Figure 1
Circadian locomotor activity profiles, and Per2 and Pai-1 mRNA expression pattern in peripheral tissues. Circadian activities were measured for approximately 4 weeks using a nano tag. Actograms in the left and right panels represent the Control and Shift groups, respectively (n = 4, A). Locomotor activity levels were measured at 5-min intervals and are indicated by black bars. Per2 and Pai-1 expression levels in atria (B and E), ventricle (C and F) and liver (D and G) were analyzed at ZT2, 6, 11, 18, and 23. Values were normalized using Actb mRNA levels. Control gene expression levels at ZT2 were defined at as 1. Data represent mean ± S.E.M. (n = 4). *, P < 0.05; **, P < 0.01.
Figure 2
Disturbance of the light/dark cycle does not affect tumor growth and decrease survival. Schematic representation of the experimental plan and light/dark conditions (A). Female BALB/c mice were implanted with BJMC3879Luc2 cells in the right fourth mammary fat pad and then maintained under each light/dark cycle condition for 3 to 8 weeks. The lighting conditions of each group were set as follows: Control group, 12:12 h light/dark cycle (lights on at 8:00; ZT0); Shift group, light on ZT0 for 2 days and then conversely on the following 2 days. The star mark (★) indicates the timing of body weight and tumor volume measurement (between ZT7 and ZT8). Mice were killed between ZT7 and ZT8 at 3, 5, or 8 weeks after tumor cell inoculation. Effect of light/dark shifts on body weight (B), tumor volume (C), tumor mass (D), and survival (E). Data represent mean ± S.E.M. (n = 10–30).
Figure 3
Bioluminescent imaging of metastatic lesion areas of mice housed under normal or light/dark cycle shift conditions. Representative bioluminescent images of metastatic lesions obtained at 8 weeks after inoculation (A). Bioluminescent images were obtained 7 min after intraperitoneal administration of luciferin to mice. Quantification of bioluminescent intensities in each tissue taken from mice (B). Regions of interest (ROI) from displayed images were identified in the primary tumor, lung, kidney lymph node, and axillary lymph node, and quantified as photons per second. Data represent mean ± S.E.M. (n = 13–15).
Figure 4
Frequency of metastasis in lymph nodes. Number of lymph node metastasis in each group (A) and frequency of metastasis in distant lymph nodes (B). Data represent mean ± S.E.M. (n = 18). *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Figure 5
Total white blood cells (WBCs) and neutrophil-to-lymphocyte ratio (NLR). Total WBCs were measured at 5- or 8-weeks post-inoculation (A). NLR was calculated from the neutrophil and lymphocyte percentage of WBCs at 5- or 8-weeks post-inoculation (B). Data represent mean ± S.E.M. (n = 6). *, P < 0.05.
Figure 6
Change in tumor NA levels. Intratumoral NA concentration was measured at 5- or 8-weeks post-inoculation. Data represent mean ± S.E.M. (n = 5–10).
Figure 7
Effect of 0.3% quercetin diet on the lymph node metastasis. All animals were given normal MF diet or 0.3% quercetin-containing MF diet over the experimental period. Food intake (A), body weight (B), and tumor volume (C) were measured once a week (n = 6–10). Tumor weight was measured at the final necropsy (D, n = 6–10). Data represent mean ± S.E.M. The lymph node metastasis inhibition rates in 0.3% Q-Ctrl and 0.3% Q-Sh were calculated using the frequency of metastasis occurrence in the Control or Shift groups as the baseline data (E, n = 6–10). *, P < 0.05 vs. control; †, P < 0.05 vs. shift.