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Sulfasalazine, a therapeutic agent for ulcerative colitis, inhibits the growth of CD44v9+ cancer stem cells in ulcerative colitis-related cancer
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Sulfasalazine, a therapeutic agent for ulcerative colitis, inhibits the growth of CD44v9+ cancer stem cells in ulcerative colitis-related cancer Ryo Seishima a,b, Koji Okabayashi a,∗, Osamu Nagano b, Hirotoshi Hasegawa a, Masashi Tsuruta a, Masayuki Shimoda c, Kaori Kameyama c, Hideyuki Saya b, Yuko Kitagawa a a
Department of surgery, Keio university school of medicine, 35, Shinano-machi, 1608582 Shinjuku-ku, Tokyo, Japan b Division of gene regulation, institute for advanced medical research, Keio university school of medicine, 35, Shinano-machi, 1608582 Shinjuku-ku, Tokyo, Japan c Department of pathology, Keio university school of medicine, 35, Shinano-machi, 1608582 Shinjuku-ku, Tokyo, Japan
Summary Background and objective: Sulfasalazine reduces the risk of ulcerative colitis (UC)-related cancer through its anti-inflammatory effect and induction of oxidative stress in cancer cells by inhibiting the glutamate—cystine transporter, which is closely associated with the cancer stem cell surface marker CD44v9. This study aimed to quantify the effects of sulfasalazine on CD44v9 expression and pathological factors in colorectal cancers (CRCs) arising from UC. Methods: Twenty-six patients with UC-related cancer were classified into groups according to the length of sulfasalazine treatment as follows: (1) long-term (LT) (≥ 5 years) and (2) shortterm (ST) (< 5 years). Using immunohistochemistry, we compared CD44v9 and Ki-67 expression and pathological characteristics of each group’s tumors. In vitro assay was performed to investigate the effect of sulfasalazine on epithelial differentiation and proliferation of CD44+ cancer cells. Results: Immunohistochemical analysis revealed that CD44v9 expression tended to be lower in the LT group (LT:ST = 15.4%:46.2%, P = 0.20), and Ki-67/CD44v9 double-stained cells were significantly lower in the LT group (LT:ST = 0%:6.9%, P = 0.01). Pathologically, the frequency of well-differentiated adenocarcinomas was higher in the LT group (LT:ST = 84.6%:38.5%, P = 0.04).
∗
Corresponding author. E-mail address: [email protected] (K. Okabayashi).
http://dx.doi.org/10.1016/j.clinre.2015.11.007 2210-7401/© 2015 Elsevier Masson SAS. All rights reserved.
Please cite this article in press as: Seishima R, et al. Sulfasalazine, a therapeutic agent for ulcerative colitis, inhibits the growth of CD44v9+ cancer stem cells in ulcerative colitis-related cancer. Clin Res Hepatol Gastroenterol (2015), http://dx.doi.org/10.1016/j.clinre.2015.11.007
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In vitro assay revealed that sulfasalazine promoted the expression of epithelial differentiation markers (E-cadherin and CDX2) and inhibited the proliferation of CD44+ cancer cells. Conclusions: Long-term sulfasalazine administration reduced proliferative CD44v9+ cells and increased the degree of differentiation of adenocarcinomas. These findings indicate the importance of CD44v9+ cells in UC-related cancer progression and suggest that sulfasalazine may serve as a novel therapeutic agent that targets CD44v9+ cells. © 2015 Elsevier Masson SAS. All rights reserved.
Introduction Chronic intestinal mucosal inflammation is strongly associated with gastrointestinal carcinogenesis [1]. For example, patients with inflammatory bowel diseases (IBD), including ulcerative colitis (UC) and Crohn’s disease (CD), have a significantly higher risk of colorectal cancer (CRC). A metaanalysis reported that one in five patients with UC will develop CRC over 30 years, and that the risk of CRC is influenced by the extent of lesions and duration of the disease [2]. Chronic inflammation accumulates reactive oxygen species (ROS), which have an important role in the pathogenesis of epithelial cell cancers [3,4]. Compared with normal cells, cancer cells accumulate higher levels of ROS [5], which promote oncogenic transformation by stimulating cell proliferation or promote DNA mutations and genetic instability [6]. The interaction of CD44v with xCT (SLC7A11), a subunit of the cystine/glutamate antiporter known as system xc(−), stabilizes the latter and thereby potentiates the ability of cancer cells to increase glutathione synthesis and defend themselves against ROS [7,8]. Cancer stem cells (CSCs) can self-renew and drive tumorigenesis. Therefore, the cancer stem cell concept has important implications for cancer therapy [9,10]. The existence of CSCs is considered a cause of the emergence of drug-resistant cancers, because they eliminate sources of biochemical stress. Furthermore, the expression of CSC markers significantly associates with clinicopathological features. For example, high CD44 expression levels correlate with tumor size [11] or invasiveness [12], suggesting that CSCs act to enhance the malignant phenotype. Moreover, the expression of CD44v, a splice variant of CD44, is associated with the progression of colon [13] and gastric [14,15] cancers. 5-aminosalicylic acid (5-ASA), the most commonly used anti-inflammatory medication for IBD, is associated with variable reductions in the risk of CRC [16,17], and the effect of 5-ASA therapy on reducing the risk of CRC may be mediated by a reduction in mucosal inflammation. Sulfasalazine consists of sulfapyridine and its active moiety 5-ASA that is released in the gut. Furthermore, sulfasalazine is a specific inhibitor of xCT-mediated cystine transport and it inhibits the growth, invasion, and metastasis of several types of cancer [18—20]. Recently, intact sulfasalazine, but not its metabolites, was found to induce oxidative stress in cancer cells by inhibiting the glutamate—cystine transporter [19]. Therefore, sulfasalazine may serve as a novel therapeutic agent targeting CSCs for the prevention and treatment of
CRC. However, little evidence is available regarding the relationship between sulfasalazine and pathological findings of CRCs related to UC. This study aimed to retrospectively quantify the effect of sulfasalazine on CSCs and identify the factors related to carcinogenesis in CRCs arising from UC.
Materials and methods Patients We conducted a retrospective review of the records of 31 consecutive UC-associated CRC patients who underwent curative colorectal surgery at Keio University Hospital, Tokyo, Japan, between 1998 and 2012. All data were obtained from a prospectively maintained registry of colorectal surgery and patients’ medical records. Pathological diagnosis for UC-associated cancers have been confirmed considering the presence of over-expression of p53 and mucosal inflammation around lesions [21,22]. Five patients who lacked detailed information regarding sulfasalazine administration were excluded, and the remaining 26 UCassociated CRC patients were included. We compared patients’ clinicopathological findings according to the length of sulfasalazine administration. For this purpose, patients were classified into long-term (LT) (≥ 5 years) and shortterm (ST) (< 5 years) groups. The length of term was defined as a cumulative term treated by sulfasalazine. Severity of inflammation was diagnosed by gastroenterologist according to their endoscopic findings before surgery. The institutional ethics committee approved this study.
Histopathological examination The lesions were histologically confirmed by pathologists. All histopathological specimens were fixed in 20% buffered formalin and routinely processed for embedding in paraffin and sectioning at a thickness of 4 m. Paraffin blocks of carcinomas were examined. Sections were depleted of paraffin and then rehydrated in a graded series of ethanol solutions. For histology, sections were stained with hematoxylin and eosin. For immunohistochemistry, sections were washed with phosphate-buffered saline (PBS), subjected to antigen retrieval by heating for 10 min at 100 ◦ C in 0.01 M sodium citrate buffer (pH 6.0), and exposed to 3% hydrogen peroxide before incubation with primary antibodies. Immune complexes were detected using a Vectastain Elite
Please cite this article in press as: Seishima R, et al. Sulfasalazine, a therapeutic agent for ulcerative colitis, inhibits the growth of CD44v9+ cancer stem cells in ulcerative colitis-related cancer. Clin Res Hepatol Gastroenterol (2015), http://dx.doi.org/10.1016/j.clinre.2015.11.007
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Sulfasalazine inhibits the growth of cancer stem cells
3 The number of viable cells was then determined by using Cell Titer-Glo luminescence cell viability kit (Promega) and compared with control cells exposed to dimethyl sulfoxide (DMSO).
Quantitative RT-PCR analysis
Figure 1 A representative image of high CD44v9 expression in UC-related colorectal adenocarcinoma. Scale bar: 50 m.
Kit (Vector Laboratories, Burlingame, CA, USA) and 3,3 diaminobenzidine, and the sections were counterstained with hematoxylin. A rat monoclonal antibody (mAb) specific for human CD44v9 (1:100) was used to detect CSCs as our previous publication [7,23]. Membranous CD44v9 expression was evaluated using the Allred score [24]. Briefly, the staining results were scored according to the intensity (0: undetectable; 1: weak; 2: intermediate; 3: strong) and the proportion of stained tumor cells (0: none; 1: < 1/100; 2: 1/100−1/10; 3: 1/10−1/3; 4: 1/3−2/3; 5: > 2/3). Combined scores of ≥ 3 were defined as highly expressed. Representative image of CD44v9 expression is shown in Fig. 1.
Total RNA was extracted cultured cells using RNeasy mini kit (QIAGEN) and reverse transcribed using the ReverTraAce (TOYOBO). Quantitative RT-PCR analysis was performed with a Thermal Cycler Dice Real Time System (TaKaRa Bio, Tokyo, Japan). The amplification protocol comprised an initial incubation at 95 ◦ C for 2 min and 40 cycles of 95 ◦ C for 30 s and 60 ◦ C for 30 s, followed by dissociation-curve analysis to confirm specificity. Primers used are: CD44, 5 -AGAAGGTGTGGGCAGAAGAA-3 and 5 -AAATGCACCATTTCCTGAGA-3 ; E-cadherin, 5 -AGAACGCATTGCCACATACACTC3 and 5 -CATTCTGATCGGTTACCGTGATC-3 ; CDX2, 5 TTCACTACAGTCGCTACATCACCAT-3 and 5 -TTGTTGATTTTCCTCTCCTTTGCT-3 ; GAPDH, 5 -GTGAAGGTCGGTGTGAACG-3 and 5 -GACCATGTAGTTGAGGTCAATG-3 . All quantitative data were normalized by the abundance of GAPDH mRNAs.
Statistical analysis Statistical analyses were performed using the Chi2 and Mann—Whitney U-tests. All statistical tests were two-sided, and P < 0.05 was considered statistically significant. All calculations were performed using SPSS software (SPSS Inc., Chicago, IL, USA).
Immunofluorescence analysis
Results
Tissues were processed as for immunohistochemistry but without hydrogen peroxide. The sections were incubated with appropriate Alexa Fluor® 488-conjugated or Alexa Fluor® 594-conjugated secondary antibodies (Invitrogen, Tokyo, Japan), diluted in PBS, washed thrice with PBS, and mounted in Vectashield mounting medium (Vector Laboratories). Sections were viewed and photographed using a Biorevo BZ-9000 fluorescence microscope (Keyence, Tokyo, Japan). Anti-CD44v9 (1:100) antibody and a rabbit mAb to Ki-67 (diluted 1:100; Abcam, Cambridge, UK) were used to detect proliferating CSCs. CD44v9+ cells in a randomly selected microscopic field (magnification, ×40) per section were counted using image analysis software (BZ-9000; Keyence, Tokyo, Japan), and the percentage of Ki-67+ cells was calculated. Ki-67/CD44v9 double-stained cells were defined as proliferating CSCs.
Patient demographics
Cell culture and in vitro proliferation assay HCT116, known as a CD44 highly expressing colon cancer cell line [7], was culture in Dulbecco’s Modified Eagle’s Medium (DMEM) with high glucose, supplemented with 10% FBS. Cells were maintained under 5% CO2 at 37 ◦ C. Cells were plated in 96-well plates (2 × 103 cells per well), cultured overnight, and then exposed to sulfasalazine (Sigma) with the dose of 300 mol/L for 2 days as our previous publication [25].
We analyzed the records of 26 patients who underwent surgery for UC-associated CRC (Table 1). The length of sulfasalazine administration ranged from 0 to 32 years. There were no pStage IV patients.
Association between sulfasalazine administration and histopathological findings Comparison of patient characteristics and various histopathological factors, including CD44v9 expression, between both groups is shown in Table 2. There was no significant difference in age (P = 0.60), gender (P = 0.38), or severity of the disease (P = 1.00). The median length of sulfasalazine treatment was 0.2 and 13.7 years in the ST and LT groups, respectively, (P < 0.001). In the ST group, 5 patients did not receive sulfasalazine at all. All of these patients received 5-ASA instead. In remained 8 patients, who received sulfasalazine at least 2 months, on-off was controlled according to severity of the disease. Of these, 5 patients received other agents (steroids or immunosuppressives) and 3 patients were treated only with sulfasalazine. All patients’ data concerned with the length of sulfasalazine treatment and the period from UC diagnosis to cancer diagnosis is shown in Supplement Fig. 1. There
Please cite this article in press as: Seishima R, et al. Sulfasalazine, a therapeutic agent for ulcerative colitis, inhibits the growth of CD44v9+ cancer stem cells in ulcerative colitis-related cancer. Clin Res Hepatol Gastroenterol (2015), http://dx.doi.org/10.1016/j.clinre.2015.11.007
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contrast to 46.2% in the ST group, although the difference was not statistically significant (P = 0.20).
Patient characteristics. Ulcerative colitis-associated colorectal cancer patients (n = 26)
Age (years) Sex (Male:Female) Period from ulcerative colitis diagnosis to surgery (years) Length of sulfasalazine treatment (years) Cancer location (Rt/Lt/Rec) Histology (well differentiated/others) pT (0/1/2/3/4) pN (0/1/2) pStage (0/1/2/3/4) Lymphatic invasion (absent/present) Venous invasion
a
45 (26—70) 19:7 12.5 (0.1—32)a 4.5 (0—32)a 4/16/6 16/10 5/8/3/10/0 22/2/2 5/9/8/4/0 11/15 14/12
Rt: right-side colon (cecum, ascending colon, and transverse colon); Lt: left-side colon (descending colon and sigmoid colon); Rec: rectum. a Median (min—max).
was no significant difference in use of other therapeutic agents, such as steroids (P = 1.00), immunosuppressives (P = 1.00), and anti-TNF␣ antibody. Presence of multiple lesions or dysplasia around the main tumors was similar in both groups (P = 1.00). Well-differentiated adenocarcinomas were present in a significantly higher proportion in the LT group (LT:ST = 84.6%:38.5%, P = 0.04). There was no significant difference in other pathological factors, including pT, pN, pStage, and lymphatic and venous invasion. In the LT group, 15.4% of patients expressed high CD44v9 levels, in Table 2
CD44v9+ cell proliferation in tumors Because sulfasalazine targets the ROS tolerance of CD44v9+ cells, it may affect the proliferation of these cells. We conducted immunofluorescence assays to investigate CD44v9+ cell proliferation in tumors (Fig. 2). The median proportion of proliferating CD44v9+ cells (Ki-67/CD44v9 double-stained cells) was significantly lower in the LT group than in the ST group (LT:ST = 0%:6.9%, P = 0.01, Table 2), indicating that long-term sulfasalazine administration may down-regulate the CSC proliferation in tumors.
Effects of sulfasalazine on proliferation and differentiation marker expression in CD44+ cancer cells To validate the result of clinical data, we performed in vitro assays. Given that sulfasalazine inhibits the cell proliferation of CD44+ selectively in head and neck cancer, we aimed to examine it in CD44+ colon cancer cell line. Proliferation assay revealed that the proliferation of HCT 116 cells (CD44+ cells) was significantly suppressed by sulfasalazine treatment (Fig. 3). We next investigated whether sulfasalazine has some effect on cell differentiation. The expressions of epithelial differentiation markers (E-cadherin and CDX2) in sulfasalazine treated cells were compared with control cells [26—28]. Quantitative RT-PCR results revealed that the mRNA levels in sulfasalazine treated cells were increased in both markers, concurrently with the suppression of CD44 expression (Fig. 4). These results support our clinical findings that sulfasalazine inhibits the proliferation of CD44+ cells and promotes the tumor differentiation.
Comparison of clinicopathological findings according to the length of sulfasalazine treatment.
Age (years) Male (%) Severe activity of UC (%) Length of sulfasalazine treatment (years) Steroids use (%) Immunosuppressives use (%) Anti-TNF␣ antibody use (%) Period from ulcerative colitis diagnosis to cancer diagnosis (years) Well-differentiated adenocarcinoma (%) pT 0/1 (%) pN0 (%) pStage 0/1 (%) Lymphatic invasion (present, %) Venous invasion (present, %) Multiple lesion (present, %) Dysplasia (present, %) High CD44v9 expression (%) Proportion of proliferating CD44v9 cells (%) a
Short term (n = 13)
Long term (n = 13)
P-value
39 (26—70)a 8 (61.5) 2 (15.4) 0.2 (0—4.0)a 3 (23.1) 3 (23.1) 0 (0) 5 (0.09—21)a 5 (38.5) 8 (61.5) 11 (84.6) 11 (84.6) 7 (53.8) 6 (46.2) 2 (15.4) 6 (46.2) 6 (46.2) 6.9% (0—86.3)a
46 (30—64)a 11 (84.6) 1 (7.7) 13.7 (5—32.0)a 2 (15.4) 3 (23.1) 0 (0) 14 (9—32)a 11 (84.6) 5 (38.5) 11 (84.6) 11 (84.6) 8 (61.5) 6 (46.2) 3 (23.1) 7 (53.8) 2 (15.4) 0%a
0.60 0.38 1.00 < 0.001 1.00 1.00 0.01 0.04 0.43 1.00 1.00 1.00 1.00 1.00 1.00 0.20 0.01
Median (min—max).
Please cite this article in press as: Seishima R, et al. Sulfasalazine, a therapeutic agent for ulcerative colitis, inhibits the growth of CD44v9+ cancer stem cells in ulcerative colitis-related cancer. Clin Res Hepatol Gastroenterol (2015), http://dx.doi.org/10.1016/j.clinre.2015.11.007
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Figure 3 Time course of cell proliferation for HCT116 cells treated with control (DMSO) or sulfasalazine. Data are mean ± SD from 4 independent experiments. * P < 0.01.
Figure 2 Representative images of immunofluorescence detection of CD44v9 (green) and Ki-67 (red). CD44v9+ cells with high (A) and low (B) expression of Ki-67. Scale bar: 50 m.
Discussion Herein, we show that sulfasalazine downregulates the proliferation of CSCs that express CD44v9. Additionally, our
findings implies that long-term sulfasalazine treatment was associated with a higher incidence of well-differentiated tubular adenocarcinomas in patients with UC-related CRCs. Although controversial, there are reports showing that the expression of CSC markers influences tumor differentiation. For example, Yanagisawa et al. reported that CD44v overexpression was associated with poor differentiation of gallbladder carcinomas [29], and Chen et al. reported that in gastric cancer, higher CD44 expression was associated with poorer differentiation [30]. However, the association between CSC and the histology of CRC tumors is unknown. We showed that a CSC marker (CD44v9) was associated with tumor differentiation in UC-related CRC as well and that sulfasalazine influenced the malignant phenotype. Because well-differentiated adenocarcinomas are highly sensitive to treatment and patients with these tumors have a favorable prognosis, a novel therapy that enhances tumor differentiation may be efficacious. Chronic inflammation is strongly associated with carcinogenesis in CRC. Accumulation of ROS is an important change that occurs during malignant transformation and correlates with carcinogenesis. Among several drugs with a 5-ASA
Figure 4 Epithelial differentiation marker gene expression in sulfasalazine treated HCT116 cells relative to that in control cells treated with DMSO, as determined by quantitative RT-PCR analysis. * P < 0.01, ** P < 0.05.
Please cite this article in press as: Seishima R, et al. Sulfasalazine, a therapeutic agent for ulcerative colitis, inhibits the growth of CD44v9+ cancer stem cells in ulcerative colitis-related cancer. Clin Res Hepatol Gastroenterol (2015), http://dx.doi.org/10.1016/j.clinre.2015.11.007
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moiety, sulfasalazine weakens the resistance of CSCs to ROS. For example, sulfasalazine administration influences antitumorigenicity in mouse models of gastric carcinogenesis [23]. Furthermore, using head and neck cancer cell lines, CD44v9+ tumor cells were selectively depleted, and tumor progression was suppressed by sulfasalazine administration [25]. In this study, long-term sulfasalazine administration reduced CD44v9 expression and the proportion of proliferating CD44v9+ cells, which is consistent with the results of studies of a mouse model or in vitro experiments [23,25]. Generally, under the accumulation of ROS induced by chronic inflammation, CD44v9+ CSCs expand for the purpose of self-protection [8]. In this study, however, the proliferation of CD44v9+ cells was suppressed in the LT group, which had longer disease duration and probably exposed to inflammation longer than the ST group. This fact emphasizes the effect of sulfasalazine which is targeting CD44v9+ cells. This result suggests that sulfasalazine has a negative effect on the tolerance against ROS and the proliferation of CSCs. Therefore, we consider sulfasalazine as a clinically promising agent that targets CD44v9+ CSCs via the increase of accumulated intracellular ROS. This conclusion is based on our findings that there was no difference in lymphatic or venous invasion by the tumors. Therefore, the efficacy of sulfasalazine may be limited to inhibiting carcinogenesis and not tumor progression. We previously reported that a combination of anti-epidermal growth factor and sulfasalazine therapy dramatically suppresses tumor progression [25], indicating that sulfasalazine acts as a supportive drug that specifically targets chemotherapyresistant cancer cells. Because chemotherapy was not administered before surgery to the patients studied here, further investigation addressing the effect of chemotherapy combined with sulfasalazine is required. Additional finding from our results is that sulfasalazine may influence the tumor differentiation. From our clinical data, we considered that this mechanism was due to the reduced number of CD44v9+ CSCs by longer sulfasalazine administration. From in vitro assay, we also found that the expressions of epithelial differentiation markers were promoted by sulfasalazine treatment, synchronically with the inhibition of CD44 expression. This result implies that sulfasalazine may have an effect on differentiation markers directly or indirectly through some CD44 related genes. Further investigation should be needed to clarify this molecular mechanism. This study has some limitations. Because the study was retrospective and it included a small number of patients, it is difficult to make definite conclusions. It was also difficult to precisely record dose intensity of sulfasalazine for all enrolled patients in this retrospective study. Since there is no clear evidence of effective dose and duration of sulfasalazine for influencing CD44v9 expression, pharmacodynamic investigation should be performed. Furthermore, we limited our analyses to UC-associated CRC patients, because sulfasalazine is mainly used to treat patients with IBD but not other diseases. Watanabe et al. reported that UC-associated CRC was more malignant than sporadic CRC [31]. A difference in carcinogenesis between UC-associated CRC and sporadic CRC may exist. Therefore, it is unclear whether sulfasalazine affects sporadic cancers as well. In this study, although there was no significant difference in
5-year disease-free survival (data not shown), the protective effect of sulfasalazine on postoperative recurrence should be assessed. Further studies should be conducted with a larger number of patients to arrive at a definitive conclusion. In conclusion, long-term sulfasalazine administration reduced proliferative CD44v9+ cells and had a significant impact on tumor cell differentiation. This result indicates the importance of CD44v9+ CSCs in the progression of UCrelated cancer and suggests the possibility of developing novel targets for eliminating CSCs. The feasibility of this approach is unclear and is supported by our present results.
Grant support This work was supported by grants (to H.S.) from, as well as in part by the Project for Development of Innovative Research on Cancer Therapeutics (P-Direct [to O.N.]) of, the Ministry of Education, Culture, Sports, Science, and Technology of Japan.
Disclosure of interest The authors declare that they have no competing interest.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.clinre.2015.11.007.
References [1] Coussens LM, Werb Z. Inflammation and cancer. Nature 2002;420(6917):860—7. [2] Eaden JA, Abrams KR, Mayberry JF. The risk of colorectal cancer in ulcerative colitis: a meta-analysis. Gut 2001;48(4): 526—35. [3] Federico A, Morgillo F, Tuccillo C, Ciardiello F, Loguercio C. Chronic inflammation and oxidative stress in human carcinogenesis. Int J Cancer 2007;121(11):2381—6. [4] Waris G, Ahsan H. Reactive oxygen species: role in the development of cancer and various chronic conditions. J Carcinog 2006;5:14. [5] Pelicano H, Carney D, Huang P. ROS stress in cancer cells and therapeutic implications. Drug Resist Updat 2004;7(2): 97—110. [6] Jackson AL, Loeb LA. The contribution of endogenous sources of DNA damage to the multiple mutations in cancer. Mutat Res 2001;477(1—2):7—21. [7] Ishimoto T, Nagano O, Yae T, Tamada M, Motohara T, Oshima H, et al. CD44 variant regulates redox status in cancer cells by stabilizing the xCT subunit of system xc(−) and thereby promotes tumor growth. Cancer Cell 2011;19(3):387—400. [8] Nagano O, Okazaki S, Saya H. Redox regulation in stemlike cancer cells by CD44 variant isoforms. Oncogene 2013;32(44):5191—8. [9] O’Brien CA, Pollett A, Gallinger S, Dick JE. A human colon cancer cell capable of initiating tumour growth in immunodeficient mice. Nature 2007;445(7123):106—10. [10] Ricci-Vitiani L, Lombardi DG, Pilozzi E, Biffoni M, Todaro M, Peshle C, et al. Identification and expansion of human coloncancer-initiating cells. Nature 2007;445(7123):111—5.
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+Model CLINRE-834; No. of Pages 7
ARTICLE IN PRESS
Sulfasalazine inhibits the growth of cancer stem cells [11] Choi D, Lee HW, Hur KY, Kim JJ, Park GS, Jang SH, et al. Cancer stem cell markers CD133 and CD24 correlate with invasiveness and differentiation in colorectal adenocarcinoma. World J Gastroenterol 2009;15(18):2258—64. [12] Bendardaf R, Elzagheid A, Lamlum H, Ristamaki R, Collan Y, Pyrhonen S. E-cadherin. CD44s and CD44v6 correlate with tumour differentiation in colorectal cancer. Oncol Rep 2005;13(5):831—5. [13] Tanabe KK, Ellis LM, Saya H. Expression of CD44R1 adhesion molecule in colon carcinomas and metastases. Lancet 1993;341(8847):725—6. [14] Mayer B, Jauch KW, Gunthert U, Figdor CG, Schildberg FW, Funke I, et al. De-novo expression of CD44 and survival in gastric cancer. Lancet 1993;342(8878):1019—22. [15] Miwa T, Watanabe A, Yamada Y, Shino Y, Yamada T, Yamashita J, et al. Progression in gastric carcinoma relative to the ratio of CD44 epithelial variant transcript to CD44 hematopoietic variant transcript. Cancer 1996;77(1):25—9. [16] van Staa TP, Card T, Logan RF, Leufkens HG. 5-aminosalicylate use and colorectal cancer risk in inflammatory bowel disease: a Large Epidemiological Study. Gut 2005;54(11):1573—8. [17] Velayos FS, Terdiman JP, Walsh JM. Effect of 5-aminosalicylate use on colorectal cancer and dysplasia risk: a systematic review and metaanalysis of observational studies. Am J Gastroenterol 2005;100(6):1345—53. [18] Chen RS, Song YM, Zhou ZY, Tong T, Li Y, Fu M, et al. Disruption of xCT inhibits cancer cell metastasis via the caveolin-1/betacatenin pathway. Oncogene 2009;28(4):599—609. [19] Gout PW, Buckley AR, Simms CR, Bruchovsky N. Sulfasalazine, a potent suppressor of lymphoma growth by inhibition of the x(c)− cystine transporter: a new action for an old drug. Leukemia 2001;15(10):1633—40. [20] Lo M, Wang YZ, Gout PW. The x(c)− cystine/glutamate antiporter: a potential target for therapy of cancer and other diseases. J Cell Physiol 2008;215(3):593—602. [21] Brentnall TA, Crispin DA, Rabinovitch PS, Haggitt RC, Rubin CE, Stevens AC, et al. Mutations in the p53 gene: an early marker of neoplastic progression in ulcerative colitis. Gastroenterology 1994;107:369—78.
7 [22] Yin J, Harpaz N, Tong Y, Huang Y, Laurin J, Greenwald BD, et al. p53 point mutations in dysplastic and cancerous ulcerative colitis lesions. Gastroenterology 1993;104:1633—9. [23] Wada T, Ishimoto T, Seishima R, Tsuchihashi K, Yoshikawa M, Oshima H, et al. Functional role of CD44v-xCT system in the development of spasmolytic polypeptide-expressing metaplasia. Cancer Sci 2013;104(10):1323—9. [24] Allred DC, Harvey JM, Berardo M, Clark GM. Prognostic and predictive factors in breast cancer by immunohistochemical analysis. Mod Pathol 1998;11(2):155—68. [25] Yoshikawa M, Tsuchihashi K, Ishimoto T, Yae T, Motohara T, Sugihara E, et al. xCT inhibition depletes CD44v-expressing tumor cells that are resistant to EGFR-targeted therapy in head and neck squamous cell carcinoma. Cancer Res 2013;73(6):1855—66. [26] Keiichiro S, Masahiro A, Reiji K. Transcription factors c-Myc and CDX2 mediate E-selectin ligand expression in colon cancer cells undergoing EGF/bFGF-induced epithelial—mesenchymal transition. PNAS 2012;109(20):7776—81. [27] Mizoshita T, Inada K, Tsukamoto T, Kodera Y, Yamamura Y, Hirai T, et al. Expression of Cdx1 and Cdx2 mRNAs and relevance of this expression to differentiation in human gastrointestinal mucosa. Gastric Cancer 2001;4(4):185—91. [28] Guo RJ, Suh ER, Lynch JP. The role of Cdx proteins in intestinal development and cancer. Cancer Biol Ther 2004;3(7): 593—601. [29] Yanagisawa N, Mikami T, Mitomi H, Saegusa M, Koike M, Okayasu I. CD44 variant overexpression in gallbladder carcinoma associated with tumor dedifferentiation. Cancer 2001;91(2): 408—16. [30] Chen S, Hou JH, Feng XY, Zhang XS, Zhou ZW, Yun JP, et al. Clinicopathologic significance of putative stem cell marker, CD44 and CD133, in human gastric carcinoma. J Surg Oncol 2013;107(8):799—806. [31] Watanabe T, Konishi T, Kishimoto J, Kotake K, Muto T, Sugihara, et al. Ulcerative colitis-associated colorectal cancer shows a poorer survival than sporadic colorectal cancer: a Nationwide Japanese Study. Inflamm Bowel Dis 2011;17(3):802—8.
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Sulfasalazine, a therapeutic agent for ulcerative colitis, inhibits the growth of CD44v9+ cancer stem cells in ulcerative colitis-related cancer Ryo Seishima a,b, Koji Okabayashi a,∗, Osamu Nagano b, Hirotoshi Hasegawa a, Masashi Tsuruta a, Masayuki Shimoda c, Kaori Kameyama c, Hideyuki Saya b, Yuko Kitagawa a a
Department of surgery, Keio university school of medicine, 35, Shinano-machi, 1608582 Shinjuku-ku, Tokyo, Japan b Division of gene regulation, institute for advanced medical research, Keio university school of medicine, 35, Shinano-machi, 1608582 Shinjuku-ku, Tokyo, Japan c Department of pathology, Keio university school of medicine, 35, Shinano-machi, 1608582 Shinjuku-ku, Tokyo, Japan
Summary Background and objective: Sulfasalazine reduces the risk of ulcerative colitis (UC)-related cancer through its anti-inflammatory effect and induction of oxidative stress in cancer cells by inhibiting the glutamate—cystine transporter, which is closely associated with the cancer stem cell surface marker CD44v9. This study aimed to quantify the effects of sulfasalazine on CD44v9 expression and pathological factors in colorectal cancers (CRCs) arising from UC. Methods: Twenty-six patients with UC-related cancer were classified into groups according to the length of sulfasalazine treatment as follows: (1) long-term (LT) (≥ 5 years) and (2) shortterm (ST) (< 5 years). Using immunohistochemistry, we compared CD44v9 and Ki-67 expression and pathological characteristics of each group’s tumors. In vitro assay was performed to investigate the effect of sulfasalazine on epithelial differentiation and proliferation of CD44+ cancer cells. Results: Immunohistochemical analysis revealed that CD44v9 expression tended to be lower in the LT group (LT:ST = 15.4%:46.2%, P = 0.20), and Ki-67/CD44v9 double-stained cells were significantly lower in the LT group (LT:ST = 0%:6.9%, P = 0.01). Pathologically, the frequency of well-differentiated adenocarcinomas was higher in the LT group (LT:ST = 84.6%:38.5%, P = 0.04).
∗
Corresponding author. E-mail address: [email protected] (K. Okabayashi).
http://dx.doi.org/10.1016/j.clinre.2015.11.007 2210-7401/© 2015 Elsevier Masson SAS. All rights reserved.
Please cite this article in press as: Seishima R, et al. Sulfasalazine, a therapeutic agent for ulcerative colitis, inhibits the growth of CD44v9+ cancer stem cells in ulcerative colitis-related cancer. Clin Res Hepatol Gastroenterol (2015), http://dx.doi.org/10.1016/j.clinre.2015.11.007
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In vitro assay revealed that sulfasalazine promoted the expression of epithelial differentiation markers (E-cadherin and CDX2) and inhibited the proliferation of CD44+ cancer cells. Conclusions: Long-term sulfasalazine administration reduced proliferative CD44v9+ cells and increased the degree of differentiation of adenocarcinomas. These findings indicate the importance of CD44v9+ cells in UC-related cancer progression and suggest that sulfasalazine may serve as a novel therapeutic agent that targets CD44v9+ cells. © 2015 Elsevier Masson SAS. All rights reserved.
Introduction Chronic intestinal mucosal inflammation is strongly associated with gastrointestinal carcinogenesis [1]. For example, patients with inflammatory bowel diseases (IBD), including ulcerative colitis (UC) and Crohn’s disease (CD), have a significantly higher risk of colorectal cancer (CRC). A metaanalysis reported that one in five patients with UC will develop CRC over 30 years, and that the risk of CRC is influenced by the extent of lesions and duration of the disease [2]. Chronic inflammation accumulates reactive oxygen species (ROS), which have an important role in the pathogenesis of epithelial cell cancers [3,4]. Compared with normal cells, cancer cells accumulate higher levels of ROS [5], which promote oncogenic transformation by stimulating cell proliferation or promote DNA mutations and genetic instability [6]. The interaction of CD44v with xCT (SLC7A11), a subunit of the cystine/glutamate antiporter known as system xc(−), stabilizes the latter and thereby potentiates the ability of cancer cells to increase glutathione synthesis and defend themselves against ROS [7,8]. Cancer stem cells (CSCs) can self-renew and drive tumorigenesis. Therefore, the cancer stem cell concept has important implications for cancer therapy [9,10]. The existence of CSCs is considered a cause of the emergence of drug-resistant cancers, because they eliminate sources of biochemical stress. Furthermore, the expression of CSC markers significantly associates with clinicopathological features. For example, high CD44 expression levels correlate with tumor size [11] or invasiveness [12], suggesting that CSCs act to enhance the malignant phenotype. Moreover, the expression of CD44v, a splice variant of CD44, is associated with the progression of colon [13] and gastric [14,15] cancers. 5-aminosalicylic acid (5-ASA), the most commonly used anti-inflammatory medication for IBD, is associated with variable reductions in the risk of CRC [16,17], and the effect of 5-ASA therapy on reducing the risk of CRC may be mediated by a reduction in mucosal inflammation. Sulfasalazine consists of sulfapyridine and its active moiety 5-ASA that is released in the gut. Furthermore, sulfasalazine is a specific inhibitor of xCT-mediated cystine transport and it inhibits the growth, invasion, and metastasis of several types of cancer [18—20]. Recently, intact sulfasalazine, but not its metabolites, was found to induce oxidative stress in cancer cells by inhibiting the glutamate—cystine transporter [19]. Therefore, sulfasalazine may serve as a novel therapeutic agent targeting CSCs for the prevention and treatment of
CRC. However, little evidence is available regarding the relationship between sulfasalazine and pathological findings of CRCs related to UC. This study aimed to retrospectively quantify the effect of sulfasalazine on CSCs and identify the factors related to carcinogenesis in CRCs arising from UC.
Materials and methods Patients We conducted a retrospective review of the records of 31 consecutive UC-associated CRC patients who underwent curative colorectal surgery at Keio University Hospital, Tokyo, Japan, between 1998 and 2012. All data were obtained from a prospectively maintained registry of colorectal surgery and patients’ medical records. Pathological diagnosis for UC-associated cancers have been confirmed considering the presence of over-expression of p53 and mucosal inflammation around lesions [21,22]. Five patients who lacked detailed information regarding sulfasalazine administration were excluded, and the remaining 26 UCassociated CRC patients were included. We compared patients’ clinicopathological findings according to the length of sulfasalazine administration. For this purpose, patients were classified into long-term (LT) (≥ 5 years) and shortterm (ST) (< 5 years) groups. The length of term was defined as a cumulative term treated by sulfasalazine. Severity of inflammation was diagnosed by gastroenterologist according to their endoscopic findings before surgery. The institutional ethics committee approved this study.
Histopathological examination The lesions were histologically confirmed by pathologists. All histopathological specimens were fixed in 20% buffered formalin and routinely processed for embedding in paraffin and sectioning at a thickness of 4 m. Paraffin blocks of carcinomas were examined. Sections were depleted of paraffin and then rehydrated in a graded series of ethanol solutions. For histology, sections were stained with hematoxylin and eosin. For immunohistochemistry, sections were washed with phosphate-buffered saline (PBS), subjected to antigen retrieval by heating for 10 min at 100 ◦ C in 0.01 M sodium citrate buffer (pH 6.0), and exposed to 3% hydrogen peroxide before incubation with primary antibodies. Immune complexes were detected using a Vectastain Elite
Please cite this article in press as: Seishima R, et al. Sulfasalazine, a therapeutic agent for ulcerative colitis, inhibits the growth of CD44v9+ cancer stem cells in ulcerative colitis-related cancer. Clin Res Hepatol Gastroenterol (2015), http://dx.doi.org/10.1016/j.clinre.2015.11.007
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Sulfasalazine inhibits the growth of cancer stem cells
3 The number of viable cells was then determined by using Cell Titer-Glo luminescence cell viability kit (Promega) and compared with control cells exposed to dimethyl sulfoxide (DMSO).
Quantitative RT-PCR analysis
Figure 1 A representative image of high CD44v9 expression in UC-related colorectal adenocarcinoma. Scale bar: 50 m.
Kit (Vector Laboratories, Burlingame, CA, USA) and 3,3 diaminobenzidine, and the sections were counterstained with hematoxylin. A rat monoclonal antibody (mAb) specific for human CD44v9 (1:100) was used to detect CSCs as our previous publication [7,23]. Membranous CD44v9 expression was evaluated using the Allred score [24]. Briefly, the staining results were scored according to the intensity (0: undetectable; 1: weak; 2: intermediate; 3: strong) and the proportion of stained tumor cells (0: none; 1: < 1/100; 2: 1/100−1/10; 3: 1/10−1/3; 4: 1/3−2/3; 5: > 2/3). Combined scores of ≥ 3 were defined as highly expressed. Representative image of CD44v9 expression is shown in Fig. 1.
Total RNA was extracted cultured cells using RNeasy mini kit (QIAGEN) and reverse transcribed using the ReverTraAce (TOYOBO). Quantitative RT-PCR analysis was performed with a Thermal Cycler Dice Real Time System (TaKaRa Bio, Tokyo, Japan). The amplification protocol comprised an initial incubation at 95 ◦ C for 2 min and 40 cycles of 95 ◦ C for 30 s and 60 ◦ C for 30 s, followed by dissociation-curve analysis to confirm specificity. Primers used are: CD44, 5 -AGAAGGTGTGGGCAGAAGAA-3 and 5 -AAATGCACCATTTCCTGAGA-3 ; E-cadherin, 5 -AGAACGCATTGCCACATACACTC3 and 5 -CATTCTGATCGGTTACCGTGATC-3 ; CDX2, 5 TTCACTACAGTCGCTACATCACCAT-3 and 5 -TTGTTGATTTTCCTCTCCTTTGCT-3 ; GAPDH, 5 -GTGAAGGTCGGTGTGAACG-3 and 5 -GACCATGTAGTTGAGGTCAATG-3 . All quantitative data were normalized by the abundance of GAPDH mRNAs.
Statistical analysis Statistical analyses were performed using the Chi2 and Mann—Whitney U-tests. All statistical tests were two-sided, and P < 0.05 was considered statistically significant. All calculations were performed using SPSS software (SPSS Inc., Chicago, IL, USA).
Immunofluorescence analysis
Results
Tissues were processed as for immunohistochemistry but without hydrogen peroxide. The sections were incubated with appropriate Alexa Fluor® 488-conjugated or Alexa Fluor® 594-conjugated secondary antibodies (Invitrogen, Tokyo, Japan), diluted in PBS, washed thrice with PBS, and mounted in Vectashield mounting medium (Vector Laboratories). Sections were viewed and photographed using a Biorevo BZ-9000 fluorescence microscope (Keyence, Tokyo, Japan). Anti-CD44v9 (1:100) antibody and a rabbit mAb to Ki-67 (diluted 1:100; Abcam, Cambridge, UK) were used to detect proliferating CSCs. CD44v9+ cells in a randomly selected microscopic field (magnification, ×40) per section were counted using image analysis software (BZ-9000; Keyence, Tokyo, Japan), and the percentage of Ki-67+ cells was calculated. Ki-67/CD44v9 double-stained cells were defined as proliferating CSCs.
Patient demographics
Cell culture and in vitro proliferation assay HCT116, known as a CD44 highly expressing colon cancer cell line [7], was culture in Dulbecco’s Modified Eagle’s Medium (DMEM) with high glucose, supplemented with 10% FBS. Cells were maintained under 5% CO2 at 37 ◦ C. Cells were plated in 96-well plates (2 × 103 cells per well), cultured overnight, and then exposed to sulfasalazine (Sigma) with the dose of 300 mol/L for 2 days as our previous publication [25].
We analyzed the records of 26 patients who underwent surgery for UC-associated CRC (Table 1). The length of sulfasalazine administration ranged from 0 to 32 years. There were no pStage IV patients.
Association between sulfasalazine administration and histopathological findings Comparison of patient characteristics and various histopathological factors, including CD44v9 expression, between both groups is shown in Table 2. There was no significant difference in age (P = 0.60), gender (P = 0.38), or severity of the disease (P = 1.00). The median length of sulfasalazine treatment was 0.2 and 13.7 years in the ST and LT groups, respectively, (P < 0.001). In the ST group, 5 patients did not receive sulfasalazine at all. All of these patients received 5-ASA instead. In remained 8 patients, who received sulfasalazine at least 2 months, on-off was controlled according to severity of the disease. Of these, 5 patients received other agents (steroids or immunosuppressives) and 3 patients were treated only with sulfasalazine. All patients’ data concerned with the length of sulfasalazine treatment and the period from UC diagnosis to cancer diagnosis is shown in Supplement Fig. 1. There
Please cite this article in press as: Seishima R, et al. Sulfasalazine, a therapeutic agent for ulcerative colitis, inhibits the growth of CD44v9+ cancer stem cells in ulcerative colitis-related cancer. Clin Res Hepatol Gastroenterol (2015), http://dx.doi.org/10.1016/j.clinre.2015.11.007
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contrast to 46.2% in the ST group, although the difference was not statistically significant (P = 0.20).
Patient characteristics. Ulcerative colitis-associated colorectal cancer patients (n = 26)
Age (years) Sex (Male:Female) Period from ulcerative colitis diagnosis to surgery (years) Length of sulfasalazine treatment (years) Cancer location (Rt/Lt/Rec) Histology (well differentiated/others) pT (0/1/2/3/4) pN (0/1/2) pStage (0/1/2/3/4) Lymphatic invasion (absent/present) Venous invasion
a
45 (26—70) 19:7 12.5 (0.1—32)a 4.5 (0—32)a 4/16/6 16/10 5/8/3/10/0 22/2/2 5/9/8/4/0 11/15 14/12
Rt: right-side colon (cecum, ascending colon, and transverse colon); Lt: left-side colon (descending colon and sigmoid colon); Rec: rectum. a Median (min—max).
was no significant difference in use of other therapeutic agents, such as steroids (P = 1.00), immunosuppressives (P = 1.00), and anti-TNF␣ antibody. Presence of multiple lesions or dysplasia around the main tumors was similar in both groups (P = 1.00). Well-differentiated adenocarcinomas were present in a significantly higher proportion in the LT group (LT:ST = 84.6%:38.5%, P = 0.04). There was no significant difference in other pathological factors, including pT, pN, pStage, and lymphatic and venous invasion. In the LT group, 15.4% of patients expressed high CD44v9 levels, in Table 2
CD44v9+ cell proliferation in tumors Because sulfasalazine targets the ROS tolerance of CD44v9+ cells, it may affect the proliferation of these cells. We conducted immunofluorescence assays to investigate CD44v9+ cell proliferation in tumors (Fig. 2). The median proportion of proliferating CD44v9+ cells (Ki-67/CD44v9 double-stained cells) was significantly lower in the LT group than in the ST group (LT:ST = 0%:6.9%, P = 0.01, Table 2), indicating that long-term sulfasalazine administration may down-regulate the CSC proliferation in tumors.
Effects of sulfasalazine on proliferation and differentiation marker expression in CD44+ cancer cells To validate the result of clinical data, we performed in vitro assays. Given that sulfasalazine inhibits the cell proliferation of CD44+ selectively in head and neck cancer, we aimed to examine it in CD44+ colon cancer cell line. Proliferation assay revealed that the proliferation of HCT 116 cells (CD44+ cells) was significantly suppressed by sulfasalazine treatment (Fig. 3). We next investigated whether sulfasalazine has some effect on cell differentiation. The expressions of epithelial differentiation markers (E-cadherin and CDX2) in sulfasalazine treated cells were compared with control cells [26—28]. Quantitative RT-PCR results revealed that the mRNA levels in sulfasalazine treated cells were increased in both markers, concurrently with the suppression of CD44 expression (Fig. 4). These results support our clinical findings that sulfasalazine inhibits the proliferation of CD44+ cells and promotes the tumor differentiation.
Comparison of clinicopathological findings according to the length of sulfasalazine treatment.
Age (years) Male (%) Severe activity of UC (%) Length of sulfasalazine treatment (years) Steroids use (%) Immunosuppressives use (%) Anti-TNF␣ antibody use (%) Period from ulcerative colitis diagnosis to cancer diagnosis (years) Well-differentiated adenocarcinoma (%) pT 0/1 (%) pN0 (%) pStage 0/1 (%) Lymphatic invasion (present, %) Venous invasion (present, %) Multiple lesion (present, %) Dysplasia (present, %) High CD44v9 expression (%) Proportion of proliferating CD44v9 cells (%) a
Short term (n = 13)
Long term (n = 13)
P-value
39 (26—70)a 8 (61.5) 2 (15.4) 0.2 (0—4.0)a 3 (23.1) 3 (23.1) 0 (0) 5 (0.09—21)a 5 (38.5) 8 (61.5) 11 (84.6) 11 (84.6) 7 (53.8) 6 (46.2) 2 (15.4) 6 (46.2) 6 (46.2) 6.9% (0—86.3)a
46 (30—64)a 11 (84.6) 1 (7.7) 13.7 (5—32.0)a 2 (15.4) 3 (23.1) 0 (0) 14 (9—32)a 11 (84.6) 5 (38.5) 11 (84.6) 11 (84.6) 8 (61.5) 6 (46.2) 3 (23.1) 7 (53.8) 2 (15.4) 0%a
0.60 0.38 1.00 < 0.001 1.00 1.00 0.01 0.04 0.43 1.00 1.00 1.00 1.00 1.00 1.00 0.20 0.01
Median (min—max).
Please cite this article in press as: Seishima R, et al. Sulfasalazine, a therapeutic agent for ulcerative colitis, inhibits the growth of CD44v9+ cancer stem cells in ulcerative colitis-related cancer. Clin Res Hepatol Gastroenterol (2015), http://dx.doi.org/10.1016/j.clinre.2015.11.007
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Figure 3 Time course of cell proliferation for HCT116 cells treated with control (DMSO) or sulfasalazine. Data are mean ± SD from 4 independent experiments. * P < 0.01.
Figure 2 Representative images of immunofluorescence detection of CD44v9 (green) and Ki-67 (red). CD44v9+ cells with high (A) and low (B) expression of Ki-67. Scale bar: 50 m.
Discussion Herein, we show that sulfasalazine downregulates the proliferation of CSCs that express CD44v9. Additionally, our
findings implies that long-term sulfasalazine treatment was associated with a higher incidence of well-differentiated tubular adenocarcinomas in patients with UC-related CRCs. Although controversial, there are reports showing that the expression of CSC markers influences tumor differentiation. For example, Yanagisawa et al. reported that CD44v overexpression was associated with poor differentiation of gallbladder carcinomas [29], and Chen et al. reported that in gastric cancer, higher CD44 expression was associated with poorer differentiation [30]. However, the association between CSC and the histology of CRC tumors is unknown. We showed that a CSC marker (CD44v9) was associated with tumor differentiation in UC-related CRC as well and that sulfasalazine influenced the malignant phenotype. Because well-differentiated adenocarcinomas are highly sensitive to treatment and patients with these tumors have a favorable prognosis, a novel therapy that enhances tumor differentiation may be efficacious. Chronic inflammation is strongly associated with carcinogenesis in CRC. Accumulation of ROS is an important change that occurs during malignant transformation and correlates with carcinogenesis. Among several drugs with a 5-ASA
Figure 4 Epithelial differentiation marker gene expression in sulfasalazine treated HCT116 cells relative to that in control cells treated with DMSO, as determined by quantitative RT-PCR analysis. * P < 0.01, ** P < 0.05.
Please cite this article in press as: Seishima R, et al. Sulfasalazine, a therapeutic agent for ulcerative colitis, inhibits the growth of CD44v9+ cancer stem cells in ulcerative colitis-related cancer. Clin Res Hepatol Gastroenterol (2015), http://dx.doi.org/10.1016/j.clinre.2015.11.007
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moiety, sulfasalazine weakens the resistance of CSCs to ROS. For example, sulfasalazine administration influences antitumorigenicity in mouse models of gastric carcinogenesis [23]. Furthermore, using head and neck cancer cell lines, CD44v9+ tumor cells were selectively depleted, and tumor progression was suppressed by sulfasalazine administration [25]. In this study, long-term sulfasalazine administration reduced CD44v9 expression and the proportion of proliferating CD44v9+ cells, which is consistent with the results of studies of a mouse model or in vitro experiments [23,25]. Generally, under the accumulation of ROS induced by chronic inflammation, CD44v9+ CSCs expand for the purpose of self-protection [8]. In this study, however, the proliferation of CD44v9+ cells was suppressed in the LT group, which had longer disease duration and probably exposed to inflammation longer than the ST group. This fact emphasizes the effect of sulfasalazine which is targeting CD44v9+ cells. This result suggests that sulfasalazine has a negative effect on the tolerance against ROS and the proliferation of CSCs. Therefore, we consider sulfasalazine as a clinically promising agent that targets CD44v9+ CSCs via the increase of accumulated intracellular ROS. This conclusion is based on our findings that there was no difference in lymphatic or venous invasion by the tumors. Therefore, the efficacy of sulfasalazine may be limited to inhibiting carcinogenesis and not tumor progression. We previously reported that a combination of anti-epidermal growth factor and sulfasalazine therapy dramatically suppresses tumor progression [25], indicating that sulfasalazine acts as a supportive drug that specifically targets chemotherapyresistant cancer cells. Because chemotherapy was not administered before surgery to the patients studied here, further investigation addressing the effect of chemotherapy combined with sulfasalazine is required. Additional finding from our results is that sulfasalazine may influence the tumor differentiation. From our clinical data, we considered that this mechanism was due to the reduced number of CD44v9+ CSCs by longer sulfasalazine administration. From in vitro assay, we also found that the expressions of epithelial differentiation markers were promoted by sulfasalazine treatment, synchronically with the inhibition of CD44 expression. This result implies that sulfasalazine may have an effect on differentiation markers directly or indirectly through some CD44 related genes. Further investigation should be needed to clarify this molecular mechanism. This study has some limitations. Because the study was retrospective and it included a small number of patients, it is difficult to make definite conclusions. It was also difficult to precisely record dose intensity of sulfasalazine for all enrolled patients in this retrospective study. Since there is no clear evidence of effective dose and duration of sulfasalazine for influencing CD44v9 expression, pharmacodynamic investigation should be performed. Furthermore, we limited our analyses to UC-associated CRC patients, because sulfasalazine is mainly used to treat patients with IBD but not other diseases. Watanabe et al. reported that UC-associated CRC was more malignant than sporadic CRC [31]. A difference in carcinogenesis between UC-associated CRC and sporadic CRC may exist. Therefore, it is unclear whether sulfasalazine affects sporadic cancers as well. In this study, although there was no significant difference in
5-year disease-free survival (data not shown), the protective effect of sulfasalazine on postoperative recurrence should be assessed. Further studies should be conducted with a larger number of patients to arrive at a definitive conclusion. In conclusion, long-term sulfasalazine administration reduced proliferative CD44v9+ cells and had a significant impact on tumor cell differentiation. This result indicates the importance of CD44v9+ CSCs in the progression of UCrelated cancer and suggests the possibility of developing novel targets for eliminating CSCs. The feasibility of this approach is unclear and is supported by our present results.
Grant support This work was supported by grants (to H.S.) from, as well as in part by the Project for Development of Innovative Research on Cancer Therapeutics (P-Direct [to O.N.]) of, the Ministry of Education, Culture, Sports, Science, and Technology of Japan.
Disclosure of interest The authors declare that they have no competing interest.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.clinre.2015.11.007.
References [1] Coussens LM, Werb Z. Inflammation and cancer. Nature 2002;420(6917):860—7. [2] Eaden JA, Abrams KR, Mayberry JF. The risk of colorectal cancer in ulcerative colitis: a meta-analysis. Gut 2001;48(4): 526—35. [3] Federico A, Morgillo F, Tuccillo C, Ciardiello F, Loguercio C. Chronic inflammation and oxidative stress in human carcinogenesis. Int J Cancer 2007;121(11):2381—6. [4] Waris G, Ahsan H. Reactive oxygen species: role in the development of cancer and various chronic conditions. J Carcinog 2006;5:14. [5] Pelicano H, Carney D, Huang P. ROS stress in cancer cells and therapeutic implications. Drug Resist Updat 2004;7(2): 97—110. [6] Jackson AL, Loeb LA. The contribution of endogenous sources of DNA damage to the multiple mutations in cancer. Mutat Res 2001;477(1—2):7—21. [7] Ishimoto T, Nagano O, Yae T, Tamada M, Motohara T, Oshima H, et al. CD44 variant regulates redox status in cancer cells by stabilizing the xCT subunit of system xc(−) and thereby promotes tumor growth. Cancer Cell 2011;19(3):387—400. [8] Nagano O, Okazaki S, Saya H. Redox regulation in stemlike cancer cells by CD44 variant isoforms. Oncogene 2013;32(44):5191—8. [9] O’Brien CA, Pollett A, Gallinger S, Dick JE. A human colon cancer cell capable of initiating tumour growth in immunodeficient mice. Nature 2007;445(7123):106—10. [10] Ricci-Vitiani L, Lombardi DG, Pilozzi E, Biffoni M, Todaro M, Peshle C, et al. Identification and expansion of human coloncancer-initiating cells. Nature 2007;445(7123):111—5.
Please cite this article in press as: Seishima R, et al. Sulfasalazine, a therapeutic agent for ulcerative colitis, inhibits the growth of CD44v9+ cancer stem cells in ulcerative colitis-related cancer. Clin Res Hepatol Gastroenterol (2015), http://dx.doi.org/10.1016/j.clinre.2015.11.007
+Model CLINRE-834; No. of Pages 7
ARTICLE IN PRESS
Sulfasalazine inhibits the growth of cancer stem cells [11] Choi D, Lee HW, Hur KY, Kim JJ, Park GS, Jang SH, et al. Cancer stem cell markers CD133 and CD24 correlate with invasiveness and differentiation in colorectal adenocarcinoma. World J Gastroenterol 2009;15(18):2258—64. [12] Bendardaf R, Elzagheid A, Lamlum H, Ristamaki R, Collan Y, Pyrhonen S. E-cadherin. CD44s and CD44v6 correlate with tumour differentiation in colorectal cancer. Oncol Rep 2005;13(5):831—5. [13] Tanabe KK, Ellis LM, Saya H. Expression of CD44R1 adhesion molecule in colon carcinomas and metastases. Lancet 1993;341(8847):725—6. [14] Mayer B, Jauch KW, Gunthert U, Figdor CG, Schildberg FW, Funke I, et al. De-novo expression of CD44 and survival in gastric cancer. Lancet 1993;342(8878):1019—22. [15] Miwa T, Watanabe A, Yamada Y, Shino Y, Yamada T, Yamashita J, et al. Progression in gastric carcinoma relative to the ratio of CD44 epithelial variant transcript to CD44 hematopoietic variant transcript. Cancer 1996;77(1):25—9. [16] van Staa TP, Card T, Logan RF, Leufkens HG. 5-aminosalicylate use and colorectal cancer risk in inflammatory bowel disease: a Large Epidemiological Study. Gut 2005;54(11):1573—8. [17] Velayos FS, Terdiman JP, Walsh JM. Effect of 5-aminosalicylate use on colorectal cancer and dysplasia risk: a systematic review and metaanalysis of observational studies. Am J Gastroenterol 2005;100(6):1345—53. [18] Chen RS, Song YM, Zhou ZY, Tong T, Li Y, Fu M, et al. Disruption of xCT inhibits cancer cell metastasis via the caveolin-1/betacatenin pathway. Oncogene 2009;28(4):599—609. [19] Gout PW, Buckley AR, Simms CR, Bruchovsky N. Sulfasalazine, a potent suppressor of lymphoma growth by inhibition of the x(c)− cystine transporter: a new action for an old drug. Leukemia 2001;15(10):1633—40. [20] Lo M, Wang YZ, Gout PW. The x(c)− cystine/glutamate antiporter: a potential target for therapy of cancer and other diseases. J Cell Physiol 2008;215(3):593—602. [21] Brentnall TA, Crispin DA, Rabinovitch PS, Haggitt RC, Rubin CE, Stevens AC, et al. Mutations in the p53 gene: an early marker of neoplastic progression in ulcerative colitis. Gastroenterology 1994;107:369—78.
7 [22] Yin J, Harpaz N, Tong Y, Huang Y, Laurin J, Greenwald BD, et al. p53 point mutations in dysplastic and cancerous ulcerative colitis lesions. Gastroenterology 1993;104:1633—9. [23] Wada T, Ishimoto T, Seishima R, Tsuchihashi K, Yoshikawa M, Oshima H, et al. Functional role of CD44v-xCT system in the development of spasmolytic polypeptide-expressing metaplasia. Cancer Sci 2013;104(10):1323—9. [24] Allred DC, Harvey JM, Berardo M, Clark GM. Prognostic and predictive factors in breast cancer by immunohistochemical analysis. Mod Pathol 1998;11(2):155—68. [25] Yoshikawa M, Tsuchihashi K, Ishimoto T, Yae T, Motohara T, Sugihara E, et al. xCT inhibition depletes CD44v-expressing tumor cells that are resistant to EGFR-targeted therapy in head and neck squamous cell carcinoma. Cancer Res 2013;73(6):1855—66. [26] Keiichiro S, Masahiro A, Reiji K. Transcription factors c-Myc and CDX2 mediate E-selectin ligand expression in colon cancer cells undergoing EGF/bFGF-induced epithelial—mesenchymal transition. PNAS 2012;109(20):7776—81. [27] Mizoshita T, Inada K, Tsukamoto T, Kodera Y, Yamamura Y, Hirai T, et al. Expression of Cdx1 and Cdx2 mRNAs and relevance of this expression to differentiation in human gastrointestinal mucosa. Gastric Cancer 2001;4(4):185—91. [28] Guo RJ, Suh ER, Lynch JP. The role of Cdx proteins in intestinal development and cancer. Cancer Biol Ther 2004;3(7): 593—601. [29] Yanagisawa N, Mikami T, Mitomi H, Saegusa M, Koike M, Okayasu I. CD44 variant overexpression in gallbladder carcinoma associated with tumor dedifferentiation. Cancer 2001;91(2): 408—16. [30] Chen S, Hou JH, Feng XY, Zhang XS, Zhou ZW, Yun JP, et al. Clinicopathologic significance of putative stem cell marker, CD44 and CD133, in human gastric carcinoma. J Surg Oncol 2013;107(8):799—806. [31] Watanabe T, Konishi T, Kishimoto J, Kotake K, Muto T, Sugihara, et al. Ulcerative colitis-associated colorectal cancer shows a poorer survival than sporadic colorectal cancer: a Nationwide Japanese Study. Inflamm Bowel Dis 2011;17(3):802—8.
Please cite this article in press as: Seishima R, et al. Sulfasalazine, a therapeutic agent for ulcerative colitis, inhibits the growth of CD44v9+ cancer stem cells in ulcerative colitis-related cancer. Clin Res Hepatol Gastroenterol (2015), http://dx.doi.org/10.1016/j.clinre.2015.11.007