Fig 1.
Daily combination treatments of M4N with secondary anticancer drugs synergistically induced strong tumoricidal activity.
(A) Effects of combination treatment of M4N with TMZ on tumor growth in nu/nu mice implanted with glioblastoma LN229 cells. LN229 xenograft mice (N = 5/group) were treated with or without M4N by daily oral administration for 35 days. Meanwhile, another set of LN229 xenograft mice (N = 5/group) were treated with TMZ only or M4N+TMZ by daily oral administration for 25 days, and the examination of tumor growth was continued even after the termination of drug treatments. The tumor volumes of M4N, TMZ, and M4N+TMZ groups were significantly different compared to the control group after day 23 (*p<0.05). The bars indicate standard deviations. (B-C) The effects of combination treatments of M4N with sorafenib on the survival rates of nu/nu mice (N = 5) implanted with various tumors were examined (A-B). Drugs were administered daily via intravenous tail vein injection (B-C). (B) AsPC-1 pancreatic tumors. (C) HepG2 hepatic tumors. (D) Effect of combination treatments of M4N with etoposide, rapamycin, or UCN-01 on HL-1 mouse heart cells. Cell death was examined by the Trypan blue exclusion assay in HL-1 cells treated with combination treatments for 24 h. The concentrations of M4N are shown in the figure. The concentrations of etoposide, rapamycin, and UCN-01 were 10, 20, and 5 μM.
Fig 2.
M4N prevents obesity in mice consuming HF diets.
(A) M4N prevented obesity in mice that consumed HF diets. Male C57BL/6J mice (N = 5) consumed HF diets containing corn oil, either HFM diets (6.83 mg M4N/g food) or food without M4N. The body weights were measured periodically. The bars indicate standard deviations. The points designated by ‘*’ or ‘**’ indicate that the differences between the control and the group whose food contained M4N were statistically significant by the Student’s t-test (p<0.05). (B) Total food intake of male C57BL/6J mice (N = 5) during long-term systemic oral administration of either HFM or control diets were measured periodically. The bars indicate standard deviations. Food intake did not significantly differ between the control mice and the mice that consumed HFM diets. (C) A C57BL/6J mouse consumed food containing 25 mg/mL M4N (6.83 mg M4N/g food) for 16 weeks and the amount of M4N in the tissues was measured. M4N concentrations were calculated based on the amount of M4N extracted from the dry tissue samples, and then were correlated back to the wet weight of the original tissue sample. The standard curve for these calculations ranged from 0.1 to 10 ng/mL.
Fig 3.
Effects of M4N combination treatment on glycolysis, the TCA cycle, the salvage pathway of nicotinamide adenine dinucleotide synthesis, and the production of flavin adenine dinucleotide.
(A) The box figures show the amounts of metabolites in the treated tumors on an arbitrary scale. The upper edge of each box represents a limit of the upper quartile, whereas the lower edge represents a limit of the lower quartile. The line in the middle of each box represents a median value. Asterisks show that the difference between the LN229 tumors treated with TMZ (T) alone and those with TMZ+M4N (TM) were statistically significant by the Student’s t-test (*p<0.05, **p<0.02, ***p<0.01, and ****p<0.1). Sharp marks show that the difference between the LN229 tumors treated with vehicle alone and either those with TMZ (T) alone or those with TM were statistically significant by the Student’s t-test (##p<0.01). The amounts of lactate, α-ketoglutarate, fumarate, and malate were smaller in the TM than T group (indicated by blue downward arrows). Right inlet figure: TM combination treatments suppressed the expression of LDHA in LN229 tumors transplanted in xenograft mice. Lower right inlet figure: M4N suppressed the O2 consumption of LN229 cells. LN229 cells were treated with M4N (30 μM) for 24 h. O2 consumption, which is an indicator of the activity of mitochondrial oxidative phosphorylation, was measured by an O2 consumption rate assay kit (Cayman Chemicals, Ann Arbor, MI, USA). When the concentration of O2 is lower, the intensity of the fluorescence becomes stronger. The bars in the figure indicate the standard deviations. There were statistically significant differences between the control and M4N-treated cells at all the time points later than 10 min by the Student’s t-test (p<0.05). (B) Effect of M4N (M) and/or T on the intracellular contents of NAD+, nicotinamide, and FAD. The data points are from the tumors of five mice. One asterisk (*) indicates that there was a significant difference between either control and T, or control and TM by the Student’s t-test (p<1%), whereas one sharp mark (#) indicates that there was a significant difference between T and TM by the Student’s t-test (p<5%). The upper edge of each box represents a limit of the upper quartile, whereas the lower edge represents a limit of the lower quartile. The line in the middle of each box represents a median value. (C) Effect of M and/or T on the expression of NAMPT. (D) Schematic showing the effect of the combination treatment of TM on the mechanisms of NAD+ synthesis. See S2 Table for metabolite abbreviations.
Fig 4.
M4N treatment promoted the degradation of HIF1A.
(A) Left panel: M4N+TMZ (TM) combination treatment synergistically reduced the viability of cultured LN229 cells. LN229 cells were treated with M4N (M) and/or TMZ (T) at the concentrations indicated in the figure. Cell viability was examined by the MTT assay at 72 h after treatment. The bars indicate standard deviations (N = 8). Right panel: A combination index (CI) plot obtained by the CompuSyn software for the experiment shown in the left panel. A CI less than 1.0 indicates that there is a synergy between two drugs. (B) M or TM combination treatment reduced HIF1A contents in cultured LN229 cells. LN229 cells were treated with M (40 μM) and/or T (30 μM) in the presence or absence of 50 or 150 μM CoCl2, which mimics hypoxic conditions. The cells were collected at 16 h after the treatment and the contents of HIF1A were examined by Western blotting. β-actin was used as a control. The arrows indicate the bands for HIF1A and β-actin. Control–vehicle only (C), M, T, and TM. (C) M4N treatment induces the rapid degradation of HIF1A in cultured HeLa cervical cancer cells during moderate and intermittent hypoxia. HeLa cells were exposed to either moderate hypoxia (4.0% O2) for 10 h or intermittent hypoxia (see Materials and Methods) for 7 h in the presence or absence of 60 μM M4N, and HIF1A protein levels were determined by Western blot analysis (Ca). The extent of HIF1A protein loss due to PHD-dependent proteasome-mediated degradation was ascertained by co-treatment of the cells with DFO (150 μM) (Ca). β-actin was used as a control (Ca). Levels of HIF1A mRNA were assessed by Northern blot analysis (Cb). The blot was re-probed with a β-actin cDNA probe to control for gel loading and transfer (Cb).
Fig 5.
M4N+TMZ synergistically induced stress-related proteins, ATF4, and CHAC1 in tumors from LN229 xenograft mice and synergistically generated superoxide in LN229 tissue culture cells.
(A) Two stress-related proteins, ATF4 and CHAC1, were induced in LN229 tumors implanted in nu/nu mice treated orally with M4N (M) and TMZ (T) for 25 days. The proteins were detected by Western blotting. (B) TMZ+M4N (TM) combination treatment synergistically induced the production of superoxide in cultured LN229 cells. The superoxide assay using MitoROS 580 dyes was performed in LN229 cells treated with M (40 μM) and/or T (30 μM) for 2, 4, 24, or 48 h. Control—vehicle only (C), M, T, or TM. The bars indicate standard deviations (N = 8). Asterisks show that the difference between the control and the LN229 tumors treated with M, T, or TM were statistically significant by the Student’s t-test (*p<0.05, **p<0.01, and ***p<0.001).
Fig 6.
Lactate and 2-HG were suppressed in LN229 tumors from xenograft mice treated with M4N and TMZ for 25 days, and itaconate was induced in macrophages infiltrated in these tumors.
The box figures showed the amounts of metabolites in the tumors (which included both LN229 cancer cells and their associated cells such as macrophages infiltrated in the tumors) treated with drug on an arbitrary scale. The difference in amount of 2-hydroxyglutarate (2-HE) between the tumors treated with TMZ (T) alone and those with TMZ+M4N (TM) was statistically significant by the Student’s t-test (**p<0.05). The difference in amount of itaconate between the control and the tumor treated with M4N (M) was statistically significant by the Student’s t-test (***p<0.01). The data points are from tumors of five mice. The upper edge of each box represents a limit of the upper quartile, whereas the lower edge represents a limit of the lower quartile. The line in the middle of each box represents a median value. The green downward arrows indicate that the contents in lactate, α-ketoglutarate, and LDHA were reduced by TM, compared with T alone. The red upward arrows indicate that the content of itaconate was increased by M, compared with the control or that the content of pyruvate was increased by T alone and TM than the control. Itaconate was produced from macrophage-related cells only (indicated by the designation ‘macrophage’), whereas 2-HE and lactate were produced by any cells including LN229 cells (indicated by the designation ‘LN229’). See S2 Table for enzyme abbreviations.
Fig 7.
A presumptive schematic about the mechanism underlying the ability of M4N combination treatment to induce tumoricidal activity.
The arrow accompanying each item indicates the effect of M4N (upward arrow in red: augmentation by M4N; downward arrow in green: suppression by M4N). See S2 Table for metabolite abbreviations.