Cancer stem-like sphere cells induced from de-differentiated hepatocellular carcinoma-derived cell lines possess the resistance to anti-cancer drugs

BMC Cancer

Noriaki Hashimoto¹  Ryouichi Tsunedomi¹  Kiyoshi Yoshimura¹  Yusaku Watanabe¹  Shoichi Hazama¹  Masaaki Oka¹ 

[1] Department of Digestive Surgery and Surgical Oncology, Yamaguchi University Graduate School of Medicine, 1-1-1 Minami-Kogushi, Ube, Yamaguchi 755-8505, Japan

关键词: Chemoresistance;    Sphere;    HCC;    Cancer stem cell;   

Others  :  1121018
DOI  :  10.1186/1471-2407-14-722

 received in 2014-04-08, accepted in 2014-09-17,  发布年份 2014

【 摘 要 】

Background

Cancer stem cells (CSCs) are thought to play important roles in therapy-resistance. In this study, we induced cancer stem-like cells from hepatocellular carcinoma (HCC) cell lines using a unique medium, and examined their potential for resistance to anti-cancer drugs.

Methods

The human HCC cell lines SK-HEP-1 (SK), HLE, Hep 3B, and HuH-7 were used to induce cancer stem-like cells with our sphere induction medium supplemented with neural survival factor-1. NANOG and LIN28A were examined as stemness markers. Several surface markers for CSC such as CD24, CD44, CD44 variant, and CD90 were analyzed by flow-cytometry. To assess the resistance to anti-cancer drugs, the MTS assay, cell cycle analysis, and reactive oxygen species (ROS) activity assay were performed.

Results

Poorly differentiated HCC derived SK and undifferentiated HCC derived HLE cell lines efficiently formed spheres of cells (SK-sphere and HLE-sphere), but well-differentiated HCC-derived HuH-7 and Hep 3B cells did not. SK-spheres showed increased NANOG, LIN28A, and ALDH1A1 mRNA levels compared to parental cells. We observed more CD44 variant-positive cells in SK-spheres than in parental cells. The cell viability of SK-spheres was significantly higher than that of SK cells in the presence of several anti-cancer drugs except sorafenib (1.7- to 7.3-fold, each P < 0.05). The cell cycle of SK-spheres was arrested at the G0/G1 phase compared to SK cells. SK-spheres showed higher ABCG2 and HIF1A mRNA expression and lower ROS production compared to parental cells.

Conclusion

Our novel method successfully induced cancer stem-like cells, which possessed chemoresistance that was related to the cell cycle, drug efflux, and ROS.

【 授权许可】

   
2014 Hashimoto et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

Figure 7.

Figure 8.

Figure 9.

Figure 10.

【 参考文献 】

• [1]Forner A, Llovet JM, Bruix J: Hepatocellular carcinoma. Lancet 2012, 379:245-255.
• [2]Tung-Ping PR, Fan ST, Wong J: Risk factors, prevention, and management of postoperative recurrence after resection of hepatocellular carcinoma. Ann Surg 2000, 232:10-24.
• [3]Chang CH, Chau GY, Lui WY, Tsay SH, King KL, Wu CW: Long-term results of hepatic resection for hepatocellular carcinoma originating from the noncirrhotic liver. Arch Surg 2004, 139:320-325.
• [4]Clarke MF, Dick JE, Dirks PB, Eaves CJ, Jamieson CH, Jones DL, Visvader J, Weissman IL, Wahl GM: Cancer stem cells–perspectives on current status and future directions: AACR Workshop on cancer stem cells. Cancer Res 2006, 66:9339-9344.
• [5]Singh SK, Hawkins C, Clarke ID, Squire JA, Bayani J, Hide T, Henkelman RM, Cusimano MD, Dirks PB: Identification of human brain tumour initiating cells. Nature 2004, 432:396-401.
• [6]Ho MM, Ng AV, Lam S, Hung JY: Side population in human lung cancer cell lines and tumors is enriched with stem-like cancer cells. Cancer Res 2007, 67:4827-4833.
• [7]Endo K, Terada T: Protein expression of cd44 (standard and variant isoforms) in hepatocellular carcinoma: relationships with tumor grade, clinicopathologic parameters, p53 expression, and patient survival. J Hepatol 2000, 32:78-84.
• [8]Ma S, Chan KW, Hu L, Lee TK, Wo JY, Ng IO, Zheng BJ, Guan XY: Identification and characterization of tumorigenic liver cancer stem/progenitor cells. Gastroenterology 2007, 132:2542-2556.
• [9]Ma S, Lee TK, Zheng BJ, Chan KW, Guan XY: CD133+ HCC cancer stem cells confer chemoresistance by preferential expression of the akt/pkb survival pathway. Oncogene 2008, 27:1749-1758.
• [10]Yang ZF, Ho DW, Ng MN, Lau CK, Yu WC, Ngai P, Chu PW, Lam CT, Poon RT, Fan ST: Significance of CD90+ cancer stem cells in human liver cancer. Cancer Cell 2008, 13:153-166.
• [11]Yang W, Yan HX, Chen L, Liu Q, He YQ, Yu LX, Zhang SH, Huang DD, Tang L, Kong XN, Chen C, Liu SQ, Wu MC, Wang HY: Wnt/beta-catenin signaling contributes to activation of normal and tumorigenic liver progenitor cells. Cancer Res 2008, 68:4287-4295.
• [12]Kimura O, Takahashi T, Ishii N, Inoue Y, Ueno Y, Kogure T, Fukushima K, Shiina M, Yamagiwa Y, Kondo Y, Inoue J, Kakazu E, Iwasaki T, Kawagishi N, Shimosegawa T, Sugamura K: Characterization of the epithelial cell adhesion molecule (EpCAM) + cell population in hepatocellular carcinoma cell lines. Cancer Sci 2010, 101:2145-2155.
• [13]Fan L, He F, Liu H, Zhu J, Liu Y, Yin Z, Wang L, Guo Y, Wang Z, Yan Q, Huang G: CD133: a potential indicator for differentiation and prognosis of human cholangiocarcinoma. BMC Cancer 2011, 11:320.
• [14]Kim HM, Haraguchi N, Ishii H, Ohkuma M, Okano M, Mimori K, Eguchi H, Yamamoto H, Nagano H, Sekimoto M, Doki Y, Mori M: Increased CD13 expression reduces reactive oxygen species, promoting survival of liver cancer stem cells via an epithelial-mesenchymal transition-like phenomenon. Ann Surg Oncol 2012, 3:539-548.
• [15]Shen G, Shen F, Shi Z, Liu W, Hu W, Zheng X, Wen L, Yang X: Identification of cancer stem-like cells in the C6 glioma cell line and the limitation of current identification methods. In Vitro Cell Dev Biol Anim 2008, 44:280-289.
• [16]Yu SC, Ping YF, Yi L, Zhou ZH, Chen JH, Yao XH, Gao L, Wang JM, Bian XW: Isolation and characterization of cancer stem cells from a human glioblastoma cell line U87. Cancer Lett 2008, 265:124-134.
• [17]Cao L, Zhou Y, Zhai B, Liao J, Xu W, Zhang R, Li J, Zhang Y, Chen L, Qian H, Wu M, Yin Z: Sphere-forming cell subpopulations with cancer stem cell properties in human hepatoma cell lines. BMC Gastroenterol 2011, 11:71.
• [18]O’Brien CA, Kreso A, Jamieson CH: Cancer stem cells and self-renewal. Clin Cancer Res 2010, 16:3113-3120.
• [19]Chiba T, Kita K, Zheng YW, Yokosuka O, Saisho H, Iwama A, Nakauchi H, Taniguchi H: Side population purified from hepatocellular carcinoma cells harbors cancer stem cell-like properties. Hepatology 2006, 44:240-251.
• [20]Harris MA, Yang H, Low BE, Mukherjee J, Guha A, Bronson RT, Shultz LD, Israel MA, Yun K: Cancer stem cells are enriched in the side population cells in a mouse model of glioma. Cancer Res 2008, 68:10051-10059.
• [21]Yamashita T, Ji J, Budhu A, Forgues M, Yang W, Wang HY, Jia H, Ye Q, Qin LX, Wauthier E, Reid LM, Minato H, Honda M, Kaneko S, Tang ZY, Wang XW: EpCAM-positive hepatocellular carcinoma cells are tumor-initiating cells with stem/progenitor cell. Gastroenterology 2009, 136:1012-1024.
• [22]Chen X, Lingala S, Khoobyari S, Nolta J, Zern MA, Wu J: Epithelial mesenchymal transition and hedgehog signaling activation are associated with chemoresistance and invasion of hepatoma subpopulations. J Hepatol 2011, 55:838-845.
• [23]Mani SA, Guo W, Liao MJ, Eaton EN, Ayyanan A, Zhou AY, Brooks M, Reinhard F, Zhang CC, Shipitsin M, Campbell LL, Polyak K, Brisken C, Yang J, Weinberg RA: The epithelial-mesenchymal transition generates cells with properties of stem cells. Cell 2008, 133:704-715.
• [24]Chiou SH, Yu CC, Huang CY, Lin SC, Liu CJ, Tsai TH, Chou SH, Chien CS, Ku HH, Lo JF: Positive correlations of Oct-4 and Nanog in oral cancer stem-like cells and high-grade oral squamous cell carcinoma. Clin Cancer Res 2008, 14:4085-4095.
• [25]Thon N, Damianoff K, Hegermann J, Grau S, Krebs B, Schnell O, Tonn JC, Goldbrunner R: Presence of pluripotent CD133+ cells correlates with malignancy of gliomas. Mol Cell Neurosci 2010, 43:51-59.
• [26]Tsunedomi R, Iizuka N, Tamesa T, Sakamoto K, Hamaguchi T, Somura H, Yamada M, Oka M: Decreased ID2 promotes metastatic potentials of hepatocellular carcinoma by altering secretion of vascular endothelial growth factor. Clin Cancer Res 2008, 14:1025-1031.
• [27]Zhong Y, Guan K, Guo S, Zhou C, Wang D, Ma W, Zhang Y, Li C, Zhang S: Spheres derived from the human SK-RC-42 renal cell carcinoma cell line are enriched in cancer stem cells. Cancer Lett 2010, 299:150-160.
• [28]Bao S, Wu Q, McLendon RE, Hao Y, Shi Q, Hjelmeland AB, Dewhirst MW, Bigner DD, Rich JN: Glioma stem cells promote radio-resistance by preferential activation of the DNA damage response. Nature 2006, 444:756-760.
• [29]Lee J, Kotliarova S, Kotliarov Y, Li A, Su Q, Donin NM, Pastorino S, Purow BW, Christopher N, Zhang W, Park JK, Fine HA: Tumor stem cells derived from glioblastomas cultured in bFGF and EGF more closely mirror the phenotype and genotype of primary tumors than do serum-cultured cell lines. Cancer Cell 2006, 9:391-403.
• [30]Chambers I, Colby D, Robertson M, Nichols J, Lee S, Tweedie S, Smith A: Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells. Cell 2003, 113:643-655.
• [31]Yang DH, Moss EG: Temporally regulated expression of Lin-28 in diverse tissues of the developing mouse. Gene Expr Patterns 2003, 3:719-726.
• [32]Ma S, Chan KW, Lee TK, Tang KH, Wo JY, Zheng BJ, Guan XY: Aldehyde dehydrogenase discriminates the CD133 liver cancer stem cell populations. Mol Cancer Res 2008, 6:1146-1153.
• [33]Ma I, Allan AL: The role of human aldehyde dehydrogenase in normal and cancer stem cells. Stem Cell Rev 2011, 7:292-306.
• [34]Gao C, Guo H, Downey L, Marroquin C, Wei J, Kuo PC: Osteopontin-dependent CD44v6 expression and cell adhesion in HepG2 cells. Carcinogenesis 2003, 24:1871-1878.
• [35]Phillips TM, McBride WH, Pajonk F: The response of CD24(-/low)/CD44+ breast cancer-initiating cells to radiation. J Natl Cancer Inst 2006, 98:1777-1785.
• [36]Scheel C, Weinberg RA: Phenotypic Plasticity and Epithelial-Mesenchymal Transitions in Cancer - and Normal Stem Cells? Int J Cancer 2011, 129:2310-2314.
• [37]Szakacs G, Paterson JK, Ludwig JA, Booth-Genthe C, Gottesman MM: Targeting multidrug resistance in cancer. Nat Rev Drug Discov 2006, 5:219-234.
• [38]Sukowati CH, Rosso N, Crocè LS, Tiribelli C: Hepatic cancer stem cells and drug resistance: Relevance in targeted therapies for hepatocellular carcinoma. World J Hepatol 2010, 2:114-126.
• [39]Gillet JP, Gottesman MM: Advances in themolecular detection of ABC transporters involved in multidrug resistance in cancer. Curr Pharm Biotechnol 2011, 12:686-692.
• [40]Gottesman MM: Mechanisms of cancer drug resistance. Annu Rev Med 2002, 53:615-627.
• [41]Sherr CJ, Roberts JM: CDK inhibitors: positive and negative regulators of G1-phase progression. Genes Dev 1999, 13:1501-1512.
• [42]Coqueret O: New roles for p21 and p27 cell-cycle inhibitors: a function for each cell compartment? Trends Cell Biol 2003, 13:65-70.
• [43]Semenza GL: HIF-1 and tumor progression: pathophysiology and therapeutics. Trends Mol Med 2002, 8:62-67.
• [44]Rohwer N, Cramer T: HIFs as central regulators of gastric cancer pathogenesis. Cancer Biol Ther 2010, 10:383-385.
• [45]Nagano O, Okazaki S, Saya H: Redox regulation in stem-like cancer cells by CD44 variant isoforms. Oncogene 2013, 32:5191-5198.
• [46]Chang A, Kang Y, Lin D, Park J, Kudo M, Qin S, Omata M, Pitman Lowenthal SW, Lanzalone S, Yang L, Lechuga M, Raymond E: Phase III trial of sunitinib (Su) versus sorafenib (So) in advanced hepatocellular carcinoma (HCC). J Clin Oncol 2011, 29(suppl):256s. Abstract 4000