Seliciclib (CYC202; r-roscovitine) in Combination with Cytotoxic Agents in Human Uterine Sarcoma Cell Lines

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1 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 (CDKs) has recently emerged as an interesting approach to treat human malignancies. This was explored in human leiomyosarcoma (LMS) lines, which represent a tumour associated with poor survival, chemo-unresponsiveness and deregulation of cell cycle components. Materials and Methods: Using isobologram analysis with MTT chemosensitivity testing, the effects of the CDK inhibitor seliciclib (CYC202, R-roscovitine) when used alone or in combination with paclitaxel was studied in uterine cancer cell lines. Apoptotic endpoints were also examined via Annexin V assay using flow cytometry and Western blotting. Results: Overall seliciclib combined with paclitaxel proved synergistic for all cell lines. This was concomitant with an enhanced apoptotic effect and downregulation of the IAP survivin. Conclusion: Our data support the use of seliciclib as part of combination therapy for uterine cancer. Human uterine sarcomas, including leiomyosarcoma (LMS) and mixed mesodermal tumours (MMT), are amongst the most chemorefractory types of cancers with high rates of local recurrence and distant metastasis. For example, for patients with uterine LMS the 5-year survival rate is 15-25% (1). Amongst the chemotherapeutic agents with reported activity in uterine sarcomas, taxanes and ifosfamide have been used as combination therapy (2, 3), although response rates overall are disappointingly low. Thus, there is an urgent need for the introduction of new therapies for uterine sarcomas. Deregulated cyclins and cyclin-dependent kinases (CDK) conferring a selective growth advantage is a common feature of human cancer, including LMS (4). These observations have formed the rationale for large drug development programs that have been designed to target Correspondence to: Helen M. Coley, Room 26PGM02, Postgraduate Medical School, Daphne Jackson Road, Manor Park, Guildford, Surrey GU2 7WG, U.K. Tel: , Fax: , h.coley@surrey.ac.uk Key Words: Uterine cancer, seliciclib, paclitaxel, combination therapy, apoptosis. the cell cycle as an approach to treat cancer. Seliciclib (CYC202), the r-enantiomer of the cell cycle inhibitory agent roscovitine has been developed as a potent CDK2 inhibitor and is currently in phase II clinical trials. Preclinical studies involving other CDK inhibitors, such as flavopiridol have demonstrated their interaction with a number of different cytotoxic agents in a synergistic manner (5). We have explored this approach by examining the effects of seliciclib combined with paclitaxel in three human uterine sarcoma cell line models in terms of any synergy and effects on apoptosis. Materials and Methods Chemicals and reagents. Seliciclib (CYC202) kindly provided by 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 iodide (Sigma Aldrich, Poole, UK) was stored as a 1 mg/ml aqueous solution, protected from light at 4ÆC; dimethylthiazol-2-yl-diphenyl tetrazolium bromide MTT (Sigma Aldrich) was made up as a 5 mg/ml solution in sterile phosphate-buffered saline (PBS). Cell culture. Cell culture reagents were from Sigma Aldrich (unless stated otherwise). SK-UT-1 human uterine grade III LMS and the SK-LMS-1 human vulval primary LMS were obtained from Dr. Janet Shipley, Institute of Cancer Research, Sutton, UK; the SK- UT-1b cell line, a subline of SK-UT-1 was kindly provided by Dr. Doris Germain (formerly of the Peter MacCallum Institute, East Melbourne, Australia). Cell lines were maintained as monolayers in Minimal Essential Medium, supplemented with non-essential amino acids, sodium pyruvate, glutamine and 10% heat-inactivated foetal bovine serum (Invitrogen, Paisley, UK). Cells were trypsinized for use in experiments using Trypsin-EDTA solution. 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 8x10 3 cells per well. Drugs were diluted in tissue culture medium containing 10% foetal calf serum (FCS), with untreated wells being used as controls. In order to select the appropriate drug concentration ranges, the IC 50 of each drug was assessed in preliminary assays using MTT and gave the following results: seliciclib (ÌM) IC 50 values SK-LMS (7.8); SK-UT (3.9); SK-UT-1b 6.9 (1.0) and for paclitaxel (nm) SK-LMS (3.6); SK-UT (2.9); SK-UT-1b 2.5 (0.9). Results are the means and /2007 $

2 Figure 1. Bar graphs (left hand panel) showing the change in IC 50 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 IC 50 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 dose range of seliciclib used was ÌM for all cell lines; paclitaxel doses were nm for the SK-UT-1 and SK-UT-1b lines and nm for SK-LMS-1. Cells were treated either with single agents or in combination simultaneously or sequentially. For treatment of cells with the simultaneous combination, drugs were added simultaneously 24 h after cell seeding. For sequential treatments the first drug was added 24 h following seeding of cells (with single drug treatment wells being set up alongside) and this was followed by the second drug addition 24 h later, (with single drug treatment wells set up in other trays to facilitate isobologram analysis, see below). In wells that contained a single drug addition, an appropriate volume of tissue culture medium diluent was added to enable valid comparison of wells that contained combined (dual) drug treatments. Cells were exposed to drug over a 72 h period and then subjected to the MTT assay as in (6). Isobologram analysis. The interaction of seliciclib with paclitaxel was studied using the CalcuSyn software program (Cambridge Biosoft, Cambridge, UK). This program uses the Combination Index (CI) method (7) based on the multiple drug effect equation. The constant ratio approach was used to assess the effect of both drugs in combination, whereby dose-response curves were determined with both drugs in combination, at a fixed ratio that was equivalent to their IC 50 values. CI values of <1.0 indicate greater than additive effects (synergism where the greater the synergy the smaller the value), CI values equal to 1.0 indicate additivity and CI values >1 indicate antagonism. Each CI ratio represented in the data is the mean derived from at least 3 independent experiments. The "IC 50 relative to control" values are shown in Figure 1. Relative paclitaxel IC 50 values were obtained by dividing the IC 50 obtained from the drug combination by the corresponding value obtained for paclitaxel when used singly. These particular data were subjected to a statistical analysis to look for significant differences in the shift of the IC 50 value from that seen with the paclitaxel alone. A Student s (paired, 1 tailed) t-test was used and significant values were <0.05, as indicated by *, using the SPSS program. 274

3 Coley et al: Seliciclib with Paclitaxel in Uterine Cancer Cell Lines Figure 2. Bar graphs (left hand panel) show the changes in IC 50 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 flasks to give a density approximately 30% confluence, allowed to attach for 2-3 h and then treated with the appropriate compounds either simultaneously or in sequence (for SK-LMS-1). An Annexin V-FITC (fluorescein-isothiocyanate) conjugated apoptosis detection kit incorporating PI (propidium iodide) was used as described by the manufacturer s protocol (supplied by CN Biosciences, Beeston, UK). Samples were analysed using flow cytometry, using the FL1 (FITC) and FL3 (PI) lines and each reading was taken using 10,000 events. Western immunoblotting. Exponentially growing cells were treated with either seliciclib or paclitaxel singly or in combination for a total of 72 h for survivin analysis. Sequential drug treatment involved adding the first drug at 24 h followed by the second drug at 48 h. Whole cell lysates were obtained by scraping the monolayer of adherent cells, combining them with the floating cell population and washing with PBS at 4ÆC. Cell lysates were processed and subjected to SDS-PAGE electrophoresis under standard conditions (8) using 10% NuPAGE gels with a MES buffer system (Invitrogen, Paisley, UK). Antibodies used were survivin (FL-142) rabbit polyclonal antibody (Autogen Bioclear, UK) and actin (Ab-1) rabbit polyclonal antibody (CN Biosciences, Beeston, UK). Results Drug sensitivity and isobologram analyses presented in Figures 1 and 2 show the median effect plots obtained for the isobologram analyses. The data indicate synergistic effects for paclitaxel in combination with seliciclib. There was evidence of sequence dependence for the administration of paclitaxel and seliciclib combination in SK-LMS-1 cells (Figure 1 bar graph). There were significant differences in the IC 50 values obtained for paclitaxel followed by seliciclib treatment schedule which were significantly higher than those seen for paclitaxel alone (p 0.02). For the reverse combination when seliciclib was administered followed by paclitaxel the IC 50 values obtained were significantly lower than those with paclitaxel alone (p 0.02). Data in Figure 1 indicate simultaneous addition of both agents to be synergistic up to 20 ÌM seliciclib (Figure 1A), with CI values from 0.6 to 0.9 (obtained using the Calcusyn program). At 40 ÌM seliciclib the effect of the combination was additive.in the SK-LMS-1 cells. The sequence of paclitaxel followed by seliciclib was slightly 275

4 Figure 3. Bar graphs showing % of cells in various stages of cell death (measured using Annexin V and PI with flow cytometric analysis) following treatment with seliciclib and paclitaxel alone and in combination. Key: Black solid = viable population; white solid = early apoptosis; gray solid = midphase apoptosis; vertical black line = late stage apoptosis/necrosis. SK-LMS-1: A = untreated control; B = seliciclib; C = paclitaxel; D = simultaneous combination; E = paclitaxel followed by seliciclib; F = seliciclib followed by paclitaxel. SK-UT-1 and SK-UT-1b: A-D as for SK-LMS-1. Data shown are the means with error bars indicating standard deviation for >3 repeat experiments. antagonistic in the SK-LMS-1 line (Figure 1B), with CI values typically varying from 1.3 to 2.1. In contrast, the cytotoxicity of the drug combination could be enhanced by the use of seliciclib followed by paclitaxel (Figure 1C), CI values varying from 0.9 at low doses decreasing to 0.4 at the higher drug concentrations, indicative of a synergistic effect. Figure 2 shows that there is no real sequence dependence for the paclitaxel and seliciclib combination in the SK-UT-1 cells, but paclitaxel followed by seliciclib was again antagonistic as shown by the changes in IC 50 values compared with paclitaxel alone. None of the drug combination schedules were shown to give rise to significantly different 276

5 Coley et al: Seliciclib with Paclitaxel in Uterine Cancer Cell Lines treated cells (20 ÌM SK-LMS-1; 10 ÌM SK-UT-1 and SK-UT- 1b) were shown to vary according to treatment schedule. For cells treated in combination the biggest decrease in survivin levels was seen when seliciclib was added first followed by paclitaxel. In contrast, when paclitaxel was added first followed by seliciclib or with a simultaneous combination then survivin levels showed little change when compared to levels of expression in untreated control cells. Discussion Figure 4. Western immunoblotting of drug-treated uterine cancer cells for the detection of survivin. Key: for SK-UT-1 and SK-UT-1b lines (1) Control; (2) paclitaxel 5 nm; (3) seliciclib 10 ÌM; (4) simultaneous combination. For SK-LMS-1 cells (1) Control; (2) paclitaxel 10 nm; (3) seliciclib 20 ÌM; (4) simultaneous combination; (5) seliciclib followed by paclitaxel; (6) paclitaxel followed by seliciclib. IC 50 values compared with paclitaxel alone using a Student s t-test. The paclitaxel and seliciclib concomitant combination appeared slightly less effective in the SK-UT-1 cell line than for SK-LMS-1 (Figure 2A) with typical CI values varying from 0.5 to 0.9. For SK-UT-1b, there was a weakly synergistic/ additive effect with concomitant administration of paclitaxel and seliciclib (Figure 2B) with typical CI values varying from 0.8 to 1.4. Preliminary experiments showed no evidence of synergy with sequential drug administration in SK-UT-1b cells (data not shown). Annexin-V assay for apoptosis. The biggest effect was seen in SK-LMS1 cells treated with seliciclib followed by paclitaxel (Figure 3F). The number of viable cells left following the combination treatment was shown to statistically significantly reduced with the seliciclib followed by paclitaxel schedule compared with the simultaneous drug schedule, p= For the same comparison there was no significant difference between the number of viable cells remaining following the schedule of paclitaxel followed by seliciclib, p=0.33. The effect of drug combination was also somewhat growth inhibitory as the overall cell number was significantly reduced (data not shown) for the simultaneous and seliciclib followed by paclitaxel combinations. These data are overall in line with the isobologram data shown in Figures 1 and 2. Survivin expression. In order to look further at the apoptotic effects of seliciclib and its possible involvement in the sequence dependence effects we saw in drug treated SK-LMS-1 cells we carried out further experiments. Survivin levels in seliciclib We looked at the potential for the developmental CDK inhibitor seliciclib to be used as part of a combination therapy for the treatment of uterine sarcomas. Data shown indicate synergism when seliciclib is combined with paclitaxel. Concomitant combined drug treatment was shown to be effective in all models, although there were cell line specific effects, the best combination was the sequence of seliciclib followed by paclitaxel in SK-LMS-1. Bible and Kauffman (5) used a combination of paclitaxel with flavopiridol and found marked sequence dependence, with synergy being seen when paclitaxel was added first followed by flavopiridol. Marked antagonism was seen with either concomitant treatment or flavopiridol followed by paclitaxel. The authors suggested that the reduced S-phase in the cell cycle gives rise to a concomitant reduction in cells undergoing mitosis, therefore, rendering subsequent paclitaxel treatment less effective (5, 9). We have seen a G 2 M blockade following treatment with seliciclib, particularly in SK-LMS-1 cells (manuscript in preparation). Hence, an increase in cells at this phase of the cell cycle may explain the enhancement seen in paclitaxel-induced apoptosis as this cytotoxic agent is a known mitotic poison. Survivin can inhibit apoptosis induced by paclitaxel and this could be relevant to the effects we saw with the paclitaxel and seliciclib combination treatments. Survivin is cell cycle regulated, being predominantly expressed in the G 2 M phase, and is also a suppressor of apoptosis (10). Therefore, with reduced survivin there is increased apoptosis and consequently enhancement of paclitaxel-induced cytotoxicity given as part of the combination. Our findings are in agreement with a recent report by Tirado et al. (11) who showed that following treatment with roscovitine, survivin levels were significantly reduced in Ewing s sarcoma cells. Both roscovitine and seliciclib have been shown to behave as inhibitors of RNA synthesis (12, 13). Indeed, a rapid down-regulation of the anti-apoptotic factor Mcl-1mRNA and protein levels in myeloma cells has been observed correlating with induction of apoptosis (13). This may also be relevant to the reduction in survivin levels we saw in SK- LMS-1 cells treated with combinations of seliciclib and paclitaxel. Our data showed that seliciclib whilst being effective as monotherapy also interacts synergistically with paclitaxel in uterine sarcoma cells. 277

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