Irinotecan was purchased from Jiangsu Hengrui Medicine Co., Ltd. (Jiangsu, China), dissolved in dimethyl sulfoxide to obtain a 100 mg/ml stock solution and stored at −20°C.

The HT29 and SW620 cell lines were purchased from the Biochemistry and Cell Biology Institute of Shanghai, Chinese Academy of Sciences, within 3 months of the experiments. The HT29 cells were cultured in McCoy's 5A medium, and the SW620 cells were cultured in DMEM medium, both containing 10% heat-inactivated fetal bovine serum, 1% penicillin (final concentration, 100 U/ml) and streptomycin (final concentration, 0.1 mg/ml). The cells were maintained at 37°C in a humidified atmosphere containing 5% CO₂. The experiments were conducted in the exponential phase of growth.

The HT29 (8×10³) and SW620 (8×10³) cells in 100 μl of culture medium were seeded onto 96-well plates (Corning Inc., Corning, NY, USA) and cultured overnight. The cells were treated with irinotecan by exchanging the medium premixed with various drug concentrations (0, 6.25, 12.5, 25, 50, 100, 200 and 400 μg/ml) for 24 h. Cell viability was evaluated using a Cell Counting kit-8 (CCK-8) assay (Dojindo Laboratories, Kumamoto, Japan), according to the manufacturer's instructions. The absorbance at 450 nm was then measured using a BioTek microplate reader and analyzed by Gen5 2.0 microplate software (BioTek Instruments, Inc., Winooski, VT, USA). The cell growth inhibition curve was presented, and the 24-h half-maximal inhibitory concentration (IC₅₀) of irinotecan was calculated. A chemotherapeutic drug exerts cytotoxic effects on cells, which can influence the accuracy of the results when it is combined with radiation. To examine the radiosensitizing effects of irinotecan, the concentrations need to be low in order to decrease the cytotoxic effects of the drug itself. According to past practice (19), low drug concentrations (<10% of the IC₅₀ value or IC₁₀ value) were considered for use in subsequent experiments. According to the IC₅₀ values in this study (shown in the Results section below), low concentrations <4 μg/ml (0, 0.5, 1, 1.5, 2, 3 and 4 μg/ml) were used to examine the inhibitory effects of irinotecan as a single agent on clonogenic survival. Finally, we selected the doses of 0, 1 and 2 μg/ml as the experimental concentrations for use in combination with radiation.

The cells were irradiated with 6-MV X-rays using a linear accelerator (Varian Medical Systems, Palo Alto, CA, USA) at 3 Gy/min. Irradiation was performed at the Experimental Irradiation Core of the Fudan University Shanghai Cancer Center, Shanghai, China. In clonogenic assays, the cells were irradiated with X-rays at a dose of 0, 2, 4, 6 and 8 Gy. However, in the experiments comparing the results of the control, irinotecan, radiation and combined groups, there are no criteria on radiation doses. Preliminary experiments (data not shown) based on low radiation doses (2 and 4 Gy) revealed relatively small absolute values of the results and the differences among groups were not so obvious. In addition, preliminary experiments (data not shown) using high radiation doses (8-16 Gy) revealed severe toxicity of the radiation itself and it was hard to determine the differences between the radiation group and the combination group. Thus, we finally selected the dose of 6 Gy as the experimental radiation dose so that we could obtain obvious and high-quality results.

A clonogenic assay is the standard method to examine the radiosensitizing effects of a drug. The cells were pre-seeded in 6-well plates (Corning Inc., Corning, NY, USA) at several densities (300-4,000 cells for the HT29 and 300-10,000 cells for the SW620 cells) overnight. After exchanging with drug-containing medium and incubation for 24 h, the cells were irradiated with X-rays at a series of dose levels (0, 2, 4, 6 and 8 Gy). The medium was then exchanged with culture medium without drugs, and the cells were incubated for 10 to 14 days to allow for colony formation. The colonies were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet (100% methanol solution) prior to counting. The number of clones containing ≥50 cells was counted under a stereomicroscope (Leica Microsystems, Wetzlar, Germany). The plating efficiency (PE) was calculated in the following manner: PE = number of colonies formed without irradiation/number of cells inoculated ×100%. The cell survival fraction (SF) was calculated at each irradiation dose in the following manner: SF = (number of colonies formed at a certain irradiation dose)/(number of cells inoculated × PE). SF curve fitting was conducted with a linear-quadratic model (20) via the equation y = exp[ −(a x x + b x x²)]. Currently, the therapeutic regimen of 45-50 Gy/25-28 fractions (1.8-2.0 Gy per fraction) is widely used in the neoadjuvant chemoradio-therapy of locally advanced rectal cancers (21). In the present study, SFs at 2 Gy were used to determine the sensitivity enhancement ratio (SER), which was consistent with clinical radiotherapy. In this study, SER referred to the fractional ratio of counts of surviving colonies between cells treated with only 2 Gy X-rays compared to cells treated with both 2 Gy X-rays and irinotecan.

The HT29 (4×10³) and SW620 (3×10³) cells with 100 μl of culture medium were seeded onto 96-well plates (Corning Inc., Corning, New York, NY, USA) and cultured overnight. After exchanging the medium with drug-containing medium and incubating for 24 h, the cells were irradiated with X-rays at 0 or 6 Gy. The medium was then exchanged with culture medium without drugs, and the cells were incubated for 5 days. The cell growth assay was performed daily using the CCK-8 assay kit as described above.

The HT29 and SW620 cells were seeded onto 60-mm plastic Petri dishes (Corning Inc.) and cultured overnight. After exchanging the medium with drug-containing medium and incubating the cells for 24 h, the cells were irradiated with X-rays at 0 or 6 Gy. The medium was then exchanged with culture medium without drugs, and cells were incubated for 3 and 24 h. The cells (discarding the culture medium) were harvested by trypsinization at 3 and 24 h following radiation. After rinsing 2 times with PBS, the cells were fixed with 75% ethanol at −20°C overnight. The fixed cells were incubated in PBS for 15 min and subsequently stained with propidium iodide (PI) staining solution (50 μg/ml) for 30 min. The cell cycle was assayed with a flow cytometer (FACSCalibur; BD Biosciences, Franklin Lakes, NJ, USA) at an excitation wavelength of 488 nm and an emission wavelength of 585 nm and analyzed using BD CellQuest Pro software (643274 Rev. A; BD Biosciences). A doublet discrimination module was used when using flow cytometry to remove the doublets that can end up in the G2/M phase.

The HT29 and SW620 cells were seeded onto 60-mm plastic Petri dishes (Corning Inc.) and cultured overnight. After exchanging the medium with drug-containing medium and incubating the cells for 24 h, the cells were irradiated with X-rays at 0 or 6 Gy. The medium was then exchanged with culture medium without drugs, and the cells were incubated for 24 h to 3 days. The cells (including cells in the culture medium) were harvested by trypsinization at 24, 48 and 72 h following radiation. After rinsing twice with PBS, the cells were suspended in a binding buffer, stained with both 4 μl of PI and 4 μl of Annexin V-FITC per sample and loaded onto a flow cytometer (FACSCalibur; BD Biosciences) for FL1 (Annexin V) and FL2 (PI) bivariate analysis. Untreated samples stained with only PI or Annexin V-FITC were also prepared. The data from 20,000 cells/sample were collected, and the quadrants were set according to the population of viable, unstained cells in the untreated samples. The percentage of cells in the respective quadrants was calculated and analyzed using BD CellQuest Pro software (643274 Rev. A; BD Biosciences).

A total of 5×10⁴ HT29 and SW620 cells were seeded separately onto 24-well plates (Corning Inc.) containing a glass coverslip in each well and cultured overnight. After exchanging the medium with drug-containing medium and incubating the cells for 24 h, the cells were irradiated with X-rays at 0 or 6 Gy. The medium was subsequently exchanged with culture medium without the drug, and cells were incubated for 24 h. The slides were then rinsed with PBS and fixed for 30 min in 4% paraformal-dehyde. The cells were rinsed, treated with 1% Triton X-100 for permeabilization of the cell membrane, and rinsed again. The slides were blocked for 1 h in 5% bovine serum albumin (BSA) and finally incubated with phospho-histone H2A.X (Ser139) (20E3) rabbit monoclonal antibody (Alexa Fluor^® 555 Conjugate; #8228S, diluted 1:100 in 5% BSA; Cell Signaling Technology, Danvers, MA, USA) at 4°C overnight. The slides were rinsed, mounted with ProLong Diamond Antifade Mountant with DAPI (Thermo Fisher Scientific Inc., Waltham, MA, USA) and sealed. Images were aquired under a laser scanning confocal microscope (Leica Microsystems, Wetzlar, Germany). The numbers of ^Ser139p-γH2AX foci in the nuclei of at least 50 cells were counted in a double-blinded manner to calculate the average number of foci per nucleus.

The HT29 and SW620 cells were seeded onto 60-mm plastic Petri dishes (Corning Inc.) and cultured overnight. After exchanging the medium with drug-containing medium and incubating cells for 24 h, the cells were irradiated with X-rays at 0 or 6 Gy. The medium was then exchanged with culture medium without drugs, and cells were incubated for 3 and 24 h. The cells were lysed at 3 and 24 h following radiation with M-PER Mammalian Protein Extraction Reagent (Thermo Fisher Scientific Inc.) with the addition of protease inhibitors (cOmplete Tablets Mini; Roche, South San Francisco, CA, USA) and phosphatase inhibitors (PhosSTOP; Roche). Following the quantitation of the protein concentration using a Pierce BCA Protein Assay kit (Thermo Fisher Scientific Inc.) and protein degeneration at 100°C for 10 min, the protein samples were resolved on SDS-polyacrylamide gels (^Ser1981p-ATM, ATM in 7.5% resolving gels, ^Ser345p-Chk1, Chk1, ^Thr68p-Chk2, Chk2, Cyclin B1, ^Ser216p-Cdc25C, Cdc25C, β-actin in 12.5% or 10% resolving gels, ^Tyr15p-Cdc2, Cdc2, ^Ser139p-_γH2AX, ^Ser10p-Histone H3, Histone H3 in 12.5% resolving gels) by electrophoresis and subsequently transferred onto PVDF membranes (Millipore, Darmstadt, Germany). The membranes were blocked with 5% BSA and probed with primary antibodies at 4°C overnight. The membranes were washed with Tris-buffered saline and Tween-20 (TBST) for 5×5 min, incubated with goat anti-mouse and goat anti-rabbit secondary antibodies for 1 h and washed again with TBST for 5×5 min. Antibodies against ^Ser1981p-ATM, ATM, ^Ser345p-Chk1 (#2348; dilution, 1:1,000), Chk1 (#2360; dilution, 1:1,000), ^Thr68p-Chk2 (#2197; dilution, 1:1,000), Chk2 (#6334; dilution, 1:1,000), ^Ser139p-γH2AX (#9718; dilution, 1:1,000), ^Ser216p-Cdc25C (#4901; dilution, 1:1,000), Cdc25C (#4688; dilution, 1:1,000), ^Tyr15p-Cdc2 (#4539; dilution, 1:1,000), Cdc2 (#9116; dilution, 1:1,000), cyclin B1 (#12231; dilution, 1:1,000), ^Ser10p-Histone H3 (#53348; dilution, 1:1,000), Histone H3 (#4499; dilution, 1:1,000) and β-actin, as well as goat anti-mouse (#7056; dilution, 1:10,000) and goat anti-rabbit (#7074; dilution, 1:10,000) secondary antibodies were purchased from Cell Signaling Technology (Cell Signaling Technology). The protein bands were visualized using an enhanced chemiluminescence detection system (Thermo Fisher Scientific Inc.). Protein bands were analyzed by ImageJ software version 1.8.0 (National Institutes of Health, Bethesda, MD, USA).

All experiments were repeated independently at least 3 times. SPSS version 22.0 software (SPSS Inc., Chicago, IL, USA) was used for the statistical analysis. The data are presented as the means ± standard deviation. Comparisons among multiple groups were conducted using the one-way ANOVA or two-way ANOVA method for quantitative data. Turkey's test was used for the post hoc multiple comparisons. A P-value <0.05 was considered to indicate a statistically significant difference. All curves and plots were generated using GraphPad Prism 7.0 software (GraphPad Software Inc., San Diego, CA, USA).

To verify the cytotoxic effects of irinotecan on colorectal cancer cells, we first treated both cell lines with irinotecan as a single agent at several increasing concentrations (0, 6.25, 12.5, 25, 50, 100, 200 and 400 μg/ml) for 24 h. Cell viability was examined using the CCK-8 assay kit. As the scatter grams shown in Fig. 1 A and B, the inhibition rates increased with the increasing drug concentration (0, 6.25, 12.5, 25, 50, 100, 200 and 400 μg/ml) in both cell lines; the HT29 cells seemed to be more sensitive to irinotecan than the SW620 cells. We then transformed the drug concentrations into log μg/ml style. The drug inhibition curves (shown in Fig. 1C and D) exhibited an S-like shape, from which IC₅₀ values at 24 h for both cell lines were calculated. For the HT29 cells, the IC₅₀ value was 39.84 μg/ml (95% CI, 38.27-41.48) and for the SW620 cells, it was 96.86 μg/ml (95% CI, 89.04-105.4). Concentrations of irinotecan <10% of the IC₅₀ value were considered for use in the subsequent experiments.

Subsequently, we examined the inhibitory effects of irinotecan on clonogenic survival at low concentrations (0, 0.5, 1, 1.5, 2, 3 and 4 μg/ml) for 24 h (Fig. 1E and F). The results revealed that the clonogenic survival rates were very low when the concentration was >2 μg/ml; thus, we selected the dose of 0, 1 and 2 μg/ml as the experimental concentrations for use in combination with radiation.

Both cell lines were treated with low concentrations of irinotecan (0, 1 and 2 μg/ml) as a single agent. The cells were harvested after 24 h of incubation with irinotecan, and the protein expression of ^Ser1981p-ATM, ATM and ^Ser139p-γH2AX was measured by western blot analysis. The cells were also submitted to flow cytometry to analyze changes in the cell cycle and apoptosis.

Low levels of irinotecan induced a statistically significant increase in the expression of ^Ser1981p-ATM/ATM and ^Ser139p-γH2AX (Fig. 2 A–C and F–H) and a marked increase in the proportion of cells in the G2/M phase, with a corresponding decrease in the proportion of cells in the G1 phase, all in a concentration-dependent manner (Fig. 2D and I). The results indicated that SW620 cells were more sensitive to delay at the G2/M phase than the HT29 cells. However, only a significant increase (P=0.022, 2 μg/ml vs. 1 μg/ml) in apoptosis was detected at 2 μg/ml in the SW620 cells and no increase was detected in the HT29 cells after 24 h of irinotecan treatment (Fig. 2E and J).

These findings suggest that irinotecan activates the DNA damage response system, induces potent G2/M phase arrest and minimal apoptosis of the HT29 and SW620 cells, consistent with the findings of previous studies (22,23). These changes may potentially represent important mechanisms for radiosensitization.

We selected 1 and 2 μg/ml of irinotecan for use in the radiosensitization experiments. The cells were treated with irinotecan for 24 h, followed by treatment with radiation at different doses of up to 8 Gy. The culture medium was then exchanged with irinotecan-free medium for 10-14 days to enable colony formation. Following linear-quadratic analysis (LQ model), we found that irinotecan effectively sensitized the HT29 and SW620 cells to radiation, and the SER at 2 Gy increased with the increasing drug concentration. For the HT29 cells, the SER at 2 Gy was 1.16 and 1.41 at 1 and 2 μg/ml, respectively, and for the SW620 cells, the SER at 2 Gy was 1.13 and 1.87 at 1 and 2 μg/ml, respectively (Fig. 3). Thus, we concluded that irinotecan is a good radiosensitizer against p53-mutant colorectal cancer cells.

Subsequently, we verified the radiosensitizing effects of irinotecan by drawing cell growth curves. The cells were incubated for 5 days following 24 h of drug treatment, followed by radiation treatment. Cell viability was measured daily using the CCK-8 assay kit. As expected, irinotecan in combination with radiation resulted in a reduced growth of both the HT29 and SW620 cells compared with growth in the irinotecan, radiation and control groups, with most high significance observed on days 4 and 5 (Fig. 4).

The cells were treated with irinotecan (2 μg/ml), radiation (6 Gy) or irinotecan (2 μg/ml) in combination with radiation (6 Gy). The formation of ^Ser139p-γH2AX foci was illustrated by immunofluorescence staining. Subsequently, the expression levels of proteins related to the DNA damage response, such as ^Ser1981p-ATM, ATM, ^Ser345p-Chk1, Chk1, ^Thr68p-Chk2, Chk2 and ^Ser139p-γH2AX, were measured by western blot analysis.

The results of immunofluorescence microscopy revealed that the ^Ser139p-γH2AX foci in the nuclei of both the HT29 and SW620 cells at 24 h following radiation were the most obvious in the combination group. The immunofluorescence staining images of the HT29 cells are presented in Fig. 5; however, the images of the SW620 cells are not presented (data not shown).

The results of western blot analysis also verified these findings. Compared with results from the irinotecan and radiation groups, irinotecan in combination with radiation resulted in a marked increase in the phosphorylation levels of ATM at Ser1981 and γH2AX at Ser139 at 3 and 24 h. Combined treatment also increased the activation of Chk1 at Ser345 and Chk2 at Thr68 at 3 h, with significantly increased ratios of ^Ser1981p-ATM/ATM, ^Ser345p-Chk1/Chk1 and ^Thr68p-Chk2/Chk2, and the activation of these proteins persisted for >24 h (Fig. 6A–E and F–J). These results indicate that irinotecan could likely enhance radiation-induced DNA double-strand damage and the DNA damage response process through the ATM/Chk signaling pathway.

The cells were treated with irinotecan (2 μg/ml), radiation (6 Gy) or irinotecan (2 μg/ml) combined with radiation (6 Gy). The cell cycle distribution was measured by flow cytometry at 3 and 24 h following radiation. Subsequently, the expression of levels proteins related to G2/M phase arrest, such as ^Tyr15p-Cdc2, Cdc2 and cyclin B1, were measured by western blot analysis.

The proportions of cells in the G2/M phase were elevated to a fairly high level following pre-treatment with irinotecan. Cells in the G2/M phase are more sensitive to radiation (24). Thus, it was suggested that treatment with irinotecan prior to radiation could produce a greater number of radiosensitized cells.

At 3 h following radiation, the combination group exhibited a slight increase in the number of cells undergoing G2/M phase arrest compared with the irinotecan group (data not shown). However, 24 h later, a marked increase in the number of cells undergoing G2/M phase arrest was observed in the radiation group and the combination group, indicating that it took some time for radiation to induce cell cycle arrest. As expected, the combination group exhibited the highest rates of G2/M phase arrest at 24 h when compared with those in the other 3 groups. The rates of G2/M phase arrest were 66.08±1.42% for the HT29 cells and 79.35±1.49% for the SW620 cells, indicating that the addition of irinotecan enhanced the activation of the cell cycle checkpoint and delayed the growth of the cells. The cell cycle distributions at 24 h are presented in Fig. 7A and B.

Cdc2 and cyclin B1 are two key regulators of the G2-to-M phase transition. We found a significant increase in the expression levels of ^Tyr15p-Cdc2 (and in the ratio of ^Tyr15p-Cdc2/Cdc2) and cyclin B1 at 3 h in the combination group compared with values in the other 3 groups, and this increase was enhanced over 24 h (Fig. 7C–E and F–H).

The tumor suppressor p53 is a key protein in checkpoint pathways in normal cells, and the mutation of the p53 gene is considered an important step in colorectal cancer formation. There are two pathways related to G2/M phase arrest, the p21-dependent pathway in p53-wild-type cancer cells and the Chk-dependent pathway in p53-deficient cancer cells (24-27). For p53-mutant HT29 and SW620 cells, we hypothesized that the Chk-dependent pathway plays an important role in irinotecan-induced or/and radiation-induced G2/M phase arrest. The key intermediate factor between Chk proteins and the Cdc2-cyclin B1 complex is Cdc25. The negative regulation of Cdc25C by phosphorylation at Ser216 can subsequently inhibit the activity of the Cdc2-cyclin B1 complex and lead to G2/M phase arrest. Therefore, we examined the effects of irinotecan and radiation on the expression of ^Ser216p-Cdc25C and Cdc25C in p53-mutant HT29 and SW620 cells.

As shown in Fig. 8A and B, and C and D, for both cell lines, irinotecan and radiation as single treatments slightly increased the expression of ^Ser216p-Cdc25C; however, combined treatment significantly enhanced phosphorylation, with an increasing ratio of p-Cdc25C/Cdc25C. These results suggest that the addition of irinotecan to radiation treatment promotes cycle arrest through the negative regulation of Cdc25C by phosphorylation at Ser216, and the ATM/Chk/Cdc25C/Cdc2/cyclin B1 pathway likely plays an important role in the mechanism of G2/M phase arrest.

Histone H3 phosphorylation is a well-established marker of mitosis (28). In this study, to determine whether radiation combined with irinotecan causes the arrest of cells in the G2 phase or M phase, we examined the expression of ^Ser10p-Histone H3 at 3 h and 24 h following treatment.

As shown in Fig. 9A and B, and C and D, for both cell lines, the cells in the control group exhibited some degree of expression of ^Ser10p-Histone H3. Of note, the effects of irinotecan alone on the HT29 cells and SW620 cells were not consistent. For the HT29 cells, irinotecan alone led to an enhanced expression of ^Ser10p-Histone H3 at 3 and 24 h. However, for the SW620 cells, irinotecan alone reduced the expression of ^Ser10p-Histone H3 at 3 h and lasted for >24 h.

For both cell lines, treatment with radiation alone inhibited the expression of ^Ser10p-Histone H3 for a short period of time, showing a low-level expression of ^Ser10p-Histone H3 at 3 h. However, its expression increased in the following 24 h, showing an increased level at 24 h after treatment. When radiation was combined with irinotecan, we found the same trend at 3 and 24 h following combined treatment. For the SW620 cells, the expression of ^Ser10p-Histone H3 in the combination group at 24 h was higher than that in the irinotecan, radiation and control groups. However, for the HT29 cells, the expression of ^Ser10p-Histone H3 in the combination group at 24 h was lower than that in the radiation group, but higher than that in the irinotecan and control group. However, the expression of ^Ser10p-Histone H3 in the combination group exhibited an increasing trend, probably exceeding the radiation group in the following hours. Thus, we found that irinotecan in combination with radiation resulted in a decreased and then increased M phase arrest.

It has been previously demonstrated that the DNA damage induced by the TOP I inhibitor, irinotecan, triggers the long-term cell cycle arrest of p53-wild-type colorectal carcinoma cells and transient arrest followed by apoptosis in p53-mutant cells (29). Thus, for the two p53-mutant cell lines used in the present study, we examined whether the G2/M phase arrest caused by irinotecan and radiation was followed by apoptosis.

The cells were treated with irinotecan (2 μg/ml), radiation (6 Gy) or irinotecan (2 μg/ml) in combination with radiation (6 Gy). Cell apoptosis was measured by flow cytometry at a series of time points (24, 48 and 72 h) following radiation.

As expected, the rates of cell apoptosis were highest in the combination group, compared with the control group, irinotecan group and radiation group. However, at 24 h, the rates of apoptosis were relatively low and increased significantly when the cells were incubated for a further 24 or 48 h. The rates of apoptosis at 7 h were 26.25±1.77% for the HT29 and 49.40±4.45% for the SW620 cells (Fig. 10). Thus, the radiosensitizing effects of irinotecan are attributable to short-term DNA damage-induced G2/M cycle arrest, followed by enhanced apoptosis, which significantly inhibits cell viability and proliferation.