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Experimental Therapeutics, Preclinical Pharmacology |
Fédération des Services des Maladies Sanguines, Immunitaires, et Tumorales, Hôpital Paul Brousse, 94804 Villejuif [N. Z-S., E. C., F. G.]; and Unité INSERM U55, Hôpital St. Antoine, 75012 Paris [E. R.], France
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We explored the cellular pharmacology of the combination of SN-38 and oxaliplatin, both in view of the therapeutic potential of the combination in the clinic and in an attempt to investigate the cellular interactions between camptothecin derivatives and platinum derivatives.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell Culture.
HT29 human colon adenocarcinoma cells were provided by Dr Y. Pommier (National Cancer Institute, NIH, Bethesda, MD) and were grown in monolayer cultures in RPMI 1640 supplemented with 5% heat inactivated FCS and 2 mM glutamine. No antibiotics were added to the medium. The cells were trypsinized and passed twice a week.
Cytotoxicity Assays.
Cytotoxicity was determined by colony formation assays. Briefly, exponentially growing cells (2 x 105 cells/ml) were plated 24 h before drug treatment to allow them to attach as single cells. Following drug treatment, cells were washed twice with 5 ml of PBS preheated at 37°C and trypsinized. Colony-forming ability was determined in triplicate by cloning 103 cells into 25-cm2 flasks. Colonies were stained with methylene blue and counted after 14 days. Cytotoxicity was assessed from at least two different experiments. The cloning efficiency for untreated cells was 75%.
Growth inhibition was measured using the MTT (Sigma Chemical Co., St. Louis, MO) dye assay described previously (21) to evaluate the growth-inhibitory effects of the cytotoxic agents. Cell suspensions containing 2 x 104 viable cells/ml were seeded into 96-well microtiter plates. After a 48-h incubation, the cells were treated for 24 h with various concentrations of SN-38 (5 x 10-3 to 102 µM), oxaliplatin (10-2 to 103 µM). Combination assays were performed using 0.05 µM SN-38 with various concentrations of oxaliplatin. In the sequential schedule, SN-38 was administered 24 h prior to or after oxaliplatin. Seventy-two h following exposure to the drugs, 50 µl of a 125 µM MTT solution were added to each well, and the plates were incubated at 37°C for another 3 h to allow MTT metabolism into formazan crystals. The formazan crystals were finally solubilized adding 50 µl of 25% SDS solution (Sigma) into each well. Absorbance at 540 nm was measured using a Delta Soft ELISA analysis program for a Macintosh computer interfaced with a Bio-Tek microplate reader (model EL-340; BioMetallics, Princeton, NJ). Wells containing only RPMI 1640-fetal bovine serum and MTT were used as controls. Each experiment was performed using eight replicated wells for each drug concentration and carried out independently at least three times. The GI50 was defined as the concentration that reduced the absorbance in each test by 50%. The absorbance was calculated as: [(mean absorbance of eight wells containing drug) - (absorbance of eight control wells)]/[(mean absorbance of eight drug-free wells) - (absorbance of eight control wells)] x 100.
Analysis of Combination Effects.
Combination analysis was performed using the method described by Chou et al. (21
, 22)
and a Calcusyn software program for automated analysis (Biosoft, Cambridge, United Kingdom). The influence on the combination of the two drugs was evaluated by comparing the sequential assays with assays involving oxaliplatin or SN-38 exposures alone. The combination effect was evaluated from isoeffect analysis CIs, calculated as follows: CI = Coxaliplatin/Cxoxaliplatin + CSN-38/CxSN-38, where Cxoxaliplatin and CxSN-38 are the concentrations of oxaliplatin and SN-38 alone, respectively, needed to achieve a given effect (x%) and Coxaliplatin and CSN-38 are the concentrations of oxaliplatin and SN-38 needed for the same effect (x%) when the drugs were combined. These concentrations were calculated for each experiment and for each combination experiment at a fixed concentration ratio. The CIs were calculated under the assumption of a mutually exclusive drug interaction, i.e., that the effect of SN-38 on topo I may influence the effect of oxaliplatin on DNA and vice versa. The combination was considered as positive (synergistic) when the combination index was <1 and negative (antagonistic) when it was >1.
DNA Synthesis Inhibition.
Cellular DNA of exponentially growing cells was labeled with 0.005 µCi/ml [14C]thymidine (53.6 mCi/mmol) for 48 h at 37°C. The rate of DNA synthesis was measured by 10-min pulses with 1 µCi/ml [methyl-3H] thymidine (80.9 Ci/mmol). Tritiated thymidine incorporation was stopped by washing cell cultures twice in ice-cold HBSS and then scraping in 4 ml of ice-cold HBSS. One ml aliquots were transferred in Eppendorf tubes and precipitated with 100 µl of 100% trichloroacetic acid. Samples were vortexed, mixed, and centrifuged for 10 min at high speed in microfuge at 4°C. The precipitates were dissolved overnight at 37°C in 0.5 ml of 0.4 M NaOH. Samples were counted by dual-label liquid scintillation counting, and 3H values were normalized using 14C counts. Inhibition of DNA synthesis was calculated as the ratio of 3H/14C ratio in treated samples to 3H/14C in control cells.
RNA Synthesis Inhibition.
The assay was performed as described in the paragraph above for the DNA synthesis inhibition assay. Pulses of [3H]uridine at 1 µCi/ml were used to label RNA.
Measurement of DNA ISCs.
ISCs were measured using the alkaline elution technique (23)
. DNA ISCs were assayed after a 300-rad irradiation of the cells. After irradiation, the filter elution assay was performed as described previously (23)
. The relative ISC frequency was obtained by the formula: ISC = {[(1 - R0)/(1 - R1)]1/2 - 1} x 300 rad-eq, where 300 represents the standard irradiation dose (3 Gy = 300 rad) used to introduce random DNA single-strand breaks, R0 represents the DNA retention of control cells treated only with the standard irradiation at a fixed elution end point, and R1 represents the DNA retention of experimental cells treated with oxaliplatin and the standard dose of radiation at the same fixed elution end point.
Measurement of DNA Elongation.
DNA elongation was measured using pulse-labeling alkaline elution, as described previously (24, 25, 26)
. Exponentially growing HT29 cells were pulse-labeled for 15 min with 1 µCi/ml [methyl-3H] thymidine, washed twice in preheated PBS, and incubated in radioactivity-free medium with 1 µM SN-38 at 37°C. After 1 h, the cells were washed twice in preheated PBS and maintained at 37°C. The aliquots were removed and analyzed by alkaline elution as described above, except that [14C]thymidine-labeled cells were used as internal controls. The extent of DNA elongation, expressed as a percentage of control, was calculated as: [(R0 - r) /(R0 - rx)] x 100, where rx and r are the fractions of pulse-labeled [methyl-3H] DNA retained on filter for control and SN-38 treated cells, respectively, and R0 is the fraction of [14C]DNA retained on filter for control cells. rx, r, and R0 were all determined at the same time elution point. Several time elution points were used to determine the kinetics of SN-38-induced DNA elongation. Thereafter, the effect of oxaliplatin alone or in combination with SN-38 was determined using 6- and 12-h elution time points.
Cell Cycle Determination.
Cells were harvested, washed twice in ice-cold PBS (pH 7.4), fixed in 1 ml of 70% ethanol for 30 min at 4°C, washed twice in ice-cold PBS, treated for 30 min at 37°C with PBS containing 200 units/ml RNase (Sigma), and cellular DNA was stained with 1 ml of 500 µg/ml propidium iodide (Sigma). Cells were stored at 4°C prior to analysis. Cell cycle determinations were performed using a FACScan flow cytometer (Coulter Electronics, Paris, France), and data were interpreted using the multicycle AV model program, provided by the manufacturer. Each experiment was performed in triplicate.
Filter Binding Assay.
This filter methodology has been described previously and allows the quantitation of DNA fragmentation (27)
. Briefly, cells were deposited on a protein-adsorbing filter (Metricel; Gelman Science, Ann Harbor, MI), washed with 3 ml of 0.02 mM Na2EDTA (pH 10), and lysed with 5 ml of LS10 [2 M NaCl, 0.04 M Na2EDTA, and 0.2% sarkosyl (pH 10)], followed by washing with 5 ml of 0.02 mM Na2EDTA (pH 10). Lysis fraction (L), Na2EDTA for washing (E), and filters (F) were collected and counted by liquid scintillation (28)
. DNA fragmentation was calculated as the percentage of DNA eluting from the filter as: DNA fragmentation = 100 x (L + E)/(L + E + F).
In Vivo Antitumor Activity.
The antitumor effect of CPT-11 and oxaliplatin was investigated in the GR-1 mouse mammary tumor model. GR-1 mice were obtained from M. Sluyser (NKI, Amsterdam, The Netherlands). Mice care was in agreement with institution guidelines. Treatments were administered by i.p. injection. The MTDs of oxaliplatin and CPT-11 alone and in combination were determined. The MTD was defined as the dose leading to reversible 25–30% body weight loss, without toxic death. The GR-1 human xenografts (1 x 106–2 x 106 cells) were transplanted s.c. into the hind limbs. Treatment started by day 13, after the tumor became palpable. Treatments were performed on day 10 after transplantation. The mean tumor volume on day 10 was 100 ± 50 mm3. The animals were weighed concurrently with tumor measurements. In all cases, the mice were sacrificed before the tumor load or their general condition reduced their mobility. At the time of sacrifice, the tumor was excised, weighed, and, when required, embedded in paraffin and microscopically analyzed. Antitumor activity was defined following the recommendations of Schabel et al. (28)
. Two separate experiments were conducted using six or seven animals for each experimental group.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, the concentrations of SN-38 and oxaliplatin that inhibited the growth of 50% of the cells (GI50) when given alone for 24 h were 0.7 and 15 µM, respectively. Both results were in agreement with previous studies on DNA topo I inhibitors (29)
or oxaliplatin (30
, 31)
in HT29 cells. Greater than additive growth inhibition was observed with the SN-38-oxaliplatin combination. Consistent results were also obtained using colony formation assays (Table 1)
, indicating that the drug interaction led to cell lethality rather than growth arrest. The CI isobologram equation was used in the analysis of the SN-38-oxaliplatin combination data. Fig. 1C
illustrates the results in the case of 0.01 µM SN-38 combined with oxaliplatin. Similar results were obtained with various SN-38 concentrations in addition to a fixed dose of oxaliplatin, as summarized in Table 1
. Our data extend the synergy previously described between DNA topo I inhibitors and cisplatin (23
, 32)
to the DACH platinum derivative oxaliplatin.
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. Moreover, using the administration sequence oxaliplatin followed by SN-38, separated by a short time interval (3 h), there was higher cytotoxicity than either simultaneous administration or sequential administration with a 24-h interval (Table 1)
. This latter unexpected result led us to explore the molecular and cellular interactions likely to occur within a few hours of drug exposure and to reverse within 24 h.
Kinetics of Oxaliplatin-induced ISCs in HT29 Cells.
The effect of SN-38 on the kinetics of oxaliplatin-induced ISCs was measured in cells by alkaline elution (Fig. 2)
. Very few ISCs were detected in cells when exposed to 10 µM oxaliplatin for 1 h. The exposure to 0.01 to 10 µM SN-38 failed to elicit ISC formation (data not shown). SN-38 coexposure did not affect the formation of oxaliplatin ISCs but was associated with a 2-fold increase in ISCs 15 h after oxaliplatin removal. SN-38 preexposure had less effect on the reversion of oxaliplatin-ISCs than simultaneous exposure, whereas this effect was even more marked if SN-38 was given 6 h after oxaliplatin treatment. These results indicate that SN-38 slows down the reversion of ISCs. This effect is time dependent, with maximal inhibition of ISCs reversion obtained when SN-38 is administered close to the peak of oxaliplatin-induced ISCs.
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. Oxaliplatin inhibited DNA synthesis less profoundly but for a longer period of time than SN-38. SN-38 inhibited DNA and RNA synthesis for 4–6 h, with both DNA and RNA synthesis recovering fully 8 h after SN-38 removal. The addition of SN-38 to oxaliplatin increased the duration and extent of DNA and RNA synthesis inhibition (Fig. 3)
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. DNA elongation inhibition after a 1-h exposure to 0.1 µM SN-38 lasted for 6–8 h. DNA elongation was still inhibited when SN-38 was administered 0 to 6 h prior to oxaliplatin ISC formation (during or within 6 h after exposure to oxaliplatin), indicating that it could contribute to the longer half-life and toxicity of oxaliplatin ISCs. On the other hand, SN-38 treatment resulted in a longer DNA elongation inhibition, in the presence of oxaliplatin ISCs, with profound DNA elongation inhibition 12 h after SN-38 removal, suggesting irreversible DNA breakage.
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, the cells treated with oxaliplatin prior to SN-38 did not arrest in G2 but accumulated in S phase (Fig. 6)
. This S-phase block was consistent with the previous observation of prolonged SN-38-induced DNA elongation inhibition after oxaliplatin DNA adduct formation (Fig. 4)
. Because S-phase arrest is a common hallmark of the death induced by DNA topo I inhibitors (24
, 26)
, apoptotic DNA fragmentation was measured in cells treated with the combination of SN-38 and oxaliplatin. A high rate of DNA fragmentation was detectable in cells at 48 h, confirming that S-phase arrested cells were undergoing apoptosis (Fig. 7)
. These results suggested that preexposure to oxaliplatin enhanced SN-38 cytotoxicity.
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. These results indicated that active doses of CPT-11 and oxaliplatin could be achievable in combination and suggested antitumor activity in vivo in GR-1 mouse mammary tumors.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Low levels of resistance to oxaliplatin are associated with the increased expression and activity of 
-glutamyl transpeptidase and glutathione levels (33)
. Because SN-38 did not affect the peak level of ISCs, the increased cytotoxicity could not be explained by changes in oxaliplatin accumulation or in detoxification enzymes.
Topo I inhibitors have been found to potentiate the cytotoxicity of other DNA-damaging agents, such as ionizing radiation, and alkylating agents (34) . However, the mechanism of the synergy remained poorly understood. Synergism has also been found with cisplatin, associated with slower reversion of cisplatin-induced ISCs (23 , 32) . Cisplatin interacts synergistically with DNA synthesis inhibitors, whatever the mechanism of action leading to DNA synthesis interruption. Aracytin (35) , 5-fluorouracil (36) , camptothecin derivatives (23 , 32) , and, more recently, gemcitabine (37) have been shown to have supra-additive in vitro cytotoxicity when combined with cisplatin. Our results indicate that DNA and RNA synthesis inhibition following topo I-mediated DNA damage may also slow down the reversion of oxaliplatin-induced ISCs. Maximal cytotoxicity is observed with simultaneous DNA and RNA synthesis inhibition and the onset of oxaliplatin DNA ISCs formation.
The cytotoxicity of platinum compounds is believed to be a consequence of the formation of platinum-DNA adducts. However, DACH platinum DNA adduct structures differ from those of cisplatin and carboplatin, given the nature of the nonleaving ligand, with the DACH carrier ligand modifying the N
PtN bond angle (14)
. Loss of DNA mismatch repair may increase the tolerance to cisplatin-induced DNA adducts. The failure of the mismatch repair proteins to recognize the oxaliplatin DNA adducts may account for the antitumor activity of oxaliplatin in mismatch repair-deficient cells (16)
. Topo I may act as a specific mismatch site-nicking enzyme (38)
and can be trapped on DNA depending on the location of a given DNA damage relative to the topo I cleavage site (38)
. An interaction between the oxaliplatin ISCs repair enzyme machinery and topo I is a possibility to consider for the increased duration of oxaliplatin ISCs in the presence of a topo I inhibitor.
Oxaliplatin-induced DNA damage sensitized the cells to the toxic effects of SN-38. Both the duration of SN-38 induced DNA elongation and the percent of cells arrested in S-phase increased when the cells were exposed to oxaliplatin prior to SN-38. The increase in topo I activity after DNA damage observed in vitro (38) might be translated in vivo by a transient increase in topo I activity and/or topo I-cleavable complex formation, resulting in more topo I-mediated DNA damage and increased cytotoxicity. Unfortunately, it was not possible to detect topo I-cleavable complexes after oxaliplatin exposure because oxaliplatin also induced high levels of DNA-protein crosslinks (data not shown). S-phase block is not observed after oxaliplatin treatment but is a characteristic feature of topo I inhibitor cytotoxicity (24 , 26) . Thus, the enhanced S-phase block only observed with the sequence oxaliplatin followed by SN-38 suggests that SN-38 cytotoxicity is higher in presence of oxaliplatin-induced DNA damage. This effect might be the result of DNA breakage in the vicinity of oxaliplatin ISCs, making de novo DNA elongation impossible.
Direct correlations between quantitative DNA damage and cytotoxicity are seldom elicited, underlying the role of the cellular response to DNA damage for cytotoxicity (16 , 26 , 27 , 29) . We found that oxaliplatin may increase the cytotoxicity of topo I-mediated DNA damage and that SN-38 may interfere with the repair of oxaliplatin DNA ISCs.
Combinations of CPT-11 with any DNA damaging agent are expected to be supra-additive, and thus its association with oxaliplatin is of particular interest in colorectal cancer patients. Several clinical trials have explored the combination of cisplatin and CPT-11 (39, 40, 41) . Interaction between cisplatin and CPT-11 was suggested by the marked decrease in the MTD of each compound given in combination (39 , 40) . A Phase I clinical trial combining oxaliplatin and CPT-11, given once every 3 weeks, has already been completed (20) . A similar pharmacodynamic potentiation was observed in the absence of detectable pharmacokinetic interaction. Thus, the interaction between cisplatin or oxaliplatin and CPT-11 is likely to occur at the cellular level. Our findings reinforce this hypothesis showing that SN-38 and oxaliplatin reciprocally affect the cellular response to DNA damage. These in vitro findings may contribute to a better knowledge of the combination of topo I inhibitors and platinum derivatives cellular pharmacology, contributing to a rationale for the optimal development of the combination of CPT-11 and oxaliplatin.
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FOOTNOTES |
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1 Supported in part by a Grant from Rhône-Poulenc Rorer (Antony, France). 
2 Present address: Institut Gustave Roussy, 94800 Villejuif, France.
3 To whom requests for reprints should be addressed, at Experimental Therapeutics and Translational Research Section, Fédération des Services des Maladies Sanguines, Immunitaires, et Tumorales, Hôpital Paul Brousse, 94804 Villejuif, France. Phone: 33 1 45 59 30 00; Fax: 33 1 45 59 37 37; E-mail: F.Goldwasser{at}wanadoo.fr
4 The abbreviations used are: topo I, topoisomerase I; DACH, trans-L-1,2-diaminocyclohexane; MTT, (4,5 dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide; CI, combination index; ISC, interstrand cross-link; MTD, maximal tolerated dose.
Received 9/ 2/98; revised 1/20/99; accepted 2/12/99.
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) and oxaliplatin plus 0.01 µM SN-38 (
). Data points, means of three to six experiments; bars, SD. B, cytotoxicity of SN-38 alone. Bars, SD. C, synergy was studied when oxaliplatin and SN-38 were combined, giving SN-38 before (
), or after (
). Bars, SD.
