UCN-01 induces S and G2/M cell cycle arrest through the p53/p21waf1 or CHK2/CDC25C pathways and can suppress invasion in human hepatoma cell lines

BMC Cancer

Bo Liu¹  Nan Lin²  Linan Xu³  Guoyi Wu¹ 

[1]Department of General Surgery, the Lingnan Hospital, the Third Affiliated Hospital, Sun Yat-Sen University, GuangZhou, 510630, PR China

[2]Department of Hepatobiliary Surgery, the Third Affiliated Hospital, Sun Yat-Sen University, GuangZhou, 510630, PR China

[3]Department of Gynecology and Obstetrics, the First Affiliated Hospital, Sun Yat-Sen University, GuangZhou, 510089, PR China

关键词: Tumour invasion and migration;    Beta-catenin;    Anti-cancer drug;    Hepatocellular carcinoma;    S arrest;    G2/M arrest;    7-hydroxystaurosporine (UCN-01);   

Others  :  1079823
DOI  :  10.1186/1471-2407-13-167

 received in 2012-11-21, accepted in 2013-03-11,  发布年份 2013

【 摘 要 】

Background

UCN-01 (7-hydroxystaurosporine), a protein kinase inhibitor, has attracted a great deal of attention as a potent antitumour agent. Several clinical trials of UCN-01 alone or in combination with other agents for different tumour types are currently underway, and some of these trials have had positive results. Hepatocellular carcinoma has high incidence rates and is associated with poor prognosis and high mortality rates.

Methods

Three different hepatoma cell lines (Huh7, HepG2, and Hep3B) were treated with different concentrations of UCN-01, and the anti-tumour effects of UCN-01 were evaluated. Following UCN-01 treatment, cell growth was measured using an MTT assay, cell cycle arrest was assayed using flow cytometry, and the mechanisms of cell cycle arrest and invasion inhibition were investigated through western blotting and a Matrigel invasion assay.

Results

After a 72-h UCN-01 treatment, the growth of different hepatoma cell lines was significantly inhibited in a dose-dependent manner, with IC50 values ranging from 69.76 to 222.74 nM. Flow cytometry results suggested that UCN-01 inhibits proliferation in the hepatoma cells by inducing S and G2/M phase arrest, but not G1/S arrest, which differs from previous reports that used other tumour cell lines. Western blot results illustrated that UCN-01 induces a G2/M phase arrest, regardless of the status of the p53/P21^waf1 pathway, whereas the CHK2/CDC25C pathway and the p53/p21^waf1 pathway were involved in the UCN-01-induced S phase arrest. UCN-01 remarkably inhibited Huh7 cell invasion in a time-dependent manner. Suppression of Huh7 cell invasion may be due to the down-regulation of phosphorylated β-catenin by UCN-01.

Conclusions

These findings suggest that UCN-01 induces hepatoma cell growth inhibition by regulating the p53/p21^waf1 and CHK2/CDC25 pathways. Suppression of Huh7 cell invasion by UCN-01 may be due to the down-regulation of phosphorylated β-catenin. These data lend support for further studies on UCN-01 as a promising anti-HCC candidate.

【 授权许可】

   
2013 Wu et al.; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]El-Serag HB, Rudolph KL: Hepatocellular carcinoma: epidemiology and molecular carcinogenesis. Gastroenterology 2007, 132(7):2557-2576.
• [2]Ercolani G, Grazi GL, Ravaioli M, Del GM, Gardini A, Cescon M, Varotti G, Cetta F, Cavallari A: Liver resection for hepatocellular carcinoma on cirrhosis: univariate and multivariate analysis of risk factors for intrahepatic recurrence. Ann Surg 2003, 237(4):536-543.
• [3]Takayama T, Sekine T, Makuuchi M, Yamasaki S, Kosuge T, Yamamoto J, Shimada K, Sakamoto M, Hirohashi S, Ohashi Y: Adoptive immunotherapy to lower postsurgical recurrence rates of hepatocellular carcinoma: a randomised trial. Lancet 2000, 356(9232):802-807.
• [4]Mow BM, Blajeski AL, Chandra J, Kaufmann SH: Apoptosis and the response to anticancer therapy. Curr Opin Oncol 2001, 13(6):453-462.
• [5]Mizuno K, Noda K, Ueda Y, Hanaki H, Saido TC, Ikuta T, Kuroki T, Tamaoki T, Hirai S, Osada S: UCN-01, an anti-tumor drug, is a selective inhibitor of the conventional PKC subfamily. FEBS Lett 1995, 359(2–3):259-261.
• [6]Graves PR, Yu L, Schwarz JK, Gales J, Sausville EA, O'Connor PM, Piwnica-Worms H: The Chk1 protein kinase and the Cdc25C regulatory pathways are targets of the anticancer agent UCN-01. J Biol Chem 2000, 275(8):5600-5605.
• [7]Husain A, Yan XJ, Rosales N, Aghajanian C, Schwartz GK, Spriggs DR: UCN-01 in ovary cancer cells: effective as a single agent and in combination with cis-diamminedichloroplatinum(II)independent of P53 status. Clin Cancer Res 1997, 3(11):2089-2097.
• [8]Hirose Y, Berger MS, Pieper RO: Abrogation of the Chk1-mediated G(2) checkpoint pathway potentiates temozolomide-induced toxicity in a P53-independent manner in human glioblastoma cells. Cancer Res 2001, 61(15):5843-5849.
• [9]Levesque AA, Fanous AA, Poh A, Eastman A: Defective P53 signaling in P53 wild-type tumors attenuates P21waf1 induction and cyclin B repression rendering them sensitive to Chk1 inhibitors that abrogate DNA damage-induced S and G2 arrest. Mol Cancer Ther 2008, 7(2):252-262.
• [10]Kaina B: DNA damage-triggered apoptosis: critical role of DNA repair, double-strand breaks, cell proliferation and signaling. Biochem Pharmacol 2003, 66(8):1547-1554.
• [11]Nakanishi M, Shimada M, Niida H: Genetic instability in cancer cells by impaired cell cycle checkpoints. Cancer Sci 2006, 97(10):984-989.
• [12]Koff A, Giordano A, Desai D, Yamashita K, Harper JW, Elledge S, Nishimoto T, Morgan DO, Franza BR, Roberts JM: Formation and activation of a cyclin E-cdk2 complex during the G1 phase of the human cell cycle. Science 1992, 257(5077):1689-1694.
• [13]Morgan DO: Cyclin-dependent kinases: engines, clocks, and microprocessors. Annu Rev Cell Dev Biol 1997, 13:261-291.
• [14]Elledge SJ, Harper JW: Cdk inhibitors: on the threshold of checkpoints and development. Curr Opin Cell Biol 1994, 6(6):847-852.
• [15]Sandal T: Molecular aspects of the mammalian cell cycle and cancer. Oncologist 2002, 7(1):73-81.
• [16]Shiloh Y: ATM and related protein kinases: safeguarding genome integrity. Nat Rev Cancer 2003, 3(3):155-168.
• [17]Abraham RT: Cell cycle checkpoint signaling through the ATM and ATR kinases. Genes Dev 2001, 15(17):2177-2196.
• [18]Murphy D, Detjen KM, Welzel M, Wiedenmann B, Rosewicz S: Interferon-alpha delays S-phase progression in human hepatocellular carcinoma cells via inhibition of specific cyclin-dependent kinases. Hepatology 2001, 33(2):346-356.
• [19]Takahashi I, Kobayashi E, Asano K, Yoshida M, Nakano H: UCN-01, a selective inhibitor of protein kinase C from Streptomyces. J Antibiot (Tokyo) 1987, 40(12):1782-1784.
• [20]Akiyama T, Yoshida T, Tsujita T, Shimizu M, Mizukami T, Okabe M, Akinaga S: G1 phase accumulation induced by UCN-01 is associated with dephosphorylation of Rb and CDK2 proteins as well as induction of CDK inhibitor P21/Cip1/WAF1/Sdi1 in P53-mutated human epidermoid carcinoma A431 cells. Cancer Res 1997, 57(8):1495-1501.
• [21]Akiyama T, Sugiyama K, Shimizu M, Tamaoki T, Akinaga S: G1-checkpoint function including a cyclin-dependent kinase 2 regulatory pathway as potential determinant of 7-hydroxystaurosporine (UCN-01)-induced apoptosis and G1-phase accumulation. Jpn J Cancer Res 1999, 90(12):1364-1372.
• [22]Akiyama T, Shimizu M, Okabe M, Tamaoki T, Akinaga S: Differential effects of UCN-01, staurosporine and CGP 41 251 on cell cycle progression and CDC2/cyclin B1 regulation in A431 cells synchronized at M phase by nocodazole. Anticancer Drugs 1999, 10(1):67-78.
• [23]Akinaga S, Nomura K, Gomi K, Okabe M: Enhancement of antitumor activity of mitomycin C in vitro and in vivo by UCN-01, a selective inhibitor of protein kinase C. Cancer Chemother Pharmacol 1993, 32(3):183-189.
• [24]Wang Q, Fan S, Eastman A, Worland PJ, Sausville EA, O'Connor PM: UCN-01: a potent abrogator of G2 checkpoint function in cancer cells with disrupted P53. J Natl Cancer Inst 1996, 88(14):956-965.
• [25]Varmark H, Kwak S, Theurkauf WE: A role for Chk2 in DNA damage induced mitotic delays in human colorectal cancer cells. Cell Cycle 2010, 9(2):312-320.
• [26]Li T, Christensen SD, Frankel PH, Margolin KA, Agarwala SS, Luu T, Mack PC, Lara PJ, Gandara DR: A phase II study of cell cycle inhibitor UCN-01 in patients with metastatic melanoma: a California Cancer Consortium trial. Invest New Drugs 2012, 30(2):741-748.
• [27]Kummar S, Gutierrez ME, Gardner ER, Figg WD, Melillo G, Dancey J, Sausville EA, Conley BA, Murgo AJ, Doroshow JH: A phase I trial of UCN-01 and prednisone in patients with refractory solid tumors and lymphomas. Cancer Chemother Pharmacol 2010, 65(2):383-389.
• [28]Fracasso PM, Williams KJ, Chen RC, Picus J, Ma CX, Ellis MJ, Tan BR, Pluard TJ, Adkins DR, Naughton MJ: A Phase 1 study of UCN-01 in combination with irinotecan in patients with resistant solid tumor malignancies. Cancer Chemother Pharmacol 2011, 67(6):1225-1237.
• [29]Jimeno A, Rudek MA, Purcell T, Laheru DA, Messersmith WA, Dancey J, Carducci MA, Baker SD, Hidalgo M, Donehower RC: Phase I and pharmacokinetic study of UCN-01 in combination with irinotecan in patients with solid tumors. Cancer Chemother Pharmacol 2008, 61(3):423-433.
• [30]Bhonde MR, Hanski ML, Magrini R, Moorthy D, Muller A, Sausville EA, Kohno K, Wiegand P, Daniel PT, Zeitz M: The broad-range cyclin-dependent kinase inhibitor UCN-01 induces apoptosis in colon carcinoma cells through transcriptional suppression of the Bcl-x(L) protein. Oncogene 2005, 24(1):148-156.
• [31]Abe S, Kubota T, Otani Y, Furukawa T, Watanabe M, Kumai K, Akiyama T, Akinaga S, Kitajima M: UCN-01 (7-hydoxystaurosporine) inhibits in vivo growth of human cancer cells through selective perturbation of G1 phase checkpoint machinery. Jpn J Cancer Res 2001, 92(5):537-545.
• [32]Ullal AJ, Pisetsky DS: The release of microparticles by Jurkat leukemia T cells treated with staurosporine and related kinase inhibitors to induce apoptosis. Apoptosis 2010, 15(5):586-596.
• [33]Blagosklonny MV, Dixon SC, Robey R, Figg WD: Resistance to growth inhibitory and apoptotic effects of phorbol ester and UCN-01 in aggressive cancer cell lines. Int J Oncol 2001, 18(4):697-704.
• [34]Shimizu E, Zhao MR, Nakanishi H, Yamamoto A, Yoshida S, Takada M, Ogura T, Sone S: Differing effects of staurosporine and UCN-01 on RB protein phosphorylation and expression of lung cancer cell lines. Oncology-Basel 1996, 53(6):494-504.
• [35]Syljuasen RG, Sorensen CS, Hansen LT, Fugger K, Lundin C, Johansson F, Helleday T, Sehested M, Lukas J, Bartek J: Inhibition of human Chk1 causes increased initiation of DNA replication, phosphorylation of ATR targets, and DNA breakage. Mol Cell Biol 2005, 25(9):3553-3562.
• [36]Usuda J, Saijo N, Fukuoka K, Fukumoto H, Kuh HJ, Nakamura T, Koh Y, Suzuki T, Koizumi F, Tamura T: Molecular determinants of UCN-01-induced growth inhibition in human lung cancer cells. Int J Cancer 2000, 85(2):275-280.
• [37]Didier C, Cavelier C, Quaranta M, Galcera MO, Demur C, Laurent G, Manenti S, Ducommun B: G2/M checkpoint stringency is a key parameter in the sensitivity of AML cells to genotoxic stress. Oncogene 2008, 27(27):3811-3820.
• [38]Steinmeyer K, Maacke H, Deppert W: Cell cycle control by P53 in normal (3T3) and chemically transformed (Meth A) mouse cells. I. Regulation of P53 expression. Oncogene 1990, 5(11):1691-1699.
• [39]Sturzbecher HW, Maimets T, Chumakov P, Brain R, Addison C, Simanis V, Rudge K, Philp R, Grimaldi M, Court W: P53 interacts with p34cdc2 in mammalian cells: implications for cell cycle control and oncogenesis. Oncogene 1990, 5(6):781-795.
• [40]McDonald AC, Brown R: Induction of P53-dependent and P53-independent cellular responses by topoisomerase 1 inhibitors. Br J Cancer 1998, 78(6):745-751.
• [41]Baum N, Schiene-Fischer C, Frost M, Schumann M, Sabapathy K, Ohlenschlager O, Grosse F, Schlott B: The prolyl cis/trans isomerase cyclophilin 18 interacts with the tumor suppressor P53 and modifies its functions in cell cycle regulation and apoptosis. Oncogene 2009, 28(44):3915-3925.
• [42]Sausville EA: Cell cycle regulatory kinase modulators: interim progress and issues. Curr Top Med Chem 2005, 5(12):1109-1117.
• [43]Kawabe T: G2 checkpoint abrogators as anticancer drugs. Mol Cancer Ther 2004, 3(4):513-519.
• [44]Vermeulen K, Van Bockstaele DR, Berneman ZN: The cell cycle: a review of regulation, deregulation and therapeutic targets in cancer. Cell Prolif 2003, 36(3):131-149.
• [45]Mani S, Wang C, Wu K, Francis R, Pestell R: Cyclin-dependent kinase inhibitors: novel anticancer agents. Expert Opin Investig Drugs 2000, 9(8):1849-1870.
• [46]Fang D, Hawke D, Zheng Y, Xia Y, Meisenhelder J, Nika H, Mills GB, Kobayashi R, Hunter T, Lu Z: Phosphorylation of beta-catenin by AKT promotes beta-catenin transcriptional activity. J Biol Chem 2007, 282(15):11221-11229.
• [47]Hu J, Shao S, Song Y, Zhao J, Dong Y, Gong L, Yang P: Hepatocyte growth factor induces invasion and migration of ovarian cancer cells by decreasing the expression of E-cadherin, beta-catenin, and caveolin-1. Anat Rec (Hoboken) 2010, 293(7):1134-1139.
• [48]Du R, Xia L, Sun S, Lian Z, Zou X, Gao J, Xie H, Fan R, Song J, Li X: URG11 promotes gastric cancer growth and invasion by activation of beta-catenin signalling pathway. J Cell Mol Med 2010, 14(3):621-635.
• [49]Bandapalli OR, Dihlmann S, Helwa R, Macher-Goeppinger S, Weitz J, Schirmacher P, Brand K: Transcriptional activation of the beta-catenin gene at the invasion front of colorectal liver metastases. J Pathol 2009, 218(3):370-379.