SC-III3, a novel scopoletin derivative, induces cytotoxicity in hepatocellular cancer cells through oxidative DNA damage and ataxia telangiectasia-mutated nuclear protein kinase activation

BMC Cancer

Peng Zhao³  Li Chen²  Lin-Hu Li²  Zhi-Feng Wei³  Bei Tong³  Yu-Gai Jia³  Ling-Yi Kong²  Yu-Feng Xia¹  Yue Dai³ 

[1] Department of Chinese Materia Medica Analysis, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, China

[2] Department of Natural Medicinal Chemistry, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, China

[3] Department of Pharmacology of Chinese Materia Medica, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, China

关键词: Cell cycle arrest;    DNA damage;    ROS;    Hepatocellular cancer;    SC-III3;   

Others  :  1106810
DOI  :  10.1186/1471-2407-14-987

 received in 2014-08-09, accepted in 2014-12-11,  发布年份 2014

【 摘 要 】

Background

Natural products from plants have been proven to be important resources of antitumor agents. In this study, we exploited the antitumor activity of (E)-3-(4-chlorophenyl)-N-(7-hydroxy-6-methoxy-2-oxo-2H-chromen-3-yl) acrylamide (SC-III3), a newly synthesized derivative of scopoletin, by in vitro and in vivo experiments.

Methods

Human hepatocellular carcinoma cell line HepG2 cells and xenograft of HepG2 cells in BALB/c nude mice were used to investigate the effects of SC-III3 on hepatocellular cancers. Cell cycle arrest and apoptosis were analyzed by flow cytometry. Cell cycle arrest, apoptosis and ATM-Chk pathway-related proteins were characterized by western blot.

Results

SC-III3 selectively inhibited the viability of HepG2 cells without significant cytotoxicity against human normal liver cells LO2. In mouse xenograft model of HepG2 cells, SC-III3 showed a marked inhibition of tumor growth in a dose-dependent manner. Cell cycle analysis revealed that SC-III3 induced cells to accumulate in S phase, which was accompanied by a marked decrease of the expressions of cyclin A, cyclin B, cyclin E and Cdk2 proteins, the crucial regulators of S phase cell cycle. SC-III3 treatment resulted in DNA breaks in HepG2 cells, which might contribute to its S phase arrest. The S arrest and the activation of ATM-Chk1/Chk2-Cdc25A-Cdk2 pathways induced by SC-III3 in HepG2 cells could be efficiently abrogated by pretreatments of either Ku55933 (an inhibitor of ATM) or UCN-01 (an inhibitor of Chk1/Chk2). The activation of p53-p21 pathway by SC-III3 was also reversed by Ku55933 treatment. SC-III3 led to significant accumulation of intracellular reactive oxygen species (ROS), a breaker of DNA strand, in HepG2 cells but not LO2 cells. Pretreatment with N-acetyl-l-cysteine (NAC), a ROS scavenger, could reverse SC-III3-caused ROS accumulation, DNA damage, activation of signal pathways relevant to DNA damage, S phase arrest and cell viability decrease in HepG2 cells.

Conclusion

SC-III3 is able to efficiently inhibit the growth of hepatocellular carcinoma through inducing the generation of intracellular ROS, DNA damage and consequent S phase arrest, but lack of significant cytotoxicity against normal liver cells. This compound deserves further studies as a candidate of anticancer drugs.

【 授权许可】

   
2014 Zhao et al.; licensee BioMed Central.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

Figure 7.

Figure 8.

【 参考文献 】

• [1]El-Serag HB: Hepatocellular carcinoma. N Engl J Med 2011, 365:1118-1127.
• [2]Germano D, Daniele B: Systemic therapy of hepatocellular carcinoma: Current status and future perspectives. World J Gastroenterol 2014, 20:3087-3099.
• [3]Wilson JF: Liver cancer on the rise. Ann Intern Med 2005, 142:1029-1032.
• [4]Bruix J, Llovet JM: Major achievements in hepatocellular carcinoma. Lancet 2009, 373(9664):614-616.
• [5]Bruix J, Gores GJ, Mazzaferro V: Hepatocellular carcinoma: clinical frontiers and perspectives. Gut 2014, 63(5):844-855.
• [6]Raoul JL, Sangro B, Forner A, Mazzaferro V, Piscaglia F, Bolondi L, Lencioni R: Evolving strategies for the management of intermediate-stage hepatocellular carcinoma: available evidence and expert opinion on the use of transarterial chemoembolization. Cancer Treat Rev 2011, 37(3):212-220.
• [7]Cooke MS, Evans MD, Dizdaroglu M, Lunec J: OxidativeDNA damage: mechanisms, mutation, and disease. FASEB J 2003, 17(10):1195-1214.
• [8]Ray PD, Huang BW, Tsuji Y: Reactive oxygen species (ROS) homeostasis and redox regulation in cellular signaling. Cell Signal 2012, 24(5):981-990.
• [9]Wang HC, Choudhary S: Reactive oxygen species-mediated therapeutic control of bladder cancer. Nat Rev Urol 2011, 8(11):608-616.
• [10]Gorrini C, Harris IS, Mak TW: Modulation of oxidative stress as an anticancer strategy. Nat Rev Drug Discov 2013, 12(12):931-947.
• [11]Iaccarino I, Martins LM: Therapeutic targets in cancer cell metabolism and death. Cell Death Differ 2011, 18(3):565-570.
• [12]Guachalla LM, Rudolph KL: ROS induced DNA damage and checkpoint responses: influences on aging. Cell Cycle 2010, 9:4058-4060.
• [13]Liu Q, Guntuku S, Cui XS, Matsuoka S, Cortez D, Tamai K, Luo G, Carattini-Rivera S, DeMayo F, Bradley A, Donehower LA, Elledge SJ: Chk1 is an essential kinase that is regulated by Atr and required for the G(2)/M DNA damage checkpoint. Genes Dev 2000, 14:1448-1459.
• [14]Zhao H, Piwnica-Worms H: ATR-mediated checkpoint pathways regulate phosphorylation and activation of human Chk1. Mol Cell Biol 2001, 21:4129-4139.
• [15]Jackson SP, Bartek J: The DNA-damage response in human biology and disease. Nature 2009, 461:1071-1078.
• [16]Lindsey-Boltz LA, Sancar A: Tethering DNA damage checkpoint mediator proteins topoisomerase II beta-binding protein 1 (TopBP1) and Claspin to DNA activates ataxia-telangiectasia mutated and RAD3-related (ATR) phosphorylation of checkpoint kinase 1 (Chk1). J Biol Chem 2011, 286(22):19229-19236.
• [17]Branzei D, Foiani M: Regulation of DNA repair throughout the cell cycle. Nat Rev Mol Cell Biol 2008, 9:297-308.
• [18]Pan R, Gao XH, Li Y, Xia YF, Dai Y: Anti-arthritic effect of scopoletin, a coumarin compound occurring in Erycibe obtusifolia Benth stems, is associated with decreased angiogenesis in synovium. Fundam Clin Pharmacol 2010, 24(4):477-490.
• [19]Pan R, Dai Y, Gao XH, Lu D, Xia YF: Inhibition of vascular endothelial growth factor-induced angiogenesis by scopoletin through interrupting the autophosphorylation of VEGF receptor 2 and its downstream signaling pathways. Vascul Pharmacol 2011, 54(1–2):18-28.
• [20]Ding ZQ, Dai Y, Wang ZT: Hypouricemic acion of scopoletin arising from xanthine oxidase inhibition and uricosuric activity. Planta Med 2005, 71(2):183-185.
• [21]Kim EK, Kwon KB, Shin BC, Seo EA, Lee YR, Kim JS, Park JW, Park BH, Ryu DG: Scopoletin induces apoptosis in human promyeloleukemic cells, accompanied by activations of nuclear factor kappaB and caspase-3. Life Sci 2005, 77(7):824-836.
• [22]Liu XL, Zhang L, Fu XL, Chen K, Qian BC: Effect of scopoletin on PC3 cell proliferation and apoptosis. Acta Pharmacol Sin 2001, 22(10):929-933.
• [23]Bhattacharyya SS, Paul S, Dutta S, Boujedaini N, Khuda-Bukhsh AR: Anti-oncogenic potentials of a plant coumarin (7-hydroxy-6-methoxy coumarin) against 7, 12-dimethylbenz [a] anthracene-induced skin papilloma in mice: the possible role of several key signal proteins. Zhong Xi Yi Jie He Xue Bao 2010, 8(7):645-654.
• [24]Bhattacharyya SS, Paul S, De A, Das D, Samadder A, Boujedaini N, Khuda-Bukhsh AR: Poly (lactide-co-glycolide) acid nanoencapsulation of a synthetic coumarin: cytotoxicity and bio-distribution in mice, in cancer cell line and interaction with calf thymus DNA as target. Toxicol Appl Pharmacol 2011, 253(3):270-281.
• [25]Lluis JM, Buricchi F, Chiarugi P, Morales A, Fernandez-Checa JC: Dual role of mitochondrial reactive oxygen species in hypoxia signaling: activation of nuclear factor-{kappa}B via c-SRC and oxidant-dependent cell death. Cancer Res 2007, 67(15):7368-7377.
• [26]Zhang Y, Zhao L, Li X, Wang Y, Yao J, Wang H, Li F, Li Z, Guo Q: V8, a newly synthetic flavonoid, induces apoptosis through ROS-mediated ER stress pathway in hepatocellular carcinoma. Arch Toxicol 2014, 88(1):97-107.
• [27]Li Y, Yang DQ: The ATM inhibitor KU-55933 suppresses cell proliferation and induces apoptosis by blocking Akt in cancer cells with over activated Akt. Mol Cancer Ther 2010, 9(1):113-125.
• [28]Nguyen D, Zajac-Kaye M, Rubinstein L, Voeller D, Tomaszewski JE, Kummar S, Chen AP, Pommier Y, Doroshow JH, Yang SX: Poly (ADP-ribose) polymerase inhibition enhances p53-dependent and -independent DNA damage responses induced by DNA damaging agent. Cell Cycle 2011, 10(23):4074-4082.
• [29]Zhao H, Dobrucki J, Rybak P, Traganos F, Dorota Halicka H, Darzynkiewicz Z: Induction of DNA damage signaling by oxidative stress in relation to DNA replication as detected using "click chemistry". Cytometry A 2011, 79(11):897-902.
• [30]Motola-Kuba D, Zamora-Valdes D, Uribe M, Mendez-Sanchez N: Hepatocellular carcinoma. An overview. Ann Hepatol 2006, 5(1):16-24.
• [31]Diaz-Moralli S, Tarrado-Castellarnau M, Miranda A, Cascante M: Targeting cell cycle regulation in cancer therapy. Pharmacol Ther 2013, 138(2):255-271.
• [32]Norbury C, Nurse P: Animal cell cycles and their control. Annu Rev Biochem 1992, 61:441-470.
• [33]Ortega S, Prieto I, Odajima J, Martin A, Dubus P, Sotillo R: Cyclin-dependent kinase 2 is essential for meiosis but not for mitotic cell division in mice. Nat Genet 2003, 35(1):25-31.
• [34]Eliaš J, Dimitrio L, Clairambault J, Natalini R: The p53 protein and its molecular network: modelling a missing link between DNA damage and cellfate. Biochim Biophys Acta 2014, 1844(1 Pt B):232-247.
• [35]Zhu H, Miao ZH, Huang M, Feng JM, Zhang ZX, Lu JJ, Cai YJ, Tong LJ, Xu YF, Qian XH, Ding J: Naphthalimides induce G(2) arrest through the ATM-activated Chk2-executed pathway in HCT116 cells. Neoplasia 2009, 11(11):1226-1234.
• [36]Trachootham D, Alexandre J, Huang P: Targeting cancer cells by ROS-mediated mechanisms: a radical therapeutic approach? Nat Rev Drug Discov 2009, 8(7):579-591.
• [37]Pelicano H, Carney D, Huang P: ROS stress in cancer cells and therapeutic implications. Drug Resist Updat 2004, 7(2):97-110.
• [38]Ichikawa Y, Ghanefar M, Bayeva M, Wu R, Khechaduri A, Naga Prasad SV, Mutharasan RK, Naik TJ, Ardehali H: Cardiotoxicity of doxorubicin is mediated through mitochondrial iron accumulation. J Clin Invest 2014, 124(2):617-630.
• [39]Raj L, Ide T, Gurkar AU, Foley M, Schenone M, Li X, Tolliday NJ, Golub TR, Carr SA, Shamji AF, Stern AM, Mandinova A, Schreiber SL, Lee SW: Selective killing of cancer cells by a small molecule targeting the stress response to ROS. Nature 2011, 475(7355):231-234.