Cell Cycle Arrest and Apoptosis Induced by Oxaliplatin (L-OHP) on Four Human Cancer Cell Lines

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1 Cell Cycle Arrest and Apoptosis Induced by Oxaliplatin (L-OHP) on Four Human Cancer Cell Lines SARA WILLIAM-FALTAOS 1, DANY ROUILLARD 2, PHILIPPE LECHAT 1 and GÉRARD BASTIAN 1 1 Pitie Salpetriere Hospital, Pharmacology Department, Paris; 2 Curie Institute, Paris, France Abstract. Background: The effects of oxaliplatin (L-OHP) on cell cycle and apoptosis were examined. Materials and Methods: The HT29, MCF7, Hela and A549 cell lines were treated and dual parameter flow cytometry BrdU/PI was then performed to monitor cell cycle modifications. Annexin V-FITC and PI probes were used for the detection of apoptosis. Results: The cells were treated for 3 h followed or not by 24 and 72 h of post-incubation. No changes were observed after 3 h of treatment, while after 24 h of postincubation, all the cells except A549 were predominantly accumulated in the G2/M-phase; this accumulation was time-dependent. Apoptosis induction was late and moderate and was observed only after 12 h of treatment and 24 h of post-incubation. Conclusion: L-OHP induced cell cycle arrest and moderate apoptosis; these two activities were timedependent. These findings warrant further investigation into the potential antitumour activity of L-OHP in MCF7 and Hela cells and in lengthening the infusion duration in order to achieve the acquired cellular modifications. Oxaliplatin, L-OHP, is a third-generation platinum antitumour analogue in which the 1, 2-diaminocyclohexane (DACH) ligand substitutes for the amino groups of cisplatin (1). Oxaliplatin, in combination with 5-Fluorouracil (5-FU) and folinic acid (FA) is indicated as first-line therapy for metastatic colorectal cancer (2). It is given at the recommended doses of 130 mg/m 2 q3 weeks or 85 mg/m 2 q2 weeks, as a 2- to 6-h intravenous infusion (3). Studies carried out in cell lines of the NCI anticancer drug screening panel comparing oxaliplatin and other Pt agents have shown that cisplatin and oxaliplatin have different sensitivity profiles, suggesting that the two complexes may have different mechanism(s) of action and/ or resistance (4). Correspondence to: Bastian Gérard, Faculty of Medicine Pitie- Salpetriere, Pharmacology Department, 91 Boulevard de l Hôpital, Door: , Paris cedex 13, France. Tel: (33) , Fax: (33) , bastian@chups.jussieu.fr Key Words: Oxaliplatin, cell cycle, apoptosis. In general, the cytotoxicity of platinum compounds in cancer cells can be related to the inhibition of DNA synthesis or to saturation of the cellular capacity to repair Pt-DNA. The cytotoxic activity of L-OHP is initiated by the formation of DNA adducts. L-OHP causes more DNA strand-breaks compared to cisplatin (5). DACHplatinum adducts formed by L-OHP are more bulky and more hydrophobic than platinum adducts formed by cisplatin and carboplatin. As a result, the DACHplatinum adducts are more effective in inhibiting DNA synthesis (6). When DNA is damaged, the cells must be able to prevent progression through the cell cycle; this is ensured by the existence of checkpoints, mechanisms that arrest the cell cycle in the G1-, S- or G2-phases after activation by damaged DNA. In addition, cells can undergo apoptosis if the damage cannot be repaired (7). Most previous studies focused their interest on the effect of L-OHP on cell cycle modifications and apoptosis induction in colon and gastric cancer cells (8-10). This underlines the importance of a better understanding of the cell effects of L-OHP in other tumour cell lines, to further explore its rational use and to improve knowledge regarding its possible use in other pathologies. To this purpose, a panel of four human tumour cell lines, (HT29) colon cancer, (MCF7S) an anthracycline-sensitive human breast cancer cell line, (A549S) a taxane-sensitive human lung adenocarcinoma and the (Hela) uterine cervix cell line were used. The effects of L-OHP on the cell cycle and DNA synthesis were analyzed using bromodeoxyuridine (BrdUrd) and propidium iodide (PI). The induction of cell death, namely apoptosis, was then assessed with Annexin-V and PI probes and by performing double parameter flow cytometry. Materials and Methods Drug. Crystalline L-OHP was a gift from Sanofi. Ten mm stock solutions were prepared by dissolving an appropriate amount of the drug in water with ultrasound. Aqueous L-OHP was stored at 4ÆC, protected from light. The drug was diluted in the culture medium immediately before use /2006 $

2 Cell lines. All the cell lines used were of human origin. The HT29 colon cancer cell line was a gift from Dr. M. F. Poupon (Curie Institute, Paris). The sensitive breast cancer cell line MCF7S and the sensitive lung adenocarcinoma cell line A549S were obtained from Dr. F. Calvo (Saint-Louis Hospital, Paris). The Hela cell line was provided by Pr. P. Leroy (Nancy 1 University, France). The cells were maintained in exponential growth phase in 25-cm 2 flasks in RPMI 1640 (Gibco, France) medium supplemented with 10% heat-inactivated foetal calf serum (Gibco), 2 ÌM glutamine, 100 U/ml penicillin-streptomycin (Gibco) and 2.5 Ìg/ml fungizone (Gibco) and were incubated at 37ÆC in a 5% CO 2-95% air, humidified incubator. All the experiments were performed in triplicate and the results are expressed as the mean of three experiments. Cell cycle measurements. Cells pulse-labelled with the thymidine analogue (BrdU) were detected by use of monoclonal rat antibody anti-brdu (Seralab, France) and a fluorescein-isothiocyanate goat anti-rat (FITC)-conjugated antibody. The use of PI (Sigma, France) as a second probe allowed for the simultaneous quantification of the cellular DNA content by dual-parameter flow cytometry. This approach was employed to selectively label and determine the proportion of replicating S-phase cells following L-OHP treatment. The cell cycle studies were performed on cells exposed to various concentrations of L-OHP, (25, 50 and 100 ÌM) previously tested for cytotoxicity studies (submitted for publication, 2005) during 3-h exposure and various post-incubation times (24 or 72 h). The appropriate dilutions of L-OHP were made from the stock solution directly in the fresh culture medium. An adequate volume of BrdU was added at the end of L-OHP incubation for a final concentration of 30 ÌM and the cells were incubated for 15 min at 37ÆC. All the cells were trypsinised into single cells using trypsin/ethylene diamine tetra acetic acid (EDTA, Gibco), washed twice in cold 1 x PBS, fixed in 2 ml cold 70% ethanol and stored at 4ÆC until cytometric analysis. Prior to staining with the anti-brdu antibody, the fixed cells were washed once in 2 ml cold PBS and were then subjected to digestion in 0.3 mg/ml pepsin (Sigma) and 0.1 N HCl for 20 min at 37ÆC to remove histones. Subsequently, the cells were washed with PBS resuspended in 0.5 ml 2 N HCl and incubated for 20 min at room temperature to obtain partially denaturated DNA. The HCl was removed and the cells were washed with 5 ml PBS and resuspended in 3 ml PBS/BU buffer (PBS containing 0.5%, Tween 20, 0.5% goat serum and Hepes). BrdU detection was carried out by incubation in the same PBS/BU solution containing 20 Ìl anti-brdu monoclonal antibody. After 45-min incubation at room temperature, the cells were washed with PBS, resuspended in PBS/BU solution containing goat anti-rat IgG FITC and incubated for 30 min at room temperature in the dark. The cells were then pelleted and resuspended in 300 Ìl PBS containing 10 Ìg/ml PI. Flow cytometry analysis were carried out using a FACSORT (Becton Dickinson) flow cytometer. Two parameters were simultaneously monitored in list mode and analysed using Cell Quest Pro analysis software. Green fluorescence (BrdU content) was detected between 510 and 560 nm and red fluorescence (DNA content) was measured between 620 and 740 nm. Finally, at least 10 4 cells were analysed and cell doublets, triplets, etc., were excluded from the analysis. Annexin V-FITC assays. Staining for Annexin V-FITC was performed using an apoptotic detection kit (Beckman Coulter, France). Double-staining for annexin V-FITC binding and for cellular DNA using PI was performed in triplicate. After washing three times in PBS, the cultured cells were resuspended in 100 Ìl of 1x binding buffer. Two Ìl of Annexin V-FITC (25 Ìg/ml) and 5 Ìl PI (250 Ìg/ml in 1x binding buffer) were added to each sample. The cells were incubated in the dark for 10 min. Prior to flow cytometric analysis, 400 Ìl of ice-cold 1x binding buffer were added. The samples were all analysed within 1 h. Human Jurkat cells derived from T-cell leukaemia were used as the positive control. After 30-min incubation on ice with PBS containing 3% formaldehyde, the cells were washed and resuspended in culture medium. Induction of apoptosis by ligation of Fas (CD95) on Jurkat cells was done by the addition of 100 ng/ml anti-fas / CD95 antibody to the culture medium followed by incubation of the cells for 16 h at 37ÆC (5% CO 2 ). Following centrifugation, the cells were resuspended in 1x binding buffer. All the samples were analysed using a FACSORT (Becton Dickinson) flow cytometer. Results Cell cycle phase distribution after 3-h exposure and postincubation time. The BrdU/PI method provided relatively precise information on the transit of the cells through the different phases. It also allowed for the assessment of the DNA synthesis rate of cells through the S-phase (11). The cell cycle modifications after 3 h of L-OHP treatment followed or not by 24 or 72 h of post-incubation were assessed (Figures 1, 2). Three L-OHP concentrations (25, 50 and 100 ÌM) were used for each of the four cell lines to make comparisons between cells at the same L-OHP concentrations. After 3 h of treatment, there was no predominant accumulation of cells in any phase of the cell cycle (data not shown). After 24- or 72-h post-incubation, the cells showed different distribution patterns in comparison to the pattern prior to treatment. For the HT29 cell line, a transitional S-phase delay was observed with maximum values (84%±5), which decreased after further post-incubation (7.6%±3), whereas cells predominantly accumulated in the G2/M-phase at all L-OHP concentrations, where maximum values reached (55.9%±4) relative to the control (8.9%±1) (Figure 1A). The G0/G1-phase accumulation increased for the MCF7 cells (60.6%±1.4, 55.7%±6 and 61.7%±0.3) at 25, 50 and 100 ÌM, respectively, relative to 34%±3 for the control cells. This blockage was overcome after an additional 48 h of incubation. Meanwhile, the G2/M populations increased at all drug concentrations, (25.1%±4.2, 34.5%±5.7 and 31.4%±4) relative to the control 10%±0.6. Hence, the cells were transiently blocked in the G1-phase before accumulating in G2/M (Figure 1B). With the Hela cells, an increase in the S-phase reaching 85.1%±5.4 relative to 74%±8.4, followed by a recovery 2094

3 William-Faltaos et al: Cell Cycle Arrest and Apoptosis Induced by Oxaliplatin Figure 1. Cell cycle distribution after exposure to L-OHP, for the HT29 (A) and MCF7 (B) cell lines. H0=control, W24= 24-h post-incubation and W72=72-h post-incubation. 2095

4 Figure 2. Cell cycle distribution after exposure to L-OHP, for the Hela (A) and A549 (B) cell lines. H0=control, W24=24-h post-incubation and W72=72-h post-incubation. 2096

5 William-Faltaos et al: Cell Cycle Arrest and Apoptosis Induced by Oxaliplatin Table I. Flow cytometric analysis of apoptosis in the four cell lines stained with annexin V-FITC and PI after 12-h treatment. 12-h Live Live / Late apoptotic / treatment (A /PI ) early apoptotic early necrotic (A + /PI ) (A + /PI + ) Jurkat 77±3.6 2± ±0.7 HT29 control 91±5.8 1± ± ÌM 88± ± ± ÌM 87±3.0 3± ± ÌM 88± ±0 3.3±1.2 MCF7 control 82±1.3 1± ± ÌM 88± ± ± ÌM 87± ±0 5.6± ÌM 88± ± ±0.7 Hela control 92± ± ± ÌM 93± ± ± ÌM 93± ± ± ÌM 78± ± ±0.6 A549 control 95± ± ± ÌM 97± ± ± ÌM 96± ± ± ÌM 95± ± ±0.2 Table II. Flow cytometric analysis of apoptosis in the four cell lines stained with annexin V-FITC and PI after 12-h treatment and 24-h post-incubation. 24-h post- Live Live / early Late apoptotic / incubation (A PI ) apoptotic early necrotic (A + /PI ) (A + /PI + ) Jurkat 69± ± ±3.0 HT29 control 93± ± ± ÌM 85± ± ± ÌM 83± ± ± ÌM 70± ± ±6.8 MCF7 control 88± ± ± ÌM 91± ± ± ÌM 88± ± ± ÌM 86± ± ±1.4 Hela control 94± ± ± ÌM 91± ± ± ÌM 87± ± ± ÌM 73± ± ±1.1 A549 control 96± ± ± ÌM 92± ± ± ÌM 91± ± ± ÌM 86± ± ±3.6 from S-phase accumulation and an increase in G2/M by 48 h were observed at all L-OHP concentrations, with maximum values 71.1%±3.1 ( Figure 2A). The A549 cells showed an accumulation in the G0/G1- phase (73.5%±0.2, 71.1%±6.7 and 72.4%±9.4) relative to 42%±11 which persisted after an additional 48 h of drug release (67.5%±7, 71.7%±8.5 and 74.5%±3.9) with a relative increase in control to 54%±7.2. Hence, these cells had almost the same distribution profile as the control cells (Figure 2B). Overall, after 24 h of post-incubation, the cells exhibited different cell cycle distribution patterns: for HT29 and Hela cell lines the S-phase was transitionally delayed, meanwhile the MCF7 cell line accumulated in the G0/G1-phase. After 72 h of post-incubation, all cell lines were blocked in the G2/M-phase, while no notable changes were observed for A549 relative to control cells (lung carcinoma has never shown in vitro or in vivo sensitivity to L-OHP). Identification of viable, apoptotic populations by doubleparameter flow cytometry. In this study, the percentage of apoptosis in the four cell lines was analysed after different treatment times and a 24-h post-incubation using AnnexinV-PI. This test discriminates between viable cells (FITC /PI ), early apoptotic cells (FITC + /PI ) and late apoptotic cells (FITC + /PI + ). No apoptosis induction was observed after 3-h treatment at all the tested doses. In order to investigate whether apoptosis needed more contact time between the cells and drug, cells were exposed to L-OHP for 12 h and then remained in drugfree medium for another 24 h. After 12 h of treatment, only the MCF7 and Hela cells showed a slight late apoptosis increase (5.6%±0.6, 7%±0.6) at 50 and 100 ÌM, respectively (Table I). When the cells were left for 24 h after drug removal (Table II), late apoptosis increased for HT29, reaching 16%±6.8 and 20%±1.1 for Hela at 100 ÌM in comparison with the 12-h treatment, where the maximum values were 3.3%±1.2 and 7%±0.6, respectively. The mean percentage of apoptosis in the control cells did not exceed 3.5%. Positive control, Jurkat cells showed late apoptosis of 16%±0.7 and 15%±3 after 12 h of treatment and 24 h of post-incubation, respectively. Discussion Oxaliplatin is a novel platinum derivative with remarkable activity in advanced colorectal cancer. L-OHP, in combination with 5-FU and FA, is indicated for first-line (Europe/Australia/Asia) or second-line (US) therapy of metastatic colorectal cancer (12). L-OHP should be administered as a 2- to 6-h infusion. L-OHP is a watersoluble platinum compound characterised by a DACH platinum carrier which causes increased cytotoxicity due to more effective inhibition of DNA synthesis and DNA repair (13). Recent data suggest that, despite quantitatively lower levels of L-OHP-DNA adducts compared with cisplatin, these DACH-Pt adducts may induce cell death more efficiently 2097

6 than cisplatin-dna adducts in cultured cancer cells (14). Previous studies explored the effect of L-OHP on the cell cycle in the HT29 cell line after 24 h of treatment (8, 9). In the present study, the distribution of a panel of four human cancer cell lines in the different phases of the cell cycle were investigated after 3 h of L-OHP treatment. Previous reports indicated a block in the G1-phase after 24 h of 5 ÌM L-OHP treatment in the HT29 cell line (8), or G2/M arrest at 15.1 ÌM and a small decrease in the S-phase (9). To our knowledge, no previous studies have indicated the cellular effect of 3-h treatment with L-OHP followed or not by post-incubation. We used the 3-h exposure time, with or without 24 or 72 h of post-incubation. The post-incubation allows determination of whether the growth inhibition was cell cycle phase-specific, and whether the cells released from growth arrest resumed the cycle or proceeded to apoptosis. Several modifications were observed after drug release: after 24 h of post-incubation, the HT29 and Hela cell lines showed a transitional S-phase delay which recovered after an additional 48 h, whereas the MCF7 and A549 lines exhibited arrest in the G1-phase. The MCF7 and Hela cell lines showed slight accumulation in the G2/M-phase. Overall after 72 h of post-incubation, all cell lines, except for A549, were blocked in the G2/M-phase. The arrest in G2 prevents damaged DNA from entering the next phase of the cell cycle and M arrest traps damaged cells that have escaped G2 arrest (7). The results of a previous study demonstrated that cells which have initiated a G2/M check-point in response to DNA damage can succumb to a variety of fates, including apoptosis, prolonged permanent arrest, recovery after repair of DNA damage or adaptation to the damage, allowing progression through the cell cycle with the DNA damage that initially evoked the arrest (15). Several reports proposed that G2 cell arrest correlates with survival and is a better indicator of cytotoxicity than inhibition of DNA synthesis. The G2 block has been proposed to be a major decision point for damaged cells. According to some models, cells emerging in G2 arrest either progress through the cell cycle or go to apoptosis, depending on the extent to which the damage can be repaired during G2 arrest (16, 17). In our study, MCF7 and Hela cells showed a prolonged permanent arrest, persisting for long after drug removal. Hence, the difference in the extent and duration of G2/M arrest in the different cell lines observed in this study could depend on the difference in cellular capacities for DNA repair. A significant population of A549 cells remained in G1-phase, suggesting that a fraction had been able to start another cell cycle due to repair (18). The cytotoxicity of L-OHP is currently explained by the fact that it acts as an alkylating agent which penetrates cells and is delivered to the nucleus where it forms adducts with DNA able to block or delay replication. At this point, cells have at least two probable pathways: either adducts are detected by a system capable of generating a signal that activates an apoptotic pathway followed by cell death, or they are able to activate the NER pathway in order to repair DNA and thus escape cell death (19). To answer the question of whether apoptosis is the main event related to cell cycle modifications, the AnnexinV-PI test was performed following L-OHP treatment after exposure and post-incubation. Apoptosis induction was initially assessed after 3 h of treatment, with or without 24 h of post-incubation, but none was observed. These results are consistent with earlier studies, which demonstrated (1) that, given the reduced level of Pt-DNA adducts, L-OHP displayed a disproportionately greater ability to induce secondary lesions in DNA, which are apoptosis precursors. These lesions might need a longer exposure time to appear and, thus, to induce apoptosis. To further support this argument, cells were exposed to L-OHP for the longer duration of 12 h, followed or not by 24 h of post-incubation. It was then possible to detect apoptotic signalling generated by L-OHP, which was late and moderate. This induction was more pronounced for the Hela and HT29 cell lines after post-incubation. These data are in accordance with cell cycle modifications. It has been demonstrated that cell lines that do not arrest in G1 demonstrate delay in the S-phase and undergo apoptosis more efficiently than do cells that first arrest in G1 (20). The pharmacological results presented here also suggest that the L-OHP infusion should be lengthened to 6 h or more, rather than 2 h, in order to allow the drug to effectively modify the cell cycle and induce apoptosis. Cancer treatments are extremely costly and therefore a tendency to administer antitumoral drugs over a short period of time in the out-patient unit has arisen so as to diminish costs. However, the fact that apoptosis and cell cycle modification seem to depend on the duration of contact between the drug and the cells make investigations of extended exposure times to drugs worthwhile. An ongoing clinical trial, in which L-OHP and 5-FU or 5-FU precursors are administered to patients with gastrointestinal tumours over a 6-h infusion, appears to produce very little neurological limiting toxicity compared to the 2-h infusion. Despite the length of the L-OHP infusion, it can be easily conducted in an out-patient day unit. 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