Agent for treating colorectal cancer, and agent for suppressing increase in intestinal polyps and onset of cachexia

Abstract

To identify a component that effectively contributes to the treatment (suppression) of colorectal cancer, the risk of developing which increases with aging.SOLUTION: In the present invention, whether coumestrol effectively contributes to the treatment (suppression) of colorectal cancer is investigated using ApcMin / + mice, which are a model of familial intestinal tumor disease. The ApcMin / + mice showed an age-related increase in the number of intestinal polyps and developed cachexia. In contrast, in mice to which coumestrol is administered, the age-related increase in the number of intestinal polyps and the onset of cachexia were found to be significantly suppressed. From these findings, it can be concluded that coumestrol effectively contributes as a component for treating (suppressing) the onset of colorectal cancer, and is useful as an agent for treating (suppressing) colorectal cancer.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a therapeutic agent for colon cancer and a drug for suppressing the increase of intestinal polyps and the onset of cachexia. [Background technology]

[0002] It is known that the risk of developing colorectal cancer increases with age.

[0003] In recent years, as seen in Patent Document 1 and the like, research has been conducted on therapeutic agents for colon cancer. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Application Laid-Open No. 2015-163590 Summary of the Invention [Problem to be solved by the invention]

[0005] The object of the present invention is to find a component that effectively contributes to the treatment (suppression) of colon cancer. [Means for solving the problem]

[0006] The present invention provides a therapeutic agent for colon cancer containing coumestrol as an active ingredient.

[0007] The present invention also provides a drug for inhibiting the growth of intestinal polyps, which contains coumestrol as an active ingredient.

[0008] Furthermore, the present invention provides a drug for suppressing the onset of cachexia, which contains coumestrol as an active ingredient. [Effects of the Invention]

[0009] According to the present invention, coumestrol can suppress the increase of intestinal polyps and the onset of cachexia, and can treat (suppress) colon cancer. [Brief explanation of the drawings]

[0010] [Figure 1] Table showing body weight, intestinal length, and number of intestinal polyps in wild-type and Apc Min / + mice at 12 weeks of age. [Figure 2] Western blotting analysis of HASPIN expression in the testes and intestines of wild-type and Apc Min / + mice. DETAILED DESCRIPTION OF THE INVENTION

[0011] In the present invention, we investigated whether coumestrol effectively contributes to the treatment (suppression) of colon cancer by suppressing the increase of intestinal polyps and the onset of cachexia in wild-type mice and Apc mice, a familial colon tumor disease model. Min / + This was investigated using mice.

[0012] Here, coumestrol is a natural organic compound (phytochemical) contained in plants, and it has been confirmed that commercially available bean sprouts (bean sprouts such as soybean sprouts, mung bean sprouts, and black bean sprouts) contain a very large amount of coumestrol. Min / + The mouse is a familial colorectal cancer model mouse with a nonsense mutation at codon 850 of the Apc gene. The Apc gene is a causative gene for familial polyposis coli, which is involved in the early stage of colorectal cancer development. The Apc mouse was developed by Jackson Laboratories (Bar Harbor, Maine, USA). Min / + Mice (C57BL / 6J) were used.

[0013] C57BL / 6J mice used in this study were purchased from Japan SLC, Inc. (Shizuoka, Japan) and fed 5L37 LABDIET (Japan SLC, Inc., Shizuoka, Japan) as standard diet. To prepare the coumestrol-containing diet, soybean sprouts (see JP 2022-58105 A) were grown to be high in coumestrol. These were dried at 80°C for 12 hours, pulverized in a mill, and mixed with the standard diet to achieve a coumestrol concentration of 200 μg / g (coumestrol / diet). This coumestrol-containing diet was prepared by mixing 15% coumestrol powder by weight with 10% potato starch by weight as a binder and drying at 80°C for 10 hours. Mice were fed the coumestrol-containing diet from the 6th week of age and used in the experiments from the 12th week of age.

[0014] The number of intestinal polyps in this study was counted as follows: after the intestine was removed and its length was measured, the small intestine was divided into five equal parts, each part was incised longitudinally, washed with phosphate-buffered saline, laid flat on filter paper, and fixed in 10% neutral buffered formalin for 24 hours, then stained with 1% methylene blue, and examined for tumors by macroscopic and light microscopy.

[0015] Histological observations of the testes and epididymis were performed as follows: Testes were removed from the scrotum, and the weights of the testes, epididymis, and epididymal fat were measured. Testes fixed in Bouin's solution were then cut into 7-μm-thick sections and mounted on silane-coated slides. The slides were then stained with hematoxylin and eosin and examined microscopically.

[0016] In this study, testosterone levels (serum testosterone levels) were measured by liquid chromatography-tandem mass spectrometry (LC-MS / MS) (Oriental Yeast Co., Ltd., Shiga).

[0017] Western blot analysis in this study was performed as follows. Organ protein samples were sonicated in TBS buffer (10 mM Tris-HCl [pH 7.5]) on ice, centrifuged, and the protein concentration of the supernatant was estimated by Bradford protein assay (Nacalai Tesque, Kyoto, Japan). Extracts containing approximately 50 μg of protein were separated by polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were transferred to filters and blocked with blocking solution (Nacalai Tesque, Kyoto, Japan). The filters were then incubated overnight at 4°C with primary antibodies diluted 1:1000 in CanGetSignal (registered trademark, Toyobo Co., Ltd., Osaka, Japan). After washing with TBS buffer, the filters were incubated for 1 hour at room temperature with peroxidase-conjugated anti-immunoglobulin (secondary antibody) diluted 1:1000 in CanGetSignal. Furthermore, after washing, reactive bands were visualized by development using a POD staining kit (Fujifilm Wako Pure Chemical, Osaka, Japan).

[0018] Data are expressed as mean ± standard deviation, means were compared using Student's t test, and significance was determined at the P<0.05 level.

[0019] The results of this study are shown in Figure 1.

[0020] Intestinal polyps were induced in 6- to 12-week-old wild-type or Apc mice fed a standard diet or a coumestrol-containing diet. Min / + was observed in mice.

[0021] The number of intestinal polyps was significantly increased in Apc mice fed a standard diet. Min / + In mice, the number of Apcs was 66.0±11.7 (females) and 55.8±8.6 (males), whereas in mice fed a diet containing coumestrol, the number of Apcs was 66.0±11.7 (females) and 55.8±8.6 (males), respectively. Min / + In mice, the number was 24.1±10.7 (females) and 19.7±13.1 (males).

[0022] Thus, Apc fed a diet containing coumestrol Min / + In mice, Apc fed a standard dietMin / + A significantly lower number of intestinal polyps was observed in mice than in controls.

[0023] In addition, the length of the intestine was significantly longer than that of Apc fed a standard diet. Min / + In mice, the mean diameter was 38.2 ± 4.50 mm (females) and 38.0 ± 3.47 mm (males), whereas in Apc mice fed a diet containing coumestrol, the mean diameter was 38.2 ± 4.50 mm (females) and 38.0 ± 3.47 mm (males). Min / + In mice, the mean diameter was 48.3 ± 3.06 mm (females) and 51.6 ± 2.74 mm (males). In wild-type mice fed a standard diet, the mean diameter was 40.5 ± 5.19 mm (females) and 38.3 ± 3.30 mm (males), while in wild-type mice fed a coumestrol-containing diet, the mean diameter was 48.4 ± 3.27 mm (females) and 50.9 ± 1.70 mm (males).

[0024] Thus, wild-type mice and Apc mice fed a diet containing coumestrol Min / + In mice, wild-type mice and Apc mice fed a standard diet were Min / + The intestinal length was observed to be significantly longer (approximately 20%) than in mice.

[0025] Furthermore, the body weight of wild-type mice fed standard chow was 23.1±0.87 g (females) and 28.6±1.24 g (males), while that of wild-type mice fed chow containing coumestrol was 22.9±1.47 g (females) and 25.6±0.97 g (males), indicating a significant decrease in body weight.

[0026] In addition, the body weight was measured using standard diet-fed Apc Min / + In mice, the weight was 19.9±1.78g (female) and 18.8±1.78g (male), and the weight was 18.8±1.78g (Apc Min / + It was observed that mice lost significant weight and developed cachexia, but the Apc mice fed a diet containing coumestrol Min / + The weights of the mice were 23.0±1.47g (female) and 24.5±2.42g (male). When the weights were measured at 12 weeks of age, the weights of the Apc mice fed a diet containing coumestrol were 23.0±1.47g (female) and 24.5±2.42g (male). Min / + In mice, Apc fed a standard diet Min / + Significant weight recovery was observed in mice.

[0027] As shown in Figure 2, HASPIN is strongly expressed in germ cells. However, in this study, HASPIN was expressed in Apc cells, which do not show germ cell differentiation. Min / + HASPIN expression was not observed in the testes of mice, whereas it was observed in the intestine where polyps developed. Min / + In mice, HASPIN expression was similar to that in wild-type mice.

[0028] As explained above, Apc fed standard diet Min / + Mice showed an increase in the number of intestinal polyps and developed cachexia with age, whereas Apc mice fed a diet containing coumestrol showed no significant difference. Min / + In mice, it was found that the age-related increase in the number of intestinal polyps and cachexia were significantly suppressed.

[0029] Based on these findings, coumestrol functions as a drug for suppressing the increase in intestinal polyps and the onset of cachexia, and can be said to effectively contribute as an ingredient for treating (suppressing) the onset of colon cancer, making it useful as a colon cancer treatment agent (colon cancer inhibitor).

Claims

1. A colon cancer treatment agent containing coumestrol as an active ingredient.

2. A drug containing coumestrol as an active ingredient for inhibiting the growth of intestinal polyps.

3. A drug containing coumestrol as an active ingredient for suppressing the onset of cachexia.

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