Apigenin inhibits growth and induces apoptosis in human cholangiocarcinoma cells

Subhasitanont,Pantipa; Chokchaichamnankit,Daranee; Chiablaem,Khajeelak; Keeratichamroen,Siriporn; Ngiwsara,Lukana; Paricharttanakul,N.  Monique; Lirdprapamongkol,Kriengsak; Weeraphan,Churat; Svasti,Jisnuson; Srisomsap,Chantragan

doi:10.3892/ol.2017.6705

Apigenin inhibits growth and induces apoptosis in human cholangiocarcinoma cells

Authors:
- Pantipa Subhasitanont
- Daranee Chokchaichamnankit
- Khajeelak Chiablaem
- Siriporn Keeratichamroen
- Lukana Ngiwsara
- N. Monique Paricharttanakul
- Kriengsak Lirdprapamongkol
- Churat Weeraphan
- Jisnuson Svasti
- Chantragan Srisomsap
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Affiliations: Laboratory of Biochemistry, Chulabhorn Research Institute, Bangkok 10210, Thailand
Published online on: August 2, 2017 https://doi.org/10.3892/ol.2017.6705
Pages: 4361-4371

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Abstract

A promising nutraceutical, apigenin, was recently revealed to exhibit biological activity in inhibiting several types of cancer. The effects of apigenin on the growth inhibition and apoptosis of the cholangiocarcinoma HuCCA‑1 cell line were investigated. Protein alterations subsequent to apigenin treatment were studied using a proteomic approach. The values of 20, 50 and 90% inhibition of cell growth (IC20, IC50 and IC90) were determined by MTT cell viability assay. Apoptotic cell death was detected using two different methods, a flow cytometric analysis (Muse Cell Analyzer) and DNA fragmentation assay. A number of conditions including attached and detached cells were selected to perform two-dimensional gel electrophoresis (2‑DE) to study the alterations in the expression levels of treated and untreated proteins and identified by liquid chromatography (LC)/tandem mass spectrometry (MS/MS). The IC20, IC50 and IC90 values of apigenin after 48 h treatment in HuCCA‑1 cells were 25, 75 and 200 µM, respectively, indicating the cytotoxicity of this compound. Apigenin induced cell death in HuCCA‑1 cells via apoptosis as detected by flow cytometric analysis and exhibited, as confirmed with DNA fragmentation, characteristics of apoptotic cells. A total of 67 proteins with altered expression were identified from the 2‑DE analysis and LC/MS/MS. The cleavage of proteins involved in cytoskeletal, cytokeratin 8, 18 and 19, and high expression of S100‑A6 and S100‑A11 suggested that apoptosis was induced by apigenin via the caspase‑dependent pathway. Notably, two proteins, heterogeneous nuclear ribonucleoprotein H and A2/B1, disappeared completely subsequent to treatment, suggesting the role of apigenin in inducing cell death. The present study indicated that apigenin demonstrates an induction of growth inhibition and apoptosis in cholangiocarcinoma cells and the apoptosis pathway was confirmed by proteomic analysis.

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1	Kalra EK: Nutraceutical-definition and introduction. AAPS PharmSci. 5:E252003. View Article : Google Scholar : PubMed/NCBI
2	Manach C, Scalbert A, Morand C, Rémésy C and Jiménez L: Polyphenols: Food sources and bioavailability. Am J Clin Nutr. 79:727–747. 2004.PubMed/NCBI
3	Birt DF, Walker B, Tibbels MG and Bresnick E: Anti-mutagenesis and anti-promotion by apigenin, robinetin and indole-3-carbinol. Carcinogenesis. 7:959–963. 1986. View Article : Google Scholar : PubMed/NCBI
4	Shukla S and Gupta S: Apigenin: A promising molecule for cancer prevention. Pharm Res. 27:962–978. 2010. View Article : Google Scholar : PubMed/NCBI
5	Khan SA, Toledano MB and Taylor-Robinson SD: Epidemiology, risk factors, and pathogenesis of cholangiocarcinoma. HPB (Oxford). 10:77–82. 2008. View Article : Google Scholar : PubMed/NCBI
6	Sonakul D, Koompirochana C, Chinda K and Stitnimakarn T: Hepatic carcinoma with opisthorchiasis. Southeast Asian J Trop Med Public Health. 9:215–219. 1978.PubMed/NCBI
7	Kobayashi M, Ikeda K, Saitoh S, Suzuki F, Tsubota A, Suzuki Y, Arase Y, Murashima N, Chayama K and Kumada H: Incidence of primary cholangiocellular carcinoma of the liver in japanese patients with hepatitis C virus-related cirrhosis. Cancer. 88:2471–2477. 2000. View Article : Google Scholar : PubMed/NCBI
8	Yamamoto S, Kubo S, Hai S, Uenishi T, Yamamoto T, Shuto T, Takemura S, Tanaka H, Yamazaki O, Hirohashi K and Tanaka T: Hepatitis C virus infection as a likely etiology of intrahepatic cholangiocarcinoma. Cancer Sci. 95:592–595. 2004. View Article : Google Scholar : PubMed/NCBI
9	Shaib YH, El-Serag HB, Davila JA, Morgan R and McGlynn KA: Risk factors of intrahepatic cholangiocarcinoma in the United States: A case-control study. Gastroenterology. 128:620–626. 2005. View Article : Google Scholar : PubMed/NCBI
10	Sorensen HT, Friis S, Olsen JH, Thulstrup AM, Mellemkjaer L, Linet M, Trichopoulos D, Vilstrup H and Olsen J: Risk of liver and other types of cancer in patients with cirrhosis: A nationwide cohort study in Denmark. Hepatology. 28:921–925. 1998. View Article : Google Scholar : PubMed/NCBI
11	Srisomsap C, Sawangareetrakul P, Subhasitanont P, Panichakul T, Keeratichamroen S, Lirdprapamongkol K, Chokchaichamnankit D, Sirisinha S and Svasti J: Proteomic analysis of cholangiocarcinoma cell line. Proteomics. 4:1135–1144. 2004. View Article : Google Scholar : PubMed/NCBI
12	Srisomsap C, Subhasitanont P, Sawangareetrakul P, Chokchaichamnankit D, Ngiwsara L, Chiablaem K and Svasti J: Comparison of membrane-associated proteins in human cholangiocarcinoma and hepatocellular carcinoma cell lines. Proteomics Clin Appl. 1:89–106. 2007. View Article : Google Scholar : PubMed/NCBI
13	Elmore S: Apoptosis: A review of programmed cell death. Toxicol Pathol. 35:495–516. 2007. View Article : Google Scholar : PubMed/NCBI
14	Martin SJ and Green DR: Protease activation during apoptosis: Death by a thousand cuts? Cell. 82:349–352. 1995. View Article : Google Scholar : PubMed/NCBI
15	Thornberry NA and Lazebnik Y: Caspases: Enemies within. Science. 281:1312–1316. 1998. View Article : Google Scholar : PubMed/NCBI
16	Jaattela M: Escaping cell death: Survival proteins in cancer. Exp Cell Res. 248:30–43. 1999. View Article : Google Scholar : PubMed/NCBI
17	Levy-Strumpf N and Kimchi A: Death associated proteins (DAPs): From gene identification to the analysis of their apoptotic and tumor suppressive functions. Oncogene. 17:3331–3340. 1998. View Article : Google Scholar : PubMed/NCBI
18	Brockstedt E, Rickers A, Kostka S, Laubersheimer A, Dörken B, Wittmann-Liebold B, Bommert K and Otto A: Identification of apoptosis-associated proteins in a human Burkitt lymphoma cell line. Cleavage of heterogeneous nuclear ribonucleoprotein A1 by caspase 3. J Biol Chem. 273:28057–28064. 1998. View Article : Google Scholar : PubMed/NCBI
19	Prasad SC, Soldatenkov VA, Kuettel MR, Thraves PJ, Zou X and Dritschilo A: Protein changes associated with ionizing radiation-induced apoptosis in human prostate epithelial tumor cells. Electrophoresis. 20:1065–1074. 1999. View Article : Google Scholar : PubMed/NCBI
20	Gupta S, Afaq F and Mukhtar H: Selective growth-inhibitory, cell-cycle deregulatory and apoptotic response of apigenin in normal versus human prostate carcinoma cells. Biochem Biophys Res Commun. 287:914–920. 2001. View Article : Google Scholar : PubMed/NCBI
21	Choi EJ and Kim GH: Apigenin induces apoptosis through a mitochondria/caspase-pathway in human breast cancer MDA-MB-453 cells. J Clin Biochem Nutr. 44:260–265. 2009. View Article : Google Scholar : PubMed/NCBI
22	Xu Y, Xin Y, Diao Y, Lu C, Fu J, Luo L and Yin Z: Synergistic effects of apigenin and paclitaxel on apoptosis of cancer cells. PLoS One. 6:e291692011. View Article : Google Scholar : PubMed/NCBI
23	Khan A, Gillis K, Clor J and Tyagarajan K: Simplified evaluation of apoptosis using the Muse cell analyzer. Postepy Biochem. 58:492–496. 2012.PubMed/NCBI
24	Svasti J, Srisomsap C, Subhasitanont P, Keeratichamroen S, Chokchaichamnankit D, Ngiwsara L, Chimnoi N, Pisutjaroenpong S, Techasakul S and Chen ST: Proteomic profiling of cholangiocarcinoma cell line treated with pomiferin from Derris malaccensis. Proteomics. 5:4504–4509. 2005. View Article : Google Scholar : PubMed/NCBI
25	Ruela-de-Sousa RR, Fuhler GM, Blom N, Ferreira CV, Aoyama H and Peppelenbosch MP: Cytotoxicity of apigenin on leukemia cell lines: Implications for prevention and therapy. Cell Death Dis. 1:e192010. View Article : Google Scholar : PubMed/NCBI
26	Vargo MA, Voss OH, Poustka F, Cardounel AJ, Grotewold E and Doseff AI: Apigenin-induced-apoptosis is mediated by the activation of PKCdelta and caspases in leukemia cells. Biochem Pharmacol. 72:681–692. 2006. View Article : Google Scholar : PubMed/NCBI
27	Creagh EM, Conroy H and Martin SJ: Caspase-activation pathways in apoptosis and immunity. Immunol Rev. 193:10–21. 2003. View Article : Google Scholar : PubMed/NCBI
28	Caulin C, Salvesen GS and Oshima RG: Caspase cleavage of keratin 18 and reorganization of intermediate filaments during epithelial cell apoptosis. J Cell Biol. 138:1379–1394. 1997. View Article : Google Scholar : PubMed/NCBI
29	Leers MP, Kolgen W, Björklund V, Bergman T, Tribbick G, Persson B, Björklund P, Ramaekers FC, Björklund B, Nap M, et al: Immunocytochemical detection and mapping of a cytokeratin 18 neo-epitope exposed during early apoptosis. J Pathol. 187:567–572. 1999. View Article : Google Scholar : PubMed/NCBI
30	Schutte B, Henfling M, Kölgen W, Bouman M, Meex S, Leers MP, Nap M, Björklund V, Björklund P, Björklund B, et al: Keratin 8/18 breakdown and reorganization during apoptosis. Exp Cell Res. 297:11–26. 2004. View Article : Google Scholar : PubMed/NCBI
31	Mitamura S, Ikawa H, Mizuno N, Kaziro Y and Itoh H: Cytosolic nuclease activated by caspase-3 and inhibited by DFF-45. Biochem Biophys Res Commun. 243:480–484. 1998. View Article : Google Scholar : PubMed/NCBI
32	Joo JH, Yoon SY, Kim JH, Paik SG, Min SR, Lim JS, Choe IS, Choi I and Kim JW: S100A6 (calcyclin) enhances the sensitivity to apoptosis via the upregulation of caspase-3 activity in Hep3B cells. J Cell Biochem. 103:1183–1197. 2008. View Article : Google Scholar : PubMed/NCBI
33	Makino E, Sakaguchi M, Iwatsuki K and Huh NH: Introduction of an N-terminal peptide of S100C/A11 into human cells induces apoptotic cell death. J Mol Med (Berl). 82:612–620. 2004. View Article : Google Scholar : PubMed/NCBI
34	Hamada S, Satoh K, Hirota M, Kanno A, Ishida K, Umino J, Ito H, Kikuta K, Kume K, Masamune A, et al: Calcium-binding protein S100P is a novel diagnostic marker of cholangiocarcinoma. Cancer Sci. 102:150–156. 2011. View Article : Google Scholar : PubMed/NCBI
35	Nakanuma Y and Sato Y: Hilar cholangiocarcinoma is pathologically similar to pancreatic duct adenocarcinoma: Suggestions of similar background and development. J Hepatobiliary Pancreat Sci. 21:441–447. 2014. View Article : Google Scholar : PubMed/NCBI
36	Wu Z, Boonmars T, Nagano I, Boonjaraspinyo S, Srinontong P, Ratasuwan P, Narong K, Nielsen PS and Maekawa Y: Significance of S100P as a biomarker in diagnosis, prognosis and therapy of opisthorchiasis-associated cholangiocarcinoma. Int J Cancer. 138:396–408. 2016. View Article : Google Scholar : PubMed/NCBI
37	Noolu B, Ajumeera R, Chauhan A, Nagalla B, Manchala R and Ismail A: Murraya koenigii leaf extract inhibits proteasome activity and induces cell death in breast cancer cells. BMC Complement Altern Med. 13:72013. View Article : Google Scholar : PubMed/NCBI
38	Beyer AL, Christensen ME, Walker BW and LeStourgeon WM: Identification and characterization of the packaging proteins of core 40S hnRNP particles. Cell. 11:127–138. 1977. View Article : Google Scholar : PubMed/NCBI
39	Cooper TA, Wan L and Dreyfuss G: RNA and disease. Cell. 136:777–793. 2009. View Article : Google Scholar : PubMed/NCBI
40	Arango D, Morohashi K, Yilmaz A, Kuramochi K, Parihar A, Brahimaj B, Grotewold E and Doseff AI: Molecular basis for the action of a dietary flavonoid revealed by the comprehensive identification of apigenin human targets. Proc Natl Acad Sci USA. 110:E2153–E2162. 2013. View Article : Google Scholar : PubMed/NCBI
41	Lindquist S and Craig EA: The heat-shock proteins. Annu Rev Genet. 22:631–677. 1988. View Article : Google Scholar : PubMed/NCBI
42	Calderwood SK and Ciocca DR: Heat shock proteins: Stress proteins with Janus-like properties in cancer. Int J Hyperthermia. 24:31–39. 2008. View Article : Google Scholar : PubMed/NCBI
43	Takayama S, Reed JC and Homma S: Heat-shock proteins as regulators of apoptosis. Oncogene. 22:9041–9047. 2003. View Article : Google Scholar : PubMed/NCBI
44	Kubota H, Yamamoto S, Itoh E, Abe Y, Nakamura A, Izumi Y, Okada H, Iida M, Nanjo H, Itoh H and Yamamoto Y: Increased expression of co-chaperone HOP with HSP90 and HSC70 and complex formation in human colonic carcinoma. Cell Stress Chaperones. 15:1003–1011. 2010. View Article : Google Scholar : PubMed/NCBI
45	Sun W, Xing B, Sun Y, Du X, Lu M, Hao C, Lu Z, Mi W, Wu S, Wei H, et al: Proteome analysis of hepatocellular carcinoma by two-dimensional difference gel electrophoresis: Novel protein markers in hepatocellular carcinoma tissues. Mol Cell Proteomics. 6:1798–1808. 2007. View Article : Google Scholar : PubMed/NCBI
46	Erlich RB, Kahn SA, Lima FR, Muras AG, Martins RA, Linden R, Chiarini LB, Martins VR and Moura Neto V: STI1 promotes glioma proliferation through MAPK and PI3K pathways. Glia. 55:1690–1698. 2007. View Article : Google Scholar : PubMed/NCBI
47	Wang TH, Chao A, Tsai CL, Chang CL, Chen SH, Lee YS, Chen JK, Lin YJ, Chang PY, Wang CJ, et al: Stress-induced phosphoprotein 1 as a secreted biomarker for human ovarian cancer promotes cancer cell proliferation. Mol Cell Proteomics. 9:1873–1884. 2010. View Article : Google Scholar : PubMed/NCBI
48	Tanioka T, Nakatani Y, Semmyo N, Murakami M and Kudo I: Molecular identification of cytosolic prostaglandin E2 synthase that is functionally coupled with cyclooxygenase-1 in immediate prostaglandin E2 biosynthesis. J Biol Chem. 275:32775–32782. 2000. View Article : Google Scholar : PubMed/NCBI
49	Simpson NE, Lambert WM, Watkins R, Giashuddin S, Huang SJ, Oxelmark E, Arju R, Hochman T, Goldberg JD, Schneider RJ, et al: High levels of Hsp90 cochaperone p23 promote tumor progression and poor prognosis in breast cancer by increasing lymph node metastases and drug resistance. Cancer Res. 70:8446–8456. 2010. View Article : Google Scholar : PubMed/NCBI
50	Gausdal G, Gjertsen BT, Fladmark KE, Demol H, Vandekerckhove J and Doskeland SO: Caspase-dependent, geldanamycin-enhanced cleavage of co-chaperone p23 in leukemic apoptosis. Leukemia. 18:1989–1996. 2004. View Article : Google Scholar : PubMed/NCBI