Fig 1.
Metabolic and antitumor effect of the LDH inhibitor NCI-006 and the mitochondrial complex I inhibitor IACS-010759 in HCT116 and MKN45 cells.
(A)The metabolic activities in response to treatment were determined according to the OCR/ECAR levels of HCT116 and MKN45 cells with or without treatment. Dashed lines connect the baseline activity (0 min; open symbols) and metabolic activity after treatment (100 min; closed symbols); IACS-010759 at 2 μM and/or NCI-006 at 5 μM were applied. Data are displayed as the mean ± SD. (B)The ATP production rate was measured using the XF Real-Time ATP Rate Assay Kit (Agilent Technologies, Santa Clara, CA, USA) according to the OCR/ECAR levels in HCT116 and MKN45 cells with or without treatment. Data are displayed as mean ± SD (*p < 0.05, **p < 0.01, two-way ANOVA Tukey test). (C)HCT116 and MKN45 cells were treated with NCI-006 (1 μM) and/ or IACS-010759 (1 μM) for 48 h, and cell proliferation was assessed. Data are displayed as means ± SD (n = 18 for each group; **p < 0.01, two-way ANOVA Tukey test). ECAR, Extracellular Acidification Rate.
Fig 2.
Effect of OXPHOS and glycolytic inhibition on tumor growth in HCT116 and MKN45 xenografts.
(A) HCT116 cells (5×106) were subcutaneously injected into the back of athymic nude. mice. After the size of the subcutaneous tumor reached 100 mm3, NCI-006 (40 mg/[kg•dose] body weight, red arrowheads) was administered intravenously thrice a week, and IACS-010759 (20 mg/[kg•dose] body weight, green arrowheads) was administered orally every day for a week. Data are displayed as means ± standard error of the mean (SEM) (n = 12 for each group; *p < 0.05, **p < 0.01, two-way ANOVA Tukey test). (B) MKN45 xenograft mice were generated as described in (A). NCI-006 (40. mg/[kg•dose] body weight, red arrowheads) was administered intravenously twice a week, whereas IACS-010759 (20 mg/[kg•dose] body weight, green arrowheads) was administered orally five times a week for two weeks. Data are displayed as means ± SEM (n = 6 for each group; *p < 0.05, **p < 0.01, two-way ANOVA Tukey test). (C) The body weight change of the mice in the experiment (A). (D) The body weight change of the mice in the experiment (B). Abbreviation: IACS, IACS-010759.
Fig 3.
Effect of metabolic changes on tumor microenvironment.
(A)The pH of the medium 4h after treatment (NCI-006 [1 μM] and/or IACS-010759 [1 μM]) was measured to evaluate pH changes in vitro. The pH changes in vivo were measured by homogenizing tumors from carcinoma-bearing mice 4 h after treatment and dilution with ultrapure water. Data are presented as means ± SEM (n = 3 for each group, *p < 0.05, **p < 0.01, t-test). (B)Oxidative stress in the HCT116 xenografts. Mice were injected with ROS Brite 700 nm dye 4 h after the administration of NCI-006 and/or IACS-010759, and 20 min later, the tumors were removed, and fluorescence imaging was immediately performed. Data are displayed as the means ± SEM (n = 4 for each group, *p < 0.05, t-test).
Fig 4.
Monitoring dynamic metabolic changes in tumor xenografts using 13C-MRSI with hyperpolarized 1-13C pyruvate.
(A)Schematic representation of HP 1-13C pyruvate MR study: 13C MR was performed using mice bearing HCT116 and MKN45 xenografts. Each mouse was imaged 30 minutes after NCI-006 administration and/or 3 h after IACS-010759 administration. (B)13C chemical shift of 1-13C lactate, 1-13C hydrate pyruvate, and 1-13C pyruvate. (C)Representative 13C-MR signal intensity curves of 1-13C pyruvate and 1-13C lactate detected in HCT116 and MKN45 xenografts after hyperpolarized 1-13C pyruvate injection. (D)The 1-13C-Lactate/Pyruvate ratio in ex vivo experiments. The ex vivo data were obtained from tumor tissue samples extracted and homogenized after each treatment. The 1-13C Lac/Pyr ratio was calculated from the Area Under Curve (AUC) using time-intensity data. (E)The 1-13C-Lactate/Pyruvate ratio in vivo experiments. HP-MRSI, hyperpolarized 13C-magnetic resonance spectroscopic imaging.