Heavy ion radiation exposure triggered higher intestinal tumor frequency and greater β-catenin activation than γ radiation in APC(Min/+) mice

Abstract

Risk of colorectal cancer (CRC) after exposure to low linear energy transfer (low-LET) radiation such as γ-ray is highlighted by the studies in atom bomb survivors. On the contrary, CRC risk prediction after exposure to high-LET cosmic heavy ion radiation exposure is hindered due to scarcity of in vivo data. Therefore, intestinal tumor frequency, size, cluster, and grade were studied in APC(Min/+) mice (n = 20 per group; 6 to 8 wks old; female) 100 to 110 days after exposure to 1.6 or 4 Gy of heavy ion (56)Fe radiation (energy: 1000 MeV/nucleon) and results were compared to γ radiation doses of 2 or 5 Gy, which are equitoxic to 1.6 and 4 Gy (56)Fe respectively. Due to relevance of lower doses to radiotherapy treatment fractions and space exploration, we followed 2 Gy γ and equitoxic 1.6 Gy (56)Fe for comparative analysis of intestinal epithelial cell (IEC) proliferation, differentiation, and β-catenin signaling pathway alterations between the two radiation types using immunoblot, and immunohistochemistry. Relative to controls and γ-ray, intestinal tumor frequency and grade was significantly higher after (56)Fe radiation. Additionally, tumor incidence per unit of radiation (per cGy) was also higher after (56)Fe radiation relative to γ radiation. Staining for phospho-histone H3, indicative of IEC proliferation, was more and alcian blue staining, indicative of IEC differentiation, was less in (56)Fe than γ irradiated samples. Activation of β-catenin was more in (56)Fe-irradiated tumor-free and tumor-bearing areas of the intestinal tissues. When considered along with higher levels of cyclin D1, we infer that relative to γ radiation exposure to (56)Fe radiation induced markedly reduced differentiation, and increased proliferative index in IEC resulting in increased intestinal tumors of larger size and grade due to preferentially greater activation of β-catenin and its downstream effectors.

Figure 1. Heavy ion radiation-induced intestinal tumorigenesis in APC^Min/+ mice.

Intestinal tumor induction after 1.6 Gy or 4 Gy of ⁵⁶Fe radiation is compared to respective equitoxic doses γ radiation (2 Gy γ equitoxic to 1.6 Gy ⁵⁶Fe and 5 Gy γ equitoxic to 4 Gy ⁵⁶Fe) as well as to sham-irradiated controls.

Figure 2. Tumor clusters and segmental distribution of tumors in intestine.

A) Distribution of small intestinal tumors in duodenum, jejunum, and ilium after exposure to 5 Gy γ and equitoxic 4 Gy ⁵⁶Fe. B) Distribution of small intestinal tumors in duodenum, jejunum, and ilium after exposure to 2 Gy γ and equitoxic 1.6 Gy ⁵⁶Fe. C) Occurrence of tumor clusters defined as 5 or more tumors without intervening normal tissue was dependent on radiation quality and clusters were more after both 1.6 and 4 Gy ⁵⁶Fe relative to respective equitoxic γ radiation doses.

Figure 3. ⁵⁶Fe radiation-induced larger and higher-grade intestinal tumors.

A) Compared to γ radiation, exposure to ⁵⁶Fe radiation led to greater number of tumors whose size was ≥3 mm. B) H&E stained intestinal tumors showing crypt penetration of mucularis mucosa (arrow) indicating invasive adenocarcinoma after ⁵⁶Fe radiation. Tumors in control and γ irradiated mice were mostly adenomas. C) Percent of invasive adenocarcinoma in control, γ, and ⁵⁶Fe irradiated tumors.

Figure 4. Alcian blue staining of intestinal sections showed reduced goblet cells after ⁵⁶Fe radiation.

A) Representative images of alcian blue stained tumor free (normal) areas of intestinal sections from control, γ, and ⁵⁶Fe-irradiated mice. B) Quantification of alcian blue positive cells in tumor free areas of control, γ, and ⁵⁶Fe-irradiated sections. C) Representative images of alcian blue stained tumor bearing areas of intestinal sections from control, γ, and ⁵⁶Fe-irradiated mice. D) Quantification of alcian blue positive cells in tumor bearing areas of control, γ, and ⁵⁶Fe-irradiated sections.

Figure 5. Greater phospho-histone H3 staining after ⁵⁶Fe radiation.

A) Representative images of phospho-histone H3 stained tumor free (normal) areas of intestinal sections from control, γ, and ⁵⁶Fe-irradiated mice. B) Quantification of phospho-histone H3 positive nuclei in tumor free areas of control, γ, and ⁵⁶Fe-irradiated sections. C) Representative images of phospho-histone H3 stained tumor bearing areas of intestinal sections from control, γ, and ⁵⁶Fe-irradiated mice. D) Quantification of phospho-histone H3 positive nuclei in tumor bearing areas of control, γ, and ⁵⁶Fe-irradiated sections.

Figure 6. Increased activation of β-catenin after ⁵⁶Fe radiation.

A) Representative images of β-catenin stained tumor free (normal) areas of intestinal sections from control, γ, and ⁵⁶Fe irradiated mice. B) Quantification of total β-catenin staining in tumor free areas of control, γ, and ⁵⁶Fe irradiated sections. C) Representative images of β-catenin stained tumor-bearing areas of intestinal sections from control, γ, and ⁵⁶Fe irradiated mice. D) Quantification of total β-catenin staining in tumor-bearing areas of control, γ, and ⁵⁶Fe irradiated sections. E) Quantification of nuclear β-catenin staining in tumor-bearing areas of control, γ, and ⁵⁶Fe irradiated sections. F) Immunoblots showing increased β-catenin, decreased phospho-β-catenin, and increased phospho-GSK3β after ⁵⁶Fe radiation. Panel 1: β-catenin, 2: phospho-β-catenin, 3: phospho-GSK3β, 4: β-actin G) Quantification of immunoblots, normalized to β-actin band intensity, showed greater increase of β-catenin, decrease of phospho-β-catenin, and increase of phospho-GSK3β after ⁵⁶Fe radiation relative to control and γ radiation.

Figure 7. Cyclin D1 staining is greater after ⁵⁶Fe radiation.

A) Representative images of cyclin D1 stained tumor free (normal) areas of intestinal sections from control, γ, and ⁵⁶Fe-irradiated mice. B) Quantification of cyclin D1 staining in tumor free areas of control, γ, and ⁵⁶Fe-irradiated sections. C) Representative images of cyclin D1 stained tumor bearing areas of intestinal sections from control, γ-, and ⁵⁶Fe-irradiated mice. D) Quantification of cyclin D1 staining in tumor bearing areas of control, γ, and ⁵⁶Fe-irradiated sections.