Single-chain Antibody Against Reg4 Suppresses Gastric Cancer Cell Growth and Enhances 5-FU-induced Cell Death in vitro

Author(s): Xue-Qing Zhang, Lu-Ting Yu, Pei Du, Tian-Qi Yin, Zhi-Yuan Zhang, Ying Xu, Xiang Li, You-Jie Li, Min Wang*, Chen Luo*.

Journal Name: Anti-Cancer Agents in Medicinal Chemistry
(Formerly Current Medicinal Chemistry - Anti-Cancer Agents)

Volume 19 , Issue 5 , 2019

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Graphical Abstract:


Background: Regenerating islet-derived gene family member 4 (Reg4), a well-investigated growth factor in the regenerative pancreas, has recently been reported to be highly associated with a majority of gastrointestinal cancers. Pathological hyper-expression or artificial over-expression of Reg4 causes acceleration of tumor growth, migration, and resistance to chemotherapeutic 5-Fluorouracil (5-FU). Until now, no method has been successfully established for eliminating the effects of Reg4 protein.

Methods: This study reports the production of an engineered immunoglobin, a single-chain variable fragment (scFv-Reg4), to specifically bind Reg4 and block the bioactivity. The complementary-determining regions (CDRs) against Reg4 were assigned using MOE and ZDOCK servers. The binding affinity (KD) was determined by bio-layer interferometry (BLI). MKN45 and AGS cell proliferation was determined by Thiazolyl blue tetrazolium bromide (MTT) method and the cell apoptosis was detected by flow cytometry assay.

Results: The KD of scFv-Reg4 to Reg4 was determined to be 1.91×10-8. In MKN45 and AGS cell lines, scFv- Reg4 depressed Reg4-stimulated cell proliferation and the inhibitory rates were 27.7±1.5% and 17.3±2.6%, respectively. Furthermore, scFv significantly enhanced 5-FU-induced cell death, from 23.0±1.0% to 28.4±1.2% in MKN45 and 28.2±0.7% to 36.6±0.6% in AGS cells. Treatment with scFv alone could lyse cancer cells to a certain extent, but no significance has been observed.

Conclusion: The single-chain antibody (scFv-Reg4) significantly inhibited gastric cancer cell proliferation and synergistically enhanced the lethal effect of 5-FU. Thus, traditional chemo-/radio- therapeutics supplemented with scFv-Reg4 may provide advances in the strategy for gastrointestinal cancer treatment.

Keywords: Gastrointestinal cancers, Reg4 protein, 5-FU, cell proliferation, phage library, single-chain antibody.

Ali, I.; Lone, M.N.; Al-Othman, Z.A.; Al-Warthan, A.; Sanagi, M.M. Heterocyclic scaffolds: Centrality in anticancer drug development. Curr. Drug Targets, 2015, 16, 711-734.
Morawska, K.; Goirand, F.; Marceau, L.; Devaux, M.; Cueff, A.; Bertaut, A.; Vincent, J.; Bengrine-Lefevre, L.; Ghiringhelli, F.; Schmitt, A. 5-FU therapeutic drug monitoring as a valuable option to reduce toxicity in patients with gastrointestinal cancer. Oncotarget, 2018, 9, 11559-11571.
Ali, I.; Wani, W.A.; Saleem, K.; Haque, A. Platinum compounds: a hope for future cancer chemotherapy. Anticancer. Agents Med. Chem., 2013, 13, 296-306.
Saleem, K.; Wani, W.; Haque, A.; Milhotra, A.; Ali, I. Nanodrugs: Magic bullets in cancer chemotherapy. Topics Anti-Cancer Res., 2013, 58(2), 437-494.
Wang, H.; Hu, L.; Zang, M.; Zhang, B.; Duan, Y.; Fan, Z.; Li, J.; Su, L.; Yan, M.; Zhu, Z.; Liu, B.; Yang, Q. REG4 promotes peritoneal metastasis of gastric cancer through GPR37. Oncotarget, 2016, 7, 27874-27888.
Kawasaki, Y.; Matsumura, K.; Miyamoto, M.; Tsuji, S.; Okuno, M.; Suda, S.; Hiyoshi, M.; Kitayama, J.; Akiyama, T. REG4 is a transcriptional target of GATA6 and is essential for colorectal tumorigenesis. Sci. Rep., 2015, 5, 14291.
Lehtinen, L.; Vesterkvist, P.; Roering, P.; Korpela, T.; Hattara, L.; Kaipio, K.; Mpindi, J.P.; Hynninen, J.; Auranen, A.; Davidson, B.; Haglund, C.; Iljin, K.; Grenman, S.; Siitari, H.; Carpen, O. REG4 Is highly expressed in mucinous ovarian cancer: A potential novel serum biomarker. PLoS One, 2016, 11e0151590
He, X.J.; Jiang, X.T.; Ma, Y.Y.; Xia, Y.J.; Wang, H.J.; Guan, T.P.; Shao, Q.S.; Tao, H.Q. REG4 contributes to the invasiveness of pancreatic cancer by upregulating MMP-7 and MMP-9. Cancer Sci., 2012, 103, 2082-2091.
Huang, Q.; Chen, X.; Lu, W.; Lai, M.; Lu, B. Expression of REG4 in ovarian mucinous tumors. Appl. Immunohistochem. Mol. Morphol., 2014, 22, 295-301.
Katsuno, Y.; Ehata, S.; Yashiro, M.; Yanagihara, K.; Hirakawa, K.; Miyazono, K. Coordinated expression of REG4 and aldehyde dehydrogenase 1 regulating tumourigenic capacity of diffuse-type gastric carcinoma-initiating cells is inhibited by TGF-beta. J. Pathol., 2012, 228, 391-404.
Li, Q.; Wang, H.; Zogopoulos, G.; Shao, Q.; Dong, K.; Lv, F.; Nwilati, K.; Gui, X.Y.; Cuggia, A.; Liu, J.L.; Gao, Z.H. Reg proteins promote acinar-to-ductal metaplasia and act as novel diagnostic and prognostic markers in pancreatic ductal adenocarcinoma. Oncotarget, 2016, 7(47), 77838.
Zhu, X.; Han, Y.; Yuan, C.; Tu, W.; Qiu, G.; Lu, S.; Lu, H.; Peng, Z.; Zhou, C. Overexpression of Reg4, alone or combined with MMP-7 overexpression, is predictive of poor prognosis in colorectal cancer. Oncol. Rep., 2015, 33, 320-328.
Bishnupuri, K.S.; Luo, Q.; Murmu, N.; Houchen, C.W.; Anant, S.; Dieckgraefe, B.K. Reg IV activates the epidermal growth factor receptor/Akt/AP-1 signaling pathway in colon adenocarcinomas. Gastroenterology, 2006, 130, 137-149.
Kadowaki, Y.; Ishihara, S.; Miyaoka, Y.; Rumi, M.A.; Sato, H.; Kazumori, H.; Adachi, K.; Takasawa, S.; Okamoto, H.; Chiba, T.; Kinoshita, Y. Reg protein is overexpressed in gastric cancer cells, where it activates a signal transduction pathway that converges on ERK1/2 to stimulate growth. FEBS Lett., 2002, 530, 59-64.
Hu, G.; Shen, J.; Cheng, L.; Guo, C.; Xu, X.; Wang, F.; Huang, L.; Yang, L.; He, M.; Xiang, D.; Zhu, S.; Wu, M.; Yu, Y.; Han, W.; Wang, X. Reg4 protects against acinar cell necrosis in experimental pancreatitis. Gut, 2011, 60, 820-828.
Mitani, Y.; Oue, N.; Matsumura, S.; Yoshida, K.; Noguchi, T.; Ito, M.; Tanaka, S.; Kuniyasu, H.; Kamata, N.; Yasui, W. Reg IV is a serum biomarker for gastric cancer patients and predicts response to 5-fluorouracil-based chemotherapy. Oncogene, 2007, 26, 4383-4393.
Ying, L.S.; Yu, J.L.; Lu, X.X.; Ling, Z.Q. Enhanced RegIV expression predicts the intrinsic 5-fluorouracil (5-FU) resistance in advanced gastric cancer. Dig. Dis. Sci., 2013, 58, 414-422.
Rahbarnia, L.; Farajnia, S.; Babaei, H.; Majidi, J.; Veisi, K.; Tanomand, A.; Akbari, B. Invert biopanning: A novel method for efficient and rapid isolation of scFvs by phage display technology. Biologicals, 2016, 44, 567-573.
Shaw, I.; Kane, M. The isolation of scFvs against small target molecules. Methods Mol. Biol., 2009, 562, 83-97.
Ali, I.; Wani, W.A.; Saleem, K.; Hseih, M.F. Design and synthesis of thalidomide based dithiocarbamate Cu(II), Ni(II) and Ru(III) complexes as anticancer agents. Polyhedron, 2013, 56, 134-143.
Yu, L.T.; Yang, M.Q.; Liu, J.L.; Alfred, M.O.; Li, X.; Zhang, X.Q.; Zhang, J.; Wu, M.Y.; Wang, M.; Luo, C. Recombinant Reg3alpha protein protects against experimental acute pancreatitis in mice. Mol. Cell. Endocrinol., 2016, 422, 150-159.
Vanderlaag, K.; Wang, W.; Fayadat-Dilman, L.; Wagner, J.; Bald, L.; Grein, J.; Janatpour, M.J. Regenerating islet-derived family member, 4 modulates multiple receptor tyrosine kinases and mediators of drug resistance in cancer. Int. J. Cancer, 2012, 130, 1251-1263.
Duan, Y.; Hu, L.; Liu, B.; Yu, B.; Li, J.; Yan, M.; Yu, Y.; Li, C.; Su, L.; Zhu, Z.; Xiang, M.; Yang, Q. Tumor suppressor miR-24 restrains gastric cancer progression by downregulating RegIV. Mol. Cancer, 2014, 13, 127.
Luo, D.; Mah, N.; Krantz, M.; Wishart, D.; Jacobs, F.; Martin, L. High level secretion of single-chain antibody in pichia expression system. Biotechnol. Tech., 1997, 11, 759-761.
Miao, X.; Li, A.; Chen, W.; Qi, H.; Qiu, Z.; Zhang, Y.; Zhang, J.; Wang, M. Optimization and modification of anti-rhTNF-alpha single chain variable fragment antibody: Effective in vitro affinity maturation and functional expression of chimeric Fab. Biomed. Pharmacother., 2013, 67, 437-444.
Wu, Q.; Zhang, J.; Luo, C.; Zhang, T.; Wang, T.; Wang, M. Expression and characterization of recombinant interleukin-21 receptor and its targeting single-chain variable fragment antibodies selected from a human phage display library. DNA Cell Biol., 2012, 31, 1541-1548.
Konishi, H.; Matsumoto, S.; Namikawa, K.; Kiyama, H. N-terminal cleaved pancreatitis-associated protein-III (PAP-III) serves as a scaffold for neurites and promotes neurite outgrowth. J. Biol. Chem., 2013, 288, 10205-10213.
Ma, X.; Wu, D.; Zhou, S.; Wan, F.; Liu, H.; Xu, X.; Zhao, Y.; Tang, M. The pancreatic cancer secreted REG4 promotes macrophage polarization to M2 through EGFR/AKT/CREB pathway. Oncol. Rep., 2016, 35, 189-196.
Bishnupuri, K.S.; Sainathan, S.K.; Bishnupuri, K.; Leahy, D.R.; Luo, Q.; Anant, S.; Houchen, C.W.; Dieckgraefe, B.K. Reg4-induced mitogenesis involves Akt-GSK3beta-beta-Catenin-TCF-4 signaling in human colorectal cancer. Mol. Carcinog., 2014, 53, E169-E180.
Bishnupuri, K.S.; Luo, Q.; Sainathan, S.K.; Kikuchi, K.; Sureban, S.M.; Sabarinathan, M.; Gross, J.H.; Aden, K.; May, R.; Houchen, C.W.; Anant, S.; Dieckgraefe, B.K. Reg IV regulates normal intestinal and colorectal cancer cell susceptibility to radiation-induced apoptosis. Gastroenterology, 2010, 138, 616-626.
He, H.L.; Lee, Y.E.; Shiue, Y.L.; Lee, S.W.; Lin, L.C.; Chen, T.J.; Wu, T.F.; Hsing, C.H.; Huang, H.Y.; Wang, J.Y.; Li, C.F. Overexpression of REG4 confers an independent negative prognosticator in rectal cancers receiving concurrent chemoradiotherapy. J. Surg. Oncol., 2014, 110, 1002-1010.
Kobayashi, Y.; Niwa, Y.; Tajika, M.; Kawai, H.; Kondo, S.; Hara, K.; Mizuno, N.; Hijioka, S.; Sawaki, A.; Matsuo, K.; Nakagawa, H.; Nakamura, Y.; Yamao, K. Serum tumor antigen REG4 as a useful diagnostic biomarker in gastric cancer. Hepatogastroenterology, 2010, 57, 1631-1634.
Takayama, R.; Nakagawa, H.; Sawaki, A.; Mizuno, N.; Kawai, H.; Tajika, M.; Yatabe, Y.; Matsuo, K.; Uehara, R.; Ono, K.; Nakamura, Y.; Yamao, K. Serum tumor antigen REG4 as a diagnostic biomarker in pancreatic ductal adenocarcinoma. J. Gastroenterol., 2010, 45, 52-59.
Oue, N.; Noguchi, T.; Anami, K.; Sentani, K.; Sakamoto, N.; Uraoka, N.; Wakamatsu, Y.; Sasaki, H.; Yasui, W. Serum concentration and expression of Reg IV in patients with esophageal cancer: Age-related elevation of serum Reg IV concentration. Oncol. Lett., 2011, 2, 235-239.
Tao, H.Q.; He, X.J.; Ma, Y.Y.; Wang, H.J.; Xia, Y.J.; Ye, Z.Y.; Zhao, Z.S. Evaluation of REG4 for early diagnosis and prognosis of gastric cancer. Hum. Pathol., 2011, 42, 1401-1409.
Oue, N.; Sentani, K.; Noguchi, T.; Ohara, S.; Sakamoto, N.; Hayashi, T.; Anami, K.; Motoshita, J.; Ito, M.; Tanaka, S.; Yoshida, K.; Yasui, W. Serum olfactomedin 4 (GW112, hGC-1) in combination with Reg IV is a highly sensitive biomarker for gastric cancer patients. Int. J. Cancer, 2009, 125, 2383-2392.

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Year: 2019
Page: [610 - 619]
Pages: 10
DOI: 10.2174/1871520619666181122104720
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