Differentially expressed proteins in the human esophageal cancer cell line Eca‑109, in the presence and absence of gemcitabine

Ma,Zenghuang; Xue,Xiaojie

doi:10.3892/mmr.2017.8041

Differentially expressed proteins in the human esophageal cancer cell line Eca‑109, in the presence and absence of gemcitabine

Authors:
- Zenghuang Ma
- Xiaojie Xue
View Affiliations

Affiliations: Huangshi Center for Clinical Laboratory, Huangshi, Hubei 435000, P.R. China, Department of Clinical Laboratory, Huangshi Central Hospital of Edong Healthcare Group, Hubei Polytechnic University, Huangshi, Hubei 435000, P.R. China
Published online on: November 14, 2017 https://doi.org/10.3892/mmr.2017.8041
Pages: 1873-1878

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

The present study aimed to screen and study the roles of differentially expressed proteins in the human esophageal cancer cell line Eca‑109, in the presence and absence of gemcitabine (GEM). The 3‑(4,5)‑dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT) method was used to assay the vitality of the Eca‑109 cells following treatment with GEM (1‑16 µg/ml). The cell apoptosis was measured by using fluorescence activated cell sorting. The proteins in the treated Eca‑109 cells were extracted, validated, and assayed via two‑dimensional gel electrophoresis combined with matrix‑assisted laser desorption/ionization time of flight mass spectrometry (MALDI‑TOF‑MS). The differentially expressed proteins were then determined by western blotting. Furthermore, alterations in mitochondrial ultrastructure of the treated cells were observed under a transmission electron microscope. GEM significantly inhibited the growth of the Eca‑109 cells in a concentration‑ and time‑dependent manner, and the 50% inhibition concentration (IC50) value was 3.87 µg/ml. The MALDI‑TOF‑MS analysis revealed that there were three differentially expressed proteins following the GEM treatment, compared with the control. The differential proteins were verified to be B cell lymphoma‑2 associated X, apoptosis regulator (Bax)‑α, apoptosis‑associated speck‑like protein containing a CARD (ASC) and myeloid cell leukemia sequence (Mcl)‑1. Western blotting revealed that the expression levels of ASC and Bax‑α proteins in the treated cancer cells were significantly upregulated, whereas the Mcl‑1 protein expression was markedly downregulated compared with the control. Furthermore, the GEM treatment destroyed the mitochondrial ultrastructure of the cancer cells, leaving swelled mitochondria, a fading matrix and destroyed the mitochondrial cristae. GEM significantly inhibits the growth and promotes apoptosis of the Eca‑109 cells, due to the alterations in the expression levels of the differential proteins, including ASC, Mcl‑1 and Bax‑α.

View Figures

View References

1	Schutte B and Ramaekers FC: Molecular switches that govern the balance between proliferation and apoptosis. Prog Cell Cycle Res. 4:207–217. 2000. View Article : Google Scholar
2	Jaleco S, Swainson L, Dardalhon V, Burjanadze M, Kinet S and Taylor N: Homeostasis of naive and memory CD4+ T cells: IL-2 and IL-7 differentially regulate the balance between proliferation and Fas-mediated apoptosis. J Immunol. 171:61–68. 2003. View Article : Google Scholar
3	Mommers EC, van Diest PJ, Leonhart AM, Meijer CJ and Baak JP: Balance of cell proliferation and apoptosis in breast carcinogenesis. Breast Cancer Res Treat. 58:163–169. 1999. View Article : Google Scholar
4	Colitti M, Stefanon B and Wilde CJ: Apoptotic cell death, bax and bcl-2 expression during sheep mammary gland involution. Anat Histol Embryol. 28:257–264. 1999. View Article : Google Scholar
5	Heermeier K, Benedict M, Li M, Furth P, Nuñez G and Hennighausen L: Bax and Bcl-xs are induced at the onset of apoptosis in involuting mammary epithelial cells. Mech Dev. 56:197–207. 1996. View Article : Google Scholar
6	Kastan MB, Canman CE and Leonard CJ: P53, cell cycle control and apoptosis: Implications for cancer. Cancer Metastasis Rev. 14:3–15. 1995. View Article : Google Scholar
7	Leonard CJ, Canman CE and Kastan MB: The role of p53 in cell-cycle control and apoptosis: Implications for cancer. Important Adv Oncol. 33–42. 1995.
8	Van Parijs L, Ibraghimov A and Abbas AK: The roles of costimulation and Fas in T cell apoptosis and peripheral tolerance. Immunity. 4:321–328. 1996. View Article : Google Scholar
9	Akiyama K, Chen C, Wang D, Xu X, Qu C, Yamaza T, Cai T, Chen W, Sun L and Shi S: Mesenchymal-stem-cell-induced immunoregulation involves FAS-ligand-/FAS-mediated T cell apoptosis. Cell Stem Cell. 10:544–555. 2012. View Article : Google Scholar :
10	Schorr K, Li M, Krajewski S, Reed JC and Furth PA: Bcl-2 gene family and related proteins in mammary gland involution and breast cancer. J Mammary Gland Biol Neoplasia. 4:153–164. 1999. View Article : Google Scholar
11	Evan G, Harrington E, Fanidi A, Land H, Amati B and Bennett M: Integrated control of cell proliferation and cell death by the c-myc oncogene. Philos Trans R Soc Lond B Biol Sci. 345:269–275. 1994. View Article : Google Scholar
12	Hoffman B and Liebermann DA: The proto-oncogene c-myc and apoptosis. Oncogene. 17:3351–3357. 1998. View Article : Google Scholar
13	Evan GI, Wyllie AH, Gilbert CS, Littlewood TD, Land H, Brooks M, Waters CM, Penn LZ and Hancock DC: Induction of apoptosis in fibroblasts by c-myc protein. Cell. 69:119–128. 1992. View Article : Google Scholar
14	Igney FH and Krammer PH: Immune escape of tumors: Apoptosis resistance and tumor counterattack. J Leukoc Biol. 71:907–920. 2002.
15	El Hage F, Abouzahr-Rifai S, Meslin F, Mami-Chouaib F and Chouaib S: Immune response and cancer. Bull Cancer. 95:57–67. 2008.(In French).
16	Zhao Y and Rangnekar VM: Apoptosis and tumor resistance conferred by Par-4. Cancer Biol Ther. 7:1867–1874. 2008. View Article : Google Scholar :
17	Li J, Yu W, Tiwary R, Park SK, Xiong A, Sanders BG and Kline K: α-TEA-induced death receptor dependent apoptosis involves activation of acid sphingomyelinase and elevated ceramide-enriched cell surface membranes. Cancer Cell Int. 10:402010. View Article : Google Scholar :
18	Olson M and Kornbluth S: Mitochondria in apoptosis and human disease. Curr Mol Med. 1:91–122. 2001. View Article : Google Scholar
19	Cappello F, Bellafiore M, Palma A and Bucchieri F: Defective apoptosis and tumorigenesis: Role of p53 mutation and Fas/FasL system dysregulation. Eur J Histochem. 46:199–208. 2002. View Article : Google Scholar
20	Ding HF and Fisher DE: Induction of apoptosis in cancer: New therapeutic opportunities. Ann Med. 34:451–469. 2002. View Article : Google Scholar
21	Pan G, Vickers SM, Pickens A, Phillips JO, Ying W, Thompson JA, Siegal GP and McDonald JM: Apoptosis and tumorigenesis in human cholangiocarcinoma cells. Involvement of Fas/APO-1 (CD95) and calmodulin. Am J Pathol. 155:193–203. 1999. View Article : Google Scholar :
22	Fuxius S, Mross K, Mansouri K and Unger C: Gemcitabine and interferon-alpha 2b in solid tumors: A phase I study in patients with advanced or metastatic non-small cell lung, ovarian, pancreatic or renal cancer. Anticancer Drugs. 13:899–905. 2002. View Article : Google Scholar
23	Morisaki T, Hirano T, Koya N, Kiyota A, Tanaka H, Umebayashi M, Onishi H and Katano M: NKG2D-directed cytokine-activated killer lymphocyte therapy combined with gemcitabine for patients with chemoresistant metastatic solid tumors. Anticancer Res. 34:4529–4538. 2014.
24	Li Q, Yuan Z, Yan H, Wen Z, Zhang R and Cao B: Comparison of gemcitabine combined with targeted agent therapy versus gemcitabine monotherapy in the management of advanced pancreatic cancer. Clin Ther. 36:1054–1063. 2014. View Article : Google Scholar
25	Sanna V, Pala N and Sechi M: Targeted therapy using nanotechnology: Focus on cancer. Int J Nanomedicine. 9:467–483. 2014.
26	Xiao JY, Lee JY, Tokuhiro S, Nagataki M, Jarilla BR, Nomura H, Kim TI, Hong SJ and Agatsuma T: Molecular cloning and characterization of taurocyamine kinase from Clonorchis sinensis: A candidate chemotherapeutic target. PLoS Negl Trop Dis. 7:e25482013. View Article : Google Scholar :
27	Toyota Y, Iwama H, Kato K, Tani J, Katsura A, Miyata M, Fujiwara S, Fujita K, Sakamoto T, Fujimori T, et al: Mechanism of gemcitabine-induced suppression of human cholangiocellular carcinoma cell growth. Int J Oncol. 47:1293–1302. 2015.
28	Luo Y, Lin C, Zhang XY, Liang X, Fu M and Feng FY: Relationship between the level of RRM1 expression and the sensitivity to gemcitabine in the esophageal squamous cell carcinoma cell lines. Zhonghua Zhong Liu Za Zhi. 31:660–663. 2009.(In Chinese).
29	Ohtsuka T, Ryu H, Minamishima YA, Macip S, Sagara J, Nakayama KI, Aaronson SA and Lee SW: ASC is a Bax adaptor and regulates the p53-Bax mitochondrial apoptosis pathway. Nat Cell Biol. 6:121–128. 2004. View Article : Google Scholar
30	Liu W, Luo Y, Dunn JH, Norris DA, Dinarello CA and Fujita M: Dual role of apoptosis-associated speck-like protein containing a CARD (ASC) in tumorigenesis of human melanoma. J Invest Dermatol. 133:518–527. 2013. View Article : Google Scholar
31	Proell M, Gerlic M, Mace PD, Reed JC and Riedl SJ: The CARD plays a critical role in ASC foci formation and inflammasome signalling. Biochem J. 449:613–621. 2013. View Article : Google Scholar :