Coniferyl Aldehyde Attenuates Radiation Enteropathy by Inhibiting Cell Death and Promoting Endothelial Cell Function

<div><p>Radiation enteropathy is a common complication in cancer patients. The aim of this study was to investigate whether radiation-induced intestinal injury could be alleviated by coniferyl aldehyde (CA), an HSF1-inducing agent that increases cellular HSP70 expression. We systemically administered CA to mice with radiation enteropathy following abdominal irradiation (IR) to demonstrate the protective effects of CA against radiation-induced gastrointestinal injury. CA clearly alleviated acute radiation-induced intestinal damage, as reflected by the histopathological data and it also attenuated sub-acute enteritis. CA prevented intestinal crypt cell death and protected the microvasculature in the lamina propria during the acute and sub-acute phases of damage. CA induced HSF1 and HSP70 expression in both intestinal epithelial cells and endothelial cells <i>in vitro</i>. Additionally, CA protected against not only the apoptotic cell death of both endothelial and epithelial cells but also the loss of endothelial cell function following IR, indicating that CA has beneficial effects on the intestine. Our results provide novel insight into the effects of CA and suggest its role as a therapeutic candidate for radiation-induced enteropathy due to its ability to promote rapid re-proliferation of the intestinal epithelium by the synergic effects of the inhibition of cell death and the promotion of endothelial cell function.</p></div

CA promotes endothelial cell function via HSF1.

<p><b>A</b>. HUVECs were incubated in the presence of cycloheximide (CHX, 10 μg/ml) with or without 5 μM of CA and were analyzed by Western blotting (*<i>p</i> < 0.05 vs. control cell). <b>B</b>. HUVECs were transfected with si-con and si-HSF1, and the expression of HSF1 and eNOS was analyzed after 48 h of CA treatment and IR. <b>C</b>. A tube formation assay in Matrigel was performed using HUVECs. HUVECs were treated with 5 μM CA for 1 h before exposure to 10 Gy IR. The cells were detached from a 100-mm dish, and 4.5×10⁴ cells were seeded on Matrigel in a 96-well plate at 24 h after IR. After 16 h of incubation, the tubes on the Matrigel were counted, and the results are depicted as a graph (**<i>p</i> < 0.01, <i>n</i> = 3).</p

CA confers protection against radiation-induced cell death by inducing HSF1 and HSP70 expression.

<p><b>A</b>. HSF1 and HSP70 expression in IEC6 cells and HUVECs was detected by Western blotting after treatment with 0–10 μM of CA at the time points indicated. <b>B</b>. Apoptotic cell death was measured by Annexin V/PI assay and flow cytometry at 48 h after 15 Gy IR. <b>C</b>. IEC6 cells and HUVECs were transfected with si-con, si-HSF1 or siHSP70 and treated with 5 μM of CA and 15 Gy of IR. Western blotting was conducted to detect cleaved caspase-3 and cleaved PARP at 48 h after 15 Gy IR in IEC6 cells and HUVECs. Actin was used as a loading control. Three different experiments were performed.</p

CA treatment did not protect tumors against IR.

<p><b>A</b>. Colony formation assays were performed with human cancer cell lines. Cells were seeded and cultured 7 to 10 days following CA treatment (0 and 2.5μM) and IR (0–6 Gy). <b>B</b>. HUVECs were transfected with si-con or si-HSF1 and treated with 5 μM of CA and 15 Gy of IR. Western blotting was conducted to detect HSF1 and eNOS at 48 h after IR. Actin was used as a loading control. Three different experiments were performed. <b>C</b>. The effect of CA on tumor growth was measured in CT26 allografts in BALB/c mice. Five CA doses (10 mg/kg, <i>i</i>.<i>p</i>.) were administered to tumor-bearing mice before and after 8 or 12.5 Gy IR. Tumor volumes were measured three times per week for 21 days. The data are presented as the mean ± SEM (*<i>p</i> < 0.05, <i>n</i> = 5).</p

CA treatment inhibits apoptotic epithelial and endothelial cell death after IR.

<p><b>A</b>. Apoptosis in the jejunum at 6 h after 12.5 Gy abdominal IR. Apoptotic FITC-TUNEL (green) and endothelial rhodamine-PECAM1 (red) are shown counterstained with DAPI (blue). Scale bar = 30 μm. <b>B</b>. The distribution of apoptotic cells in crypts and lamina propria and PECAM1 in a villus unit of the jejunum after 12.5 Gy IR. The values represent the mean ± SEM (*<i>p</i> < 0.05, <i>n</i> = 6).</p

CA treatment rescues jejunal crypt survival after high-dose abdominal IR.

<p><b>A</b>. Hematoxylin and eosin (H&E)-stained jejunal sections harvested from vehicle- or CA-treated mice at 3.5 days after 12.5 Gy abdominal IR. The inserted squares depict whole cross sections of the jejunum. Original magnification, 5x. The arrows indicate crypts that survived following IR. Scale bar = 50 μm. <b>B</b>. Quantitative analysis of morphological changes in the jejunum following CA and IR. Data are presented as the mean ± SEM (*<i>p</i> < 0.05, <i>n</i> = 6). <b>C</b>. Immunohistochemical analysis of Ki-67, a proliferation marker, in a section of the jejunum at 3.5 days after IR. Ki-67 was evaluated using diaminobenzidine (DAB, brown stain) and hematoxylin counterstaining (blue). The arrows indicate crypts, representing intestinal stem cell proliferation.</p

Experimental scheme of the protective effects of CA on abdominal IR-induced radiation enteropathy.

<p>Abdominal IR was performed using X-Rad320, as shown in the photo. CA (10 mg/kg per dose) was intraperitoneally administered to C3H mice at 24 h and 1 h before and 24, 48 and 72 h after IR. The mice were euthanized at 6 h after IR for apoptosis detection and at 3.5 days after IR for crypt survival assays (<i>n</i> = 6/group). To assess sub-acute radiation enteropathy, 6 mice from the control and CA-only groups and 15 mice from the IR and CA and IR combination groups were sacrificed at 30 days after abdominal IR.</p