Med Oncol (2014) 31:273 DOI 10.1007/s12032-014-0273-4

ORIGINAL PAPER

X-linked inhibitor of apoptosis-associated factor l (XAFl) enhances the sensitivity of colorectal cancer cells to cisplatin Wen-Cui Ju • Guo-Bin Huang • Xiao-Yong Luo Wei-Hua Ren • De-Qing Zheng • Pin-Jia Chen • Yun-Feng Lou • Bin Li

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Received: 12 September 2014 / Accepted: 26 September 2014 / Published online: 1 November 2014 Ó Springer Science+Business Media New York 2014

Abstract The purpose of present study was to investigate the roles of X-linked inhibitor of apoptosis-associated factor l (XAFl) in regulation apoptosis of colorectal cancer (CRC) cells after treatment with cisplatin (DDP). A total of ten paired cancerous and non-cancerous tissues were collected from patients with CRC after surgery. The levels of XAFl protein were detected by Western blot. Primary CRC cells were separated from cancer tissues, and its viability or apoptosis after treatment with DDP was determined with MTT or Annexin V/PI assays, respectively. Furthermore, we either up-regulated transfecting a XAF1 overexpression vector or down-regulated XAF1 by siRNA interference. And then, the XAF1 levels and its sensitivity to cisplatin were assessed. XAFl had a lower expression in the cancerous tissues from samples T1, T2 and T3 than their paired non-cancerous tissues N1, N2 and N3. However, the expression of XAF1 was not detected in samples T4 and N1. XAF1 levels in cancer tissues significantly decreased in comparison with normal tissues. Cell abilities of primary cells were significantly decreased in a dose-dependent manner, after treatment with a series concentrations of cisplatin (2, 5, 10 lg/mL) for 48 h. Although, after downexpression of XAFl by siRNA, cisplatin caused a significant decreases in apoptosis rates in CRC cells. The upregulation of XAF1 distinctly increased apoptosis in CRC

W.-C. Ju  X.-Y. Luo (&)  W.-H. Ren  P.-J. Chen  Y.-F. Lou  B. Li Department of Oncology, The Affiliated Luoyang Central Hospital of Zhengzhou University, No. 288 Zhongzhou Road, Luoyang 471000, China e-mail: [email protected] G.-B. Huang  D.-Q. Zheng Department of Gastroenterology, The Affiliated Donghua Hospital of Sun Yat-sen University, Dongguan 523110, China

cells administered by cisplatin (P \ 0.001). The XAFl could promoted apoptosis and enhanced chemotherapy sensitivity to cisplatin in CRC cells. Keywords

XAF1  Cisplatin  Apoptosis  Colon cancer

Introduction Colorectal cancer (CRC) continues to be a major public health problem. Worldwide, approximately one million new cases of CRC are diagnosed each year, with nearly 500,000 deaths attributed to this disease annually [1]. China is classified as a high incidence area for CRC [2]. Recently, CRC has ranked as one of the malignant diseases with highest morbidity or mortality in China [3]. However, it should be noted that there are a growing number of young patients suffering from CRC [4]. Currently, chemotherapy with limited efficacy failed to bring patients with satisfactory clinical outcomes. It has been well known that reasons of causing dismal those outcomes mainly resulted from CRC cells resistant to chemotherapy [5]. Both the low expression of pro-apoptotic proteins and the inhibition expression of apoptosis promoting proteins fundamentally induce resistant to chemotherapy drugs in tumor cells [6]. The X-linked inhibitor of apoptosis (XIAP)-associated factor l (XAF1) is a newly identified cancer suppress gene, which could promote apoptosis of cancer cells. Furthermore, XAF1 had been indicated to involve in promoting sensitivity of cancer cells to chemotherapy [7]. Previously, studies had explored that XAF1 interacted with XIAP and migrated into nucleus. After that, XAF1 could induce cancer cell apoptosis by competitively antagonistic interacting with XIAP and preventing its role in inhibiting of caspase 3 [8]. XAF1 also has been proved

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to participate in mitochondrial apoptosis pathway. XAF1 could accelerate the relase of cytochrome c and enhance tumor necrosis factor (TNF) production, or interacting with death receptors, for promote apoptosis of tumor cells [9]. XAF1 had been previously reported with low expression among 44.9 % pancreatic cancer tissues. Moreover, low levels of XAF1 indicated with unfavorable prognosis and have a shorter survival time. Also, those patients with down-regulation of XAF1 were most likely to develop drug resistance [10]. XAF1 also commonly proved with low levels in esophageal carcinoma. The hyper-methylation in the promoter region resulted in the low expression of XAF1 in tumor tissues. The low expression of XAF1 means a higher malignant disease type of esophageal carcinoma [11]. Not only XAF1 was found with a low level in esophageal carcinoma, but also XAF1 genes had been detected with a hyper-methylation status in lung cancer, gastric cancer and bladder cancer. Furthermore, the levels of XAF1 might serve as one of the primary causes of inducing chemotherapy resistant to cancer patients [12– 14]. In 2007, Yu et al. [15] identified that XAF1 promoted cell apoptosis of CRC via activating extracellular signalregulated kinase (ERK1/2) pathway. Meanwhile, Sun et al. [16] explored that interferon b could control XAF1 transcription and promoted the apoptosis of CRC cells. Besides, Tu et al. [17] confirmed that XAF1 cooperated with TNF-related apoptosis-inducing ligand had facilitated apoptosis of CRC cells too. At present study, we aimed to explore the roles of XAF1 in regulating chemotherapy sensitivity in primary CRC cells. We firstly determined the levels of XAF1 in cancerous and paired non-cancerous tissues from patient with CRCs. Then, primary cells were separated from cancer tissues and treated with cisplatin. After that, the sensitivities of primary CRC cells to cisplatin were analyzed. We further determined changes of cisplatin sensitivity of primary culturing CRC cells after over-/down-expression, and at last, the roles of XAF1 in regulation apoptosis and chemotherapy sensitivity to cisplatin were also confirmed in SW1116 CRC cell lines.

penicillin/streptomycin (Qilu Pharmaceutical Co., LTD, Jinan China), under standard culture conditions (37 °C, 5 % CO2). On the other hand, the primary cells separated from CRC tissues were cultured in DMEM with 20 % FBS, and supplementary with 20 ng/mL EGF, 1 U/mL penicillin/streptomycin/gentamicin (Qilu Pharmaceutical Co.).

Materials and methods

MTT assay for cell viability

Cell culture

The cells in logarithmic growth phase were digested with trypsin and diluted in a concentration of 5 9 104 cells/mL. A total of 1,000 cells/well were seeded in 96-well plates, and after 48 h, cells were exposed to a series concentrations of cisplatin. Equal dimethylsulfoxide (v/v) (DMSO) (Sigma-Aldrich Corp, St. Louis, MO, USA) with maximum volume of cisplatin was used as controls, and each treatment with eight wells. After culturing cells another 48 h, the medium was removed from each well and replaced with

This paper was approved by ethics committee of Luoyang Central Hospital affiliated to Zhengzhou University. Human SW116 CRC cell lines were obtained from Shanghai cell library of Chinese Academy of Science and cultured in Dulbecco’s modified Eagle medium (DMEM) (Hyclone Laboratories Inc., Logan, UT, USA) with 10 % fetal bovine serum (FBS) (Hyclone) and 1 U/mL

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Separated primary cells from CRC tissues The samples were collected from ten patients with CRC, included six male and four females, with age ranged from 20 to 84 years old. All the patients were suffered with surgical resection during March, 2013 to April, 2013, at general surgery department of Luoyang Center hospital. All of those CRC patients were not accepted any of chemotherapy or radiotherapy before surgery. The resected CRC tissues were digested with trypsin (Invitrogen, Darmstadt, Germany) to obtain primary CRC cells. In brief, the tissues were cut into 0.5–1-mm3 blocks with scissors and washed with 19 phosphate buffered saline (PBS) for several times to disperse blocks. Then, all tissue blocks were transferred to distilled 15-mL centrifugal tubes and centrifuged at 1,000g for 5 min. After washing with PBS for two times, tissues were transferred to a new distilled 15-mL centrifugal tube and added 3–5 mL trypsin to digest at 37 °C constant temperature incubator shaker for half 1 h. Next, the supernatants were collected and centrifuged at 1,000g for 5 min and were removed. After that, precipitation with CRC cells was resuspended and washed with DMEM medium. The resuspended tissue blocks were further placed in 4 °C refrigerator until to no visible tissues blocks. Cell suspension was filtered with sterile stainless steel mesh to a distilled 15-mL centrifugal tubes. The cells were washed with PBS and collected by centrifuging at 1,000g for 5 min. In the end, the cells were resuspended with completed DMEM medium and cultured as mentioned above. After culturing for 48 h, cells were replaced with new DMEM medium, and the cells of passaging \4–5 times were subjected to the following experiments.

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new medium containing 20 lL 4, 5-Dimethylthiazol-2-yl2,5-diphenyltetrazolium bromide (MTT) solution (5 mg/ mL), incubated for 4 h at 37 °C. The medium was again replaced with 150 lL DMSO and incubated for 15 min until a purple-colored formazan product developed. Then, 96-well plate was measured at 490 nm by using a microplate reader (Synergy-HT, BioTek, USA). The cell viability was calculated as following formula: Cell viability ¼ ðmean OD values of treatment=mean OD values of controlsÞ 100 %:

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Detection of XAF1 was performed with a primary rabbit anti-XAF1 monoclonal antibody (Cell Signaling Technology, Danvers, MA, USA) and rabbit anti-beta actin monoclonal antibody (Cell Signaling Technology) as interval control. A sheep anti-rat horseradish peroxidase antibody used as secondary antibody. Protein detection was performed using the enhanced chemiluminescence Western blot detection system (ECL, GE Healthcare) and Rx-films (Fuji, Tokyo, Japan). Statistical analysis

Detection of apoptosis Cell apoptosis was detected using the FITC Annexin V Apoptosis Detection kit I (Invitrogen) according to the instruction provided by the manufacturer. The procedures were as following: At first, medium was removed, washed with PBS, digested with trypsin and collected cells to a centrifugal tube; then, cells were resuspended with PBS, added into 5 ll FITC Annexin V solutions and incubated at 20–25 °C for 10 min in dark place. At last, 10 lL propidium iodide (PI) was mixed to perform flow cytofluorometry analysis using a FACSCalibur cytometer equipped with CellQuestPro software (BD Biosciences, Heidelberg, Germany). Plasmid transfection and RNA interference The siRNA sequence of targeting XAF1 as described in Ref. [18] and negative siRNA negative (NC) sequence was 50 -AUGUUGUCCAGACUCAGAG-30 . The overexpression plasmid of XAF1 was purchased from FulenGen Co., LTD, Guangzhou, China. The opening reading fragment (ORF) was cloned into pReceiver-M2 vector. The cells were transfected with either XAF1 plasmids or siRNAs using LipofectamineTM 2000 (Invitrogen) according to the manufacturer’s protocol. The concentrations of plasmid and siRNA were 2 lg/mL and 100 nM, respectively. The mix of plasmids, siRNA and LipofectamineTM 2000 with serum medium was added into 1.4 9 104 cells/cm2. The medium was removed after transfection for 8 h and replaced with completely medium with 10 % FBS. The cells were harvested for further analysis 72 h later [19]. Western blot analysis Proteins extracted from cells after washing for three times with precooling PBS by proper volume lysis buffer [50 mM Tris–HCl pH 8.0, 150 mM NaCl, 1 % Triton 9100, 1 mM Na3VO4, 100 mM phenylmethanesulfonyl fluoride (PMSF)]. Equal amounts of proteins were then separated under reducing conditions in 12 % sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE), transferred to polyvinylidene fluoride (PVDF) membranes.

Student’s t tests were performed using SPSS for Windows (version 16.0; SPSS Inc, Chicago, IL, USA). All analyses were based on at least three independent experiments. Continuous variables were expressed as mean ± SD, and a P value of \0.05 was considered significant.

Results The expression of XAF1 proteins in CRC tissues A total of ten paired cancerous and non-cancerous tissues were collected from patients with CRC after surgery. Then, Western blotting was applied to determine the levels of XAF1 between paired cancerous and non-cancerous tissues. As shown in Fig. 1a, XAF1 had a lower expression in the cancerous tissues from samples T1, T2 and T3 than their paired non-cancerous tissues N1, N2 and N3. On the other hand, there were not found XAF1 in sample T4 and N1. After that, immunoblotting pictures of ten paired tissues were changed to grayscale values. It also reveals that the expression of XAF1 in cancerous tissues is decreased in comparison with paired normal tissues (Fig. 1b). Cisplatin inhibits viability of primary CRC cells We choose three cancerous tissues from above-mentioned samples to separate primary CRC cells for further analysis inhibition rates after treatment with cisplatin. MMT assay used to analyze viability of three types primary cells deprive from different patients. As except, cell ability of all primary cells was significantly decreased in a dosedependent manner, after treatment with a series concentration of cisplatin (2, 5, 10 lg/mL) for 48 h (Fig. 2). The growth inhibition rates of T1 primary cells were 30.1, 59.8 and 80.5 %, respectively, in comparison with controls after treatment with different concentrations cisplatin (Fig. 2a). Meanwhile, the growth inhibition rates for T2 primary cells were 17.3, 52.9 and 82.8 %, respectively (Fig. 2b). At last, T3 primary cells show growth inhibition rates with 19.8, 46.4 and 81.2 %, respectively (Fig. 2c).

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Fig. 3 Cisplatin (DDP) induces cell apoptosis of primary colorectal cancer (CRC) cells

FCM was used to analyze apoptosis of primary cells when cultured in a series concentrations of cisplatin as mentioned above for 48 h. Also, the apoptosis rates are in a dosedependent manner, and as shown in Fig 3, the higher levels of cisplatin result a larger amount of cancer cells to apoptosis. The apoptosis rates of primary CRC T1 cells are 5.2, 13.1, 25.3 and 48.5 %, respectively. In addition, primary CRC T2 cells reveal apoptosis rates of 4.8, 14.3, 26.7 and 42.1 %, respectively. In similar, CRC T3 also inhibited by cisplatin and found to be with apoptosis rates for 7.9, 16.8, 33.5 and 54.2 %, respectively. Fig. 1 The expression of XAF1 is found with low levels in colorectal cancer (CRC) tissues (T) in comparison with normal tissues (N). a Western blotting is used to determine XAF1 levels, and the results are shown with four representatives from ten patients. b Changing the XAF1 levels to grayscale value

Cisplatin induces apoptosis of primary CRC cells Even that, the inhibition of cisplatin proliferation has been extensively determine whether cisplatin could those primary cells. After staining

Fig. 2 Cisplatin (DDP) significantly reduces human primary CRC cell viability in a dose-dependent manner. Three types of primary cells T1 (a), T2 (b) and T3 (c) have similar trends after treatment with a series concentration of cisplatin (0, 2, 5, 10 lg)

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on primary CRC cell developed. We thus induce apoptosis of with PI/Annexin V,

Up-regulation of XAF1 enhances primary CRC cell sensitivity to cisplatin Initially, the down-regulation of XAF1 was found in cancerous CRC tissues in comparison with normal tissues. Given that XAF1 is one of the pro-apoptosis factors, we further identify that up-regulation of XAF1 whether enhances cell apoptosis of three primary CRC cells. After transient transfection with XAF1 vectors, the apoptosis of three cells is analyzed by FCM. As shown in Fig. 4a, after

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Fig. 4 XAF1 increases apoptosis rates of primary colorectal cancer (CRC) cells when treated with cisplatin (DDP). a The apoptosis rates of three primary CRC cells (T1, T2, T3) are stained with PI/Annexin V and analyzed by flow cytometry (FCM). Apoptosis rates of XAF1

up-regulation cells are comparing with empty vector cells after treatment with 2 lg/mL DDP, **P \ 0.01; ***P \ 0.001. b Western blotting detects XAF1 expression in up-regulation and controls cells

Fig. 5 Depletion of XAF1 attenuates cell apoptosis rates induced by cisplatin (DDP) in SW1116 cells. a Transfection with siRNA targeting XAF1 (si XAF1) significantly reduces expression of XAF1 proteins. b The apoptosis rates are decreased after downregulation of XAF1 when administrate SW1116 cells with DDP. Here

shows the mean values of apoptosis rates from three independent experiments (***P \ 0.001). c Flow cytometry analyzes cell apoptosis after treatment with DDP which is a representative picture from at least three independent experiments

treatment with 2 lg/mL cisplatin for 48 h, apoptosis rates of XAF1-up-regulated cells increased significantly compared with controls. On the other hand, Western blotting confirms that XAF1 protein levels are higher than controls in T1 primary CRC cells after transfection with XAF1 expression vector (Fig. 4b).

(Fig. 5b, c). After administration with cisplatin for 48 h, mean inhibition rates for SW116 cell lines transfection relative control siRNAs are 32.2 %. Meanwhile, mean inhibition rates for XAF1 siRNAs are 15.7 % (the results from three independent experiments). Those results indicate that down-regulation of XAF1 decreases apoptosis rates (P \ 0.001). It is meaning that the sensitivity to cisplatin is reduced by down-regulation of XAF1 in SW1116 cell lines.

Down-regulation of XAF1 decreases sensitivity of SW1116 cell lines to cisplatin Since, the overexpression XAF1 has been shown with a role in increasing sensitivity of primary CRC cells to cisplatin. We further determine the effects of XAF1 downregulation on cell sensitivity to cisplatin (Fig. 5a). SW1116 is a CRC cell lines with high levels of XAF1. The apoptosis rates of SW1116 are analyzed with FCM after transfection by siRNA targeting XAF1 to down-regulate its expression

Discussion In clinical, high-dose cisplatin might be more effective in curing patients with CRC. However, the adverse events often bring with intolerable side effects (such as vomiting, baldness and hypertension) [20]. On the other hand, low

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dose chemotherapy would had a low treatment efficaccy and could not benefit to prolong progression free survival (PFS) or overall survival (OS) [21]. Thereafter, there is an urgent need to explore the mechanism that regulates cell sensitivity to chemotherapy drugs in a low dose. There are already abundant studies to assess efficacy of chemotherapy based on the observation of cancer cell lines sensitivity to drugs [22]. The advantages for cancer cells included convenience source, limitless passages, and easily clutured in a large-scale. Nonetheless, the findings of previous studies concluded from cancer cell models did not reflect the real responsive mechanisms during CRC chemotherapy [23, 24]. The discrepancy between cancer cell lines and cancer tissues is partially resulted from variation during continuous passages [25]. At present study, we separated cell from resectable cancer tissues as cell models to analyze its sensitivity to cisplatin administration. Those primary cells from CRC patient may be superior to cancer cell lines in the determining efficacy of chemotherapy and exactly reflect response in cancer tissues. XAF1 plays an important role in leading to cell apoptosis [26]. Moreover, XAF1 also has gained a lot of attention since it has a potential to be a tumor suppressor gene [27]. In addition, XAF1 is usually expressed in low levels in several types of tumor, such as pancreatic cancer [10], gastric cancer [28] and colon cancer [29]. Our study is consistent with previous reports and detects XAF1 with a low expression in CRC tissues. The high methylation in the promoter regions of XAF1 was found to be the major reason for down-regulation in tumor tissues [29]. According to our knowledge, we firstly conducted the present study to explore the effect of chemotherapy on culturing primary CRC cells. All three establishing primary CRC cells are sensitive to cisplatin and cause a lot of cell apoptosis. However, the apoptosis rates are further promoted after up-regulation of XAF1, indicating that XAF1 plays an important roles in conducting CRC response to chemotherapy. In contrary, the down-regulation of XAF1 weakens cisplatin causing SW1116 cell apoptosis. In addition, those results confirm that XAF1 serves as a tumor suppressor gene during carcinogenesis as described by other studies [27, 29]. A previous study demonstrated that the overexpression of XIAP contributed to enhance ovarian cancer cells resistant to cisplatin [30]. Moreover, XAF1 is one of the antagonists of XIAP; the interacted complex of both proteins will prevent XIAP to induce cell apoptosis [7]. Therefore, it is necessary that detail mechanism of XAF1 interacting with XIAP in regulating CRC sensitive to chemotherapy be explored in future study. In conclusion, XAFl plays an important role in regulation apoptosis of colon cancer cell during treatment with cisplatin. Up-regulation of XAF1 enhances sensitivity of CRC to chemotherapy. In other words, XAF1 proteins

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might have the potential to be one of the biomarkers to help clinics discern who will more benefit from chemotherapy. Targeting XAF1 to enhance CRC patient responses to chemotherapy may be one of the promising strategies for advantage CRC. Acknowledgments Thanks for the support of the Bureau of Dongguan City Science and Technology 2010 Key Project Fund (201028). Conflict of interest

This article has no conflicts of interest.

References 1. Siegel R, Naishadham D, Jemal A. Cancer statistics. Cancer J Clin. 2013;63:11–30. 2. Li QG, Li P, Tang D, Chen J, Wang DR. Impact of postoperative complications on long-term survival after radical resection for gastric cancer. World J Gastroenterol. 2013;19:4060–5. 3. Yang G, Wang Y, Zeng Y, Gao GF, Liang X, Zhou M, Wan X, Yu S, Jiang Y, Naghavi M, Vos T, Wang H, Lopez AD, Murray CJ. Rapid health transition in China, 1990–2010: findings from the Global Burden of Disease Study 2010. Lancet. 2013;381: 1987–2015. 4. Schellerer VS, Merkel S, Schumann SC, Schlabrakowski A, Fortsch T, Schildberg C, Hohenberger W, Croner RS. Despite aggressive histopathology survival is not impaired in young patients with colorectal cancer: CRC in patients under 50 years of age. Int J Colorectal Dis. 2012;27:71–9. 5. Nakamura K, Abu Lila AS, Matsunaga M, Doi Y, Ishida T, Kiwada H. A double-modulation strategy in cancer treatment with a chemotherapeutic agent and siRNA. Mol Ther. 2011;19: 2040–7. 6. Troiani T, Zappavigna S, Martinelli E, Addeo SR, Stiuso P, Ciardiello F, Caraglia M. Optimizing treatment of metastatic colorectal cancer patients with anti-EGFR antibodies: overcoming the mechanisms of cancer cell resistance. Expert Opin Biol Ther. 2013;13:241–55. 7. Xia Y, Novak R, Lewis J, Duckett CS, Phillips AC. Xaf1 can cooperate with TNFalpha in the induction of apoptosis, independently of interaction with XIAP. Mol Cell Biochem. 2006; 286:67–76. 8. Liston P, Fong WG, Kelly NL, Toji S, Miyazaki T, Conte D, Tamai K, Craig CG, McBurney MW, Korneluk RG. Identification of XAF1 as an antagonist of XIAP anti-caspase activity. Nat Cell Biol. 2001;3:128–33. 9. Kim SK, Park HJ, Seok H, Jeon HS, Kim JW, Chung JH, Kwon KH, Woo SH, Lee BW, Baik HH. Missense polymorphisms in XIAP-associated factor-1 (XAF1) and risk of papillary thyroid cancer: correlation with clinicopathological features. Anticancer Res. 2013;33:2205–10. 10. Huang J, Yao WY, Zhu Q, Tu SP, Yuan F, Wang HF, Zhang YP, Yuan YZ. XAF1 as a prognostic biomarker and therapeutic target in pancreatic cancer. Cancer Sci. 2010;101:559–67. 11. Chen XY, He QY, Guo MZ. XAF1 is frequently methylated in human esophageal cancer. World J Gastroenterol. 2012;18: 2844–9. 12. Lee MG, Huh JS, Chung SK, Lee JH, Byun DS, Ryu BK, Kang MJ, Chae KS, Lee SJ, Lee CH, Kim JI, Chang SG, Chi SG. Promoter CpG hypermethylation and downregulation of XAF1 expression in human urogenital malignancies: implication for attenuated p53 response to apoptotic stresses. Oncogene. 2006;25:5807–22.

Med Oncol (2014) 31:273 13. Chen YB, Shu J, Yang WT, Shi L, Guo XF, Wang FG, Qian YY. XAF1 as a prognostic biomarker and therapeutic target in squamous cell lung cancer. Chin Med J (Engl). 2011;124:3238–43. 14. Byun DS, Cho K, Ryu BK, Lee MG, Kang MJ, Kim HR, Chi SG. Hypermethylation of XIAP-associated factor 1, a putative tumor suppressor gene from the 17p13.2 locus, in human gastric adenocarcinomas. Cancer Res. 2003;63:7068–75. 15. Yu LF, Wang J, Zou B, Lin MC, Wu YL, Xia HH, Sun YW, Gu Q, He H, Lam SK, Kung HF, Wong BC. XAF1 mediates apoptosis through an extracellular signal-regulated kinase pathway in colon cancer. Cancer. 2007;109:1996–2003. 16. Sun Y, Qiao L, Xia HH, Lin MC, Zou B, Yuan Y, Zhu S, Gu Q, Cheung TK, Kung HF, Yuen MF, Chan AO, Wong BC. Regulation of XAF1 expression in human colon cancer cell by interferon beta: activation by the transcription regulator STAT1. Cancer Lett. 2008;260:62–71. 17. Tu SP, Sun YW, Cui JT, Zou B, Lin MC, Gu Q, Jiang SH, Kung HF, Korneluk RG, Wong BC. Tumor suppressor XIAP-associated factor 1 (XAF1) cooperates with tumor necrosis factor-related apoptosis-inducing ligand to suppress colon cancer growth and trigger tumor regression. Cancer. 2010;116:1252–63. 18. Wang J, Gu Q, Li M, Zhang W, Yang M, Zou B, Chan S, Qiao L, Jiang B, Tu S, Ma J, Hung IF, Lan HY, Wong BC. Identification of XAF1 as a novel cell cycle regulator through modulating G(2)/ M checkpoint and interaction with checkpoint kinase 1 in gastrointestinal cancer. Carcinogenesis. 2009;30:1507–16. 19. Zou Z, Yuan Z, Zhang Q, Long Z, Chen J, Tang Z, Zhu Y, Chen S, Xu J, Yan M, Wang J, Liu Q. Aurora kinase A inhibitioninduced autophagy triggers drug resistance in breast cancer cells. Autophagy. 2012;8:1798–810. 20. Kim KY, Cha IH, Ahn JB, Kim NK, Rha SY, Chung HC, Roh JK, Shin SJ. Estimating the adjuvant chemotherapy effect in elderly stage II and III colon cancer patients in an observational study. J Surg Oncol. 2013;107:613–8. 21. Kurniali PC, Hrinczenko B, Al-Janadi A. Management of locally advanced and metastatic colon cancer in elderly patients. World J Gastroenterol. 2014;20:1910–22.

Page 7 of 7 273 22. Jaganathan SK, Supriyanto E, Mandal M. Events associated with apoptotic effect of p-coumaric acid in HCT-15 colon cancer cells. World J Gastroenterol. 2013;19:7726–34. 23. Weiswald LB, Richon S, Massonnet G, Guinebretiere JM, Vacher S, Laurendeau I, Cottu P, Marangoni E, Nemati F, Validire P, Bellet D, Bieche I, Dangles-Marie V. A short-term colorectal cancer sphere culture as a relevant tool for human cancer biology investigation. Br J Cancer. 2013;108:1720–31. 24. Lee J, Ballikaya S, Schonig K, Ball CR, Glimm H, Kopitz J, Gebert J. Transforming growth factor beta receptor 2 (TGFBR2) changes sialylation in the microsatellite unstable (MSI) colorectal cancer cell line HCT116. PLoS One. 2013;8:e57074. 25. Stevens JB, Horne SD, Abdallah BY, Ye CJ, Heng HH. Chromosomal instability and transcriptome dynamics in cancer. Cancer Metastasis Rev. 2013;32:391–402. 26. Long X, Li Y, Qi Y, Xu J, Wang Z, Zhang X, Zhang D, Zhang L, Huang J. XAF1 contributes to dengue virus-induced apoptosis in vascular endothelial cells. FASEB J. 2013;27:1062–73. 27. Ding Y, Cheng X, Zhu L, Qiao M, Ye J, Jiang SR, Tu S. Tu1893 tumor suppressor XAF1 and TRAIL cooperate to inhibit migration and invasion of colon cancer cells. Gastroenterology. 2013;144:S-874. 28. Wang J, He H, Yu L, Xia HH-x, Lin MC, Gu Q, Li M, Zou B, An X, Jiang B. HSF1 down-regulates XAF1 through transcriptional regulation. J Biol Chem. 2006;281:2451–9. 29. Zou B, Chim CS, Zeng H, Leung SY, Yang Y, Tu SP, Lin M, Wang J, He H, Jiang SH. Correlation between the single-site CpG methylation and expression silencing of the XAF1 gene in human gastric and colon cancers. Gastroenterology. 2006;131:1835–43. 30. Fraser M, Leung BM, Yan X, Dan HC, Cheng JQ, Tsang BK. p53 is a determinant of X-linked inhibitor of apoptosis protein/Aktmediated chemoresistance in human ovarian cancer cells. Cancer Res. 2003;63:7081–8.

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