RKO human colon cancer cells (ATCC no. CRL-257) were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), while Trr1 shRNA knockdown cells were grown in RPMI-1640 medium containing 10% FBS and 0.5 μg/ml puromycin. The cultures were incubated at 37°C in a humidified atmosphere of 5% CO₂/95% air.

The Escherichia coli clone, which harbors retroviral vector pSM2c containing the Trr1-specific short-hairpin RNA (shRNA) target, was provided from Open Biosystems (USA) and subjected to plasmid extraction using the HiSpeed Plasmid Midi kit (Qiagen, Germany). The purified plasmids were then submitted to sequencing to verify the Trr1-specific shRNA target (sense, CATCCCGGTGACAAAGAA and anti-sense, CATCCCTGGTGACAAAGAA). Stably transfected Trr1 shRNA (knockdown) cells were prepared using transfection reagent Fugen6 (Roche, Germany) according to the manufacturer's instructions. Briefly, 5×10⁴ RKO cells in a 2-ml suspension were seeded in each well of a 6-well plate. Subsequently, the 12-h cultured cells were transfected with the Trr1-specific shRNA construct using Fugen6, and selection of puromycin-resistant clones was performed in the presence of 0.5 μg/ml puromycin after 48 h of incubation. The reduced Trr1 expression level in each puromycin-resistant clone was verified by Western blotting.

The Trr1 shRNA transfectant and wild-type cells were harvested by centrifugation at 1,500 rpm for 5 min and then lysed in RIPA lysis buffer [50 mM Tris-HCl (pH 7.5), 0.5 mM ethylenediaminetetraacetic acid (EDTA), 150 mM NaCl, 1% (v/v) Triton-100, 0.1% (v/v) sodium dodecyl sulfate (SDS), 1 mM dithiothreitol (DTT) and a protease inhibitor cocktail]. Cell lysates (40 μg) were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at 60 V for 180 min and transferred onto polyvinylidene fluoride (PVDF) membranes. Blotted membranes were blocked with a blocking buffer [Tris-buffered saline (TBS), 5% skim milk, 0.02% NaN₃ and 0.001% Tween-20] at 25°C for 2 h. After washing, the Trr1 protein was immunologically detected using a goat polyclonal antibody against Trr1 at a 1:2,000 dilution ratio (Santa Cruz Biotechnology, USA). A peroxidase-conjugated donkey anti-goat IgG (Santa Cruz Biotechnology) was used as a secondary antibody. Immunoreactive bands were visualized with an enhanced chemiluminescence (ECL) Plus Western blotting detection system (GE Healthcare, UK). GADPH was used as a control for protein loading. The entire procedure was performed three times independently.

Cell viability was monitored by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay using a cell proliferation kit (Roche). Briefly, 1×10⁴ cells in a 100-μl suspension were seeded in each well of 96-well plates. After 18 h of incubation, both RKO and Trr1 shRNA knockdown cells were treated with various concentrations of MMC (0, 0.5, 1, 2.5, 5, 10 and 20 μM) for 1 h, following an additional 24-h incubation in fresh selective media. After incubation, MTT-labeling reagent was added at a final concentration of 0.5 mg/ml to each culture well and further incubated for 4 h. Subsequently, 200 μl of solubilization solution was added to dissolve formazan crystal-forming products. The percentage of cell survival was quantified by measuring the absorbance at 595 nm (A595) with a microplate reader 680 (Bio-Rad, USA).

The apoptotic fraction was evaluated as morphological change indicator of apoptosis using acridine orange (AO) staining. The RKO and Trr1 shRNA knockdown cells (6×10⁵ cells in a 3-ml suspension) were seeded in 60-mm dishes. The cells were harvested after a 24-h (5 μM) MMC treatment, following incubation in a selective fresh media for another 24 h. The cell pellets were resuspended in AO staining solution (Sigma, USA). The stained cells were dropped onto a glass slide and observed under a fluorescence microscope (Nikon). The apoptotic fraction was determined by cell counting.

The detection of ROS was determined as a measure of intracellular accumulation of free radicals in the MMC-treated Trr1 shRNA knockdown cells compared to the RKO cells. The ROS level was evaluated using 5 (and 6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA; Molecular Probes, Eugene, OR, USA) as an ROS indicator. In brief, 6×10⁵ RKO and Trr1 shRNA knockdown cells in a 3-ml suspension were seeded in 60-mm dishes and treated without and with 5 μM MMC (Sigma) for 24 h, and then exposed to 0.5 μM CM-H2DCFDA in PBS for 20 min at 37°C in the dark. Subsequently, the cells were washed with PBS and examined under a fluorescence microscope (Nikon). Image analysis was carried out with NIS-Elements BR 3.0 software (Nikon).

The formation of phosphorylated histone H2AX (γ-H2AX) foci was determined using the immunofluorescence staining approach (19). Briefly, either RKO or Trr1 shRNA knockdown cells grown on glass coverslips were treated without and with 5 μM MMC (Sigma) for 24 h, following incubation in selective fresh media for an additional 12 h. The cells were fixed with chilled methanol at −20°C for 30 min and rinsed with chilled acetone for a few seconds. After washing with PBS, the cells were incubated with an anti-γ-H2AX primary antibody (Active Motif, USA) at 4°C overnight and an anti-rabbit-cy3 secondary antibody (Jackson ImmunoResearch, USA) at room temperature for 1 h. After washing with PBS, the cells were mounted with mounting solution containing DAPI nuclear stain. The coverslips were placed face-down onto glass slides, and the foci were visualized under fluorescence microscope (Nikon).

The comet assay under neutral conditions mainly allows the detection of DNA double-strand breaks which basically trigger apoptotic processes, but not necrotic events (20,21). In brief, the RKO and Trr1 shRNA knockdown cells (6×10⁵ cells in a 3-ml suspension) were seeded in 60-mm dishes. After 24 h of 5 μM MMC treatment following an additional 24-h of fresh media incubation, the cells were harvested by trypsinization, resuspended in 1 ml resuspending buffer [Hanks' Balanced Salt Solution (HBSS), 20 mM EDTA and 10% DMSO freshly added 1 h prior to use] and chilled on ice. Cells (1×104 in 10 μl) were embedded in 90 μl low-melting point agarose (0.5% in PBS at 37°C) onto agarose-coated (1.0% in PBS) and dried glass slides that were submersed for 1 h in pre-chilled lysis buffer pH 9.5 [2.5 M NaCl, 100 mM EDTA, 10 mM Tris-base; 1 h prior to use 1% (v/v) Triton X-100 and 10% (v/v) dimethylsulphoxide (DMSO) were added]. Slides were electrophoresed at 25 V for 1 h at 4°C in electrophoresis buffer pH 9.0 [100 mM Tris-HCl (pH 9.0) and 300 mM sodium acetate]. Afterwards, DNA precipitation was carried out in 90% ethanol containing 1 M ammonium acetate at 4°C for 30 min. After ethanol dehydration, the slides were subjected to air drying and then SYBR Gold staining (1:10,000 dilution ratio in TE buffer, pH 7.6) (Invitrogen, USA). At this step, the slides could be stored in a refrigerator in light-tight boxes without any loss of assay sensitivity. Finally, the stained DNA was visualized under a fluorescence microscope (Nikon). One hundred cells per slide were scored.

Trr1 was targeted for knockdown in the RKO cells by transfection using a Trr1-specific shRNA retroviral vector. The decreased protein level of Trr1 was confirmed by Western blotting. As shown in Fig. 1A, the protein level of Trr1 was markedly reduced in the Trr1 shRNA transfectant cells relative to the wild-type RKO cells, while there was apparently no difference in the expression level of the internal control GAPDH between the Trr1 shRNA transfectant and the wild-type RKO cells. This indicated that suppression of Trr1 expression via shRNA transfection in the colon cancer RKO cells was successfully conducted.

Highly expressed Trr1 is noted in many cancer types with malignant phenotypes, including resistance to anticancer drugs (9,10). This raises the question whether Trr1 suppression via shRNA leads to enhanced MMC-mediated cytotoxicity in colon cancer RKO cells. An MTT assay was performed to evaluate cell viability towards MMC. The result showed that the Trr1 shRNA knockdown cells displayed higher sensitivity to MMC treatment compared to the wild-type RKO cells (Fig. 1B). This suggests that Trr1 may be a potent mediator for cell survival caused by MMC toxicity in colon cancer RKO cells. Additionally, the Trr1 shRNA knockdown cells also showed a higher rate of MMC-induced apoptosis, relative to the wild-type cells (Fig. 1C). This implies that the inhibition of Trr1 expression is potentially effective for inducing apoptosis in colon cancer RKO cells, as a tentative approach to enhance the antitumor effects of MMC treatment.

As MMC is one of the quinone anticancer agents to generate free radicals (22,23), enhancement of the ROS level in the Trr1-deficient cells in comparison to wild-type RKO cells was assessed. The intracellular ROS was detected using fluorescent probe CM-H2DCFDA. Exposure to 5 μM MMC for 24 h induced the generation of intracellular ROS to a greater extent in the Trr1 shRNA knockdown cells as compared to that in the wild-type RKO cells (Fig. 2). This implicates the intracellular accumulation of ROS in the Trr1 shRNA knockdown. Therefore, Trr1 deficiency may be a potent mediator by which to sensitize colon cancer RKO cells to MMC treatment via enhancement of ROS-mediated oxidative damage.

DNA is a target molecule for underlying molecular mechanisms of MMC toxicity in antitumor therapy (24,25). Accordingly, the accumulation of intracellular ROS generating oxidative damage to cellular components, particularly DNA, was observed in the Trr1 shRNA knockdown cells (Fig. 2). Hence, MMC-induced DNA damage in Trr1 shRNA knockdown cells was examined in comparison to wild-type RKO cells using γ-H2AX immunostaining. Phosphorylated nuclear histone protein variant H2AX ‘γ-H2AX’ causes recruitment of DNA repair protein complexes at sites flanking DNA strand breaks (19). As shown in Fig. 3, the Trr1 shRNA knockdown cells exhibited a marked increase in DNA strand breaks compared to that in the wild-type RKO cells after MMC treatment.

In addition to the γ-H2AX staining, a neutral comet assay was also utilized to evaluate the relative occurrence of DNA double-strand breaks at the individual cell level. After MMC exposure, the Trr1 shRNA knockdown cells showed significantly increased DNA double-strand breaks, represented by a much greater extent of stretched DNA, compared to the wild-type RKO cells (Fig. 4).

The results overall demonstrated that Trr1 suppression sensitizes colon cancer RKO cells to MMC exposure through augmentation of DNA double-strand damage, probably leading to enhanced cell death.