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

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.

2 residue for vortexed-mixed for 30 sec, and samples were centrifuged at 10,000 g for 10 min at 4 °C. Then the two extraction solutions were pooled together in a tube for evaporation till dryness under airflow and stored at -80 °C till analysis.

UPLC-MS/MS analysis
Two hundred microliter of cold solvent mixture (ACN: MeOH: H 2 O,40:40:20,v/v/v) was added to the dried residue. The mixture was vigorously vortexed for 30 s and stored at -20 C for 1 h, then centrifuged at 14,000 g for 15 min at 4 °C. The 150 L supernatant of each sample was transferred to a new glass tube for UPLC-MS/MS analysis.
Analysis was performed on a TSQ Quantum Ultra triple quadrupole mass spectrometer (Thermo Fisher Scientific) via an electrospray interface (ESI), operating in negative ionization mode and configuring in selective reaction monitoring (SRM) mode. The metabolite separation was performed using an ACQUITY UPLC ® BEH Amide (1.7 μm, 100×2.1 mm) column (Waters, Ireland). The mobile phases were consisted of acetonitrile (A) and 20 mM ammonium formate and 20 mM ammonium hydroxide in solvent mixture (water:acetonitrile, 95/5, v/v) (B). The gradient elution program initiated from 80% A; decreased to 65% A in 4 min; to 60% A in 6 min; to 55% A in 8 min; to 5% A in 10 min; and held for 13 min with a flow rate of 0.3 mL min -1 .
Mass spectrometric conditions were optimized for each metabolite by using reference standard. Spray voltage, vaporizer temperature, sheath gas, auxiliary gas and capillary temperature were set 2800 V, 350 °C, 35 arb, 8 arb and 300 °C, respectively. The LC-MS/MS data were acquired and processed with LCquan TM software version 2.5.6 (Thermo Fisher Scientific).

GC/MS analysis
An internal standard (10 μL L-4-chloro-phenylalanine in water, 5 μg mL -1 ) was added to the residue, mixed and freeze-dried. Subsequently, 80 μL of methoxylamine solution (15 mg mL -1 in pyridine) was added to each vial. The resultant mixture was vortex-mixed for 1 min and reacted at 37 °C for 24 h in order to inhibit the cyclization of reducing sugars and the decarboxylation of R-keto acids. Eighty μL BSTFA (with 1% TMCS) were added into the mixture and derivatized at 70 °C for 60 min, and vortexed-mix for 30 sec and samples were centrifuged at 10,000 g for 10 min at room temperature. The supernatant was removed to a new glass tube prior to analysis.
The derivatives were separated on a GC column DB-5MS fused-silica capillary column (30m × 250µm i.d., 0.25um film thickness, Agilent J&W Scientific, Folsom, CA). Helium as a carrier gas was used at a constant flow rate of 1 mL min -1 . One μL of derivative was injected, and the solvent delay time was set to 5.5 min. The initial oven temperature was set at 60 °C for 2 min, ramped to 280 °C at a rate of 10 °C min -1 , and finally held at 280 °C for 6 min. The temperatures of injector, transfer line, and electron impact ion source were set at 250 °C, 280 °C and 230 °C, respectively. The initial inlet gas pressure was 8.2317 psi and electron energy was 70 eV. Mass data was collected in a full scan mode from 6.5 to 28 min and the m/z range was set at 50 to 600.

Cell culture for lipidomics analysis
Human colon cancer cell line HCT116 was seeded into 10-cm dish at a density of 5×10 6 cells per dish in 5 mL medium, which maintained in high-glucose DMEM supplemented 10% FBS and 100 unites mL -1 penicillin-streptomycin. After cultured for 24 h, cells were treated with 20 nM HF for 12 h, and then removed the culture medium by vacuum. Cells were rapidly rinsed 4 twice with PBS, then adding 1 mL PBS to the dish and quickly detached from the dish using a cell lifter. Removed the liquid containing cells into a 2-mL tube and centrifuged at 10,000 g for 10 min. The supernatant were discarded and adding 400 μL cold 80% methanol (20% water) to mix cells. The mixing cells were cracked by ultrasonic extraction 2 min, and then added 1 mL Methyl tert-butyl ether (MTBE) into the tube. After shaking at room temperature for 1 h, added 250 μL water and placed 10 min, then centrifuged at 10,000 g for 10 min. The organic phase is in the upper supernatant and the water phase is in the under layer. The organic phase was transferred to a new tube and dried under gentle nitrogen stream. The water phase was also transferred to a new tube and stored at -80 C till analysis.

UPLC/LTQ-Orbitrap MS for lipidomic analysis
Thermo Fisher Accela 1250 UHPLC coupled online via ESI with an LTQ Orbitrap XL (Thermo Fisher Scientific) hybrid mass spectrometer was employed for lipidomic analysis with modification from previous literatures. External mass calibration of the Orbitrap prior to sample analysis was performed by flow injection of the calibration polytyrosine-1, 3, 6 solution according to the manufacturer's instruction. Sample aliquots were reconstituted in 200 µL solvent mixture (ACN: isopropanol: water, 65:30:5, v/v/v). A QC sample was prepared by pooling 50 L from all of the control group and treatment group of each cell line. To cover different lipid species, chromatographic separation was performed on a reversed phase UPLC ACQUITY BEH C18 column (2.1 mm×100 mm×1.7 µm) (Waters, Milford, USA) by gradient elution. Mobile phase A was 60% ACN in water containing 10 mM ammonium acetate and 0.1% acetic acid, and B was isopropanol:ACN (9:1), containing 0.1% acetic acid. The flow rate was 0.2 mL min -1 , with the gradient elution program as follows: 25% B held for 1 min, then linearly 5 increased to 70% B from 1 to 4 min, then to 97% B from 4 to 15 min and held for 8 min followed by equilibration with 25% B for 6.5 min. Mass spectrometric detection was performed in positive ion mode with ESI. High resolution data (resolution 30,000) was acquired by full scan from m/z 450 to 1500 with source voltage of 3500 V, capillary temperature of 300 C, sheath gas flow of 40 arb, auxiliary gas flow of 5 arb, ion spray temperature 350 C and tube lens of 110 V.
Prior to sample analysis external mass calibration was applied to ensure mass accuracy of the mass spectrometer.