Development of ghrelin resistance in a cancer cachexia rat model using human gastric cancer-derived 85As2 cells and the palliative effects of the Kampo medicine rikkunshito on the model

<div><p>Cancer cachexia (CC) is a multifactorial disease characterized by decreased food intake and loss of body weight due to reduced musculature with or without loss of fat mass. Patients with gastric cancer have a high incidence of cachexia. We previously established a novel CC rat model induced by human gastric cancer-derived 85As2 cells in order to examine the pathophysiology of CC and identify potential therapeutics. In patients with CC, anorexia is often observed, despite elevation of ghrelin, suggesting that ghrelin resistance may develop in these patients. In this study, we aimed to clarify the occurrence of ghrelin resistance in CC rats accompanied by anorexia and we investigated whether rikkunshito (RKT), a traditional Japanese Kampo medicine that potentiates ghrelin signaling, ameliorated CC-related anorexia through alleviation of ghrelin resistance. 85As2-tumor-bearing rats developed severe CC symptoms, including anorexia and loss of body weight/musculature, with the latter symptoms being greater in cachectic rats than in non-tumor-bearing or pair-fed rats. CC rats showed poor responses to intraperitoneal injection of ghrelin. In CC rats, plasma ghrelin levels were elevated and hypothalamic anorexigenic peptide mRNA levels were decreased, whereas hypothalamic growth hormone secretagogue receptor (<i>GHS-R</i>) mRNA was not affected. <i>In vitro</i>, RKT directly enhanced ghrelin-induced GHS-R activation. RKT administrated orally for 7 days partly alleviated the poor response to ghrelin and ameliorated anorexia without affecting the elevation of plasma ghrelin levels in CC rats. The expression of hypothalamic orexigenic neuropeptide Y mRNA but not hypothalamic <i>GHS-R</i> mRNA was increased by RKT. Thus, the 85As2 cell-induced CC rat model developed ghrelin resistance, possibly contributing to anorexia and body weight loss. The mechanism through which RKT ameliorated anorexia in the CC rat model may involve alleviation of ghrelin resistance by enhancement of ghrelin signaling. These findings suggest that RKT may be a promising agent for the treatment of CC.</p></div

Effects of RKT on tumor growth, body weight loss, and anorexia in the 85As2-induced CC model.

<p>Changes in (A) tumor volume over time and (B) body weight after RKT or DW administration. Comparison of (C) body weight gain and (D) increase in average daily food intake before and after RKT or DW administration in individual rats. Rats were implanted subcutaneously with 85As2 cells in both flanks (1 × 10⁷ cells/site) on day 0. RKT (1 g/kg/day) or DW was administered orally twice a day for 7 days from day 14. Rats inoculated with saline served as a non-tumor-bearing control group and were administered DW. Each data point represents the mean ± SEM of 14–16 rats. Differences between groups were evaluated using two-way repeated measures ANOVA followed by post-hoc Bonferroni tests; ***<i>p</i> < 0.001 versus the 85As2 + DW-treated group (B). Differences before (Pre) and after (Post) administration of RKT or DW were evaluated using paired <i>t</i>-tests; ## <i>p</i> < 0.01, ###<i>p</i> < 0.001 versus the corresponding group before administration (each baseline was set as 0) (C, D). Differences between groups were evaluated using one-way ANOVA followed by post-hoc Dunnett’s multiple comparison tests; **<i>p</i> < 0.01 versus the 85As2 + DW-treated group (D). RKT: rikkunshito; DW: distilled water; n.s.: not significant.</p

Comparison of pair-fed and 85As2-induced CC rats.

<p>Changes in (A) body weight, (B) food intake, and (C) water intake. Rats were inoculated subcutaneously with 85As2 cells in both flanks (1 × 10⁷ cells/site) on day 0. Rats inoculated with saline served as a non-tumor-bearing control group. The pair-fed rats received an amount of food that was the same as that consumed by the 85As2-induced CC rats for the previous day (days 1–15). Each data point represents the mean ± SEM of six rats. Differences between groups were evaluated using two-way repeated measures ANOVA followed by post-hoc Bonferroni tests; *<i>p</i> < 0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0.001 versus the corresponding control group.</p

Effects of long-term feeding of RKT (1% diet) from before tumor implantation on tumor growth or anorexia in the 85As2-induced CC model.

<p>Changes in (A) tumor volume, (B) body weight, and (C) average daily food intake during the experiment. Rats were implanted subcutaneously with 85As2 cells in both flanks (1 × 10⁶ cells/each site) on day 0. RKT (CE-2 diet containing 1% RKT) or CE-2 was fed for 35 days from day –7. Rats inoculated with saline served as a non-tumor-bearing control group and were fed similarly for 35 days. Each data point represents the mean ± SEM of 9–11 rats. Differences between groups in the time course of body weight and food intake were evaluated using two-way repeated measures ANOVA followed by Bonferroni post-hoc test; ***<i>p</i> < 0.001 versus the corresponding Control + CE-2 group, ##<i>p</i> < 0.01 versus the corresponding 85As2 + CE-2 group. RKT: rikkunshito.</p

Effects of RKT on attenuation of ghrelin-induced orexigenic effects in 85As2-induced CC rats.

<p>(A) Effects of RKT on attenuated ghrelin-induced orexigenic effects (increase in 1-h food intake) in 85As2-induced CC rats. (B) Cumulative food intake (22 h) following vehicle or ghrelin injection in all groups. Rats were implanted s.c. with 85As2 cells in both flanks (1 × 10⁷ cells/site). Two weeks after implantation, RKT (1 g/kg/day) or DW was administered orally twice a day for 7 days. Rats inoculated with saline served as a non-tumor-bearing control group and were administered DW. Rats were injected i.p. with ghrelin (10 nmol) after administration of RKT or DW for 7 days, and 1-h food intake and 22-h cumulative food intake were measured. The next day, all rats were injected i.p. with vehicle (saline) as a control, and 1-h and 22-h food intake was measured. Each data column represents the mean ± SEM of 5–6 rats. Differences between vehicle and ghrelin treatments in each group were evaluated using paired <i>t</i>-tests. Differences between groups were evaluated using two-way repeated measures ANOVA followed by Bonferroni post-hoc tests; *<i>p</i> < 0.05, **<i>p</i> < 0.01 versus the Control + DW-treated group. RKT: rikkunshito; DW: distilled water.</p