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Clinical Cancer Research Vol. 6, 2075-2086, May 2000
© 2000 American Association for Cancer Research

Experimental Therapeutics, Preclinical Pharmacology

Distinctive Potentiating Effects of Cisplatin and/or Ifosfamide Combined with Etoposide in Human Small Cell Lung Carcinoma Xenografts

Fariba Nemati, Alain Livartowski, Patricia De Cremoux, Yveline Bourgeois, Francisco Arvelo, Pierre Pouillart and Marie-France Poupon1

UMR 147, Centre National de la Recherche Scientifique, Institut Curie, Section de Recherche [F. N., Y. B., M-F. P.] et Section médicale [A. L., P. D. C., P. P.], Institut Curie, 75 Paris Cedex 05, France, and Faculty of Sciences, Central University of Caracas, Caracas, Venezuela [F. A.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Combined modalities are currently used for cancer therapy, although their mechanisms of activity remain incompletely deciphered. The design of new drug combinations suffers from our inability to anticipate accurately their efficacy or toxicity. They can be evaluated in vivo, using human tumors grafted into immunodeficient mice, as we did here with combined protocols used in the clinical setting. Xenografts of small cell lung carcinoma (SCLC) from eight patients were used to test the tumor sensitivity to etoposide (VP16; 12–16 mg/kg/days, days 1, 2, and 3), cisplatin (CDDP; 6–9 mg/kg/day, day 1) and ifosfamide (IFO; 90–210 mg/kg/day, days 1, 2, and 3) as single agents and to evaluate the efficacy of the two-drug or three-drug combinations. Five xenografts came from untreated patients (SCLC-61, SCLC-6, SCLC-10, SCLC-41, and SCLC-96) and three after treatment (SCLC-74, SCLC-101, and SCLC-108). p53 was inactivated in all of them. Tumor growth inhibition, growth delay, and the survival rate of tumor-bearing mice reflected individual SCLC chemosensitivity. As single agents, IFO inhibited tumor growth in a dose-dependent manner, whereas CDDP and VP16 had little or no effect. Both CDDP and IFO potentiated VP16, inducing complete regressions in the most sensitive SCLCs; VP16-IFO was more effective than VP16-CDDP, with complete regressions in six versus three of the eight tumors tested, respectively. CDDP-IFO was less effective than VP16-IFO, with three of eight SCLCs giving complete regressions. The three-drug combination led to modest improvement over the best two-drug combination but only for sensitive SCLCs. Because drug-responses distinguished two classes of SCLCs, as sensitive or refractory, MDR1, glutathione S-transferase {pi}, lung-related multidrug resistance protein, multidrug resistance protein, and topoisomerase II{alpha} mRNA expression was studied by semiquantitative reverse transcription. There was no correlation with SCLC sensitivity; topoisomerase II{alpha} and multidrug resistance protein was expressed in all cases, lung-related multidrug resistance protein and glutathione S-transferase {pi} in seven of eight, and MDR1 gene in four of eight. In conclusion, these SCLC xenografts displayed a pattern of chemotherapy response close to that observed in patients. This model confirmed that in two-drug combinations, each component potentiated the effects of the other, with VP16-IFO tending to be the best two-drug combination, both of which were more effective than VP16-CDDP and better tolerated than CDDP-IFO. The addition of a third agent gave a modest, if any, therapeutic benefit in the responders but none in refractory SCLCs. There was no correlation between the extent of response and resistance markers.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
SCLC2 represents 20% of lung cancers and is a very peculiar entity. It is characterized by a high metastatic potential, an elevated proliferation index, and chemosensitivity to several agents. The most active drugs are cyclophosphamide and its recently synthesized derivative IFO, VP16, CDDP, Adriamycin, and vincristine, variously combined. An objective response can be obtained in 85–95% of thorax-confined SCLCs and in 75–85% of diffuse cancers. Presently, the reference therapy is based on the VP16-CDDP combination. The benefit of IFO as a third drug is supported by data obtained by Loehrer et al. (1, 2, 3, 4) in diffuse SCLCs, although no differences were noticed between the responses to VP16-CDDP and to VP16-IFO (5) . In thorax-confined SCLC (stages II or III), early concomitant radiochemotherapy has led to a 40% survival at 3 years and a steady cure rate (6 , 7) . Secondary effects and morbidity are high but are justified by the therapeutic benefit.

In diffuse SCLC (stage IV), complete regressions can be obtained, although survival at 2 years is rare (<5%). Recurrences occur before the end of the first year. When cure cannot be expected by the currently available therapies, new compounds or protocols can be proposed to patients as part of clinical trials; this is justified when true therapeutic benefits are expected.

Combinations that involve CDDP allow a high rate of response, but the price to pay is significant toxicity, which necessitates intensive care in a specialized environment; this must be balanced against the short life span expectation of the patients concerned. Combination drug therapy without CDDP could be administered at home, and given greater tolerance and at least similar efficacy, it could improve the quality of life. Introducing a change in the accepted treatment principles of a disease requires a large amount of data to be collected supporting the efficacy of any newly proposed protocols. This was the aim of the present study, which compares the efficacy of various combination protocols for SCLC.

Evaluation of the therapeutic effects of drug combinations should first be tested in preclinical, experimental assays. This should allow compounds that do not potentiate the efficacy of the drugs in combination to be eliminated, as well as drugs that could decrease the tolerance of the whole. Here we have investigated three drugs that were associated in two- or three-drug combinations, the total number of combinations being 4, in mice because such comparisons of relative efficacy in humans are not ethical. Experimental assays are the means by which the contribution to antitumoral efficacy of each agent in a combination can be identified.

A series of small cell lung carcinomas, originating from chemosensitive or refractory clinical tumors, were established by us as xenografts into nude mice and used to analyze the efficacy of three compounds of VP16, CDDP, and IFO as single agents or in combination. Drug responses were measured as a function of dose; the tolerance to treatment was evaluated by the survival of mice and the loss of body weight. In these experimental assays, the dilemma was to choose the doses of drugs to combine. The strategy was to first test the dose-response of SCLC to drugs used as single agents, to define a dose range that inhibit tumor growth without any toxicity, and to combine each of the three drugs at these optimal doses. VP16 and IFO were given for three consecutive days, with CDDP as a bolus on the first day of treatment when combined.

VP16 is a DNA topoisomerase II{alpha} (topII {alpha}) poison and is a substrate of P-glycoprotein that, when increased, induces a refractory state. CDDP interacts with DNA, forming intra- and interstrand cross-links; resistance to CDDP has been attributed to different mechanisms, including high levels of GST-{pi} and/or MRP; CDDP has been combined with various drugs in an impressive number of protocols, suggesting that its mechanism of action when combined is not specific, likely because of activation of apoptosis. IFO is a DNA-alkylating agent that requires intracellular metabolism to be active. In all cases, chemotherapeutic responses are related to drug-initiated death processes; their alterations might reduce the extent of tumor responses, as has been shown in p53 mutated cancers.

The model designed here provides a potent tool to respond to a series of questions. Using three drugs that have proven to be active in SCLC, is a three-drug combination better than two drugs? Are the different two-drug combinations similar? Is coadministration of agents required? Do drug-resistance mechanisms intervene in the responses observed?

The aim of these assays was to compare the efficacy of the three different two-drug combinations: VP16-CDDP, considered as the reference for SCLC treatment; and VP16-IFO and CDDP-IFO, compared with that of the three-drug combination. All these assays were done using several SCLCs derived from both treated and untreated patients and from both localized tumors and tumor at an advanced stage, characterized for the expression of molecular determinants of chemoresistance. Our purpose was to define: whether there was a common pattern of response to the different drug-combinations tested, despite the diversity of the SCLCs tested; whether these combinations revealed synergistic or antagonistic effects; and which combinations were the most active. Ultimately, the results of this study will help to identify an optimal ratio between efficacy and toxicity and allow the rational design of new protocols for clinical trials in SCLC.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice.
Swiss nu/nu female mice, 6–10 weeks of age, were bred in the animal facilities of the Curie Institute. The animals were maintained under specified pathogen-free conditions. Their care and housing were in accordance with the institutional guidelines of the French Ethical Committee (Ministère de l’Agriculture et de la Forêt, Direction de la Santé et de la Protection Animale, Paris, France) and under the supervision of authorized investigators.

SCLC Xenografts and Tumor Growth.
Tumor specimens were obtained from patients during surgical resection with their consent. Tumor samples were established as xenografts by s.c. implantation of a tumor fragment into the scapular area of nude mice and sequentially transplanted. For therapeutic experimental assays, female mice received a s.c. graft of tumor fragments of ~15 mm3 volume. Tumors appeared at the graft site 2–5 weeks later. Mice bearing growing tumors with a volume of 60–400 mm3 were individually identified and randomly assigned to the control or treatment group (5–10 animals/group, as detailed in the tables and legends of figures), and the treatment started at day 1. Animals with tumor volumes outside this range were excluded. Mice were weighed weekly. Mice bearing tumors were sacrificed when their tumor volume reached 2500 mm3, defined as ethical sacrifice. The tumor volumes were calculated from the measurement of two perpendicular diameters using a caliper. Each tumor volume (V) was calculated according to the following formula:

where a and b are the largest and smallest perpendicular tumor diameters. RTVs were calculated from the formula:

where Vx is the tumor volume on day x and V1 is the tumor volume at the initiation of therapy (day 1). Growth curves were obtained by plotting the median of values of RTV as the Y axis against time (expressed as days after start of treatment). The antitumor activity was evaluated according to three criteria:

(a) the tumor growth inhibition, which was calculated according to the following formula: % growth inhibition = 100 - (RTVt/RTVc x 100, where RTVt are calculated for individual tumors and RTVc was the mean of the RTV in the control group at a given time; the medians of growth inhibition are calculated and reported in the tables. Calculations of growth inhibition were done as a function of time until the day of sacrifice of the first mouse, 50% of growth inhibition was considered as the limit of meaningful effect.

(b) individual growth delays were calculated as the time in days required for individual tumor to reach a 5-fold increase in volume, as published previously (8) ; growth delay median/group was calculated and reported in the tables; the growth delay index was calculated as the median growth delay in the treated group divided by the median growth delay in the control group; a growth delay index of 2 was considered as the limit of meaningful effect.

(c) complete regressions were defined as the absence of any palpable nodule at the graft site and were followed by either cure, i.e., no tumor regrowth up to 3 months, or relapse. In the latter case, the duration of regression was noticed. All data are presented as the median of values and reported in tables and figures.

A statistical analysis was performed by Student’s t test to compare the differences observed in the growth inhibitions induced by the different protocols. At given times after treatment, differences in RTV were compared. The differences were considered as significant if the Student’s t test gave a P < 0.05 for five consecutive measurements, knowing that they were done three times/week.

Formulation and Administration of Drugs.
VP16 (Vépéside-Sandoz, Novartis Pharma, Rueil-Malmaison, France) in a stock solution of VP16 was diluted in 0.9% sodium chloride solution and administered i.p. in a 0.2-ml volume to tumor-bearing mice at days 1, 2, and 3. Different daily doses were tested from 8 to 16 mg/kg/day.

IFO (Holoxan, Bordeaux, France) was supplied by Asta Medica Laboratories. A stock solution of IFO was diluted in 0.9% sodium chloride solution and administered i.p. in a 0.2-ml volume to tumor-bearing mice at days 1, 2, and 3. Different daily doses were tested from 30 to 210 mg/kg/day.

CDDP (Cisplatyl; Laboratoires Roger Bellon, Montrouge, France). A stock solution of CDDP was reconstituted by water, diluted in 0.9% sodium chloride solution, and administered i.p. in a 0.2-ml volume to tumor-bearing mice at day 1. Doses of 6 or 9 mg/kg were administered.

All drugs were extemporaneously prepared; when combined, drugs were injected separately into animals. Mice in the control groups received 0.2 ml of the drug-formulating vehicle with the same schedule as the treated animals.

RT-PCR and Yeast Functional Assay for p53.
cDNA was synthesized from 1 mg of RNA at 45°C for 1 h using 100 units of Superscript II reverse transcriptase (Life Technologies, Inc., Cergy Pontoise, France) and 200 pmol of random hexamer (Boehringer Mannheim, France). PCR was performed with primers as detailed in Table 1Citation . PCR cycles were done at 94°C for 30 s, 65°C for 30 s, and 74°C for 2 min in a thermocycler (Minicycler; MJ Research, Poly Labo, France). Two µl of the cDNA was amplified in 20 µl of Pfu buffer and of 0.5 unit of Pfu DNA polymerase (Stratagene, La Jolla, CA), 10 pmol of primers, and 50 mM dNTPs. Purified PCR products were sequenced bidirectionally by using a DyeDeoxy Terminator kit (Perkin-Elmer, Yvelines, France). Characterization of the p53 gene was conducted according to Flaman et al. (9) and an automated sequencer (ABI Prism 310 Genetic Analyser (Perkin-Elmer). We studied the transcriptional activity of the p53 protein by using the yeast functional assay described previously by Flaman et al. (9) . We visually quantified the percentage of red colonies and of the white colonies in each assay. More than 10% of red colonies is always associated with a p53 mutation. We sequenced pooled plasmids from red colonies. In all cases, the mutation matched those found by direct sequencing of the tumor cDNA.


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  		Table 1

Summary of RT-PCR parameters used

 
MDR1, MRP, LRP, GST-{pi}, and topII{alpha} PCR mRNA.
Expression Isolation of total RNA from frozen tumor samples weighing ~20 mg was done as follows. The frozen tissue was placed under liquid nitrogen and immediately pulverized with a mortar and pestle. The pulverized tissue was rapidly transferred to 1 ml of TRIzol reagent (Life Technologies, Inc.). The purification of total RNA was performed according to the TRIzol procedure. The resulting RNA pellet was resuspended in sterile deionized water and stored overnight at -80°C for complete solubilization. The RNA concentration was determined by spectrophotometry. Synthesis of cDNAs from poly(A) mRNA was done as follows. RNA was placed in a sterile 0.5-ml tube at a final concentration of 2 µg in 20 µl of sterile water and incubated at 65°C for 10 min. A mix containing 4 µl of 5x RT buffer (Life Technologies), 2 µl of DTT (100 mM), 1 µl of oligo-dT (200 ng/µl), 1 µl of a mix containing 12.5 mM of each dNTP (final concentration, 0.63 mM; Boehringer Mannheim), and 0.2 µl (200 units/µl) of RNase H reverse transcriptase (Life Technologies) was added to the RNA-containing tube. The mixture was then incubated at 45°C for 60 min. The PCR primers listed in Table 1Citation were selected for their specificity and their selectivity toward human gene sequences (purchased from Oligo Express, Paris, France). PCR conditions were as follows. The final PCR reaction volume was 50 µl. One µl (1:20) of the cDNA solution was pipetted into a sterile 0.2-ml tube, and the following mix containing 5 µl of 10x Taq buffer (Appligene-Oncor, Illkirch, France), 1 µl of dNTP (final concentration, 0.25 mM each), 1 µl of each of the 5' and 3' primers (100 ng/µl), 40.5 µl of water, and finally 0.5 µl (2.5 units) of Taq polymerase (Appligene-Oncor) were added. The tubes were placed in a Perkin-Elmer 2400 thermocycler for 5 min at 94°C (hot-start), followed by 50 s at 94°C, 1 min at 55°C, and 1 min at 72°C for a number of cycles given in Table 1Citation , and a final elongation at 72°C for 10 min. These conditions have been demonstrated to be highly efficient and reliable in the amplification of the different cDNA templates and were used with all of the primer sets (Table 1)Citation .


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Characteristics of SCLC.
SCLC xenografts were histologically either of oat cell or of variant type (Table 2)Citation , three originated from a metastasis, and five were from the primary site. Five SCLCs were obtained before any treatment, and three came from treated patients. Treatment consisted of VP16 and CCDP, and all three treated patients had received radiotherapy. The p53 gene was inactivated in all SCLCs by deletion or mutation; different codons were involved, except for codon 175, which was altered in two SCLCs. Growth rates of SCLC differed; their doubling times varied between 3 and 6 days. Survival of patients after diagnosis and treatment varied between 8 weeks and 30 months.


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  		Table 2

Clinical and biological features of SCLC xenografts tested

 
Antitumoral Effects of Drugs as Single Agents.
Tumor-bearing mice were treated when the tumors reached a volume ranging between 60 and 400 mm3; no spontaneous regression was observed. CDDP, VP16, or IFO was used first as a single agent to treat tumor-bearing mice. Their respective effects on tumor growth are summarized in Table 3Citation . CDDP delayed the growth of four of the six xenografts tested by 1.3–2 times. The corresponding percentage of optimal growth inhibition was superior to 50% for these four tumors (SCLC-61, SCLC-6, SCLC-101, and SCLC-41) and was observable between day 5 and day 13 after the start of treatment. SCLC-6 and SCLC-41 were the most sensitive to CDDP, leading to complete regressions in two of five and one of six mice, respectively; however, all tumors regrew rapidly. SCLC-100 and SCLC-74 were not sensitive at all to CDDP. At 6 mg/kg, cisplatin was well tolerated and did not lead to any loss of weight. An increase of cisplatin doses to 9 mg/kg did not improve the therapeutic index (data not shown) and was less well tolerated.


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  		Table 3

Responses of SCLC xenografts to CDDP, VP16, and IFO given to tumor-bearing mice as single agents

 
VP16 prolonged the growth delay by 1.6–2.9 times for three of the six SCLCs tested, with an optimal growth inhibition close to or >48% for all tumors. VP16 at 12 mg/kg/day was well tolerated; no loss of weight was observed. Administration of VP16 at 16 mg/kg/day did not improve the therapeutic index (data not shown) and led to a loss of weight.

IFO induced a growth delay of all of the tumors, except SCLC-74; the most sensitive tumor was SCLC-61, with complete regressions; recurrences were observed after 16 days, with a growth delay prolonged 3.3-fold. SCLC-6 responded with an optimal growth inhibition of 79% and a 2-fold increase in median growth delay. Complete regressions of short duration were obtained in SCLC-100 and SCLC-41 xenografts. SCLC-74 did not respond at all. IFO at 90 mg/kg/day (days 1, 2, and 3) was well tolerated; no loss of weight was observed. Administration of IFO at higher doses (120, 180, or 210) improved the therapeutic index (data not shown); at a dose of 210 mg/kg/day, we observed a loss of weight of 10%, 7 days after the start of treatment, and a complete recovery at day 14.

These six xenografted tumors displayed different responses to CDDP, VP16, and IFO as single agents. SCLC-61 was sensitive to the three drugs, whereas SCLC-74 was a refractory tumor; the four others responded to two drugs but not the same ones.

Antitumoral Effects of the Two-Drug Combinations.
The drugs were combined at the same doses used as above. The tolerance to combinations was acceptable (loss of weight of ~5%; no death). Combination assays were conducted simultaneously with groups treated with each drug as a single agent. Comparison between the efficacy of VP16-CDDP and VP16-IFO combinations, or as single agents, using SCLC-61 is illustrated in Fig. 1Citation . CDDP administered alone was ineffective (Fig. 1A)Citation . VP16 alone induced complete resorption of tumors recurring at day 18. VP16-CDDP led to complete regressions of tumors recurring at day 33; all tumors regrew, and no mouse was cured. IFO alone induced complete regressions of tumors in all mice recurring at day 25; after treatment with IFO-VP16, five of seven tumor-free mice survived (Fig. 1B)Citation .



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  		Fig. 1. Effects of VP16, CDDP, or IFO, as single agents or combined as VP16-CDDP or VP16-IFO administered to mice bearing SCLC-61 xenografts. A, growth curves of tumors not treated or treated with single agent VP16, CDDP, or both combined (six mice/group). B, growth curves of tumors not treated or treated with single agent VP16, IFO, or both combined (six mice/group). {square}, control group; *, VP16, 12 mg/kg/day, days 1, 2, and 3; {triangleup}, CDDP, 9 mg/kg/day, day 1; {blacktriangleup}, VP16-CDDP; {circ}, IFO, 90 mg/kg/day, days 1, 2, and 3; •, VP16-CDDP.

 
The efficacy of these two two-drug combinations were tested with seven other SCLC xenografts (Tables 4Citation and 5)Citation . As evidenced by growth inhibition, a response to both two-drug combinations was obtained in all of the SCLCs. Analysis of growth delays showed a variability among the SCLCs. VP16-IFO was clearly better than VP16-CDDP for SCLC-100, SCLC-6, and SCLC-101 (P < 0.05; Table 5Citation ). Both combinations were similarly efficient for SCLC-41. SCLC-74, SCLC-96, and SCLC-108 were similarly poor responders to both combinations. In no case was VP16-CDDP more effective than VP16-IFO. Growth delays obtained by drug combinations were quite constantly superior to those induced by single agent, showing a potentiation of their respective efficacy. The efficacy of CDDP-IFO was lower or equal to that of the two others in SCLC-100, better in SCLC-6, and similar to that of VP16-IFO in the other SCLC. All together, complete regressions were obtained in six tumors of the eight treated with VP16-IFO versus three of eight treated with VP16-CDDP or with CDDP-IFO; however, only VP16-IFO induced durable complete regressions.


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  		Table 4

Comparative responses of eight different SCLC xenografts to two-drug or three-drug combinations

 

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  		Table 5

Statistical analysis of the differences between the growth inhibition induced by two-drug or three-drug combinations of SCLC-6 and SCLC-101

 
Effect of Fractionation, Dose Intensification, and Coadministration of Drugs in Combination.
The effect of fractionation of drug dosage in the VP16-IFO combination was tested using the SCLC-6 tumor. The total doses of both drugs (VP16 and IFO) were administered either in three or five daily doses, with the total dosage being similar. The two schedules of treatment were effective (Table 6)Citation ; the standard fractionation of drug dosage in three gave the best response, as compared with that obtained by fractionation by 5. The growth delay index was longer, 8.3 versus 2.9 times, for three and five fractions, respectively, with a similar optimal growth inhibition. Fractionation of drug dosage did not improve the antitumoral effect of VP16-CDDP when VP16 was given at 8 mg/kg/day for 5 days combined with cisplatin in five daily doses of 1.2 mg/kg/day (data not shown).


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  		Table 6

Effects of dosage fractionation in the VP16-IFO combination for the treatment of the SCLC-6 xenografts

 
Intensification of dosage of IFO in the two combinations VP16-IFO and IFO-CDDP was tested using SCLC-101 xenografts; a dose-dependent effect of IFO was observed (Table 7)Citation . Growth of SCLC-101 xenografts was slowed down by VP16-IFO, even at the lowest dosage of IFO; however, the gain of response was not impressive. When combined with CDDP, increasing the dosage of IFO from 90 to 180 mg/kg in daily doses led to more complete regressions (five of five versus one of nine) and longer growth delays (4.2 versus 2.7). These associations were tolerated, and no death was registered, despite a loss of weight >10% at the two highest dosages. An increase of CDDP or VP16 dosage in any combination did not improve the xenograft response (data not shown).


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  		Table 7

Dose-response effects of IFO combined with VP16 or CDDP for the treatment of SCLC-101 xenografts

 
The importance of coadministration of drugs in the two-drug combination was tested. SCLC-101 tumor-bearing mice first received either VP16 or IFO at days 1, 2, and 3, and then mice received the other drug either from day 4 (no arrest of treatment) or from day 8 (arrest of 4 days between the two treatments). Results are presented in Fig. 2Citation . When IFO and VP16 were injected simultaneously or IFO first and VP16 immediately after, or IFO first and VP16 after an interval of 4 days, tumor growth of SCLC-101 was similarly and strongly delayed. When IFO was injected after VP16 with an interval of 4 days, the tumors continued to grow as in the controls, and then growth inhibition occurred, reaching 98% at day 18. However, the tumors regrew rapidly. IFO appeared to be the key-drug of the VP16-IFO combination in rapidly initiating the antitumoral response. However, VP16 had a potentiating effect, leading to complete regressions, which was not possible with either agent used alone, as reported in Table 3Citation .



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  		Fig. 2. Effects of simultaneous or alternate administration of VP16 and IFO on the growth of SCLC-101 xenografts. Growth curves of treated and untreated tumors (six mice/group). VP16 was given at 12 mg/kg/day and IFO at 180 mg/kg/day. {square}, control group; {diamondsuit}, VP16, day 1, 2, and 3 plus IFO, days 1, 2, and 3; {diamond}, IFO, days 1, 2, and 3 and then VP16, days 4, 5, and 6; {triangleup}, VP16, days 1, 2, and 3 and then IFO, days 4, 5, and 6; •, IFO, days 1, 2, and 3 and then VP16, days 8, 9, and 10; {circ}, VP16, days 1, 2, and 3 plus IFO, days 8, 9, and 10.

 
Sensitivity of SCLC Xenografts after a First Course of Two-Drug Treatment.
The response of SCLC-61 xenografts in mice treated previously with a two-drug combination (VP16-CDDP or VP16-IFO) to a second course of treatment was studied. This was done to evaluate the putative acquisition of resistance after treatment. For this purpose, two tumors, one recurrent tumor originating from a SCLC-61-bearing mouse treated with VP16-IFO and another tumor coming from a mouse treated with VP16-CDDP, were grafted into five nude mice. Then these newly growing tumors were grafted into 25 mice and treated either with the same two-drug association or with the alternative association; mice bearing VP16-IFO-treated xenografts were treated either with VP16-IFO or with VP16-CDDP. A complete regression of tumors and a prolongation of survival were obtained with the two different protocols. At day 106, there were five tumor-free mice of seven in the group treated with VP16-IFO versus four of seven treated with VP16-CDDP.

Mice bearing VP16-CDDP-treated xenografts were treated either with VP16-CDDP or with VP16-IFO. Complete regressions of tumors and prolongation of survival were obtained with the two protocols. At day 70, six tumor-free mice of seven treated with VP16-IFO were alive versus five of seven treated with VP16-CDDP.

Antitumoral Effects of Three-Drug Combinations.
The effects of the three-drug combination were analyzed in detail using SCLC-6 xenografts (Fig. 3)Citation . VP16 was given at 12 mg/kg/day at days 1, 2, and 3, CDDP at 6 mg/kg day 1, and IFO at 90 mg/kg/day at days 1, 2, and 3. This triple association was tolerated. Growth curves (Fig. 3)Citation show the efficacy of the three-drug combination. A 98% growth inhibition was obtained, leading to a 4.7-fold increase in the growth delay (Table 4)Citation ; however, no complete regression was obtained. As compared with the efficacy of the two-drug combinations, VP16-IFO and CDDP-IFO, the responses were similar (ratio of 3.2 and 3.8, respectively; not statistically significant, see Table 5Citation ), although tumors regrew slightly earlier, after 19 and 23 days. VP16-CDDP showed no activity at all.



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  		Fig. 3. Response of SCLC-6 xenografts to three-drug or two-drug combinations. VP16 was given in all groups at 12 mg/kg/day, IFO at 90 mg/kg/day, and CDDP at 6 mg/kg/day (8 mice/group). Tumor growth curves: {square}, control group; {oplus}, VP16-CDDP-IFO; +, CDDP-IFO; •, VP16-IFO; and {triangleup}, VP16-CDDP.

 
The three-drug combination was highly effective for treating mice bearing SCLC-101 xenografts. The efficacy of the three-drug combination was compared with that of VP16-CDDP, VP16-IFO (Fig. 4A)Citation , and CDDP-IFO (Fig. 4B)Citation . Inhibition of tumor growth was obtained in all groups treated. The three-drug association inhibited and delayed tumor growth to the same degree as CDDP-IFO and more than VP16-IFO or VP16-CDDP (P < 0.05; Table 5Citation ).



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  		Fig. 4. Response of SCLC-101 xenografts to three-drug or two-drug combinations. VP16 was given in all groups at 12 mg/kg/day, IFO at 90 mg/kg/day, and CDDP at 6 mg/kg/day (eight mice/group). A, growth curves of untreated tumors and those treated with VP16-CDDP, VP16-IFO, and the three drugs (six mice/group). B, growth curves of untreated tumors and those treated with CDDP-IFO or the three agents combined. {square}, control group; {oplus}, VP16-CDDP-IFO; •, VP16-IFO; {blacktriangleup}, VP16-CDDP; +, CDDP-IFO.

 
Combination of the three drugs was tested with the other SCLC xenografts, and its efficacy was compared with that of the two-drug combinations (Table 4)Citation . SCLC-61 and SCLC-100 tumors were cured by the three-drug combination as they were by VP16-IFO. Growth of SCLC-96 and SCLC-74 was modestly inhibited by the different combinations, leading to similar growth delays, whatever the combination used. No complete regression was obtained with the three-drug association. SCLC-108 xenografts showed inhibition of tumor growth after the different treatments, which did not delay their progression. All data as growth delay indexes induced by the different treatments tested are shown in Fig. 5Citation .



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  		Fig. 5. Comparative scale of responses of the different SCLCs tested with the various protocols.

 
Expression of Different Genes Known to Be Implicated in Drug Resistance.
Total RNA isolated from xenografts and expression of different genes of molecular determinants involved in drug resistance was analyzed by semiquantitative RT-PCR. Relative expression of the different genes was determined with respect to the ß2-microglobulin (ß2m) gene, chosen as the internal standard (Table 8)Citation . The relative expression of the genes of interest was calculated within the linear amplification range. Levels of expression of topII{alpha}, the pharmacological target of VP16, differed from one tumor to another. It was low in SCLC-74 and SCLC-101. SCLC-6 and SCLC-61 expressed the highest levels. SCLC-100, SCLC-108, and SCLC-41 had detectable expression. The MDR1 gene was expressed in four of the eight tumors tested. Expression of MRP was found in all xenografts; it was elevated in SCLC-61 and SCLC-100 tumors. The gene encoding LRP was highly expressed in SCLC-100 and SCLC-74, as drug sensitive and drug refractory, respectively and much weaker in SCLC-6, SCLC-61, and SCLC-41. Expression of the gene encoding GST-{pi} was expressed in SCLC-61, SCLC-100, SCLC-101, and SCLC-74.


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  		Table 8

Relative expression of chemoresistance-related genes as compared with that of ß2-microglobulin in SCLC xenografts by RT-PCR

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Human tumor xenografts constitute a powerful tool for evaluating the in vivo efficacy of therapies. As preclinical assays, they are a prerequisite for any clinical use of new compounds. They are usually used to test pharmacological compounds as single agents and are especially used for testing drug combinations. The aim of this study was to demonstrate the usefulness of such an approach in deciding which drug combinations to use for subsequent clinical purpose.

Polychemotherapy has increased the median survival of patients with SCLC. The currently accepted treatment for limited-stage SCLC is combined chemotherapy using CDDP-based regimens, with concurrent radiotherapy. For advanced-stage disease, a response is still achievable, but no particular combination chemotherapy has established its superiority over another, as reported by Sandler (10) . CDDP is the most widely used compound in diverse combinations for SCLC. In the report by Miyamoto et al. (11) of a randomized trial comparing VP16-CDDP, with or without IFO, in patients with both advanced- and limited-stage SCLC, both treatments were effective with a median survival of 34 and 33 weeks, respectively. More recently, the Hoosier Oncology Group compared VP16-CDDP alone or with IFO in patients with advanced-stage SCLC, with 67 and 73% of responses, respectively, and an improved survival rate in the latter after 2 years (5% versus 13%) and after 3 years (0% versus 5%; Ref. 1 ). Overall, these data indicated a limited advantage for IFO combined with CDDP-based combined therapy. There have been few reports looking at the use of IFO-VP16 as compared with VP16-CDDP. In a German multicenter trial including 144 patients with SCLC and comparing the efficacy of VP16-CDDP to that of VP16-IFO, Wolf et al. (5) concluded that both treatments were effective, but VP16-CDDP was modestly superior (statistically significant) to VP16-IFO for limited-stage SCLC, and there were no differences for the advanced stages. To date, the available data do not identify which of the different combinations is the best. If these were differences, it is likely that they were small; otherwise, they would already have been detected. Our study, limited to an experimental model and a short series of xenografts, supports this assumption. Therefore, if these different combined protocols were demonstrated to be similarly effective, the choice for treatment would be based on which causes the least harm to the patient.

The human SCLC xenograft model was used here to compare the responses to the different combination chemotherapies. The response of eight different SCLC xenografts to three drugs, VP16, CDDP, and IFO, indicates, as expected, that the three-drug combination is highly effective. Despite their limitations, in vivo experimental assays allow a detailed analysis of the different aspects of the drug responses. Their main value is that it is possible to simultaneously compare the drug response with different combinations or agents using transplantable tumors that are expanded as much as necessary. In addition, acquisition of data are rapid. Such a comparison cannot be done in the clinical setting.

Several SCLC xenografts used have already been studied (12, 13, 14, 15, 16) but needed to be validated for the present analysis. Our series comprises five untreated and three previously treated SCLC patients. All were histologically characterized as SCLCs (both the clinical samples as well as the xenografts). Four of the five untreated SCLC xenografts and one of the treated xenografts were responders, the drug combination leading to growth inhibitions, complete regressions, and cures for two SCLCs. Two of them originated from high responder patients with a survival superior to 2 years; the three others came from patients who survived 8 months after a complete remission. SCLC-96, not previously treated, and two previously treated SCLC xenografts were refractory to chemotherapy, regardless of the protocol used. The corresponding patients did not respond at all to treatment. The diversity of drug responses observed in patients was reproduced by the xenografts, as observed by us previously (14) .

Potentiation of drug activity is the basis for using combined-protocol treatments. Our objective was to compare the potentiating effects of CDDP and/or IFO with VP16 in evaluating each two-drug combination with the three-drug combination. This was done keeping in mind that the three-drug combination would obviously be less tolerable by the patients than two-drug combinations at equal dosage of active agents. Analysis of drug responses of SCLC xenografts was conducted by comparing their sensitivity to each of the three two-drug combinations. To determine the part of drug interactions in the final response, each drug was tested as a single agent.

As single agents, IFO was effective in five of the six SCLCs tested, inducing complete regressions in 30% of the mice. Three of the seven SCLCs tested responded to VP16, and only one responded to CDDP as a single agent. These responses were dose dependent for IFO (30–210 mg/kg daily for 3 consecutive days) but not for VP16 (in a range of 8–20 mg/kg daily for 3 consecutive days) or CDDP (1–10 mg/kg; data not shown). The effects of two-drug combinations markedly contrasted with the modest effects of single agents. An impressive example was the response of SCLC-61 to VP16-IFO; this combination cured all of the mice, whereas none was cured by the same drugs used alone. Looking at the growth delay index, which indicates the increased survival of treated mice, all drug combinations were effective but to various degrees, depending on the agents combined and the SCLC tested.

VP16-IFO was the most effective combination, leading to complete regressions in six different SCLCs. In the same conditions, VP16-CDDP and CDDP-IFO gave complete regressions in three SCLC and no cures. The three-drug combination gave a score of complete responses inferior to that of VP16-IFO, with four responders of the eight tested. SCLC xenografts responded in a similar manner to the different drug combinations (Fig. 5)Citation , but the degree of response was individual to each tumor.

Some biological parameters putatively involved in drug response were analyzed. Alteration of p53 gene product was found in all of the xenografts. p53 inactivation is a frequent event in the progression of lung cancer, but its role in proliferation and response to chemotherapy is still debated (17 , 18) . Some studies demonstrated that mutated p53 tumors grew faster than wild-type p53 tumors and that a mutated p53 confers a relative resistance to radio and chemotherapy, as shown in SCLC xenografts (19 , 20) or in clinical samples of non-SCLCs (21) . Other studies found that p53 did not predict for chemotherapy response. Recently, Blandino et al. (22) suggested that the nature of the p53 mutation could diversely affects its drug sensitivity, with some mutations leading to a gain of function. In our study, it is clear that p53 mutations are diverse in nature but consistently present and detected in responders as well as in refractory tumors.

The expression of other genes involved in drug responses could explain the diversity of the responses of SCLC xenografts. topII{alpha} expression is required for VP16 efficacy, and topII{alpha} mRNA transcript was expressed in all of the SCLCs tested, but only one SCLC responded to VP16. topII{alpha} expression was low in SCLC-74 and SCLC-101 and two pretreated tumors, which did not respond to VP16. The topII{alpha} content by itself is not enough to explain resistance to topoisomerase II poisons (23) . Mutations, inactivation of this ATP-dependent enzyme, and lack of cleavage complexes could contribute to the refractory state of SCLC (24) . Multidrug resistance can also be associated with overexpression of the MDR1- or MRP-encoding genes or with an increase of LRP (25 , 26) . Lack of response to CDDP could be explained by a high GST content and related enzymes, which is found in SCLC (27, 28, 29, 30) . In our study, each SCLC tested expressed more than one molecular determinant of drug resistance, and it was not possible to relate their expression to SCLC sensitivity.

Polychemotherapy aims at achieving potentiation or synergy between antitumoral compounds, and this is indeed observed; however, investigating the underlying mechanisms is difficult. Drug combinations could lead to accumulated random lesions. Alteration of repair processes frequent in cancer cells could contribute to an increase of their chemosensitivity. Pharmacokinetic drug interactions may be divided in two types: induction or inhibition of enzymes involved in the metabolism of the drug (31) . Little information is available about interactions of cancer drugs in human tumors. Combined drug efficacy depends on interactions that require to be further studied (32) .

The specific metabolism of cancer cells should be a key area for the analysis of such mechanisms. In the present study, it has to be underlined that in the variable responses observed, the variable parameter was the tumors and not the murine hosts. In such studies, the variability of the responses depends exclusively on the human tumors. The metabolism of the SCLC used has not yet been characterized. Among the enzymes that could influence drug activity, molecules involved in the detoxification of xenobiotics are good candidates. CYP450 constitutes a large family of enzymes, and one member, 3A4, is required for the activation of IFO, a key agent in the combined therapy of SCLC. On the other hand, some agents strongly inhibit CYP450-mediated metabolism (33) . VP16 is also metabolized by the same 3A4, and the pharmacokinetics of these metabolites is strongly influenced by preadministration of CDDP. Normal and tumoral lung tissue synthesize several types of CYP450 (34) , which could influence the effects of combined treatment. This model of human SCLC offers the opportunity to study such chemosensitivity mechanisms.

To conclude, this study clearly showed that IFO-based two-drug combinations are similarly or more effective than the CDDP-based ones. Adding a third agent to the two-drug combinations was not beneficial in terms of response, except when the added agent was IFO, which increased the efficacy of CDDP-VP16. More importantly, the limited extent of the gain has to be balanced against the resulting toxicity, which, in the clinical setting, will inevitably increase, leading sometimes to a decrease in the dosage of drugs and thereby a concomitant decrease in the expected benefit. Moreover, in refractory SCLC, the addition of a third drug had no effect on survival.


   ACKNOWLEDGMENTS
 
We thank Prof. T. Soussi (Institut Curie, Paris, France) and Dr. J-F. Gimonet (Laboratoires Asta Medica, Bordeaux, France) for their stimulating and very supportive discussions. We are grateful to S. Liva, V. Bordier, and C. Alberti for excellent technical assistance. We thank Dr. S. Agrawal for reviewing the English used in this report.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 To whom requests for reprints should be addressed, at Institut Curie, Laboratoire de Cytogénétique Moléculaire et Oncologie, UMR 147 du Centre National de la Recherche Scientifique, 26 rue d’Ulm, 75248 Paris Cedex 05, France. Back

2 The abbreviations used are: SCLC, small cell lung cancer; VP16, etoposide; IFO, ifosfamide; CDDP, cisplatin; RTV, relative tumor volume; MDR, multidrug resistance; GST, glutathione S-transferase; LRP, lung-related multidrug resistance protein; MRP, multidrug resistance-related protein; RT-PCR, reverse transcription-PCR; RTV, relative tumor volume; dNTP, deoxynucleotide triphosphate; CYP450, cytochrome P450. Back

Received 11/22/99; revised 2/ 2/00; accepted 2/ 2/00.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Loehrer P. J., Sr. The role of ifosfamide in small cell lung cancer. Semin. Oncol., 23: 40-44, 1996.[Medline]
  2. Loehrer P. J., Sr., Ansari R., Gonin R., Monaco F., Fisher W., Sandler A., Einhorn L. H. Cisplatin plus etoposide with and without ifosfamide in extensive small-cell lung cancer: a Hoosier Oncology Group study. J. Clin. Oncol., 13: 2594-2599, 1995.[Abstract]
  3. Loehrer P. J., Sr., Rynard S., Ansari R., Songer J., Pennington K., Einhorn L. Etoposide, ifosfamide, and cisplatin in extensive small cell lung cancer. Cancer (Phila.), 69: 669-673, 1992.[CrossRef][Medline]
  4. Loehrer P. J., Einhorn L. H., Williams S. D., Hui S. L., Estes N. C., Pennington K. Cisplatin plus 5-FU for the treatment of adenocarcinoma of the colon. Cancer Treat. Rep., 69: 1359-1363, 1985.[Medline]
  5. Wolf M., Havemann K., Holle R., Gropp C., Drings P., Hans K., Schroeder M., Heim M., Dommes M., Mende S., et al Cisplatin/etoposide versus ifosfamide/etoposide combination chemotherapy in small-cell lung cancer: a multicenter German randomized trial. J. Clin. Oncol., 5: 1880-1889, 1987.[Abstract/Free Full Text]
  6. Arriagada R., Le Chevalier T., Pignon J. P., Riviere A., Monnet I., Chomy P., Tuchais C., Tarayre M., Ruffie P. Initial chemotherapeutic doses and survival in patients with limited small-cell lung cancer. N. Engl. J. Med., 329: 1848-1852, 1993.[Abstract/Free Full Text]
  7. Turrisi A. T. R., Kim K., Blum R., Sause W. T., Livingston R. B., Komaki R., Wagner H., Aisner S., Johnson D. H. Twice-daily compared with once-daily thoracic radiotherapy in limited small-cell lung cancer treated concurrently with cisplatin and etoposide. N. Engl. J. Med., 340: 265-271, 1999.[Abstract/Free Full Text]
  8. Houghton P. J., Cheshire P. J., Hallman J. D., II, Lutz L., Friedman H. S., Danks M. K., Houghton J. A. Efficacy of topoisomerase I inhibitors, topotecan and irinotecan, administered at low dose levels in protracted schedules to mice bearing xenografts of human tumors. Cancer Chemother. Pharmacol., 36: 393-403, 1995.[Medline]
  9. Flaman J. M., Frebourg T., Moreau V., Charbonnier F., Martin C., Chappuis P., Sappino A. P., Limacher I. M., Bron L., Benhattar J., et al A simple p53 functional assay for screening cell lines, blood, and tumors. Proc. Natl. Acad. Sci. USA, 92: 3963-3967, 1995.[Abstract/Free Full Text]
  10. Sandler A. B. Etoposide plus ifosfamide plus cisplatin in the treatment of small cell lung cancer. Semin. Oncol., 25: 38-41, 1998.[Medline]
  11. Miyamoto H., Nakabayashi T., Isobe H., Akita H., Kawakami Y., Arimoto T., Asakawa M., Suzuki A., Fujikane T., Shimizu T., et al A Phase III comparison of etoposide/cisplatin with or without added ifosfamide in small-cell lung cancer. Oncology, 49: 431-435, 1992.[Medline]
  12. Thomas F., Arvelo F., Antoine E., Jacrot M., Poupon M. F. Antitumoral activity of bombesin analogues on small cell lung cancer xenografts: relationship with bombesin receptor expression. Cancer Res., 52: 4872-4877, 1992.[Abstract/Free Full Text]
  13. Arvelo F., Poupon M. F., Goguel A. F., Lizard G., Bourgeois Y., Arriagada R., Le Chevalier T. Response of a multidrug-resistant human small-cell lung cancer xenografts to chemotherapy. J. Cancer Res. Clin. Oncol., 120: 17-23, 1993.[CrossRef][Medline]
  14. Poupon M. F., Arvelo F., Goguel A. F., Bourgeois Y., Jacrot M., Hanania N., Arriagada R., Le Chevalier T. Response of small-cell lung cancer xenografts to chemotherapy: multidrug resistance and direct clinical correlates. J. Natl. Cancer Inst., 85: 2023-2029, 1993.[Abstract/Free Full Text]
  15. Arvelo F., Poupon M. F., Bichat F., Grossin F., Bourgeois Y., Jacrot M., Bastian G., Le Chevalier T. Adding a reverser (verapamil) to combined chemotherapy overrides resistance in small cell lung cancer xenografts. Eur. J. Cancer, 31A: 1862-1868, 1995.[CrossRef]
  16. Canitrot Y., Bichat F., Cole S. P., Deeley R. G., Gerlach J. H., Bastian G., Arvelo F., Poupon M. F. Multidrug resistance genes (MRP) and MDR1 expression in small cell lung cancer xenografts: relationship with response to chemotherapy. Cancer Lett., 130: 133-141, 1998.[CrossRef][Medline]
  17. Aas T., Borresen A. L., Geisler S., Smith-Sorensen B., Johnsen H., Varhaug J. E., Akslen L. A., Lonning P. E. Specific p53 mutations are associated with de novo resistance to doxorubicin in breast cancer patients. Nat. Med., 2: 811-814, 1996.[CrossRef][Medline]
  18. Safran H., King T., Choy H., Gollerkeri A., Kwakwa H., Lopez F., Cole B., Myers J., Tarpey J., Rosmarin A. p53 mutations do not predict response to paclitaxel/radiation for nonsmall cell lung carcinoma. Cancer (Phila.), 78: 1203-1210, 1996.[CrossRef][Medline]
  19. Perdomo J. A., Naomoto Y., Haisa M., Fujiwara T., Hamada M., Yasuoka Y., Tanaka N. In vivo influence of p53 status on proliferation and chemoradiosensitivity in non-small-cell lung cancer. J. Cancer Res. Clin. Oncol., 124: 10-18, 1998.[CrossRef][Medline]
  20. Galimberti S., Marchetti A., Buttitta F., Carnicelli V., Pellegrini S., Bevilacqua G., Petrini M. Multidrug resistance related genes and p53 expression in human non small cell lung cancer. Anticancer Res., 18: 2973-2976, 1998.[Medline]
  21. Kawasaki M., Nakanishi Y., Kuwano K., Yatsunami J., Takayama K., Hara N. The utility of p53 immunostaining of transbronchial biopsy specimens of lung cancer: p53 overexpression predicts poor prognosis and chemoresistance in advanced non-small cell lung cancer. Clin. Cancer Res., 3: 1195-1200, 1997.[Abstract]
  22. Blandino G., Levine A. J., Oren M. Mutant p53 gain of function: differential effects of different p53 mutants on resistance of cultured cells to chemotherapy. Oncogene, 18: 477-485, 1999.[CrossRef][Medline]
  23. Yamazaki K., Isobe H., Hanada T., Betsuyaku T., Hasegawa A., Hizawa N., Ogura S., Kawakami Y. Topoisomerase II {alpha} content and topoisomerase II catalytic activity cannot explain drug sensitivities to topoisomerase II inhibitors in lung cancer cell lines. Cancer Chemother. Pharmacol., 39: 192-198, 1997.[CrossRef][Medline]
  24. Jain N., Lam Y. M., Pym J., Campling B. G. Mechanisms of resistance of human small cell lung cancer lines selected in VP-16 and cisplatin. Cancer (Phila.), 77: 1797-1808, 1996.[CrossRef][Medline]
  25. Giaccone G., van Ark-Otte J., Rubio G. J., Gazdar A. F., Broxterman H. J., Dingemans A. M., Flens M. J., Scheper R. J., Pinedo H. M. MRP is frequently expressed in human lung-cancer cell lines, in non-small-cell lung cancer and in normal lungs. Int. J. Cancer, 66: 760-767, 1996.[CrossRef][Medline]
  26. Laurencot C. M., Scheffer G. L., Scheper R. J., Shoemaker R. H. Increased LRP mRNA expression is associated with the MDR phenotype in intrinsically resistant human cancer cell lines. Int. J. Cancer, 72: 1021-1026, 1997.[CrossRef][Medline]
  27. Doyle L. A. Mechanisms of drug resistance in human lung cancer cells. Semin. Oncol., 20: 326-337, 1993.[Medline]
  28. Sharma R., Singhal S. S., Srivastava S. K., Bajpai K. K., Frenkel E. P., Awasthi S. Glutathione and glutathione linked enzymes in human small cell lung cancer cell lines. Cancer Lett., 75: 111-119, 1993.[CrossRef][Medline]
  29. Hao X. Y., Bergh J., Brodin O., Hellman U., Mannervik B. Acquired resistance to cisplatin and doxorubicin in a small cell lung cancer cell line is correlated to elevated expression of glutathione-linked detoxification enzymes. Carcinogenesis (Lond.), 15: 1167-1173, 1994.[Abstract/Free Full Text]
  30. Savaraj N., Wu C. J., Xu R., Lampidis T., Lai S., Donnelly E., Solomon J., Feun L. G. Multidrug-resistant gene expression in small-cell lung cancer. Am. J. Clin. Oncol., 20: 398-403, 1997.[CrossRef][Medline]
  31. Kamataki T., Yokoi T., Fujita K., Ando Y. Preclinical approach for identifying drug interactions, Cancer Chemother. Pharmacol., 42: S50-S53, 1998.
  32. Relling M. V., McLeod H. L., Bowman L. C., Santana V. M. Etoposide pharmacokinetics and pharmacodynamics after acute and chronic exposure to cisplatin. Clin. Pharmacol. Ther., 56: 503-511, 1994.[Medline]
  33. Kivisto K. T., Kroemer H. K., Eichelbaum M. The role of human cytochrome P450 enzymes in the metabolism of anticancer agents: implications for drug interactions. Br. J. Clin. Pharmacol., 40: 523-530, 1995.[Medline]
  34. Kivisto K. T., Griese E. U., Fritz P., Linder A., Hakkola J., Raunio H., Beaune P., Kroemer H. K. Expression of cytochrome P 450 3A enzymes in human lung: a combined RT-PCR and immunohistochemical analysis of normal tissue and lung tumours. Naunyn-Schmiedebergs Arch. Pharmacol., 353: 207-212, 1996.[Medline]




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