SB225002 Promotes Mitotic Catastrophe in Chemo-Sensitive and -Resistant Ovarian Cancer Cells Independent of p53 Status In Vitro

et al. (2013) SB225002 Promotes Mitotic Catastrophe in Chemo-Sensitive and -Resistant Ovarian Cancer Cells Independent of p53 Status In Vitro. PLoS ONE 8(1): e54572. doi:10.1371/journal.pone.0054572 SB225002 Promotes Mitotic Catastrophe in Chemo- Sensitive and -Resistant Ovarian Cancer Cells Independent of p53 Status In Vitro Meirong Du 0 Qing Qiu 0 Andree Gruslin 0 John Gordon 0 Miao He 0 Chi Chung Chan 0 Dajin Li 0 Benjamin K. Tsang 0 Carl G. Maki, Rush University Medical Center, United States of America 0 1 Laboratory for Reproductive Immunology, Hospital and Institute of Obstetrics & Gynecology, Institute of Biomedical Sciences, Fudan University Shanghai, Medical College , Shanghai , China , 2 Departments of Cellular & Molecular Medicine and Obstetrics & Gynaecology, University of Ottawa , Ottawa , Canada , 3 Chronic Disease Program, Ottawa Hospital Research Institute , Ottawa , Canada , 4 Department of Veterinary Microbiology, University of Saskatchewan , Saskatoon , Canada , 5 WuXi AppTech Co., Ltd. , Shanghai , China , 6 Department of Obstetrics & Gynecology, Hainan Medical College Affiliated Hospital , Haikou , China , 7 World Class University (WCU) Biomodulation Major, Department of Agricultural Biotechnology, College of Agriculture and Life Sciences, Seoul National University , Republic of Korea Recent evidence indicates that CXCR2 signaling is crucial for cancer progression, and its antagonist SB225002 induces apoptosis in Wilms' tumor cells. Here, we investigated the effect of SB225002 on cell cycle progression and apoptosis induction in vitro, using CDDP-sensitive and -resistant OVCA cell lines with different p53 status (wild type, mutant or null). Adenovirus infection of wild-type p53 or transfection of p53 siRNA was used to over-express or knock-down p53. Cell cycle and apoptosis were determined by flow cytometry or Hoechst staining and observation of nuclear morphology. Our data demonstrated that SB225002 induced apoptosis in both wild-type and p53-deficient ovarian cancer (OVCA) cells through alternative mechanisms. SB225002 promoted mitotic catastrophe, as evidenced by the accumulation of mitotic cells with spindle abnormalities, chromosome mis-segregation, multi-polar cell division, multiple nuclei, aneuploidy/polyploidy and subsequent extensive apoptosis. SB225002-induced mitotic catastrophe appeared to be mediated by down-regulation of checkpoint kinase Chk1 and Cdk1-cyclin B activation. In cells expressing wild-type p53 (OV2008 and C13*), SB225002 increased total and phospho-Ser p53 levels, and p53 knock-down decreased SB225002-induced apoptosis, without affecting premature mitosis. These results suggest that SB225002 induces p53-dependent apoptosis, and provokes mitotic catastrophe in p53-independent manner in p53 wild-type cells. Reconstitution with wild-type P53 in P53-null SKOV3 cell attenuated SB225002-induced mitotic catastrophe, suggesting p53 prevented mitotic catastrophe induced by SB225002 in p53-deficient OVCA cells. Finally, the effect of SB225002 could not be prevented by pretreatment with CXCR2 ligand or its neutralizing antibody. The present studies demonstrate for the first time that SB225002 has dual actions in OVCA cells, inducing classic apoptosis through p53 activation and provoking mitotic catastrophe in both p53 wild-type and deficient cells by Chk1 inhibition and Cdk activation. These findings raise the possibility of SB225002 as a new candidate molecule for OVCA therapy independent of the p53 status. - Funding: This work was supported by Major International Joint Research Project of National Natural Science Foundation of China (30910103909 to DJ Li), National and Shanghai Leading Academic Discipline Project (211XK22 to DJL), Program for Outstanding Medical Academic Leader of Shanghai (to DJL), and National Natural Science Foundation of China (81070537 to MRD), National Natural Science Foundation of China (31171437 to MRD), and Shanghai PuJiang Talent Program (10PJ1401600 to MRD), and grants from the Canadian Institutes of Health Research (MOP-15691 to BKT) and the World Class University (WCU) program through the Ministry of Education, Science and Technology of Korea and funded by the National Research Foundation of Korea (R31-10056 to BKT). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: Although two of the authors (Miao He and Chi Chung Chan) are employees of the company WuXi AppTech Co., Ltd., this does not alter the authors adherence to all the PLOS ONE policies on sharing data and materials. The company has no declarations relating to employment, consultancy, patents, products in development or marketed products as they relate to this project/manuscript. Other authors of this manuscript (Meirong Du, Qing Qiu, Andree Gruslin, John Gordon, Dajin Li and Benjamin K. Tsang) have no financial or other interests with the company and have confirmed their declaration that they have no competing interests as defined by PLOS ONE. Ovarian cancer (OVCA) is the most fatal gynecological malignancy that has frustrated both clinicians and researchers for several decades. It is the fifth leading cause of cancer-related death among women, with estimated 21,990 newly diagnosed cases and 15,460 deaths occurring in the United States in 2011 (http://seer.cancer.gov/statfacts/html/ovary.html). Although chemotherapy and cytoreductive surgery are currently standard modalities for OVCA treatment, chemo-resistance remains a major cause of chemotherapeutic failure. Various mechanisms have been implicated for chemoresistance, including altered drug transport, enhanced DNA repair and increased tolerance to DNA damage [1,2,3]. In addition, mammalian cells exhibit complex cellular responses to genotoxic stress, including cell cycle checkpoint, DNA repair and apoptosis, and gene activation is a critical initial step during the cellular response to DNA damage. Although adjuvant chemotherapy with paclitaxel and cisplatin achieves clinical response in a high percentage of cases, the systemic toxicity severely limits treatment opportunity [4]. Thus, more effective and safe therapeutic strategies are urgently required to combat this disease. Apoptosis is characterized morphologically by cytoplasmic shrinkage, chromatin condensation and nuclear fragmentation with chromatinolysis [5], and biochemically by caspase activation and permeabilization of the outer mitochondrial membrane [6,7,8]. Mitotic catastrophe is a special case of apoptosis, and occurs during mitosis with a combination of deficient cell-cycle checkpoints and cellular damage [9]. DNA damage activates checkpoints to delay cell cycle progression. P53 regulates G1/S transition (G1-checkpoint), while CHK1 prevents the entry of DNA-damaged cells into M-phase (G2-check point). In the case of chemoresistance, DNA damage induced by chemotherapeutic agents fails to arrest the cancer cells in the G1 (...truncated)