Halofuginone inhibits colorectal cancer growth through suppression of Akt/mTORC1 signaling and glucose metabolism

Abstract

The Akt/mTORC1 pathway plays a central role in the activation of Warburg effect in cancer. Here, we present for the first time that halofuginone (HF) treatment inhibits colorectal cancer (CRC) growth both in vitro and in vivo through regulation of Akt/mTORC1 signaling pathway. Halofuginone treatment of human CRC cells inhibited cell proliferation, induced the generation of reactive oxygen species and apoptosis. As expected, reduced level of NADPH was also observed, at least in part due to inactivation of glucose-6-phosphate dehydrogenase in pentose phosphate pathway upon HF treatment. Given these findings, we further investigated metabolic regulation of HF through Akt/mTORC1-mediated aerobic glycolysis and found that HF downregulated Akt/mTORC1 signaling pathway. Moreover, metabolomics delineated the slower rates in both glycolytic flux and glucose-derived tricarboxylic acid cycle flux. Meanwhile, both glucose transporter GLUT1 and hexokinase-2 in glycolysis were suppressed in CRC cells upon HF treatment, to support our notion that HF regulates Akt/mTORC1 signaling pathway to dampen glucose uptake and glycolysis in CRC cells. Furthermore, HF retarded tumor growth in nude mice inoculated with HCT116 cells, showing the anticancer activity of HF through metabolic regulation of Akt/mTORC1 in CRC.

Keywords: Akt/mTORC1; anticancer activity; colorectal cancer; glucose metabolism; halofuginone.

Figure 1. Halofuginone inhibits colorectal cancer cell proliferation

A. Chemical structure of halofuginone. B. MTT assay of five CRC cell lines (SW480, HCT116, SW620, HT29 and DLD-1) treated with increasing concentrations of HF in a time course (12 h, 24 h and 48 h). *P < 0.05, **P < 0.01, compared with 12 h at the same concentration. C. MTT assay of non-transformed rat small intestinal epithelial cell line IEC-6 and human non-tumorigenic liver cell line MIHA treated with increasing concentrations of HF in a time course (12 h, 24 h and 48 h). D. Colony formation assay of CRC cell lines (SW480, HCT116, SW620, HT29 and DLD-1) treated with 0, 5, 10 and 20 nM of HF. E. A flow cytometry analysis of SW480 and HCT116 treated with 0, 5, 10 and 20 nM of HF for 12 h inducing cell cycle arrest in G1/G0 phase. *P < 0.05, **P < 0.01, compared with control group.

Figure 2. Halofuginone induces cell apoptosis

A. The bar chart shows the percentage of apoptotic cells in SW480 and HCT116 cell lines treated with 0, 5, 10 and 20nM of HF (upper panel), and the representative flow cytometry annexin V-PI data (lower panel). *P < 0.05, **P < 0.01, compared with control group. B. Protein expressions of cleaved Caspase-3 and cleaved PARP in SW480 and HCT116 cell lines treated with 0, 5, 10 and 20 nM of HF.

Figure 3. Halofuginone enhances ROS levels while reduces NADPH production

A. ROS levels in SW480 and HCT116 cell lines treated with 0, 5, 10, 20 nM of HF. *P < 0.05, **P < 0.01, compared with control group. B. Protein expression of mitochondrial marker VDAC in SW480 and HCT116 cell lines treated with 0, 5, 10, 20 nM of HF. C. Relative ratios of [NADP⁺]/[NADPH] in SW480 and HCT116 cell lines treated with 0, 5, 10, 20 nM of HF. *P < 0.05, **P < 0.01, compared with control group. D. Protein expression of G6PD, PGD, ME1 and IDH1 in SW480 and HCT116 cell lines treated with 0, 5, 10, 20 nM of HF (G6PD: glucose-6-phosphate dehydrogenase, PGD: 6-phosphogluconate dehydrogenase, ME1: malic enzyme, IDH1: isocitrate dehydrogenase).

Figure 4. Halofuginone suppresses Akt/mTORC1 signaling pathway and slows glycolytic flux and glucose-derived TCA cycle flux

A. Protein expressions of phosphorylation of Akt, mTORC1, p70S6 and 4EBP1 (left panel); quantitative analysis of protein expressions (right panel) in SW480 and HCT116 upon HF treatment in a dose-dependent manner for 12 h. *P < 0.05, **P < 0.01, compared with control group. B. Uniformly ¹³C-labeled glucose feeding cancer cells for intermediate metabolites in glycolysis by using UPLC-MS/MS (G6P: glucose-6-phosphate, GAP: glyceraldehyde-3-phosphate, PEP: phosphoenolpyruvate, Pyr: pyruvate). The results shown are means ± SEM, n = 5. *P < 0.05, **P < 0.01, compared with control group. C. Protein expressions of HK-II, PKM2, PDH and LDHA by Western blot (HK-II: hexokinase-2, PKM2: M2 isoform of pyruvate kinase, PDH: pyruvate dehydrogenase, LDHA: lactate dehydrogenase A). D. The GC/MS analysis of [U-¹³C₆]-glucose contribution to citrate, fumarate and malate synthesis in HCT116 cell with or without HF treatment. All GC/MS data were corrected for natural abundance isotopic contribution and normalized to cell number and internal standard 4-chloro-DL-phenylalanine. The results shown are means ± SEM, n = 5.

Figure 5. Halofuginone retards tumor growth in xenograft-bearing BALB/c nude mice

A. Tumor volumes between HF-treated and vehicle group during treatment for 14 days. *P < 0.05, **P < 0.01, compared with vehicle group. B. Change in body weight between HF-treated and vehicle group. C. Change in tumor weight between HF-treated and vehicle group. *P < 0.05 compared with vehicle group. D. The xenograft tumors were dissected and measured after two weeks and shown. E. Photos of all of the animals. F. TUNEL staining of paraffin embedded 5 micron think tumor sections of HCT116 xenograft-bearing nude mice. G. The decreased p-Akt, p-mTORC1, p-p70S6K and increased p-4EBP1 are shown in the immunofluorescence staining.

Figure 6. The proposed metabolic mechanism modulated by HF treatment in CRC cells

HF can downregulate Akt/mTORC1 signaling pathway and repress Warburg effect, particularly, inhibit GLUT1 and HK-II through activation of 4EBP1. Accordingly, the glycolytic flux and TCA cycle flux are downregulated. Meanwhile, HF can inhibit the downstream of mTORC1 p70S6K for dampening PPP and lipid biosynthesis.