Sensitization of human cancer cells to gemcitabine by the Chk1 inhibitor MK-8776: cell cycle perturbation and impact of administration schedule in vitro and in vivo

BMC Cancer

Ryan Montano¹  Ruth Thompson¹  Injae Chung²  Huagang Hou¹  Nadeem Khan¹  Alan Eastman¹ 

[1] Norris Cotton Cancer Center, Geisel School of Medicine at Dartmouth, Rubin Building Level 6, Lebanon, NH USA

[2] Duksung Women’s University, Seoul, Korea

关键词: Cell cycle perturbation;    Homologous recombination;    Pancreas cancer xenografts;    Drug combinations;    MK-8776;    Gemcitabine;    Chk1;   

Others  :  859188
DOI  :  10.1186/1471-2407-13-604

 received in 2013-08-26, accepted in 2013-12-04,  发布年份 2013

【 摘 要 】

Background

Chk1 inhibitors have emerged as promising anticancer therapeutic agents particularly when combined with antimetabolites such as gemcitabine, cytarabine or hydroxyurea. Here, we address the importance of appropriate drug scheduling when gemcitabine is combined with the Chk1 inhibitor MK-8776, and the mechanisms involved in the schedule dependence.

Methods

Growth inhibition induced by gemcitabine plus MK-8776 was assessed across multiple cancer cell lines. Experiments used clinically relevant “bolus” administration of both drugs rather than continuous drug exposures. We assessed the effect of different treatment schedules on cell cycle perturbation and tumor cell growth in vitro and in xenograft tumor models.

Results

MK-8776 induced an average 7-fold sensitization to gemcitabine in 16 cancer cell lines. The time of MK-8776 administration significantly affected the response of tumor cells to gemcitabine. Although gemcitabine induced rapid cell cycle arrest, the stalled replication forks were not initially dependent on Chk1 for stability. By 18 h, RAD51 was loaded onto DNA indicative of homologous recombination. Inhibition of Chk1 at 18 h rapidly dissociated RAD51 leading to the collapse of replication forks and cell death. Addition of MK-8776 from 18–24 h after a 6-h incubation with gemcitabine induced much greater sensitization than if the two drugs were incubated concurrently for 6 h. The ability of this short incubation with MK-8776 to sensitize cells is critical because of the short half-life of MK-8776 in patients’ plasma. Cell cycle perturbation was also assessed in human pancreas tumor xenografts in mice. There was a dramatic accumulation of cells in S/G₂ phase 18 h after gemcitabine administration, but cells had started to recover by 42 h. Administration of MK-8776 18 h after gemcitabine caused significantly delayed tumor growth compared to either drug alone, or when the two drugs were administered with only a 30 min interval.

Conclusions

There are two reasons why delayed addition of MK-8776 enhances sensitivity to gemcitabine: first, there is an increased number of cells arrested in S phase; and second, the arrested cells have adequate time to initiate recombination and thereby become Chk1 dependent. These results have important implications for the design of clinical trials using this drug combination.

【 授权许可】

   
2013 Montano et al.; licensee BioMed Central Ltd.

【 图 表 】

【 参考文献 】

• [1]Carrassa L, Damia G: Unleashing Chk1 in cancer therapy. Cell Cycle 2011, 10:2121-2128.
• [2]Chen T, Stephens PA, Middleton FK, Curtin NJ: Targeting the S and G2 checkpoint to treat cancer. Drug Discov Today 2012, 17:194-202.
• [3]Thompson R, Eastman A: The cancer chemotherapeutic potential of Chk1 inhibitors: how mechanistic studies impact clinical trial design. Br J Clin Pharmacol 2013, 76:358-369.
• [4]Kohn EA, Ruth ND, Brown MK, Livingstone M, Eastman A: Abrogation of the S phase DNA damage checkpoint results in S phase progression or premature mitosis depending on the concentration of UCN-01 and the kinetics of Cdc25C activation. J Biol Chem 2002, 277:26553-26564.
• [5]Zegerman P, Diffley JFX: DNA replication as a target of the DNA damage checkpoint. DNA Repair 2009, 8:1077-1088.
• [6]Montano R, Chung I, Garner KM, Parry D, Eastman A: Preclinical development of the novel Chk1 inhibitor SCH900776 in combination with DNA damaging agents and antimetabolites. Mol Cancer Ther 2012, 11:427-438.
• [7]Guzi T, Paruch K, Dwyer MP, Labroli M, Shanahan F, Davis N, Taricani L, Wiswell D, Seghezzi W, Penaflor E, Bhagwat B, Wang W, Gu D, Hsieh Y, Lee S, Liu M, Parry D: Targeting the replication checkpoint using SCH 900776, a potent and selective CHK1 inhibitor identified via high content functional screening. Mol Cancer Ther 2011, 10:591-602.
• [8]Karp JE, Thomas BM, Greer JM, Sorge C, Gore SD, Pratz KW, Smith BD, Flatten KS, Peterson K, Schneider P, Mackey K, Freshwater T, Levis MJ, McDevitt MA, Carraway HE, Gladstone DE, Showel MM, Loechner S, Parry DA, Horowitz JA, Isaacs R, Kaufmann SH: Phase I and pharmacologic trial of cytosine arabinoside with the selective checkpoint I inhibitor SCH 900776 in refractory acute leukemias. Clin Cancer Res 2012, 18:6723-6731.
• [9]Garner KM, Eastman A: Variations in Mre11/Rad50/Nbs1 status and DNA damage-induced S-phase arrest in cell lines of the NCI60 panel. BMC Cancer 2011, 11:206. BioMed Central Full Text
• [10]Demarcq C, Bunch RT, Creswell D, Eastman A: The role of cell cycle progression in cisplatin-induced apoptosis in Chinese hamster ovary cells. Cell Growth Differ 1994, 5:983-993.
• [11]Rao J, Otto WR: Fluorometric DNA assay for cell growth estimation. Anal Biochem 1992, 207:186-192.
• [12]Levesque AA, Kohn EA, Bresnick E, Eastman A: Distinct roles for p53 transactivation and repression in preventing UCN-01-mediated abrogation of DNA damage-induced S and G2 cell cycle checkpoints. Oncogene 2005, 24:3786-3796.
• [13]Zhang W-H, Poh A, Fanous AA, Eastman A: DNA damage-induced S phase arrest in human breast cancer depends on CHK1, but G2 arrest can occur independently of Chk1, Chk2 or MAPKAPK2. Cell Cycle 2008, 7:1668-1677.
• [14]Thompson R, Montano R, Eastman A: The Mre11 nuclease is critical for sensitivity of cells to Chk1 inhibition. PLoS One 2012, 7:e44021.
• [15]Leung-Pineda V, Ryan CE, Piwnica-Worms H: Phosphorylation of Chk1 by ATR is antagonized by a Chk1-regulated protein phosphatase 2A circuit. Mol Cell Biol 2006, 26:7529-7538.
• [16]Parsels LA, Morgan MA, Tanska DM, Parsels JD, Palmer BD, Booth RJ, Denny WA, Canman CE, Kraker AJ, Lawrence TS, Maybaum J: Gemcitabine sensitization by checkpoint kinase 1 inhibition correlates with inhibition of a Rad51 DNA damage resposne in pancreatic cancer cells. Mol Cancer Ther 2009, 8:45-54.
• [17]Sorensen CS, Hansen LT, Dziegielewski J, Syljuasen RG, Lundin C, Bartek J, Helleday T: The cell cycle checkpoint kinase Chk1 is required for mammalian homologous recombination. Nat Cell Biol 2005, 7:195-201.
• [18]Grunewald R, Kantarjian H, Du M, Faucher K, Tarassoff P, Plunkett W: Gemcitabine in leukemia: a phase I clinical, plasma, and cellular pharmacology study. J Clin Oncol 1992, 10:406-413.
• [19]Tachibana KK, Gonzalez MA, Coleman N: Cell-cycle-dependent regulation of DNA replication and its relevance to cancer pathology. J Pathol 2005, 205:123-129.
• [20]Forment JV, Blasius M, Guerini I, Jackson SP: Structure-specific DNA endonuclease Mus81/Eme1 generates DNA damage caused by Chk1 inactivation. PLoS One 2011, 6:e23517.
• [21]Bahassi EM, Ovesen JL, Risenberg AL, Bernstein WZ, Hasty PE, Stambrook PJ: The checkpoint kinases Chk1 and Chk2 regulate the functional associations between hBRCA2 and Rad51 in response to DNA damage. Oncogene 2008, 27:3977-3985.
• [22]Taricani L, Shanahan F, Parry D: Replication stress activates DNA polymerase alpha-associated Chk1. Cell Cycle 2009, 8:482-489.
• [23]Ikegami Y, Goto H, Kiyono T, Enomoto M, Kasahara K, Tomono Y, Tozawa K, Morita A, Kohri K, Inagaki M: Chk1 phosphorylation at ser286 and ser301 occurs with both stalled DNA replication and damage checkpoint stimulation. Biochem Biophys Res Commun 2008, 377:1227-1231.
• [24]Beckerman R, Donner AJ, Mattia M, Peart MJ, Manley JL, Espinosa JM, Prives C: A role for Chk1 in blocking transcriptional elongation pf p21 mRNA during S-phase checkpoint. Genes Dev 2009, 23:1364-1377.
• [25]Morgan MA, Parsels LA, Parsels JD, Mesiwala AK, Maybaum J, Lawrence TS: Role of checkpoint kinase 1 in preventing premature mitosis in response to gemcitabine. Cancer Res 2005, 65:6835-6842.
• [26]Parsels L, Qian Y, Tanska DM, Gross M, Zhao L, Hassan MC, Arumugarajah S, Parsels JD, Hylander-Gans L, Simeone DM, Morosini D, Brown JL, Zabludoff SD, Maybaum J, Lawrence TS, Morgan MA: Assessment of Chk1 phosphorylation as a pharmacodynamic biomarker of Chk1 inhibition. Clin Cancer Res 2011, 17:3706-3715.
• [27]Petermann E, Orta ML, Issaeva N, Schultz N, Helleday T: Hydroxyurea-stalled replication forks become progressively inactivated and require two different RAD51-mediated pathways for restart and repair. Mol Cell 2010, 37:492-502.
• [28]Stults DM, Killen MW, Shelton BJ, Pierce AJ: Recombination phenotypes of the NCI-60 collection of human cancer cells. BMC Mol Biol 2011, 12:23. BioMed Central Full Text
• [29]Bunch RT, Eastman A: Enhancement of cisplatin-induced cytotoxicity by 7-hydroxystaurosporine (UCN-01), a new G2-checkpoint inhibitor. Clin Cancer Res 1996, 2:791-797.
• [30]Fuse E, Tanii H, Kurata N, Kobayashi H, Shimada Y, Tamura T, Sasaki Y, Tanigawara Y, Lush RD, Headlee D, Figg WD, Arbuck SG, Senderowicz AM, Sausville EA, Akinaga S, Kuwabara T, Kobayashi S: Unpredicted clinical pharmacology of UCN-01 caused by specific binding to human a1-acid glycoprotein. Cancer Res 1998, 58:3248-3253.
• [31]Weiss GJ, Donehower RC, Iyengar T, Ramanathan RK, Lewandowski K, Westin E, Hurt K, Hynes SM, Anthony SP, McKane S: Phase I dose-escalation study to examine the safety and tolerability of LY2603618, a checkpoint I inhibitor, administered 1 day after pemetrexed 500 mg/m2 every 21 days in patients with cancer. Invest New Drugs 2013, 31:136-144.