Chemopreventive efficacy of inositol hexaphosphate against prostate tumor growth and progression in TRAMP mice

Abstract

Purpose: Herein, for the first time, we evaluated the in vivo chemopreventive efficacy of inositol hexaphosphate (IP6), a major constituent of high-fiber diets, against prostate tumor growth and progression in the transgenic adenocarcinoma of the mouse prostate (TRAMP) model.

Experimental design: Beginning at 4 weeks of age, male TRAMP mice were fed 2% (w/v) IP6 in drinking water or only drinking water till 24 weeks of age, and then sacrificed. Prostate tissue was subjected to histopathologic analysis and to immunohistochemical analyses for proliferation and apoptosis.

Results: IP6 feeding did not show any adverse effect on fluid and diet consumption and body weight. There was a significant reduction (40%; P < 0.01) in lower urogenital tract weight in IP6-fed mice. IP6 inhibited prostate cancer progression at prostatic intraepithelial neoplasia stage and strongly reduced the incidence of adenocarcinoma (prostatic intraepithelial neoplasia/adenocarcinoma, 75:25% in the IP6 group versus 39:61% in the control group; P < 0.05). The incidences of well-differentiated and poorly differentiated adenocarcinomas in the IP6-fed group were reduced by 44% and 62%, respectively. Immunohistochemical analysis of prostate tissue showed a 26% decrease (P < 0.05) in proliferation cell nuclear antigen-positive cells and a 3.5-fold increase in apoptotic cells with no effect on Tag expression by IP6.

Conclusions: These findings are both novel and highly significant in establishing for the first time that oral IP6, without any toxicity, suppresses prostate tumor growth and progression at the neoplastic stage, thereby reducing the incidence of adenocarcinoma through its antiproliferative and proapoptotic effects, and thus indicating that IP6 could have potential chemopreventive effects against human prostate cancer.

Fig. 1

(A) Effect of oral feeding of IP6 on the daily diet consumption of TRAMP mice. Food consumption by each mouse was recorded weekly throughout the feeding regimen in each group. Diet consumption (g) per mouse/ day is plotted as a function of time (weeks) for each group. (B) Effect of oral feeding of IP6 on the daily fluid consumption of TRAMP mice. Positive control mice were supplied with regular drinking water and the treatment group was fed with IP6 solution [2% IP6 (w/v) in regular drinking water] for 20 weeks. The freshly prepared solutions (as the only source of drinking water) were supplied every Monday, Wednesday and Friday; and the fluid consumption by different groups was also recorded. Fluid consumption (mL) per mouse/ day is plotted as a function of time (weeks) for each group. (C) Effect of oral feeding of IP6 on the body weight of TRAMP mice. Body weight of each mouse was recorded weekly through out the experiment. Body weights of mice are represented as average of each group and plotted as a function of time (weeks) for each group. Control, positive control (TRAMP) mice; IP6, inositol hexaphosphate.

Fig. 2

(A) Effect of IP6 feeding on the weight of the lower urogenital tract (LUT) organs. At the time of necropsy after 20 weeks of IP6 feeding, starting from 4^th week of age, each mouse was weighed and the LUT including the bladder, seminal vesicles and prostate was removed en bloc and weighed; n=18 (positive control) and n=16 (2% IP6-fed) mice per group. Error bars indicate ± SEM. The statistical significance of difference between positive controls versus IP6-fed group was analyzed by unpaired two-tailed Student’s t-test. P values <0.05 were considered significant. LUT, lower urogenital tract; Control, positive control (TRAMP) mice; IP6, inositol hexaphosphate.

Fig. 3

Inhibitory effect of IP6 feeding on prostate tumor progression in TRAMP mice. The dorsolateral prostate from the study detailed in Fig. 1, were histopathologically analyzed for the different stages of the neoplastic progression of the dorsolateral prostate. (A) Effect of IP6 feeding on the incidence of low and high grade PIN lesions in TRAMP mice. (B) Effect of IP6 on the incidence of adenocarcinoma of prostate in TRAMP mice. Fishers Exact test was used to compare incidence of PIN and adenocarcinoma in positive control versus IP6-fed group. P values <0.05 were considered significant. PIN, prostate intraepithelial neoplasia; WD, well differentiated (adenocarcinoma); MD, moderately differentiated (adenocarcinoma); PD, poorly differentiated (adenocarcinoma); Control, positive control (TRAMP) mice; IP6, inositol hexaphosphate.

Fig. 4

IP6 feeding reduces the severity of prostatic lesions (tumor grade) of dorsolateral prostate in TRAMP mice. (A) Different stages of prostate tissues were graded as described in “Results”. The maximum histological score for the individual prostate from each mouse was used to calculate a mean for the treatment group. Data is presented as mean peak histological score ± SEM (error bars) of each group. The statistical significance of difference between positive control versus IP6-fed group was analyzed by unpaired two-tailed Student’s t-test. P values <0.05 were considered significant. (B) The photomicrographs (×100 magnification) representative of a treatment group show the H&E staining of the dorsolateral prostate of positive control and IP6-fed mice sacrificed after 24 weeks of age. Control, positive control (TRAMP) mice; IP6, inositol hexaphosphate.

Fig. 5

Anti-proliferative and pro-apoptotic effects of IP6 feeding in TRAMP mice. (A) In vivo anti-proliferative effect of IP6 feeding on dorsolateral prostate of TRAMP mice. Immunohistochemical staining for PCNA in prostate was based on DAB staining as detailed in “Materials and Methods”. Representative DAB-stained tissue specimens from positive control and IP6-fed groups showing brown-colored PCNA-positive cells are depicted at ×400 magnifications. (B) Proliferation index was calculated as the number of positive cells × 100 / total number of cells counted under ×400 magnifications in 5 randomly selected areas in each sample, and shown as mean ± SEM (error bars) for each group. (C) In vivo pro-apoptotic effect of IP6 feeding on dorsolateral prostate in TRAMP mice. Apoptosis was analyzed by TUNEL staining in prostate tissues as detailed in “Materials and Methods”. Representative DAB-stained tissue specimens from positive control and IP6-fed groups showing brown-colored TUNEL-positive cells are depicted at ×400 magnifications. (D) Apoptotic index was calculated as the number of TUNEL-positive cells × 100 / total number of cells counted under ×400 magnifications in 5 randomly selected areas in each sample, and shown as mean ± SEM (error bars) for each group. For both (B) & (D), the statistical significance of difference between positive control versus IP6-fed group was analyzed by unpaired two-tailed Student’s t-test. P values <0.05 were considered significant. PCNA, proliferation cell nuclear antigen; Control, positive control (TRAMP) mice; IP6, inositol hexaphosphate.

Fig. 6

Effect of oral feeding of IP6 on the expression of SV40 large T antigen in the dorsolateral prostate of TRAMP mice. Immunohistochemical staining was based on DAB staining as detailed in “Materials and Methods”. Representative DAB-stained tissue specimens are illustrated from positive control and 2% IP6-fed groups (×400 magnifications) showing brown staining for the expression of SV40 large T antigen in prostatic intraepithelial neoplasia (PIN), well differentiated (WD) adenocarcinoma, and poorly differentiated (PD) adenocarcinoma. Control, positive control (TRAMP) mice; IP6, inositol hexaphosphate.