Yeast β-D-glucan exerts antitumour activity in liver cancer through impairing autophagy and lysosomal function, promoting reactive oxygen species production and apoptosis

Abstract

Autophagy is an evolutionarily conserved catabolic process that recycles proteins and organelles in a lysosome-dependent manner and is induced as an alternative source of energy and metabolites in response to diverse stresses. Inhibition of autophagy has emerged as an appealing therapeutic strategy in cancer. However, it remains to be explored whether autophagy inhibition is a viable approach for the treatment of hepatocellular carcinoma (HCC). Here, we identify that water-soluble yeast β-D-glucan (WSG) is a novel autophagy inhibitor and exerts significant antitumour efficacy on the inhibition of HCC cells proliferation and metabolism as well as the tumour growth in vivo. We further reveal that WSG inhibits autophagic degradation by increasing lysosomal pH and inhibiting lysosome cathepsins (cathepsin B and cathepsin D) activities, which results in the accumulation of damaged mitochondria and reactive oxygen species (ROS) production. Furthermore, WSG sensitizes HCC cells to apoptosis via the activation of caspase 8 and the transfer of truncated BID (tBID) into mitochondria under nutrient deprivation condition. Of note, administration of WSG as a single agent achieves a significant antitumour effect in xenograft mouse model and DEN/CCl₄ (diethylnitrosamine/carbon tetrachloride)-induced primary HCC model without apparent toxicity. Our studies reveal, for the first time, that WSG is a novel autophagy inhibitor with significant antitumour efficacy as a single agent, which has great potential in clinical application for liver cancer therapy.

Keywords: Antitumour; Autophagy inhibition; Hepatocellular carcinoma; Lysosomal function; β-D-glucan.

Graphical abstract

Fig. 1

WSG exerts direct antitumour effects in liver cancer cells and in nude mice. (A) The molecular structure of water-soluble yeast β-D-glucan (WSG). WSG is polymerized by glucose monomers and its main chain is linked by β-(1 → 3)-glycoside bonds. Every nine β-(1 → 3)-glucose units in the main chain linked to one glucose unit in the side chain through β-(1 → 6)-glycoside bonds. (B) Cell viability of Huh7 cells treated with different concentrations of WSG at 48 h. Different concentrations of WSG was compared with control. (C) Cell viability of Huh7 cells at different time points treated with 8 mg/ml WSG. (D) Schematic of workflow for assessment of antitumour effect of WSG in BALB/c male nude mice. Huh7 cells were subcutaneously injected into right and left flanks of nude mice (regarded as day = 0). They were randomly divided into 3 groups on the third day after injection and were administrated with vehicle (n = 9), 150 mg/kg (n = 8) and 300 mg/kg (n = 8) via i. p. Every other day, respectively. Red circles indicate Huh 7 cells. (E) Changes of body weight of nude mice during the period of administration. (F) Images of tumour-bearing nude mice and tumours. (G) The weight of tumours from mice in (F). (H) Changes of tumour volume during the period of administration. (I) Representative images of Ki67 staining in mice tumour (left) and quantification of Ki67 positive cells (right). Scale bar: 50 μm. All data are presented as mean ± SEM. **P < 0.01; ***P < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

WSG inhibits autophagic flux at its late stage. (A) The expression of autophagy-related genes from transcriptomic RNA-Seq data in control and WSG (8 mg/ml) treated Huh7 cells. (B) Fluorescence imaging and quantification of GFP-LC3 puncta in Huh7 cells treated with 8 mg/ml WSG for 48 h. The number of GFP-LC3B puncta per cell was counted in 15 cells each condition and data are representative of 3 independent experiments. Scale bar: 5 μm. (C) Expression of LC3, p62 and the proteins upstream autophagic process in Huh7 cells upon WSG treatment for 48 h. Quantification of LC3-II/GAPDH, p62/GAPDH, p-AMPK/AMPK and p-mTOR/mTOR were based on 3 independent experiments. (D) Blockage of autophagic flux by WSG. Huh7 cells were treated with WSG (8 mg/ml) for 48 h. Baf A1 (bafilomycin A1, 50 nM) treated cells for 6 h before cell lysis. Quantification of LC3-II/GAPDH and p62/GAPDH were based on 3 independent experiments. (E) Representative fluorescence images of WSG and CQ (chloroquine) on autophagosome maturation and autolysosome formation. Huh7 cells were treated with 8 mg/ml WSG for 48 h or CQ (25 mM) for 6 h. More than 15 cells were counted in each condition and data are representative of 3 independent experiments. Scale bar: 5 μm. All data are presented as mean ± SEM. *P < 0.05; **P < 0.01.

Fig. 3

WSG impairs lysosome function by increasing lysosomal pH and inhibiting cathepsins activities without affecting the fusion between autophagosomes and lysosomes. (A) The colocalization of GFP-LC3 and LAMP1. Huh7 cells transfected with GFP-LC3 were treated with WSG (8 mg/ml) for 48 h or Baf A1 (50 nM) for 6 h before fixed in total medium. And HBSS medium was changed 3 h before cells were fixed. PCC (Pearson correlation coefficient), a statistic to quantify the extent of colocalization of the two signals, is calculated using Image J software from 3 independent experiments. Scale bar: 5 μm. (B) The effect of WSG on lysosomal pH. Huh7 cells were treated with WSG (8 mg/ml) for 48 h or CQ (25 mM) for 6 h. At the end of treatment, cells were treated with 2 μM LysoSensor Green DND-189 to evaluate lysosomal pH change. The fluorescence intensity is calculated using Image J software from 3 independent experiments. Scale bar: 5 μm. (C) The protein levels of Cat D (cathepsin D) and Cat B (cathepsin B) in Huh7 cells upon WSG treatment for 48 h. (D) Representative transmission electron microscopy (TEM) images in Huh7 cells treated with WSG (8 mg/ml) for 48 h. The arrow in left panel indicates autophagic vacuole contained electron dense cellular contents or organelles at various stages of degradation. The arrows in right panel indicate autophagic vacuoles contained electron translucent and morphologically intact cellular contents or organelles. (E) Expression of ubiquitinated proteins in Huh7 cells upon WSG treatment for 48 h. All data are presented as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

Autophagy inhibition by WSG decreases the metabolism in glycolysis and the TCA cycle pathways. (A) The expression of metabolism-related genes from transcriptomic RNA-Seq data in control and WSG (8 mg/ml) treated Huh7 cells. (B) The glucose consumption in collected medium after treatment with WSG at 48 h in Huh7 cells. (C)¹³C distribution of glycolysis and the first turn of the TCA cycle with 50% U–¹³C-glucose (labelled at all six carbons) and 50% glucose (unlabelled). All colored circles indicate ¹³C-labelled carbon atoms from the labelled glucose. Striped circles indicate ¹³C-labelled carbons are converted from the labelled glucose in glycolysis, red circles indicate ¹³C-labelled carbons are obtained from pyruvate dehydrogenase (PDH), and blue circles indicate ¹³C-labelled carbons are converted from pyruvate carboxylase (PC) or malate enzyme (ME) to catalyze metabolic pathways. G-6-P, glucose-6-phosphate; F-6-P, fructose-6-phosphate; F-1,6–2P, fructose-1,6-diphosphate; GAP, glyceraldehyde; ME, malate enzyme. (D) Fraction of the labelled metabolites of M+3 from ¹³C-glucose in glycolysis by WSG treatment for 48 h in Huh7 cells. (E) Fraction of the labelled metabolites of M+2 from ¹³C-glucose in TCA cycle by WSG treatment for 48 h in Huh7 cells. (F) Fraction of the labelled metabolites of M+3 from ¹³C-glucose in TCA cycle by WSG treatment for 48 h in Huh7 cells. All data are presented as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5

WSG leads to mitochondria dysfunction and sensitizes liver cancer cells to apoptosis via the intrinsic and extrinsic pathways under nutrient deprivation. (A) Representative TEM images of mitochondria in Huh7 cells treated with WSG (8 mg/ml) for 48 h. The arrows indicate normal mitochondria (left panel) and abnormal mitochondria (right panel). (B–C) Fluorescent images of mitochondrial membrane potential (MMP) (B) and reactive oxygen species (ROS) (C) in Huh7 cells treated with WSG for 48 h. Cells were stained with 50 nM TMRM (Tetramethylrhodamine, a cell-permeant dye that accumulates in active mitochondria with intact membrane potentials) or 5 μM DHE (dihydroethidium, a fluorescent probe for the detection of ROS generation), respectively. The fluorescence intensity was calculated in 20 individual fields per well from six individual wells. (D) The ratio of ATP/ADP and ATP/AMP (normalized to control) in Huh7 cells treated with WSG for 48 h. (E) Effect of WSG on apoptosis by annexin-V and propidium iodide (PI) double staining. Huh7 cells were treated with 8 mg/ml WSG in the presence or absence of fetal bovine serum (FBS) for 48 h. (F) Expression of LC3, p62, cathepins and caspases 8 in Huh7 cells treated with 8 mg/ml WSG in the presence or absence of FBS for 48 h (left panel). Increased lysosomal pH leads to decreased lysosomal cathepsins maturation, followed by inhibition of autophagic degradation, which then leads to the accumulation of autophagic cargos and triggers caspase 8-mediated apoptosis upon WSG treatment under nutrient deprivation (right panel). (G) Caspase 8 activity in Huh7 cells treated with WSG under FBS starvation for 48 h. (H) WSG induced the truncated BID (tBID) to transfer into mitochondria. C: cytosol; M: mitochondria. All data are presented as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 6

WSG attenuates DEN-CCl₄-induced primary liver cancer tumorigenesis. (A) Schematic of workflow for assessment of antitumour effect of WSG in C57BL/6 mice. Mice were treated with CCl₄ twice a week (i.p.) for three months. WSG or CQ was administrated (i.p.) at the dose of 50 mg/kg or 25 mg/kg three times a week, respectively. (B) Representative images of livers and hematoxylin-eosin (HE) staining images of livers. Red-dotted circles indicate tumours. Black-dotted lines indicate the boundary of normal tissues and tumour tissues. N, normal tissue; T, tumour tissue. Scale bar: 50 μm. (C–F) Shows the ration of liver weight/body weight (C), total tumour number (D), the number of tumours bigger than 5 mm (E) and max tumour volume (F). (G) Representative images of Ki67 staining in liver. Scale bar: 50 μm. (H) Quantification of Ki67 positive cells in (G). More than 10 fields were counted in each mice and data are representative of 5 mice in each group. (I) Expression of LC3, p62, Cat B and caspase 8 in tumours from at least 3 different mice in each groups. All data are presented as mean ± SEM. *P < 0.05; **P < 0.01. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 7

A working model of antitumour effect of WSG by inhibiting autophagic degradation. WSG inhibits autophagy flux by inhibiting lysosome acidification and lysosome cathepsins activities without affecting the fusion between autophagosomes and lysosomes. The blockage of autophagic degradation leads to the accumulation of damaged mitochondria and reactive oxygen species (ROS) production. Furthermore, WSG sensitizes liver cancer cells to apoptosis via the cleavage of the protein BID into the truncated BID (tBID) by activated caspase 8, which results in activation of the mitochondrial apoptosis pathway under nutrient deprivation.