Traditional Chinese Medicine Curcumin Sensitizes Human Colon Cancer to Radiation by Altering the Expression of DNA Repair-related Genes

Materials and Methods

Colon cancer cells and culture. Human colon-cancer cell line (HT-29) was purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences. Cells were cultured in RPMI 1640 medium (Thermo Fisher Scientific, CA, USA) containing 10% fetal bovine serum (Thermo Fisher Scientific, CA, USA), 100 U/ml penicillin and 100 μg/ml streptomycin (Thermo Fisher Scientific, CA, USA) at 37°C with 5% CO₂.

Cell proliferation assay. Cell proliferation was determined using the MTT assay. HT-29 cells were seeded in 96-well plates with 5×10³/200 μl and cultured overnight. Cells were divided into four groups: control group; curcumin group; irradiation group (IR); and irradiation + curcumin (IR+curcumin) group. Cells treated with dimethyl sulfoxide (0.1% DMSO) were used as a vehicle control. Each treatment included six duplicate wells. The curcumin and IR+curcumin groups were treated with 2.5 μM curcumin (94% purity, Sigma, St. Louis, MO, USA). The IR and IR+curcumin groups were irradiated with X ray at 6 MV using a 600C/D linear accelerator (RS2000Pro, RADSOURCE, Buford, GA, USA) to deliver the indicated doses (10 Gy). All cells were further cultured for 24, 48, or 72 h. To measure cell viability, 20 μl 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (5 μg/μl) (ATCC, Manassas, VA, USA) were added to each well and incubated for 2 h at 37°C. An extraction buffer (20% SDS and 50% dimethylformamide) was added, and the cells were incubated overnight at 37°C. The absorbance (OD) at 490 nm was measured with a micro-plate reader to determine the cell viability. The above experiment was repeated three times. The resulting MTT absorbance in negative control cells was used as 0% cell inhibition.

Annexin V detection of apoptotic cells. Apoptotic cells were detected by Annexin V-PE/7-AAD analysis. Cells were divided into four groups (Control; curcumin; IR; and IR+curcumin group) (5×10⁵ cells/well) and seeded in 6-well plates. After attachment to the dish, the cells in the curcumin and radiation+curcumin were treated with 2.5 μM curcumin. The IR and IR+curcumin groups were treated with X ray at 6 MV using a 600C/D linear accelerator to deliver the indicated doses (10Gy). All cells were further cultured for 48h. Cells were digested by 0.25% trypsin without EDTA (Thermo Fisher Scientific, CA, USA) and then were stained using an Annexin V-PE/7-AAD apoptosis kit (Nanjing KeyGen BioTech, Nanjing, P.R.China) according to the manufacturer's instructions. Stained cells were detected and analyzed using flow cytometry (Becton Dickinson, NJ, USA). Each experiment was performed in triplicate.

Real-time PCR array analysis of gene expression. Samples from each group were submerged in 2 ml Trizol (Invitrogen, Carlsbad, CA, USA) for RNA extraction and stored at −80°C until further processed. cDNA synthesis was performed on 4 μg RNA in a 10 μl sample volume using SuperScript II reverse transcriptase (Invitrogen) as recommended by the manufacturer. Real-time PCR array analysis was performed in a total volume of 20 μl including 1 μl cDNA, 10 μl qPCR Master Mix 2x (Roche, Mannheim, Germany) and 9 μl ddH₂O. Reactions were run on an Lightcycler 480 (Roche) using universal thermal cycling parameters (95°C for 5 min, 40 cycles of 15 sec at 95°C, 15 sec at 60°C and 20 sec at 72°C; followed by a melting curve: 5 sec at 95°C, 60 sec at 60°C and continued melting). Data were obtained using the Lightcycler 480 sequence detection software and analyzed using Microsoft Excel. For quality control purposes, melting curves were acquired for all samples. The comparative Ct method was used to quantify gene expression. The target gene expression level was normalized to expression of the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) within the same sample (−ΔCt). The relative expression of each gene was calculated using Log2 (−ΔCt).

Western blotting. The differential gene expression of CCNH, LIG4, XRCC5 and PNKP in the various groups was determined by Western blotting. Cells were lysed in 100 μl RIPA lysis buffer (50 mmol/l Tris-HCl, pH 7.5, 1% NP-40, 150 mmol/l NaCl, 1 mg/ml aprotinin, 1 mg/ml leupeptin, 1 mmol/l Na₃VO₄, 1 mmol/l NaF) at 4°C for 30 min. Cell debris was removed by centrifugation at 12,000 × g for 20 min at 4°C. Protein concentrations were determined with the Bradford assay (Bio-Red, Hercules, CA, USA). An equal amount of lysate (40 μg) was resolved by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA). The membranes were blocked with 5% non-fat milk at room temperature for 1 h and then incubated for 2 h with primary antibodies to CCNH, LIG4, XRCC or PNKP (Cell Signaling Technology, Boston, MA, USA). The membranes were then incubated for 1 h with an appropriate horseradish peroxidase-linked secondary antibody (Santa Cruz Biotechnology, Dallas, TX, USA). Electro-chemi-luminescence was performed according to the manufacturer's instructions using a ChemiDoc™ Touch Imaging System (Bio-Rad, Hercules, California, USA). Quantity One software (Bio-Rad, Hercules, CA, USA) was used to quantify band density.

Animal care. Twenty-eight [28] BALB/C female nude mice aged 4-6 weeks weighing 20-25g, were purchased from Shanghai SLAC Laboratory Animal Co., Ltd., Shanghai, P.R.China (SCXK, Hu. 2007-0005). All mice were maintained in a HEPA-filtered environment at 24-25°C and humidity was maintained at 50-60%. All animals were fed with autoclaved laboratory rodent diet. Animal experiments were approved by the Animal Committee of Nanjing Origin Biosciences, P.R. China (OB1431).

In vivo anti-tumor efficacy. A mouse model of human colon cancer [14] was established with the HT-29 cancer cell line. Stocks of HT-29 tumors were established by subcutaneously injecting 5×10⁶ HT29 cells in the flank of nude mice. The resulting tumors were harvested at the exponential growth phase and resected under aseptic conditions. Necrotic tissues were removed and viable tissues were cut with scissors and minced into 1-mm³ pieces. Animals were anesthetized by injection of 0.02 ml of solution of 50% ketamine, 38% xylazine, and 12% acepromazine maleate. Two tumor fragments were transplanted to the flank of nude mice. All surgical procedures and animal manipulations were conducted under HEPA-filtered laminar-flow hoods.

Treatments were initiated when the average tumor size reached 100 mm³. The mice with tumors were randomly divided into four groups of seven. Group 1 (Control) received intraperitoneal saline injection only; Group 2 (curcumin); Group 3 received irradiation; Group 4 (irradiation and curcumin) received intraperitoneal curcumin treatment at 20 mg/kg/dose. Six hours after saline or curcumin treatment, Groups 3 and 4 received irradiation (10 Gy). For irradiation, animals were anesthetized and immobilized in the treatment position with their right legs extended. Radiation was delivered at a dose rate of 2.0 Gy/min through a single posterior to anterior collimated 3-cm cobalt beam with a 5-mm bolus placed over the tumor. Tumor growth was measured twice a week with calipers. Tumor volume was calculated using the formula (L × W²) × ½, where W and L represent the perpendicular minor dimension and major dimension, respectively. Animal body weights and clinical signs were recorded over the course of the experiments. All animals were sacrificed 15 days after treatment initiation. At autopsy the tumor was removed and weighed. The tumor tissue was formalin-fixed and paraffin-embedded for further analysis.

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Figure 1.

Curcumin sensitizes colon cancer cells to radiation (IR). Cell proliferation was measured using the MTT assay as described in the Materials and Methods. IR had significantly greater proliferation inhibition on HT-29 cells than curcumin at each time point. IR-induced proliferation inhibition was significantly increased by combination curcumin treatment compared with IR-alone at each time point. *p<0.01, when compared with curcumin. **p<0.01, when compared with IR. Cur: Curcumin; IR: irradiation.

TUNEL detection of apoptosis. Apoptosis of the tumor cells following in vivo treatment was determined by TUNEL staining, using a commercially-available kit (In Situ Cell Death Detection Kit, POD; Roche). Tumor sections were deparaffinized and dehydrated according to standard protocols. Tissue sections were incubated with Proteinase K working solution for 30 min at 21-37°C. The slides were then washed with PBS (pH 7.2-7.6) twice. The positive control was incubated with DNase I for 10 min at 15-25°C. The negative control was incubated with label solution (without terminal transferase) instead of the TUNEL reaction mixture. The slides were then washed with PBS (pH 7.2-7.6) three times. Converter-POD was added on the slides which were then incubated in a humidified chamber for 30 min at 37°C. The slides were then washed with PBS (pH 7.2-7.6) three times. DAB substrate was added on the slides which were then incubated for 10 min at 15-25°C. The slides were then washed with PBS (pH 7.2-7.6) three times. The slides were mounted and analyzed by microscopy (Olympus, Melville, NY, USA). The apoptotic index (AI) was calculated by counting the number of TUNEL⁺ nuclei visible with a high-power-field 40x objective in at least 3 fields/sample. The results were expressed as the percentage of apoptotic cells with respect to the total number of cells in each field. Apoptotic cells were identified by the appearance of brown- or tan-stained nuclei.

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Figure 2.

Curcumin potentiates IR-induced cancer-cell apoptosis. Apoptotic cells were detected with Annexin V-PE/7-AAD as described in the Materials and Methods. All treatments increased cancer-cell apoptosis compared with the control. IR-induced cancer-cell apoptosis was significantly increased by curcumin as compared with IR-alone. *p<0.01, when compared with IR-alone. **p<0.01, when compared with IR-alone. Cur: Curcumin; IR: irradiation.

Statistical analysis. Data are expressed as means ± SD, and were analyzed by one-way analysis of variance (ANOVA), using SPSS software version 19.0, where p<0.05 was considered to be statistically significant.