The combination of hydroxychloroquine and 2-deoxyglucose enhances apoptosis in breast cancer cells by blocking protective autophagy and sustaining endoplasmic reticulum stress

Abstract

2-Deoxyglucose (2-DG) can be used in antitumour research by inhibiting glycolysis and promoting the endoplasmic reticulum stress (ERS) pathway, but its clinical application is restricted due to dose-limiting side effects and survival chance for cancer cells by protective autophagy. Therefore, our research explored whether the combination of hydroxychloroquine (HCQ), an FDA-approved autophagy inhibiting drug, and 2-DG is a promising therapeutic strategy. Here, we report that HCQ combined with 2-DG can further inhibit the viability and migration and induce apoptosis of breast tumour cells compared with other individual drugs. The combination of 2-DG and HCQ can significantly reduce transplanted tumour size and tumour cell metastasis of the lung and liver in vivo. At the cellular level, HCQ suppressed autolysosome formation and terminated the autophagy process induced by 2-DG-mediated ERS, resulting in the continuous accumulation of misfolded proteins in the endoplasmic reticulum, which generated sustained ERS through the PERK-eIF2α-ATF-4-CHOP axis and triggered the transformation from a survival process to cell death. Our research reinforced the research interest of metabolic disruptors in triple-negative breast cancer and emphasized the potential of the combination of 2-DG and HCQ as an anticancerous treatment.

Fig. 1. HCQ and 2‐DG inhibit breast cancer cell viability.

A, B The 24 h IC50 values of HCQ and 2‐DG were calculated in 4T1 and CMT-7364 cells. Cell viability was determined via the Cell Counting Kit‐8. The combination of 2-DG and HCQ had a stronger cytotoxic effect on cell viability than 2-DG alone. n = 5 for each group. Data are presented as the means ± SD of three independent experiments. **p < 0.01, ****p < 0.0001, ns = not significant (p ≥ 0.05).

Fig. 2. HCQ synergizes with 2‐DG to decrease cell proliferation and promote apoptosis in 4T1 and CMT-7364 cells.

A Cell Counting Kit‐8 kits were used to assess the proliferation of 4T1 and CMT-7364 cells treated with HCQ, 2‐DG or HCQ combined with 2‐DG at 0, 12, 24, 48 and 72 h. n = 3 independent experiments performed in quintuplicate for each condition. B, C Wound-healing assays of 4T1 and CMT-7364 cells after HCQ and 2‐DG treatment alone or in combination for 24 h. Representative images depicting the beginning (t = 0 h) and the end (t = 24 h) of the recording period are shown. n = 3 independent experiments performed in triplicate for each condition. D, E Cell apoptosis assays of 4T1 and CMT-7364 cells treated with HCQ, 2‐DG or their combination using FACS. Cells were collected and labelled with Annexin V‐FITC and PI. n = 3 independent experiments performed in triplicate for each condition. F, G Western blot analyses of Bax, Bcl‐2, cleaved caspase-3 (C-Casp3), and cleaved PARP (C-PARP) in cells treated with HCQ and 2‐DG alone or in combination for 24 h. β‐actin was used as an internal control. H Immunofluorescence staining of C-Casp3 in 4T1 and CMT-7364 cells, which were treated with HCQ and 2‐DG alone or in combination for 24 h. Scale bar: 200 μm. n = 3 independent experiments performed in triplicate for each condition. All results from three independent experiments are expressed as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns = not significant (p ≥ 0.05).

Fig. 3. In vivo, HCQ combined with 2‐DG inhibits xenograft tumour growth.

BALB/c female mice were divided into five groups, and injection of the indicated drugs began on the fifteenth. A–C Every 2 days, the tumour volume and body weight were measured (n = 5 per group). D On Day 40, the mice were humanely killed, samples were collected, and the tumour weight was measured. Data are shown as the mean ± SD. The symbols ns, *, **, **** denote significant differences of not significant (p ≥ 0.05), P < 0.05, P < 0.01, P < 0.0001 versus the negative control group, respectively. E Representative panels of H&E staining of tumours and major vital organs, such as the liver, lung, spleen, heart and kidneys of mice. Scale bar: 50 μm. Blue-lined bordered boxes at the sections of the liver indicate metastatic 4T1 cells.

Fig. 4. HCQ and 2-DG synergistically promote endoplasmic reticulum stress through the PERK/eIF2α/ATF-4 pathway.

A Representative TEM images depicting the ultrastructure of 4T1 and CMT-7364 cells treated without or with HCQ, 2-DG or 2-DG combined with HCQ for 24 h. Red arrows indicate autophagic vacuoles; blue arrows indicate endoplasmic reticulum. Scale bars: 2 mm (Thumbnail), 200 nm (Enlarge image). B Western blot analyses of Grp78, PERK, p-PERK, eIF2α, p-eIF2α, ATF-4 and CHOP in cells treated with HCQ and 2‐DG alone or in combination for 24 h. β‐actin was used as an internal control. Data are represented as mean ± SD from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns = not significant (p ≥ 0.05).

Fig. 5. HCQ combined with 2-DG inhibits autophagic degradation in 4T1 cells and CMT-7364 cells.

A, B Western blot analyses of Beclin-1 and LC3B-II in 4T1 and CMT-7364 cells that were treated without or with HCQ, 2-DG or 2-DG combined with HCQ for 24 h. β‐actin was used as an internal control. Data are represented as mean ± SD from three independent experiments. **p < 0.01, ****p < 0.0001. C, D Immunofluorescence staining of LC3B-II in 4T1 and CMT-7364 cells, which were treated with HCQ and 2‐DG alone or in combination for 24 h. Scale bar: 10 μm. n = 3 independent experiments performed in triplicate for each condition and at least 30 cells scored.

Fig. 6. Schematic illustration delineating the role of HCQ/2‐DG combination therapy.

The metabolites of 2-DG interfere with the N-linked glycosylation of proteins, leading to the accumulation of unfolded/misfolded proteins in the ER, followed by ERS, apoptosis, and cytoprotective autophagy. HCQ causes deacidification of lysosomes, inhibits the fusion of autophagosomes and lysosomes to form autolysates, which results in the inhibition of misfolded protein degradation and further induces endoplasmic reticulum stress, ultimately leading to increased apoptosis.