Zhe Huang  1 , Xiang Qin  1 , Jun-He Chen  1 , Qian-Yu Lin  1 , Wan-Jun Lin  1 , Wen-Zhe Ma  2
Affiliations

Autophagy inhibitors increase the susceptibility of KRAS-mutant human colorectal cancer cells to a combined treatment of 2-deoxy-D-glucose and lovastatin

Xiao-Ming Huang et al. Acta Pharmacol Sin. 2021 Nov.

Erratum in

Abstract

RAS-driven colorectal cancer relies on glucose metabolism to support uncontrolled growth. However, monotherapy with glycolysis inhibitors like 2-deoxy-D-glucose causes limited effectiveness. Recent studies suggest that anti-tumor effects of glycolysis inhibition could be improved by combination treatment with inhibitors of oxidative phosphorylation. In this study we investigated the effect of a combination of 2-deoxy-D-glucose with lovastatin (a known inhibitor of mevalonate pathway and oxidative phosphorylation) on growth of KRAS-mutant human colorectal cancer cell lines HCT116 and LoVo. A combination of lovastatin (>3.75 μM) and 2-deoxy-D-glucose (>1.25 mM) synergistically reduced cell viability, arrested cells in the G2/M phase, and induced apoptosis. The combined treatment also reduced cellular oxygen consumption and extracellular acidification rate, resulting in decreased production of ATP and lower steady-state ATP levels. Energy depletion markedly activated AMPK, inhibited mTOR and RAS signaling pathways, eventually inducing autophagy, the cellular pro-survival process under metabolic stress, whereas inhibition of autophagy by chloroquine (6.25 μM) enhanced the cytotoxic effect of the combination of lovastatin and 2-deoxy-D-glucose. These in vitro experiment results were reproduced in a nude mouse xenograft model of HCT116 cells. Our findings suggest that concurrently targeting glycolysis, oxidative phosphorylation, and autophagy may be a promising regimen for the management of RAS-driven colorectal cancers.

Keywords: 2DG; OXPHOS; autophagy; chloroquine; glycolysis; human colorectal cancers; hydroxychloroquine; lovastatin.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. The combination of lovastatin and 2DG synergistically inhibits the proliferation of KRAS-mutant CRC cells.
a HCT116 and LoVo cells were cultured with different concentrations of lovastatin and 2DG alone or in combination for 72 h, and cell proliferation was determined. b Combination index (CI) plot. The CI was identified using CompuSyn software, where CI<1, =1, and >1 indicate the synergism, additive effect, and antagonism, respectively. c Isobologram of the combination of two drugs. The combination data (triangle, square, and circle) points on the diagonal line indicate additive effects; the lower left indicates synergism, and the upper right indicates antagonism. d Colony formation of HCT116 and LoVo cells treated with lovastatin (7.5 μM) and 2DG (2.5 mM) alone or in combination for 10 d. The data are from at least three independent experiments.
Fig. 2
Fig. 2. The combination of lovastatin and 2DG triggers significantly increased apoptosis rates.
The combination of lovastatin and 2DG triggers significantly increased apoptosis rates. After treatment with lovastatin (15 μM) and 2DG (5 mM) alone or in combination for 48 h, a HCT116 and LoVo cells were stained with FITC Annexin V/PI, and apoptosis was quantified by flow cytometry. b Quantification data of the apoptotic cells. c Representative immunoblots of cleaved caspase-3 and cleaved PARP in the HCT116 and LoVo cells. β-actin served as a loading control. d Quantified data of (c). e HCT116 and LoVo cells were treated with lovastatin (15 μM) and 2DG (5 mM) alone or in combination for 48 h. Representative immunoblots of AKT, p-AKT, p-ERK, and ERK. β-actin served as a loading control. f Quantified data of (e). All data are from at least three independent experiments. *P < 0.05, **P < 0.01.
Fig. 3
Fig. 3. The combination of lovastatin and 2DG induces G2/M cell cycle arrest.
HCT116 and LoVo cells were treated with lovastatin (15 μM) and 2DG (5 mM) alone or in combination for 24 h. a Cell cycle distribution was analyzed using flow cytometry. b Percentage of cells in the G1, S, and G2/M phase. c Representative immunoblots showing the indicated proteins. β-actin served as a loading control. d Quantified data of (c). All data are from at least three independent experiments. *P < 0.05, **P < 0.01.
Fig. 4
Fig. 4. The combination of lovastatin and 2DG depletes the intracellular ATP level and regulates the AMPK/mTOR pathway.
After 48 h of exposure to lovastatin (15 μM) and 2DG (5 mM) alone or in combination, the oxygen consumption rate (a) and extracellular acidification rate (b) of HCT116 cells were measured on a Seahorse XFp analyzer. c The relative ATP production rate by glycolysis and OXPHOS was calculated by measuring oxygen consumption and extracellular acidification with a Seahorse XFp analyzer (*P < 0.05, **P < 0.01 versus total relative ATP production rate). d After treatment with lovastatin (15 μM) and 2DG (5 mM) alone or in combination for 24 h, the intracellular ATP concentrations in HCT116 and LoVo cells were measured. e HCT116 and LoVo cells were cultured with lovastatin (15 μM) and 2DG (5 mM) alone or in combination for 72 h in either the presence or absence of 200 mM mevalonate, and cell proliferation was determined. *P < 0.05, **P < 0.01. ##P < 0.01 vs corresponding MVA (-) group. f HCT116 cells were cultured with lovastatin at the indicated concentrations in the presence or absence of 200 μM mevalonate/10 μM farnesyl pyrophosphate (FPP), and colony formation was determined. g Representative immunoblots of AMPKα, p-AMPKα Thr172, mTOR, p-mTOR Ser2448, p70S6K, p-p70S6K Thr389, 4E-BP1 and p-4E-BP1 from the HCT116 and LoVo cells treated with lovastatin (15 μM) and 2DG (5 mM) alone or in combination for 48 h. β-actin served as a loading control. h Quantified data of (g). All data are from at least three independent experiments.
Fig. 5
Fig. 5. The combination of lovastatin and 2DG enhances autophagic flux.
The combination of lovastatin and 2DG increases autophagic flux. a Representative immunoblots of LC3 and Beclin-1 in HCT116 and LoVo cells after treatment with lovastatin (15 μM) and 2DG (5 mM) alone or in combination for 48 h. β-actin served as a loading control. b Quantified data of (a). c Cells transfected with the pEGFP-LC3 plasmid were analyzed for pEGFP-LC3 puncta formation in HCT116 cells after treatment with lovastatin (15 μM) and 2DG (5 mM) alone or in combination for 48 h. Bar, 20 μm. d Representative immunoblots showing LC3 and Beclin-1 from HCT116 cells treated with either 2DG (5 mM) and different concentrations of lovastatin or lovastatin (15 μM) and different concentrations of 2DG for 48 h. e Quantified data of (d). f Representative immunoblots showing LC3 from HCT116 cells treated with either vehicle or the combination of lovastatin (15 μM) and 2DG (5 mM) for 20 h, followed by treatment with or without bafilomycin A1 (100 nM) for 4 h. g Quantified data of (f). h HCT116 cells were treated with either vehicle or a combination of lovastatin (15 μM) and 2DG (5 mM) in the presence or absence of actinomycin D (8 μM) for 24 h. Representative immunoblots of p62 protein levels. β-actin served as a loading control. i Quantified data of (h). j HCT116 cells were transduced with LAMP1-GFP and cultured for 24 h. Then, the cells were treated with vehicle, CQ (20 μM), a combination of lovastatin (15 μM) and 2DG (5 mM) or lovastatin + 2DG + CQ for 48 h. The cells were stained with 75 nM LysoTracker Red and imaged. Bar, 10 μm. k HCT116 cells expressing an autophagic flux reporter (AFR) were generated by ectopic expression of a chimeric fusion protein composed of pHluorin-mKate2-hLC3. The increased ratio of red (mKate2):green (pHluorin) fluorescence measured by flow cytometry is an indication of increased autophagic flux. Autophagic flux in HCT116 cells was also measured by flow cytometry following 48 h of treatment with either vehicle or the combination of lovastatin (15 μM) and 2DG (5 mM) and bafilomycin A1 (100 nM) as a negative control for 4 h (*P < 0.05, **P < 0.01 versus high (red)). All data are from at least three independent experiments. *P < 0.05, **P < 0.01.
Fig. 6
Fig. 6. The inhibition of autophagy increases the vulnerability of HCT116 cells and LoVo cells to combined treatment with lovastatin and 2DG.
a HCT116 and LoVo cells were treated with lovastatin (15 μM) and 2DG (5 mM) alone or in combination for 72 h in the presence or absence of chloroquine (CQ) (6.25 μM), and cell proliferation was determined (n = 3) (*P < 0.05, **P < 0.01. #P < 0.05, ##P < 0.01 versus indicated treatment group in the absence of CQ). b Colony formation of HCT116 and LoVo cells treated with lovastatin (5 μM) and 2DG (2 mM) alone or in combination in the presence or absence of CQ (400 nM) for 10 d. c Quantified data of (b) (n = 4). (*P < 0.05, **P < 0.01. #P < 0.05, ##P < 0.01 versus indicated treatment group in the absence of CQ). d HCT116 cells were injected subcutaneously into each hind limb of nude mice, and hydroxychloroquine (HCQ) (80 mg/kg every 2 d), lovastatin (25 mg/kg every 2 d), 2DG (5 mg/kg every 2 d), Comb. (lovastatin + 2DG) and HCQ + Comb. were administered orally. Ctrl n = 8, lovastatin n = 10, 2DG n = 10, HCQ n = 8, Comb. n = 9, HCQ + Comb. n = 8. Data were shown as the means ± SEM. e Tumor size in the mice shown in (d). *P < 0.05, **P < 0.01. Data are shown as the means ± SEM. f Tumors excised from mice in (d), and the dotted circle represents the tumor that completely disappeared after treatment. g Schematic representation of the combined effect of lovastatin and 2DG showing increased susceptibility of HCT116 cells to autophagy inhibition.
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