Abstract
Background: Time-dependence of cisplatin (CDDP) and oxaliplatin (L-OHP) cytotoxic effects in A431 and A431/Pt cells (sensitive and CDDP-resistant human cervical tumor cells) were investigated. Materials and Methods: The drug application modes were pulse (12.5, 25 or 50 μM up to 72 h) and pulse-plus-chase (50 μM for 2, 4 or 6h, followed by washing and 72 h-incubation in drug-free medium). Results: In the A431 cells, the pulse drug application showed time-effect curves with two plateaux; the inhibitory activity of CDDP was higher than that of L-OHP. The same growth-inhibition fraction was reached by L-OHP in a longer time than CDDP. In the A431/Pt cells, the curve shapes for both drugs were similar in both application modes and had the same general characteristics, noted in the parental cell line. CDDP appeared less active than L-OHP. Conclusion: Different cytotoxicity curves of Pt-drugs could be dictated by the presence of the bulky diaminocyclohexane (DACH) ligand, affecting the kinetics of Pt-DNA binding; mismatch repair (MSH2) protein is involved in the resistance.
- A431 and A431/Pt human cervical tumor cell lines
- platinum drugs
- prolonged drug incubation
- cytotoxic effect
The critical role of DNA-Pt adducts in the antiproliferative effects of all antitumor Pt-drugs is generally accepted (1, 2). It is not surprising that a linear correlation has been found between the gross levels of platinum bound to DNA and the extent of cytotoxicity (1, 3, 4). DNA platination can be considered as a ligand substitution reaction in Pt complexes. It is well known that the rate of nucleophilic substitution of a ligand on square planar complexes, such as cisplatin (CDDP) and oxaliplatin (L-OHP), is very sensitive to steric hindrance both in the substrate (Pt complex) and in the nucleophile (guanine, adenine DNA bases) (5). The presence of the chelated bulky ligand diaminocyclohexane (DACH) in L-OHP may interfere with the rate of DNA platination and consequently with the time of appearance of drug cytotoxicity in comparison with CDDP (6, 7). We have previously observed that in the presence of comparable levels of cellular Pt accumulation, DNA platination levels were greater for CDDP than for L-OHP (8). The relevance of kinetic parameters, such as the steric effect on the formation of Pt-DNA, can be evaluated in a clear-cut way when the cellular uptake of the tested drugs is at comparable levels and their metal-DNA adducts are similarly repaired. These particular conditions have been shown to be fulfilled for CDDP and L-OHP when applied to the sensitive human cervical tumor cells (A431) (8). Furthermore, in the CDDP-resistant counterpart (A431/Pt) the level of the DNA mismatch repair protein MSH2 is reduced (9). This feature suggests that resistant cells have an increased tolerance to DNA damage which, consequently, contributes to the expression of resistance. In our previous experimental conditions (after 1-h drug exposure) no relevance of reduced MSH2 protein level was observed and the resistance factor paralleled the reduction of drug accumulation (8).
A change in the experimental conditions, for instance prolonging the cellular exposure to CDDP and L-OHP to evaluate the time-dependence of drug cytotoxic effects, could be the way to answer both questions of the role, if any, of the steric effect of DACH on Pt-DNA adduct formation and the relevance of the reduction of MSH2 protein in A431/Pt for the L-OHP and CDDP efficacy. To our knowledge, prolonged drug exposure time is a topic which has not been extensively considered, and published literature is scarce (10-12).
In vitro this approach could be useful to study the effects of different modes of application of anticancer drugs and to gain information for the development of clinical dosing strategies (13). Moreover this methodology could increase the knowledge of the drug mechanism(s) of action in relation to the different carrier ligands, which are known to be able to confer particular physico-chemical properties (8, 14, 15).
In the present study the dependence on exposure time of the L-OHP and CDDP cytotoxic effects in A431 and A431/Pt cells were investigated, when different concentrations of the drugs were used. The concentrations of drugs were selected according to cell sensitivity determined previously (8).
Materials and Methods
Cell lines and culture conditions. The human cervical squamous cell carcinoma cell line A431 and the CDDP-resistant A431/Pt subline were used in this study (9). Both cell lines were kindly provided by Dr. P. Perego, Department of Experimental Oncology and Laboratories, Istituto Nazionale Tumori, Milano, Italy. The establishment details and biological properties have been described previously (9). The cells were grown as monolayers at 37°C in a 5% CO2 atmosphere in RPMI-1640 medium (BioWhittaker Italia S.r.L., Milano, Italy) supplemented with 10% heat-inactivated fetal calf serum (Sera-Lab, Crawley Down, Sussex, England) and 2 mM glutamine (Sigma-Aldrich S.r.L., Milano, Italy) (complete medium). Antibiotics were omitted from the medium to avoid interactions. The parental and the resistant lines were used from passage 2 to 20. Growth and morphology were monitored weekly. Platinum drugs. CDDP (Platamine®) was obtained from Pharmacia and Upjohn S.p.A., Milano, Italy and L-OHP (Eloxatine®) was a gift from Sanofi-Synthelabo S.p.A., Milano, Italy. Immediately before use, the CDDP was dissolved in 0.9% saline, while the L-OHP was dissolved in water.
Drug exposure. To develop and evaluate the assay procedure, some initial experiments were performed using the A431 and A431/Pt cell lines. In the pulse plus chase experiments (see below) it was necessary to completely remove CDDP and L-OHP before determining the drug activity. To minimize the effect of any remaining drug, the plates were washed four times with sterile PBS (13) and further incubated in complete medium for up to 72 h. The possible cell loss because of repeated washings was tested by seeding three six-well plates (9.6 cm2, Iwaki Glass Inc., Funabashi, Japan) with 1 ml of cell suspension (3·104 cells) in culture medium. The cells were incubated at 37°C and allowed to attach overnight (about 12 h). Then the medium was removed. One unwashed plate was used as control, the second plate was washed once and the third plate was washed four times. Each washing was performed using 1 ml of prewarmed PBS. In all the experiments a four-time washing procedure was applied, even if not strictly necessary. The cells were harvested using 0.5 ml of trypsin-EDTA solution (Sigma-Aldrich) and then counted with the aid of a Bürker chamber and a light microscope (8); to reduce experimenter's bias, counts were made by two independent observers and values averaged.
Drug exposure. To evaluate the dependence of drug activity on the concentration and exposure time, the plates were seeded as described previously. The medium was then removed and drugs were added (1 ml of drug solution in complete culture medium for each well). Two modes of drug exposure were used: pulse (i.e. continuous application of the drug) and pulse plus chase (i.e. application of the drug for 2, 4 or 6 h followed by washing and further incubation in drug-free medium up to 72 h).
For the pulse protocol the cells were exposed to 1 ml of 12.5, 25 or 50 μM drug for 1, 6, 15, 24, 30, 48, 54, 60 or 72 h at 37°C, then washed, harvested and counted. For CDDP in the A431 cell line an additional measurement was made after 40 h.
For the pulse plus chase protocol the cells were exposed to 1 ml of 50 μM drug solution for 2, 4 or 6 h (pulse times) at 37°C. After drug exposure, the medium was removed, the cells were washed four times with PBS and then 1 ml of fresh drug-free medium (chase) was added. The plates were further incubated to obtain a total time (pulse plus chase) of 6, 15, 24, 48, 60 or 72 h before final washing, harvesting and counting.
Statistical analysis and AUC. All the assays were performed in duplicate, and the values are the means±standard deviation (S.D.) of three independent experiments. The cell survival for each experimental drug concentration and time was expressed as the percentage of living cells in comparison to untreated control cells. The area under the curve (AUC) was calculated using the trapezoidal rule, from time 0 up to the last observed time (72 h).
Results
The loss of cells from the washing procedure was very small (≤2%) after both one and four-time washing procedures (data not shown). Furthermore, no change of the pattern of the concentration-response curve was observed when the cells, washed by the two procedures, were further incubated for 3 h, thus indicating that the Pt-drugs were removed.
Pulse drug application. i) A431 parental cell line: Figure 1 shows the time-effect curves for 12.5 μM, 25 μM and 50 μM concentrations of L-OHP and CDDP applied for up to 72 h. Both drugs showed an initial lag period of about 6 h, followed by a rapid decrease of cell survival until 25 h. After this period, further reduction in cell viability was observed with the progression of time. With the 12.5 μM concentration, a second lag period of about 24 h for L-OHP and of about 7/8 h for CDDP was observed. This phenomenon became almost imperceptible with increasing drug concentration. Consequently, the general trend of both drugs appeared to be similar, although the kinetics behaviour seemed to favor CDDP in comparison to L-OHP.
As indicated by the AUC (Figure 2), as an integrated measure of drug activity, the inhibitory activity of CDDP was higher than that of L-OHP at every concentration and seemed to be very little modulated by increasing drug concentration. The linear regression between the concentration of the Pt drugs and the AUC (Figure 3) showed a high correlation, as indicated by the r value, although the low number of points did not allow statistical significance to be reached (Table in Figure 3). The activity of both drugs appeared to be slightly dose-dependent in the range of drug concentrations used, as shown by the linear regression (Figure 3).
Time-effect curves for 12.5 μM, 25 μM and 50 μM concentrations of L-OHP and CDDP applied by the pulse method in A431 cells.
AUC at the different concentrations of drugs in the two cell lines, A431 wild type and Pt-resistant counterpart, pulse method.
ii) A431/Pt resistant cell line: Figure 4 shows the time-effect curves for 12.5 μM, 25 μM and 50 μM concentrations of L-OHP and CDDP applied for up to 72 h. The curve shape for both drugs was similar and the same characteristics that were noted in the parental cell line (see i) were also evident. It still appeared that drug-induced cell lethality was very little modulated by increasing the concentration (Figures 2 and 3).
Linear regression between concentration of Pt drugs and AUC calculated from cell viability vs time plot. The table presents the parameters of regression.
The pharmacological profile of L-OHP did not vary between the parental and CDDP-resistant cell lines (Figure 3), while CDDP appeared less active than L-OHP in the A431/Pt cells (Figure 3). The AUC analysis showed the same general behaviour found in the parental cell line.
Pulse plus chase drug application. i) A431 parental cell line: In Figure 5 the effects of CDDP and L-OHP are presented with a fixed concentration (50 μM) of Pt complex applied for 2, 4 or 6 h (pulse period) followed, after drug washing out, by an incubation period of up to 72 h (chase period).
Time-effect curves for 12.5 μM, 25 μM and 50 μM concentrations of L-OHP and CDDP applied by the pulse method in A431/Pt cells.
For both drugs the curves presented similar shapes and were steeper for incubation time <24-25 h than for longer exposure, >25 h. It appeared that the same growth-inhibition fraction (for instance IC50) with the same pulse period was reached by L-OHP treatment after a longer time than CDDP; the curves of CDDP and L-OHP were almost superimposed when higher pulse periods of the latter drug were compared with shorter pulse periods of CDDP and there were still two lag periods (as shown in the pulse mode experiment), the second of which was more evident with short pulse time.
The AUC analysis (Figures 6 and 7) showed greater activity for CDDP than L-OHP and within each drug there was low dependence on concentration.
ii) A431/Pt resistant cell line: In Figure 8 the effects of CDDP and L-OHP on the resistant cell line are presented, with a fixed concentration (50 μM) of Pt complex applied for 2, 4 or 6 h (pulse period) followed, after drug washing out, by an incubation period up to 72 h (chase period). The overall behaviour of the cells (see Figures 6 and 7) was comparable to that obtained in the pulse mode application of both drugs (Figures 2 and 3).
Discussion
Previously we found that, in A431 parental cells, the uptake levels of both drugs (after 1-h application followed by washing and further 72 h incubation in drug-free medium) were similar while the level of Pt-DNA and the IC50 were 1.5- and 5-fold, respectively, in favour of CDDP (8). Now, in the same parental cell line, using the pulse mode, the same percent cell survival was reached with L-OHP in a longer time than with CDDP treatment (Figure 1). This different behaviour between the two drugs became smaller or disappeared when the effects of higher concentrations of L-OHP in comparison to those of CDDP were considered. These results, together with those previously reported in the literature (8, 16-19), confirmed that L-OHP is less reactive than CDDP in binding cellular DNA and the steric hindrance of the DACH ligand may explain this finding, so underlining the relevance of steric effects in the modulation of L-OHP activity.
Time-effect curves for 50 μM concentration of L-OHP and CDDP applied by the pulse plus chase method in A431 cells.
AUC at the different concentrations of drugs in the two cell lines, A431 wild type and Pt-resistant counterpart, pulse plus chase method.
Linear regression between time of incubation (pulse) and AUC calculated from cell viability vs time plot. The table presents the regression parameters. The correlation was high, as indicated by the r value; *p<0.05.
In the same cell line these results were confirmed by the pulse plus chase application mode of both drugs. In particular, it should be noted that at the same accumulation time that demonstrated comparable uptake (8, 20) (pulse period) the lethality produced by 50 μM L-OHP was always lower in comparison to that of CDDP and this difference became smaller or disappeared when higher pulse periods of L-OHP were compared to shorter pulse periods of CDDP (Figure 5). Both drugs behaved similarly in the A-431 cells, the only difference being the lower cell survival at shorter times for CDDP compared to L-OHP (Figures 1 and 5).
Time-effect curves for 50 μM concentration of L-OHP and CDDP by the pulse plus chase method in A431/Pt cells.
The cell growth inhibition kinetics of both drugs and in both application modes presented two stages in which no (the first) or static (the second) cell growth inhibitory effects were shown. The first (6 h) lag period could have been associated with the time required for the drug, inter alia, to accumulate, reach the genomic DNA and to express its pharmacological activity. The second lag period, after 20-24 h of 12.5 μM treatment, when mortality (about 50%) did not change for some hours, subsequently increasing faster with CDDP than L-OHP requires further explanation. Unlike the first lag period, the second lag became almost imperceptible with increasing drug concentrations. In MCF-7 cell line treated with CDDP and L-OHP continuous incubation for 1-48 h, the accumulation did not substantially depend on the initial concentration of the drug in the medium and was rapid at the beginning of the treatment, and then smoothly reached a sort of plateau (10, 20). It has been suggested that this plateau (occurring after prolonged incubation time) is due to damage to and then to block of membrane function, as the result of extensive platination (21). However the present results indicated that the second lag in drug lethality was maintained, as a common feature, in the sensitive and CDDP-resistant A431 cell lines (Figures 1, 4, 5 and 8), even though only a cell membrane change characterizes the latter (8). Consequently it is possible that the cell membrane could be implicated in the phenomenon, but also other causes could be involved. For instance, the second lag in drug lethality could be related to mechanisms which reduce the level of active platinum drug due to a kinetics slower than that which characterizes the formation of Pt-DNA. Such kinetics could concern for instance the inactivation reaction of Pt-drugs with thiol groups, which is known to be characterized by a rate constant smaller than that of Pt (II) with DNA (22). This kinetic behaviour could explain both the fall in cell viability until 24 h and the disappearance of the second lag at the higher concentrations of platinum drugs.
Furthermore, when the general behaviour of both drugs as shown by AUC is compared, it should be noted that the proportion of surviving cells did not parallel the level of the drug in the culture medium. At present no rationale for this phenomenon is evident, but taking into account that for instance, 1 ml of 12.5 μM drug applied to 3·104 cells equates to about 1010 drug molecules/cell, it is possible that saturation of the receptor sites had already been reached. It seems clear that continuously applied high drug concentrations should be carefully considered since they do not appear necessary for increasing the pharmacological effect. Obviously, in vivo, other mechanisms for inducing arrest of tumor growth different from those of a cell line growing in monoculture could also be present.
In the CDDP-resistant cell line A431/Pt, the pulse or pulse plus chase application of both drugs, while confirming the results obtained in the sensitive cell line, showed higher activity of L-OHP in comparison to that of CDDP. This result could be related to the 2.6-fold uptake reduction of CDDP vs L-OHP but contrasts with the similar Pt-DNA levels of these drugs (8). Furthermore, since the L-OHP activity did not vary between the A431 and A431/Pt cells, the reduction of CDDP activity from the A431 to the A431/Pt cells was probably specific for this drug and related to events following the Pt-DNA formation, since, as previously reported (8), similar levels of Pt-DNA are formed by both complexes. Previously it has been noted that the mismatch repair MSH2 protein expression in A431/Pt cells is reduced in comparison to the A431 cell line (9) and, consequently, the CDDP-resistant cells are able to tolerate higher levels of CDDP-induced DNA damage than their parental sensitive counterpart. The present results confirmed this conclusion (9) which was not appreciated previously with 1 h drug incubation (8). This alteration of mismatch repair may contribute to the building up of the resistance factor and is manifested at prolonged incubation time. Moreover, the damage recognition protein MSH2 seems to present greater preference for CDDP adducts than L-OHP, possibly because L-OHP adducts are poorly recognized (23-26).
In summary, the cell growth inhibition kinetics of L-OHP and CDDP in A431 parental cells and in the CDDP-resistant counterpart, are quite similar, suggesting that a similar activity pathway characterizes both drugs. The cytotoxicity curves of L-OHP could be dictated by the bulky DACH ligand in this Pt complex, affecting the kinetics of Pt-DNA binding. After an initial lag period, the decrease of cell survival is rapid at the beginning of treatment, and then reaches a sort of a second plateau followed by a drop in cell viability. The AUC does not parallel the level of Pt-drug in the culture medium, possibly because saturation of the receptor sites is already reached. In the resistant cell line, the prolonged incubation time suggests the contribution of MSH2 protein in the building up of resistance. These almost unexpected findings suggest that the study of the prolongation of exposure time to drugs should be more extensively considered.
Acknowledgements
The authors are grateful to Dr. Lloyd Kelland (CRT Development Laboratory, Wolfson Institute for Biomedical Research, University College London, London, UK) for helpful discussion. The authors would like to thank the Anonymous Referee for the detailed review of the manuscript and comments to improve the presentation of the work.
- Received April 27, 2009.
- Revision received July 20, 2009.
- Accepted August 21, 2009.
- Copyright© 2009 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
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