Human colorectal cancer cell lines Colo205, RKO and HT29 were purchased from the American Type Culture Collection (Rockville, MD, USA) and Caco2 cells were obtained from Korea Cell Line Bank (Seoul, Korea). The cells were maintained in RPMI-1640 supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 37°C in a humidified atmosphere with 5% CO₂. RKO sub-lines with short hairpin (sh) RNA-mediated knockdown of XAF1 were established by transfection with a shXAF1 construct (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and Zeocin selection. The shRNA plasmid for XAF1 was constructed by Genolution Pharmaceuticals, Inc. (Seoul, Korea). The mitogen-activated protein kinase kinase (MEK) inhibitor U0126 was purchased from New England Biolabs, Inc. (Ipswich, MA, USA). Porcine TGF-β1 (1010-B1001) and anti-TGF-β1 neutralizing antibodies (nAb; MAB240-100) were obtained from R&D Systems, Inc. (Minneapolis, MN, USA).

To evoke apoptotic stress, cells were exposed to 5-FU (20 µM), etoposide (50 µM), H₂O₂ (50 µM), γ-IR (6 Gy) or hypoxic conditions (1% O₂). γ-irradiation and hypoxic stress assays were carried out using an irradiator (IBL-437-C; Syncor Intl. Corp., Sylmar, CA, USA) and hypoxic chamber (2000 Hypoxia Workstation; Ruskinn Technology Ltd., Leeds, UK), respectively. After 24 or 48 h exposure, the cells were harvested for further molecular analysis. TGF-β1 (2 ng/ml) was added to the cells 2 h prior to drug exposure, and TGF-β1 nAb (2 ng/ml) was added 2 h before TGF-β1 treatment.

Total cellular RNA was extracted from cells by a single-step method (30). One microgram total cellular RNA was converted to complementary DNA (cDNA) by RT using random hexamer primers and M-MLV reverse transcriptase (Invitrogen; Thermo Fisher Scientific, Inc.). PCR was initially performed over a range of cycles (24-40 cycles), and 2 µl 1:4 diluted cDNA (12.5 ng/50 µl PCR) subjected to 30-36 cycles was observed to be within the logarithmic phase of amplification with primers used for XAF1 (sense, 5′-CAGAAGTCCTCGCTGGAGTTTC-3′ and antisense, 5′-TGAAATTCTTTCCCCTTTCC-3′), PAI-1 (sense, 5′-CTGCCTAGTCCTGGGCCTGGCC-3′ and antisense, 5′-ATGAGCTCCTTGTACAGATGCC-3′) and the endogenous expression standard gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (sense, 5′-CATGTGGGCCATGAGGTCCAC CAC-3′ and antisense, 5′-AACCATGAGAAGTATGACAAC AGC-3′). PCR conditions comprised 32-38 cycles at 95°C for 1 min, 58-62°C for 0.5 min, and 72°C for 1 min in 1.5 mM MgCl₂-containing reaction buffer (PCR buffer; Takara Bio, Inc., Shiga, Japan). A total of 10-15 µl PCR product was resolved by 2% agarose gel electrophoresis. Quantitation was achieved by densitometric scanning of the ethidium bromide-stained gels. Integration and analysis were performed using Quantity One software (version 4.6.9; Bio-Rad Laboratories, Inc., Hercules, CA, USA). RT-PCR was repeated at least three times for each specimen and the mean value was determined.

Cells were lysed with buffer containing 20 mM Tris (pH 7.4), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 50 mM NaF, 2 mM sodium pyrophosphate, 1 mM sodium orthovanadate, protease inhibitor cocktail and 1 mM phenylmethylsulfonyl fluoride. The cell lysate was clarified by centrifugation (20,000 × g, 30 min) and the protein concentration was determined using a BCA Protein Assay kit (Pierce; Thermo Fisher Scientific, Inc.). Total proteins (20-40 µg) were supplemented with Laemmli buffer and loaded onto a 10% SDS-polyacrylamide gel for electrophoresis. The proteins were transferred to the nitrocellulose blotting membrane (10600001; GE Healthcare Life Sciences, Little Chalfont, UK). The membranes were incubated with specific antibodies for 12-18 h at 4°C. Antibodies specific for XAF1 (1:1,000; sc-19194) and PAI-1 (1:2,500; sc-5297) were obtained from Santa Cruz Biotechnology, Inc. and antibodies specific for cleaved poly (ADP-ribose) polymerase (1:2,000; CST no. 9541), cleaved caspase-3 (1:2,000; CST no. 9661S) and Erk (1:2,500; CST no. 910) were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibody for the loading control β-tubulin (1:10,000; T0198) was obtained from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany). Antibody binding was detected by enhanced chemiluminescence using 1 h incubation at 24°C with a secondary antibody conjugated to horseradish peroxidase (1:5,000; PI-1000/2000; Vector Laboratories, Inc., Burlingame, CA, USA).

Expression vector for green fluorescent protein (GFP)-tagged Ras was constructed using pEGFP-N3 vector (Clontech, Mannheim, Germany) and the Expand™ High Fidelity PCR System (Roche Molecular Diagnostics, Pleasanton, CA, USA). Approximately 3.3×10⁵ cells were plated per 60-mm plate in medium containing 10% FBS. When 50-60% confluence was reached, the transfection of constructs was performed using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) or Turbofect™ in vitro Transfection Reagent (Pierce; Thermo Fisher Scientific, Inc.) according to the manufacturer’s protocol. Each transfection experiment was carried out in triplicate. The transfection efficiency was monitored by fluorescence microscopy for GFP detection. The siRNA duplexes against XAF1 (5′-AUGUUGUCCAGACUCAGAG-3′), Erk1 (5′-CCC UGGAAGCCAUGAGAGAUGUCUA-3′), Erk2 (5′-CACCAU UCAAGUUCGACAU-3′) and plasminogen activator inhibitor-1 (PAI-1; 5′-AAGCACAACUCCCUUAAGGUC-3′), and control siRNA duplexes used as a negative control were synthesized by Dharmacon (D-001210-0X; Lafayette, CO, USA) or Bioneer, Inc. (SN-1001-CFG; Daejeon, Korea). The transfection of siRNAs was performed using the Neon™ Transfection System (Invitrogen; Thermo Fisher Scientific, Inc.). Transfected cells were stabilized in serum-supplemented media for 24 h before further experiments.

The XAF1 promoter region (nucleotides -221/+1) was cloned into the pGL3-basic vector (Promega Corporation, Madison, WI, USA). RKO cells were cotransfected with 0.5 µg XAF1 promoter-containing luciferase plasmid (Pro221-Luc) and 0.5 µg pPGL3-basic plasmid DNA using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). The culture medium was changed following 18 h of transfection, and the cells were maintained for another 48 h prior to lysis with 200 µl lysis buffer (E3971; Promega Corporation). Following the normalization of each extract for protein content, luciferase activity was measured using the Luciferase Assay System (Promega Corporation).

A total of 5×10⁴ cells were seeded and transfected with expression vector or siRNA. For the sub-G1 fraction analysis, cells were fixed with 70% ethanol and resuspended in 1 ml phosphate-buffered saline containing 100 mg/ml RNase and 50 mg/ml propidium iodide. (P4170; Sigma-Aldrich; Merck KGaA). The assay was performed using a FACSCalibur flow cytometer (BDBiosciences, San Jose, CA, USA) and the sub-G1 fraction was analyzed using MultiCycle software (version 3.21; Phoenix Flow Systems, San Diego, CA, USA).

Flow cytometry was performed in triplicate and data are expressed as mean ± standard deviation. Statistical analysis was performed using Student’s t-test when comparing two groups or analysis of variance with the Bonferroni correction as a post hoc test when making multiple comparisons. Statistical analyses were carried out using GraphPad Prism 6.0 software (GraphPad Software, Inc., La Jolla, CA, USA). P≤0.05 was considered to indicate a statistically significant difference.

TGF-β1 is known to promote colonic tumorigenesis partially through its protective role against apoptotic stresses. To investigate whether the anti-apoptotic function of TGF-β1 is associated with its regulation of XAF1 expression, an initial examination of the effect of TGF-β1 on the apoptosis induced by the chemotherapeutic drug 5-FU was conducted using the Colo205 cell line, which is known to have no genetic alterations of TGF-β receptors (30,31). The Colo205 cells exhibited a sensitive cytotoxic response to 5-FU (20 µM, 48 h), but this response was markedly attenuated when cells were co-treated with TGF-β1 (Fig. 1A). A flow cytometric analysis of the apoptotic sub-G1 fraction also indicated that TGF-β1 exerts a strong inhibitory effect on 5-FU-induced apoptosis (Fig. 1B). To further evaluate the cytoprotective role of TGF-β1 against various types of apoptotic stress, RKO cells, which are widely used for the study of stress-induced cell death, were utilized. RKO cells are known to express low level of TGF-β receptor II due to a premature termination mutation in one allele of the gene but have a functional TGF-β1/Smad signaling pathway (9,31,32). As shown in Fig. 1C, TGF-β1 treatment significantly attenuated the apoptotic response of RKO cells to genotoxic (5-FU, etoposide and γ-irradiation), oxidative (H₂O₂) and hypoxic (1% O₂) stresses. To further evaluate these findings, the inhibitory effects of TGF-β1 on etoposide-induced apoptosis in Colo205, RKO and two other cancer cell lines (HT29 and Caco2) that have no alterations of TGF-β receptors were compared (30,31). In all the cell lines tested, TGF-β1 evoked a significant inhibitory effect on etoposide-induced apoptosis (Fig. 1D). These results indicate that TGF-β1 evokes a strong cytoprotective effect on human colon cancer cells under various apoptotic stress conditions.

Next, the effect of TGF-β1 on XAF1 expression was investigated. Semi-quantitative RT-PCR analysis demonstrated that TGF-β1 treatment resulted in a strong reduction of XAF1 mRNA levels in all four cell lines that were tested (Fig. 2A). An IB assay revealed that the TGF-β1-induced downregulation of XAF1 mRNA expression was reflected in a clear reduction in the levels of its protein product in these cells. Furthermore, the TGF-β1-induced inhibition of XAF1 mRNA expression appeared to be concentration-dependent (Fig. 2B). In this assay, the activation of PAI-1, a transcription target of TGF-β1, as utilized as an indicator of the functionality of TGF-β1 signaling. Quantitative analysis of the RT-PCR products revealed that 4 ng/ml TGF-β1 significantly repressed ~75% of XAF1 mRNA expression in Colo205 cells (Fig. 2C). The inhibitory effect of TGF-β1 on XAF1 mRNA expression was abrogated when the cells were pretreated with TGF-β1 nAb (Fig. 2D). Next, whether TGF-β1 produced by the tumor cells exerted an inhibitory effect on XAF1 expression was examined. Notably, the basal expression level of XAF1 mRNA was elevated in all four cancer cell lines exposed to TGF-β1 nAb, supporting the hypothesis that tumor cell-produced TGF-β1 acts as a negative regulator of XAF1 expression (Fig. 2E). Together, these results indicate that XAF1 expression is repressed at the mRNA level by TGF-β1 in human colon cancer cells.

To elucidate the inhibitory effect of TGF-β1 on the pro-apoptotic function of XAF1, the role of XAF1 in stress-induced apoptosis was evaluated using the siRNA-mediated knockdown of XAF1 expression (Fig. 3A). RT-PCR analysis revealed that XAF1 mRNA expression was strongly activated in Colo205 cells exposed to 5-FU, and IB assays of cleaved PARP and caspase-3 indicated that 5-FU-induced apoptosis was attenuated by the siRNA-mediated blockade of XAF1 induction, and the attenuation appeared to be XAF1 concentration-dependent (Fig. 3B). Likewise, the etoposide-induced apoptosis of RKO cells was markedly suppressed by siXAF1 transfection in an apparently concentration-associated manner (Fig. 3C). These results indicate that XAF1 induction serves a key role in the apoptotic response of colon cancer cells to genotoxic chemotherapeutic drugs. The regulation of chemotherapeutic drug-mediated XAF1 induction by TGF-β1 was then evaluated. As shown in Fig. 3D, TGF-β1 exerted a strong inhibitory effect on the induction of XAF1 mRNA by 5-FU, etoposide and Adriamycin. To further elucidate the mechanistic basis for the TGF-β1-induced suppression of XAF1 mRNA expression, a promoter luciferase assay was performed using the XAF1-Pro221-Luc reporter, which includes the proximal region of the XAF1 promoter (nucleotides −221/+1 relative to ATG; Fig. 3E). The reporter was activated by 5-FU and this responsiveness was substantially attenuated by TGF-β1 in a dose-dependent manner, indicating that the TGF-β1-mediated inhibition of XAF1 expression occurs through transcriptional repression of the gene (Fig. 3F).

Next, whether the anti-apoptotic effect of TGF-β1 is linked to its ability to repress XAF1 was assessed. Western blot analysis revealed that the cleavage of PARP and caspase-3 triggered by the apoptosis-inducing chemotherapeutic drugs 5-FU, etoposide and Adriamycin was strongly suppressed by pretreatment with TGF-β1, and this effect was accompanied by the inhibition of XAF1 induction (Fig. 4A). A crucial link between the anti-apoptotic function of TGF-β1 and its XAF1 repressive activity was further characterized using a stable XAF1 knockdown sub-line of RKO cells constructed using a shRNA-mediated knockdown system (Fig. 4B). Compared with the shControl sub-line, the shXAF1 sub-line displayed a markedly attenuated apoptotic response to 5-FU (Fig. 4C). The anti-apoptotic effect of TGF-β1 was negligible in shXAF1 cells, further suggesting that the TGF-β1-induced inhibition of stress-induced apoptosis is highly dependent on its XAF1-repressing activity. Additionally, based on the results indicating that tumor cell-produced TGF-β1 is a negative regulator of XAF1 basal expression, whether tumor cell sensitivity to apoptotic stresses is increased by the blockade of TGF-β1 production by tumor cells was tested. As shown in Fig. 4D, RKO cells pretreated with TGF-β nAb displayed an increased apoptotic response to various apoptotic stresses compared with untreated control cells. Together these results indicate that TGF-β1 protects colon cancer cells by blocking XAF1 induction under various apoptotic stress conditions.

Activation of the Ras-Erk signaling pathway is common in colorectal tumorigenesis and serves a key role in the proliferation, survival and malignant progression of tumor cells. Studies have shown that TGF-β1 activates Ras-Erk signaling to promote tumor progression (15,33). These findings suggest that the Ras-Erk signaling pathway is involved in the TGF-β1-mediated repression of XAF1. Notably, in the present study it was observed that the expression level of XAF1 mRNA increased substantially following treatment with U0126, an inhibitor of MEK, which is upstream of Erk (Fig. 5A). Consistent with this result, XAF1 was upregulated by the siRNA-mediated knockdown of Erk1/2 expression (Fig. 5B). Furthermore, XAF1 levels were markedly decreased by the ectopic expression of the activated form of K-Ras (Ras/G12C; Fig. 5C). Together, these results indicate that XAF1 expression is suppressed at the transcription level by activation of the Ras-Erk signaling pathway.

On the basis of the findings that XAF1 mRNA expression is repressed by Ras-Erk signaling and that TGF-β1 activates the Ras-Erk pathway, the role of Ras-Erk signaling in the TGF-β1-induced repression of XAF1 by TGF-β1 was examined. The induction of XAF1 mRNA expression by 5-FU was strongly inhibited by TGF-β1, and the effect appear to be dependent on TGF-β1 concentration (Fig. 6A). Intriguingly, the inhibitory effect of TGF-β1 on 5-FU-mediated induction and apoptosis was markedly attenuated when Erk1/2 expression was depleted by siErk1/2 transfection (Fig. 6B and C). Consistently, the inhibitory effect of TGF-β1 on 5-FU-induced apoptosis was greatly abrogated by pretreatment with U0126 (Fig. 6D). This observation suggests TGF-β1 represses XAF1 mRNA expression via the Ras-Erk signaling pathway. However, the depletion of PAI-1, which has been reported to exert apoptosis-modulating activity (34,35), did not affect the TGF-β1-mediated inhibition of apoptosis and XAF1 expression. Collectively, these results demonstrate that TGF-β1 protects colorectal tumor cells from various apoptotic stresses by blocking XAF1 mRNA induction via activation of the Ras-Erk signaling pathway (Fig. 7).