Stage-Specific Effect of Inositol Hexaphosphate on Cancer Stem Cell Pool during Growth and Progression of Prostate Tumorigenesis in TRAMP Model

Abstract

Herein, we assessed the stage-specific efficacy of inositol hexaphosphate (IP6, phytic acid), a bioactive food component, on prostate cancer (PCa) growth and progression in a transgenic mouse model of prostate cancer (TRAMP). Starting at 4, 12, 20, and 30 weeks of age, male TRAMP mice were fed either regular drinking water or 2% IP6 in water for ~8-15 weeks. Pathological assessments at study endpoint indicated that tumor grade is arrested at earlier stages by IP6 treatment; IP6 also prevented progression to more advanced forms of the disease (~55-70% decrease in moderately and poorly differentiated adenocarcinoma incidence was observed in advanced stage TRAMP cohorts). Next, we determined whether the protective effects of IP6 are mediated via its effect on the expansion of the cancer stem cells (CSCs) pool; results indicated that the anti-PCa effects of IP6 are associated with its potential to eradicate the PCa CSC pool in TRAMP prostate tumors. Furthermore, in vitro assays corroborated the above findings as IP6 decreased the % of floating PC-3 prostaspheres (self-renewal of CSCs) by ~90%. Together, these findings suggest the multifaceted chemopreventive-translational potential of IP6 intervention in suppressing the growth and progression of PCa and controlling this malignancy at an early stage.

Keywords: TRAMP; cancer stem cells; chemoprevention; inositol hexaphosphate; prostate cancer.

Figure 1

Study design to assess the effect of feeding IP6 during different stages of prostate cancer growth and progression in TRAMP mice. Starting at 4, 12, 20, and 30 weeks of age, male TRAMP mice were fed either regular drinking water or 2% IP6 in water, and then sacrificed at age 12, 20, 30, and 45 weeks respectively. Depending upon the feeding period, the different groups are referred to as 4–12, 12–20, 20–30, and 30–45 week groups, respectively.

Figure 2

Stage-specific effect of IP6 feeding on pathological changes in dorsolateral prostate of TRAMP mice. (A) % incidence of normal, pre-neoplastic, and adenocarcinoma lesions in dorsolateral prostate tissues of TRAMP control and IP6-fed mice. (B) % area of dorsolateral prostate lobe displaying normal, pre-neoplastic, and adenocarcinoma lesions in TRAMP control and IP6-fed mice. LGPIN, low-grade prostatic intraepithelial neoplasia; HGPIN, high-grade prostatic intraepithelial neoplasia; WD, well-differentiated (adenocarcinoma); MD, moderately differentiated (adenocarcinoma); PD, poorly differentiated (adenocarcinoma). Quantified data are represented as columns (mean for each group); bars represent SEM. ** p ≤ 0.01, and * p ≤ 0.05.

Figure 3

Stage-specific effect of IP6 feeding on tumor grade, proliferation, and apoptosis in dorsolateral prostate of TRAMP mice. Effect on (A) Tumor grade, (B) PCNA-proliferative index, (C) TUNEL, and (D) GLUT-4 (glucose transporter) in TRAMP control and IP6-fed mice. DAB, 3,3′-diaminobenzidine; PCNA, proliferating cell nuclear antigen; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling. Quantified data are represented as mean ± SEM. *** p ≤ 0.001, ** p ≤ 0.01, and * p ≤ 0.05.

Figure 4

Stage-specific effect of IP6 feeding on angiogenic pathway in dorsolateral prostate of TRAMP mice. Effect on (A) microvessel density (MVD) as inferred by expression of PECAM-1/CD-31. MVD was determined by calculating the number of positive foci counted under ×400 magnifications in five selected areas in each section. Effect on (B) VEGF, (C) iNOS, (D) CXCR3 expression in TRAMP mice prostate and IP6-fed mice as determined by IHC. Quantified data are represented as mean ± SEM. *** p ≤ 0.001, ** p ≤ 0.01, and * p ≤ 0.05.

Figure 5

Stage-specific effect of IP6 feeding on the expansion of cancer stem cells (CSCs) pool in dorsolateral prostate of TRAMP mice. Immunofluorescence (IF) studies to determine the correlation and dual stained (BMI-1 and CD44 expression) tumor initiating cells (TICs/CSCs) pool in different pathological lesions of dorsolateral prostate of TRAMP controls and IP6-fed groups. Tissues were dual-stained for BMI-1 (green) and CD44 (red) expression. Nuclear staining was done with DAPI (blue). (A) Representative pictographs are depicted at x600 magnification and insets represent digital magnifications. (B) BMI-1 and CD44 positive foci was quantified using QuPATH analysis software. Quantified data are represented as mean ± SEM. *** p ≤ 0.001, ** p ≤ 0.01, and * p ≤ 0.05.

Figure 6

Effect of IP6 treatment on TICs/CSCs enriched prostaspheres in human prostate cancer PC-3 cancer cells. Effect of 2 mM IP6 treatment on TICs/CSCs (CD44⁺-α2β1^high) enriched prostaspheres formation, and effect on prostaspheres formation in the presence of macrophage THP-1 conditioned media and macrophage THP-1 conditioned media (pre-treated with IP6). Quantified data are represented as mean ± SEM. *** p ≤ 0.001 and ** p ≤ 0.01.

Figure 7

Stage specific effect of IP6 feeding on the expression of CSC-associated signaling molecules in the dorsolateral prostate of TRAMP mice. Pictographs and bar graphs representing the stage-specific expression of CSC-associated signaling molecules (A) cleaved-Notch-1, and (B) Shh, in WT control (T-ve), TRAMP control, and IP6-fed mice. Representative pictographs are depicted at ×100 magnification and insets represent digital magnifications. Immunoreactivity was scored as 0 (no staining), +1 (weak), +2 (moderate), +3 (strong), and +4 (very strong). The proportion area of prostate (positive for expression) was quantified as  immunopositive area score and assigned arbitrary scores as 0 (<5% area), +1 (5–25% area), +2 (26–50% area), +3 (51–75% area), and +4 (>75% area). Quantified data are represented as mean ± SEM. *** p ≤ 0.001, ** p ≤ 0.01, and * p ≤ 0.05.

Figure 8

Stage-specific effect of IP6 feeding on the expression of CSC-associated transcription factors in the dorsolateral prostate of TRAMP mice. Pictographs and bar graphs representing the stage-specific expression of CSC-associated transcription factors (A) Sox-2, and (B) Oct-4, in WT control (Tr-ve), TRAMP control, and IP6-fed mice. Representative pictographs are depicted at ×100 magnification and insets represent digital magnifications. Immunoreactivity was scored as 0 (no staining), +1 (weak), +2 (moderate), +3 (strong), and +4 (very strong). Quantified data are represented as mean ± SEM. *** p ≤ 0.001, ** p ≤ 0.01, and * p ≤ 0.05.