Polydatin Increases Radiosensitivity by Inducing Apoptosis of Stem Cells in Colorectal Cancer

Abstract

This study aimed to investigate the radiosensitizing effect of polydatin (PD) on colorectal cancer (CRC) and its underlying mechanism. The C57BL/6 mouse model of CRC was induced by treatment with azoxymethane (AOM)/dextran sodium sulfate (DSS) and then divided into four groups: control, PD alone, IR alone, and combination of PD and IR. Radiation therapy (200 cGy/min, 10Gy) was performed in mice in the experimental groups for once a week with a total of four times. Thirty minutes before IR, mice were intraperitoneally injected with PD at the dose of 25mg/kg. The number and volume of CRC xenografts were calculated. Immunohistochemical staining was performed to detect the expression of Ki67 and cleaved caspase-3 in tumor tissues samples. The effects of PD on proliferation and apoptosis were evaluated in CT26 and HCT116 colon tumor cells. Leucine-rich repeat-containing G-protein coupled receptor 5 positive (Lgr5⁺) cancer stem cells (CSCs) were sorted from CT26 cells and the effects of PD on their proliferation and apoptosis were observed to elucidate the radiosensitizing mechanism of PD in CRC cells. Combined therapy with PD and IR significantly decreased tumor volume, inhibited proliferation and induced apoptosis of tumor cells in the mouse model of CRC compared to other three groups. Compared to the IR group, in vitro assay showed that PD combined with IR inhibited proliferation and promoted apoptosis of CT26 and HCT116 colon tumor cells as well as Lgr5⁺ CSCs. However, addition of the bone morphogenetic protein (BMP) type I receptor inhibitor K02288 (6.4nM) dramatically increased proliferation of Lgr5⁺ CSCs and abolished the cytotoxic effect of PD combined with IR on Lgr5⁺ CSCs. The in vivo and in vitro experiments demonstrated that IR combined treatment with PD could inhibit proliferation and promote apoptosis of CRC cells and Lgr5⁺ CSCs, and BMP signaling pathway was involved in the radiosensitizing effect of PD.

Keywords: Lgr5+ cancer stem cells; Polydatin; colorectal cancer; radiosensitivity.

Fig 1

Establishment of orthotopic C57BL/6 mouse model of CRC and effects of PD combined with IR on survival rates of mice. (A) C57BL/6 mice aged 6 weeks were intraperitoneally injected with AOM (10 mg/kg). Subsequently, mice were given drinking water for 7 days and fed with 2% DSS dissolved in drinking water for another 7 days; this procedure was repeated for three times and lasted for 10 weeks. (B) Each group consisted of 6 mice. The radiation therapy was performed at a total dose of 10 Gy and a dose rate of 200 cGy/min, and once a week for a total of 4 times. The survival time of each mouse was recorded and analyzed for statistical significance after 200 days. PD+IR group compared with another group, P<0.05.

Fig 2

Effect of PD in combination with IR on tumor number and volume. (A) HE staining of tumor samples in the orthotopic mouse model of CRC; upper 40×, lower 200×. (B) Representative image of tumors of mice in each group. (C) Tumor number. (D) Tumor volume. ^* P<0.05, n=6.

Fig 3

Immunohistochemical staining of Ki67 and cleaved caspase-3 in CRC cells. (A) Representative images of Ki67⁺ and cleaved caspase-3⁺ cells, 400×. (B) Number of Ki67⁺ cells, "+" indicates that the number of positive cells in colorectal tissue in a microscope field is less than 20; "++" indicates that the number of positive cells is 20-50; "+++" indicates that the number of positive cells is in 50-100; "++++" indicates that positive cells are distributed throughout vision.CMHx2 test, PD+IR group vs. IR group, P<0.05. (C) Number of cleaved caspase-3⁺ cells, *P<0.05, n=6.

Fig 4

Effects of PD on CT26 cell proliferation and apoptosis. (A) Effect of PD at different doses on proliferation of CT26 cells. (B) CT26 cell proliferation was measured 24h after treatment with PD combined with IR. (C) CT26 cell proliferation was measured 48h after treatment with PD combined with IR. (D) CT26 cell proliferation was measured 72h after treatment with PD combined with IR. (E) CT26 cell proliferation was measured 48h after treatment with PD combined with IR using BrdU staining method. (F) Quantitative analysis of apoptotic rate 48h after combined treatment with PD (40μM) and IR. (G) The apoptosis of CT26 cells was detected 48h after treatment with PD (40μM) combined with IR.* P<0.05, n=3. Repeat three times for each experiment.

Fig 5

Effects of PD on HCT116 cell proliferation and apoptosis. (A) Effect of PD at different doses on proliferation of HCT116 cells. (B) HCT116 cell proliferation was measured 24h after treatment with PD combined with IR. (C) HCT116 cell proliferation was measured 48h after treatment with PD combined with IR. (D) HCT116 cell proliferation was measured 72h after treatment with PD combined with IR. (E) The apoptosis of HCT116 cell was detected 48h after treatment with PD (40μM) combined with IR. (F) Quantitative analysis of apoptotic rate 48h after combined treatment with PD (40μM) and IR. * P<0.05,n=3. Repeat three times for each experiment.

Fig 6

Effects of PD on the sphere and activity of Lgr5⁺ CSCs. (A) Representative image of Lgr5⁺ CSCs spheres at day 3, 5 and 7. PD, 40μM; radiation dose, 10Gy. (B) Effects of PD on the activity of Lgr5⁺ CSCs. The activity of Lgr5⁺ CSCs at 3, 5 and 7 days was detected using a Cell Titer-Glo®3D cell viability detection kit. * P<0.05, n=3. Repeat three times for each experiment

Fig 7

Effects of PD (40μM) combined with IR (10Gy) on Lgr5⁺ CSCs apoptosis, * P<0.05, n=3. Repeat three times for each experiment

Fig 8

BMP mediates the radiosensitizing effect of PD in CSCs. (A) Representative image of Lgr5⁺ CSCs spheres at day 3, 5 and 7 days. (B) The activity of Lgr5⁺ CSCs at 3, 5 and 7 days was detected using a Cell Titer-Glo®3D cell viability detection kit. K02288 (K), 6.4nM; radiation dose, 10Gy. (C) The contributions of Notch pathway were detected using western blot. * P<0.05, n=3. Repeat three times for each experiment.