Note
Association between Weight Gain and Sex-Related Differences through 5-Fluorouracil Administration
2024 Volume 47 Issue 8 Pages 1456-1459
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2024 Volume 47 Issue 8 Pages 1456-1459
Research on sex differences has increased across various fields, including cancer and its treatment domains. Reports have indicated sex differences in cancer incidence, survival rates, and the efficacy of anticancer drugs. However, such reports are limited, and in-depth assessments of the underlying mechanisms are still in progress. Although various chemotherapeutic regimens are applicable for breast cancer treatment, reports have surfaced regarding weight gain in female patients undergoing fluorouracil, epirubicin, cyclophosphamide (FEC) or cyclophosphamide, methotrexate, fluorouracil (CMF) therapy. We hypothesized the potential of 5-fluorouracil (5-FU) in weight gain and sex-related differences. To address this, we conducted experiments in mice to confirm weight gain and sex differences following 5-FU administration, and elucidate the underlying mechanisms. Our findings revealed weight gain and increased food intake in female mice following 5-FU administration. Additionally, female mice receiving 5-FU exhibited increased norepinephrine and α1- and α2-adrenergic receptor expression, reduced estradiol levels, and increased ghrelin levels. These results indicate 5-FU administration-induced sex differences in weight gain and implicate increased food intake because of increased norepinephrine and α1- and α2-adrenergic receptor expression, reduced estradiol levels, and a subsequent increase in ghrelin levels, which contribute to weight gain in female patients undergoing CMF therapy.
In recent years, there has been an increase in reports regarding sex differences in cancer incidence and survival, and the biological differences between women and men have garnered attention in sex-differentiated treatment strategies. However, the majority of studies have primarily focused on men.1) Additionally, there have been reports of sex differences in adverse events during cancer chemotherapy. However, these reports are limited in number and the detailed mechanisms remain unexplored.2,3)
Common chemotherapy regimens for breast cancer, including docetaxel, cyclophosphamide (TC); doxorubicin hydrochloride (Adriamycin), cyclophosphamide (AC); 5-fluorouracil (5-FU), epirubicin, cyclophosphamide (FEC); and cyclophosphamide, methotrexate, 5-FU (CMF), have been associated with weight gain in women with breast cancer.4,5) Weight gain is more commonly associated with CMF than with anthracycline-based chemotherapy regimens, such as doxorubicin.6) Therefore, it is likely that 5-FU, present in both FEC and CMF therapies, contributes to weight gain.
Because 5-FU is frequently used in combination with other drugs, its effect on weight alterations remains unclear. Additionally, because weight gain has been reported in female patients with breast cancer, we hypothesized potential sex differences in the 5-FU-induced weight alterations. In this study, we aimed to assess the effects of 5-FU monotherapy on weight alterations and sex differences in mice and elucidate the underlying mechanisms.
Sixteen Institute of Cancer Research (ICR) mice (9-weeks-old, eight males and eight females) were purchased from Japan SLC, Inc. (Hamamatsu, Shizuoka, Japan). Mice were individually housed under specific pathogen-free conditions in a room maintained at 23 ± 1 °C with a 12-h light-dark cycles, free access to drinking water, and a standard pellet diet. All experimental protocols were approved by the Ethics Committee of the Suzuka University of Medical Sciences (SUMS). This study adhered to the recommendations outlined in the Suzuka University of Medical Sciences Laboratory Animal Care and Use Guide (Approval No. 82), following the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines. All surgical procedures were performed under pentobarbital anesthesia and adjusted to minimize animal distress.
Experimental DesignEight male and eight female mice were divided into control and 5-FU-treated groups of four mice each. 5-FU (Kyowa Kirin Co., Ltd., Tokyo, Japan) dissolved in saline was intraperitoneally administered (15 mg/kg) to mice once daily for six weeks. Saline was administered to the control group. Concentrations of 5, 15, and 30 mg/kg were used, following the method of Huang et al.,7) with the lowest effective concentration selected. Body weights were measured once a week for six weeks, while food intake was monitored daily for 5 d during the last week.
Measurement of α1- and α2-Adrenergic ReceptorsThe levels of α1- and α2-adrenergic receptors in the ovaries of female mice were measured using Western blotting. Ovary samples were homogenized in lysis buffer (Kurabo, Osaka, Japan) and centrifuged (8000 × g, 10 min). The supernatant of each sample was collected and stored at −80 °C. After thawing, equal amounts of protein (5 µg/lane) were loaded onto 4–12% bis (2-hydroxyethyl) imino-tris (hydroxymethyl) methane (BIS-TRIS) Bolt gels (Life Technologies, Carlsbad, CA, U.S.A.) and electrophoresed at 200 V for 30 min. Following separation, proteins were transferred to nitrocellulose membranes using an iBlot Western blotting system (Life Technologies), followed by overnight blocking at 4 °C in 5% skim milk. Subsequently, the membranes were incubated with primary antibodies against α1- and α2-adrenergic receptors (Abcam, Cambridge, U.K.) and β-actin (Sigma-Aldrich, St. Louis, MO, U.S.A.) for 1 h at 25 °C. Membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Life Technologies) and signals were detected using ImmunoStar Zeta (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) and Lumino image analyzer (LAS-4000; FUJIFILM, Tokyo, Japan).
Measurement of Noradrenaline, Reactive Oxygen Species (ROS), Estradiol, and Ghrelin LevelsPlasma concentrations of noradrenaline, ROS, estradiol, and ghrelin were measured using commercially available enzyme-linked immunosorbent assay (ELISA) kits (noradrenaline: Abcam; ROS: Cell Biolabs Inc., San Diego, CA, U.S.A.; estradiol: Cayman Chemical, Ann Arbor, MI, U.S.A.; and ghrelin: LSI Medience Corp., Tokyo, Japan).
AnalysisAll data are expressed as the mean ± standard deviation (S.D.). Statistical analyses were performed using Microsoft Excel 2010, statistical package for the social sciences (SPSS) version 20 (SPSS Inc.), one-way ANOVA, and Tukey’s post hoc test. Statistical significance was set at p-values <0.05 and 0.01.
In male mice, there was no significant difference in weekly body weight gain between the control and 5-FU-treated groups. However, female mice in the 5-FU-treated group gained more weight per week than those in the control group (Fig. 1).
Values are expressed as the mean ± standard deviation (S.D.) of four animals. * p < 0.05, N = 4.
Blood ROS production increased in 5-FU-treated male and female mice (Fig. 2A). Blood noradrenaline levels in male mice exhibited no significant difference between the control and 5-FU-treated groups; however, in female mice, noradrenaline levels were significantly higher in the 5-FU-treated group than that in the control group (Fig. 2B).
ROS production (A) and noradrenaline concentration (B) during 5-FU administration. Values are expressed as the mean ±S.D. of four animals. ** p < 0.01, N = 4.
Both α1- and α2-adrenergic receptors in the ovaries of female mice were significantly increased in the 5-FU-treated group compared to those in the control group (Fig. 3).
α1- and α2-adrenergic receptor protein levels (A) and rates relative to β-actin (B) in the ovaries of female mice treated with 5-FU. Values are expressed as the mean ±S.D. of four animals. * p < 0.05 and ** p < 0.01; N = 4.
In male mice, blood estradiol levels exhibited no significant difference between the control and 5-FU-treated groups. However, in female mice, there was a significant reduction in estradiol levels in the 5-FU-treated group compared to that in the control group (Fig. 4A). In male mice, blood ghrelin levels exhibited no significant difference between the two groups. However, ghrelin levels in female mice were significant higher in the 5-FU-treated group compared to that in the control group (Fig. 4B). In males, there was no significant difference in food intake between the two groups; however, female mice exhibited increased food intake in the 5-FU-treated group compared to that in the control group (Fig. 4C).
Estradiol (A) and ghrelin (B) levels and food intake (C) during 5-FU administration. Values are expressed as the mean ±S.D. of four animals. ** p < 0.01, N = 4.
In this study, we demonstrated that 5-FU administration in female mice increased body weight associated with increased expression of noradrenaline and α1- and α2-adrenergic receptors, reduction in estradiol levels, and subsequent increase in ghrelin levels, resulting in increased food intake.
The sex difference in 5-FU administration-induced noradrenaline levels may be attributed to sex differences in the stress responses.8) Stress responses involve hypothalamic–pituitary–adrenal (HPA) axis and locus coeruleus (LC)-noradrenaline system, which is activated parallel to the HPA axis based on stress factors.9) Females have more LC neurons compared to males, with the LC serving as a source of noradrenaline in the brain.10,11) Therefore, the sex differences in noradrenaline levels observed in this study may be attributed to 5-FU administration-induced sex differences in the stress response.
Chemotherapy increases ROS levels, an indicator of oxidative stress. ROS activity correlates with HPA axis activity.12,13) In this study, we observed increased ROS production in the 5-FU-treated group regardless of sex, indicating that even male mice experience stress owing to 5-FU. By contrast, dihydropyrimidine dehydrogenase activity and clearance, which are involved in 5-FU metabolism, have been reported to be lower in women than in men, albeit in humans.14,15) This may suggest that sex differences in weight gain may be because of differences in metabolic capacity; however, 5-FU administration increases ROS production in a concentration-dependent manner,16) and the fact that no difference in ROS production occurred in the present study suggests that this may be because of factors other than metabolic capacity.
Noradrenaline inhibits aromatase activity and decreases estradiol production.17,18) Sympathetic nerves are abundant in the ovary, and sympathetic stimulation by stress inhibits estradiol secretion.19) Testosterone is converted to estradiol through aromatase. Inhibition of estradiol secretion involves α2 adrenergic receptors, and inhibition of testosterone secretion involves α1 adrenergic receptors.20) In the present study, ROS production was increased in the 5-FU-treated group regardless of gender, and a decrease in blood estradiol was observed with an increase in noradrenaline. Increased α1 and α2 adrenergic receptors were also observed, which may have contributed to the estradiol decrease. Furthermore, estradiol can suppress food intake via downregulation of ghrelin and its receptors.21) The increase in food intake in the 5-FU-treated female mice confirmed in the current study may be because of the increase in ghrelin caused by reduction in estradiol.
Weight gain has been observed in female patients with breast cancer undergoing FEC or CMF therapy; however, the detailed underlying mechanisms remain unclear. The findings of this study contribute to the elucidation of the mechanism of weight gain following FEC and CMF therapy. Additionally, 5-FU-induced weight gain is specific to females. These results indicate the need for novel treatment strategies that consider sex differences in cancer chemotherapy, including treatment strategies and monitoring of adverse effects. However, it should be noted that chemotherapy is commonly administered to patients with cancer, and the results of this study were obtained in mice without cancer. Therefore, future studies are required to determine whether similar results can be obtained in mice with induced cancers. In addition, the present study was conducted in mice. As noted above, sex differences in 5-FU metabolism in humans have also been reported, and results in humans may not be equivalent to those of the present study.
The authors declare no conflict of interest.
