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In Combination with Cytotoxic Agents in Human Uterine Sarcoma Cell Lines

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In Combination with Cytotoxic Agents in Human Uterine Sarcoma Cell Lines ANTICANCER RESEARCH 27: 273-278 (2007) Seliciclib (CYC202; r-Roscovitine) in Combination with Cytotoxic Agents in Human Uterine Sarcoma Cell Lines HELEN M. COLEY, CHRISTINE F. SHOTTON and HILARY THOMAS Postgraduate Medical School, University of Surrey, Guildford, Surrey GU2 7WG, U.K. Abstract. Background: Inhibition of cyclin-dependent kinases the cell cycle as an approach to treat cancer. Seliciclib (CDKs) has recently emerged as an interesting approach to treat (CYC202), the r-enantiomer of the cell cycle inhibitory human malignancies. This was explored in human leiomyo- agent roscovitine has been developed as a potent CDK2 sarcoma (LMS) lines, which represent a tumour associated with inhibitor and is currently in phase II clinical trials. poor survival, chemo-unresponsiveness and deregulation of cell Preclinical studies involving other CDK inhibitors, such as cycle components. Materials and Methods: Using isobologram flavopiridol have demonstrated their interaction with a analysis with MTT chemosensitivity testing, the effects of the CDK number of different cytotoxic agents in a synergistic manner inhibitor seliciclib (CYC202, R-roscovitine) when used alone or (5). We have explored this approach by examining the in combination with paclitaxel was studied in uterine cancer cell effects of seliciclib combined with paclitaxel in three human lines. Apoptotic endpoints were also examined via Annexin V uterine sarcoma cell line models in terms of any synergy and assay using flow cytometry and Western blotting. Results: Overall effects on apoptosis. seliciclib combined with paclitaxel proved synergistic for all cell lines. This was concomitant with an enhanced apoptotic effect Materials and Methods and downregulation of the IAP survivin. Conclusion: Our data support the use of seliciclib as part of combination therapy for Chemicals and reagents. Seliciclib (CYC202) kindly provided by uterine cancer. Cyclacel Ltd. (Dundee, UK) was made up in DMSO; paclitaxel, obtained as a pharmacy preparation (Bristol Myers Squibb Corporation), was made up into aliquots in sterile saline. Propidium Human uterine sarcomas, including leiomyosarcoma (LMS) iodide (Sigma Aldrich, Poole, UK) was stored as a 1 mg/ml aqueous and mixed mesodermal tumours (MMT), are amongst the solution, protected from light at 4ÆC; dimethylthiazol-2-yl-diphenyl most chemorefractory types of cancers with high rates of local tetrazolium bromide MTT (Sigma Aldrich) was made up as a 5 recurrence and distant metastasis. For example, for patients mg/ml solution in sterile phosphate-buffered saline (PBS). with uterine LMS the 5-year survival rate is 15-25% (1). Amongst the chemotherapeutic agents with reported activity Cell culture. Cell culture reagents were from Sigma Aldrich (unless stated otherwise). SK-UT-1 human uterine grade III LMS and the in uterine sarcomas, taxanes and ifosfamide have been used SK-LMS-1 human vulval primary LMS were obtained from Dr. as combination therapy (2, 3), although response rates overall Janet Shipley, Institute of Cancer Research, Sutton, UK; the SK- are disappointingly low. Thus, there is an urgent need for the UT-1b cell line, a subline of SK-UT-1 was kindly provided by Dr. introduction of new therapies for uterine sarcomas. Doris Germain (formerly of the Peter MacCallum Institute, East Deregulated cyclins and cyclin-dependent kinases (CDK) Melbourne, Australia). Cell lines were maintained as monolayers conferring a selective growth advantage is a common in Minimal Essential Medium, supplemented with non-essential feature of human cancer, including LMS (4). These amino acids, sodium pyruvate, glutamine and 10% heat-inactivated foetal bovine serum (Invitrogen, Paisley, UK). Cells were observations have formed the rationale for large drug trypsinized for use in experiments using Trypsin-EDTA solution. development programs that have been designed to target MTT assay for titration of drug sensitivity. The assay was used as previously described (6). Cells were seeded into 96-well plates at a density of 8x103 cells per well. Drugs were diluted in tissue culture Correspondence to: Helen M. Coley, Room 26PGM02, medium containing 10% foetal calf serum (FCS), with untreated Postgraduate Medical School, Daphne Jackson Road, Manor Park, wells being used as controls. In order to select the appropriate drug Guildford, Surrey GU2 7WG, U.K. Tel: +44 1483688617, Fax: concentration ranges, the IC50 of each drug was assessed in +44 1483688501, e-mail: [email protected] preliminary assays using MTT and gave the following results: seliciclib (ÌM) IC50 values SK-LMS-1 26.8 (7.8); SK-UT-1 9.8 (3.9); Key Words: Uterine cancer, seliciclib, paclitaxel, combination SK-UT-1b 6.9 (1.0) and for paclitaxel (nM) SK-LMS-1 8.8 (3.6); therapy, apoptosis. SK-UT-1 3.7 (2.9); SK-UT-1b 2.5 (0.9). Results are the means and 0250-7005/2007 $2.00+.40 273 ANTICANCER RESEARCH 27: 273-278 (2007) Figure 1. Bar graphs (left hand panel) showing the change in IC50 for paclitaxel alone (control) for the SK-LMS-1 line as a result of the combination therapy. *Denotes a students t-test value of <0.02 for comparison of IC50 data obtained for a particular combination versus that for paclitaxel alone. Error bars indicate the standard deviation obtained for >3 repeat analyses. Isobologram analyses (right hand panels) using a combination of paclitaxel and seliciclib. Panel A shows the effects of a concomitant (simultaneous) addition of both agents; panel B shows the effect of a sequence of paclitaxel followed by seliciclib; and panel C shows the effect of seliciclib followed by paclitaxel. The isobologram line for the drug combination is denoted by solid circles. Results shown are representative of >3 repeat experiments. standard deviation in brackets for >3 analyses. Consequently, the Biosoft, Cambridge, UK). This program uses the Combination dose range of seliciclib used was 0.625-40 ÌM for all cell lines; Index (CI) method (7) based on the multiple drug effect paclitaxel doses were 0.155-10 nM for the SK-UT-1 and SK-UT-1b equation. The constant ratio approach was used to assess the lines and 0.625-40 nM for SK-LMS-1. effect of both drugs in combination, whereby dose-response Cells were treated either with single agents or in combination curves were determined with both drugs in combination, at a simultaneously or sequentially. For treatment of cells with the fixed ratio that was equivalent to their IC50 values. CI values of simultaneous combination, drugs were added simultaneously 24 h <1.0 indicate greater than additive effects (synergism where the after cell seeding. For sequential treatments the first drug was greater the synergy the smaller the value), CI values equal to added 24 h following seeding of cells (with single drug treatment 1.0 indicate additivity and CI values >1 indicate antagonism. wells being set up alongside) and this was followed by the second Each CI ratio represented in the data is the mean derived from drug addition 24 h later, (with single drug treatment wells set up at least 3 independent experiments. The "IC50 relative to in other trays to facilitate isobologram analysis, see below). In wells control" values are shown in Figure 1. Relative paclitaxel IC50 that contained a single drug addition, an appropriate volume of values were obtained by dividing the IC50 obtained from the tissue culture medium diluent was added to enable valid drug combination by the corresponding value obtained for comparison of wells that contained combined (dual) drug paclitaxel when used singly. These particular data were treatments. Cells were exposed to drug over a 72 h period and then subjected to a statistical analysis to look for significant subjected to the MTT assay as in (6). differences in the shift of the IC50 value from that seen with the paclitaxel alone. A Student’s (paired, 1 tailed) t-test was used Isobologram analysis. The interaction of seliciclib with paclitaxel and significant values were <0.05, as indicated by *, using the was studied using the CalcuSyn software program (Cambridge SPSS program. 274 Coley et al: Seliciclib with Paclitaxel in Uterine Cancer Cell Lines Figure 2. Bar graphs (left hand panel) show the changes in IC50 value for paclitaxel alone for the SK-UT-1 cell line as a result of the combination therapy. Isobologram analyses (right hand panels) using a combination of paclitaxel and seliciclib. Panels A and B show data obtained for the SK-UT-1 and SK-UT-1b cell lines, respectively, using a simultaneous drug combination schedule. Other notes and key to symbols as for Figure 1. Annexin-V apoptosis assay. Cells were seeded into tissue culture Results flasks to give a density approximately 30% confluence, allowed to attach for 2-3 h and then treated with the appropriate compounds Drug sensitivity and isobologram analyses presented in either simultaneously or in sequence (for SK-LMS-1). An Annexin Figures 1 and 2 show the median effect plots obtained for V-FITC (fluorescein-isothiocyanate) conjugated apoptosis detection kit incorporating PI (propidium iodide) was used as the isobologram analyses. The data indicate synergistic described by the manufacturer’s protocol (supplied by CN effects for paclitaxel in combination with seliciclib. There Biosciences, Beeston, UK). Samples were analysed using flow was evidence of sequence dependence for the cytometry, using the FL1 (FITC) and FL3 (PI) lines and each administration of paclitaxel and seliciclib combination in reading was taken using 10,000 events. SK-LMS-1 cells (Figure 1 bar graph). There were significant differences in the IC values obtained for paclitaxel Western immunoblotting. Exponentially growing cells were treated 50 followed by seliciclib treatment schedule which were with either seliciclib or paclitaxel singly or in combination for a total of 72 h for survivin analysis. Sequential drug treatment significantly higher than those seen for paclitaxel alone involved adding the first drug at 24 h followed by the second drug (p≤0.02).
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