CCI-779 plus Cisplatin Is Highly Effective against Human Melanoma in a SCID Mouse Xenotranplantation Model
C. Thallingera W. Poeppla B. Pratschera M. Mayerhoferb P. Valentb
G. Tappeinerc C. Joukhadara, d
Received: November 20, 2006 Accepted: December 7, 2006 Published online: March 22, 2007
a Department of Clinical Pharmacology, Division of Clinical Pharmacokinetics, b Department of Internal Medicine I, c Department of Dermatology, Division of General Dermatology, and d Department of Internal Medicine II,
Medical University of Vienna, Vienna, Austria
Key Words
Rapamycin ti CCI-779 ti mTOR ti Melanoma ti Chemoresistance
Abstract
Background: This study set out to investigate the antitumor effects of a treatment strategy combining the mTOR inhibi- tor CCI-779 with cisplatin in vitro and in a human melanoma SCID mouse model. Methods: In vitro 518A2, Mel-JUSO or 607B cell lines were incubated with CCI-779, cisplatin and CCI-779 plus cisplatin. Based on these results, a 4-group, par- allel, controlled experimental study design was initiated in vivo. SCID mice were injected with melanoma cells, and after the development of tumors the mice received daily injec- tions of CCI-779 or solvent. On treatment days 2 and 6 cis- platin or mock solution were administered. Results: In vitro a synergistic antitumor effect was observed for the treat- ment regimen of CCI-779 plus cisplatin. In SCID mice after 2 weeks of therapy with CCI-779 plus cisplatin 4 of 6 tumors of the 518A2 cell line were completely eradicated. In the two remaining 518A2 xenografts this treatment strategy reduced the tumor weight by 94 8 9% compared to solvent. CCI-779 plus cisplatin also exerted a significant antitumor effect in Mel-JUSO and 607B xenografts compared to mock-treated
animals. Conclusion: We provide circumstantial evidence that the use of CCI-779 plus cisplatin might qualify as a prom- ising strategy in the treatment of human melanoma.
Copyright © 2007 S. Karger AG, Basel
Introduction
Metastatic malignant melanoma is an aggressive tu- mor that responds poorly to standard chemotherapeutic approaches. In general, the clinical response of metastat- ic malignant melanoma to either single or combination chemotherapy has not surpassed 25% presenting median survivals rates of only 6–12 months [1].
Due to its influence on growth arrest, apoptosis and angiogenesis, rapamycin, an approved immunosuppres- sive agent, is currently under investigation as a potential anticancer drug [2, 3]. Rapamycin forms an inhibitory complex with the immunophilin, FKBP12, which binds to and inhibits the mammalian target of rapamycin (mTOR). mTOR itself is a downstream effector of the phosphatidylinositol 3-kinase/Akt signaling pathway and regulates protein translation, cell-cycle progression and cellular proliferation. In addition, mTOR is involved in the DNA damage response [4].
© 2007 S. Karger AG, Basel 0031–7012/07/0794–0207$23.50/0
Gerhard Tappeiner
Department of Dermatology, Medical University of Vienna
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The mTOR pathway, often upregulated in cancer, is an attractive target for cancer therapeutics as effective in- hibitors such as rapamycin or the cell cycle inhibitor 779 (CCI-779) block mTOR [5]. Currently, disruption of the mTOR signaling cascade by rapamycin and CCI-779 is being tested for efficacy against a broad range of refrac- tory tumor entities [6–15]. Moreover, many tumor enti- ties appear to be highly prone to apoptosis, which has been exploited clinically through the use of DNA-dam- aging chemotherapeutic agents, such as cisplatin. Thera- peutic regimens, which sensitize tumor cells toward apoptosis-inducing agents, could be a promising strategy combating malignant tumor cell growth [4].
Against this background, the present study was car- ried out to investigate the potential of CCI-779 to increase the sensitivity to cisplatin by targeting mTOR in different melanoma cell lines in vitro and by the use of a human melanoma SCID mouse model.
Materials and Methods
Cell Lines and Culture
The human melanoma cell lines, 518A2 and 607B, were gifts from P. Schrier (University of Leiden, The Netherlands). The cell line, Mel-JUSO, was kindly provided by Dr. Judith Johnson (Uni- versity of Munich, Germany). The cell lines were maintained in Dulbecco’s modified Eagle’s culture medium supplemented with 10% fetal bovine serum (Gibco, Paisley, UK) and an antibiotic mixture containing 100 IU/ml of penicillin, 100 mg/ml of strep- tomycin, and 0.25 mg/ml of amphotericin B (all Gibco) in a fully humidified 5% CO2, 95% ambient air atmosphere at 37°C.
Cell Growth Analysis in vitro
For the 3H-thymidine incorporation assay, the tumor cell line 518A2, representative of all the cell lines used in the present study, was selected and cultured in 96-well microtiter plates (4 ! 103 cells/well) in the absence or presence of various concentrations of cisplatin and CCI-779 (25–100 nmol/l) for 48 h. 518A2 cells were incubated with a combination of cisplatin and CCI-779 (fixed ra- tio of 1:1) to analyze the potential synergistic effects between cis- platin and CCI-779. After treatment of cells, 3H-thymidine was added to each well. Twelve hours later, cells were harvested on fil- ter membranes (Packard Bioscience, Meriden, Conn., USA) in a Filtermate 196 harvester (Packard Bioscience). Filters were air- dried, and the bound radioactivity was measured in a b-counter (Top-Count NXT, Packard Bioscience). All experiments were performed in triplicate.
To determine the synergistic effects between cisplatin and CCI-779 on the growth of 518A2 cells, combination index values were calculated using a commercially available software program (Calcusyn; Biosoft, Ferguson, Mo., USA).
In vivo Experiments
The study protocol was approved by the Medical University of Vienna Animal Welfare Committee. All animal studies were per-
formed according to local guidelines for animal care and protec- tion.
The three cell lines 518A2, Mel-JUSO and 607B, were tested in the present study. Per cell line, 24 female C.B.-17 scid/scid (SCID) mice (3–4 weeks, Harlan Winkelmann, Borchen, Germany) were randomly assigned to 4 experimental groups of 6 animals each. Thus, a total of 72 SCID mice were used. SCID mice were injected subcutaneously with either 518A2, Mel-JUSO or 607B cells at an approximate total amount of 5 ! 106 cells into the lower left flank. When the animals had developed melanoma nodules of about 5 mm in diameter, they were marked by ear piercing, and study drug administration was initiated. CCI-779 solution (Wy- eth Pharma, Muenster, Germany) was prepared as previously de- scribed [3] and was administered intraperitoneally at a dose of 1.5 mg/kg daily over a period of 14 days. The dose chosen in the pres- ent study was based on previous experiments in SCID mice [9]
and is in agreement with those used in humans [16]. Cisplatin (5 mg/kg i.p.) or solvent control was administered on days 2 and 6 as previously reported [17]. During these experiments, tumor volume was assessed twice weekly by caliper measurements [18]. On treatment day 14, all animals were killed. Tumor xenotrans- plants and organs were weighed and fixed in formalin for immu- nohistochemistry and terminal deoxynucleotidyl transferase- mediated dUTP nick end labeling (TUNEL) analysis.
Assays Measuring Apoptosis and Cell Proliferation
The histochemical detection of apoptosis in formalin-fixed sections was performed by TUNEL assay using fluorescein-dUTP (Boehringer Mannheim, Mannheim, Germany). Tissue sections were counterstained using 4ti,6-diamidine-2ti-phenylindole dihy- drochloride (Roche Diagnostics, Basel, Switzerland). The expres- sion of Ki-67 in vivo was evaluated by immunohistochemistry with an anti-Ki-67 antibody, clone MIB-1 (Dianova, Hamburg, Germany) and a DAKO LSAB 2 System. TUNEL and Ki-67 label- ing indices were assessed as the mean percentage of positive cells, measured by counting defined areas in five representative high- power fields (magnification !400), employing an eyepiece grid.
Statistical Analysis
Statistical significance of differences in tumor weights among treatment groups was analyzed using Wilcoxon tests, Bonferroni adjustments and Kruskal-Wallis ANOVA (Statistica 5.1, Tulsa, USA). A two-sided p value of !0.05 was considered to be of sta- tistical significance.
Results
Inhibition of the Growth of Human Melanoma Cells in vitro
Firstly, we evaluated the antineoplastic effect of the combined treatment of the mTOR inhibitor CCI-779 and the chemotherapeutic cisplatin in vitro. We focused on 518A2 melanoma cells as we recently detected the stron- gest reduction of mTOR protein (approximately 46%) in 518A2 cells compared to Mel-JUSO and 607B melanoma cells [19]. The potential of CCI-779 to sensitize 518A2 hu-
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Cisplatin CCI-779
Cisplatin + CCI-779
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0.40
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40 60
Concentration (ng/ml)
80
100
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Cisplatin
CCI-779
Cisplatin +
CCI-779
Fig. 1. The effects of CCI-779, cisplatin and the combination of both compounds on melanoma cell killing in vitro. a 518A2 cells were incubated with various concentrations of CCI-779, cisplatin, or a combination of both compounds at a constant ratio of 1:1 for 48 h. Cell growth was determined by 3H-thymidine incorporation as- say. b 518A2 cells were incubated with cisplatin (75 ng/ml) or CCI-779 (75 ng/ml) or with a combination of both compounds (each 75 ng/ml) for 48 h. Thereafter, 3H-thymidine incorporation was determined. Each data point represents the mean 8 SD of six wells analyzed. Representative data of three independent experiments are shown.
man melanoma cells to cisplatin treatment is depicted in figure 1. The concentration versus effect relationship for CCI-779, cisplatin and the combination of both drugs was investigated and a synergistic antitumor effect was observed for the combination treatment regimen of CCI- 779 plus cisplatin (fig. 1a). The antineoplastic effects gen- erated by CCI-779 and cisplatin monotreatment were al- most similar.
Additionally, we performed a 3H-thymidine incorpo- ration assay to investigate cell proliferation (fig. 1b). 3H- Thymidine is a precursor of DNA and is incorporated into new DNA in proliferating cells. After incubation of 518A2 cells with either cisplatin, CCI-779, or a combina- tion of both compounds at a concentration of 75 ng/ml each for a period of 48 h, cell growth was 94 8 10, 31 8 15 and 9 8 5%, respectively, compared to untreated con- trol cells. Interestingly, cisplatin monotreatment exerted nearly no antiproliferative effect at the chosen dose.
Growth Inhibition of Human Melanoma Xenotransplants in SCID Mice
After 2 weeks of therapy with the study drugs the mice were killed and evaluated for tumor growth (fig. 2b; ta- ble 1). The combined treatment with CCI-779 plus cis- platin eradicated 4 of 6 established 518A2 melanomas and resulted in a significant tumor weight reduction of 94 8
9% in the remaining two 518A2 melanoma xenotrans- plants. A synergistic antitumor effect was also detected in Mel-JUSO and 607B xenografts showing a tumor growth reduction of 76 8 14 and 72 8 13%, respectively. However, no tumor was completely eradicated in these cell lines. The combination of CCI-779 plus cisplatin was statistically superior to all other treatment regimens in all cell lines tested (p ! 0.05; fig. 2b). Notably, the cisplatin dosage used was only two thirds of the commonly admin- istered and published cisplatin dosage in mice [17]. This is a probable explanation for the missing antineoplastic effect observed in the cisplatin monotherapy group.
The tumor weight data were supported by caliper mea- surements evaluating the tumor volume twice weekly during the course of the experiment and were in fine agreement with the measured tumor weights (fig. 2; ta- ble 1).
Side Effects Related to Study Drugs
Mice treated with CCI-779 plus cisplatin or cisplatin solely displayed the lowest body weights in all three cell lines (table 1). The body weights were almost identical in these 2 treatment groups, suggesting that body weight re- duction was mainly caused by cisplatin. In line with this, treatment with CCI-779 had no significant influence on mouse body weight when compared to mock-treated
mice (p 1 0.05; table 1). In mice treated with cisplatin
only or the combination (CCI-779 plus cisplatin), side ef- fects were seen, such as piloerection and ruffled fur. No histological signs of toxicity were detected by cross-path- ological examination of liver, lung, kidney and intestine. Diarrhea was not perceived in mice. No metastases were seen in any of the screened organs.
Apoptosis and Cell Proliferation in 518A2 Melanoma Xenografts
To assess whether the observed antitumor effects of
700
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0
518A2
1 5 9 14
the combination treatment regimen of CCI-779 plus cis- platin were associated with a higher rate of apoptotic cells, melanoma xenografts were examined for apoptotic cell death by TUNEL staining (fig. 3a). CCI-779 plus cis- platin increased the number of apoptotic cells approxi- mately 5-fold (15% apoptotic cells) when compared to the solvent (3%) control group in melanoma xenotransplants. Monotherapy with CCI-779 increased the amount of apoptotic cells to 6% when compared to mock-treated mice. In tumors treated only with cisplatin, the relative portion of apoptotic cells was 5%.
The evaluation of antiproliferative effects of CCI-779 treatment was performed by Ki-67 immunohistochemis- try. We detected a significant decrease in Ki-67-positive cells in tumors treated with CCI-779 when compared to solvent- or cisplatin-treated groups (fig. 3b). The com- bined treatment regimen of CCI-779 plus cisplatin result- ed in no further decrease in Ki-67-positive cells in com- parison to CCI-779 monotreatment, suggesting that an- tiproliferative effects were mainly caused by CCI-779. There was no difference in the average percentage of Ki- 67-positive cells for those xenotransplants treated with
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Time (days) Mel-JUSO
Time (days)
607B
8
9
14
14
cisplatin compared to the solvent control tumor speci- mens.
a
Time (days)
Fig. 2. The effects of CCI-779, cisplatin and the combination of both compounds on melanoma growth in SCID mice. SCID mice with established 518A2, Mel-JUSO or 607B tumors received a 14- day treatment with CCI-779 (1.5 mg/kg i.p.) or solvent control (6 animals/group). In addition, animals received cisplatin (Cis; 5 mg/kg i.p.) on days 2 and 6. a During the course of the experi- ment tumor size was assessed by caliper measurements twice weekly and is depicted in cubic millimeters as the mean 8 SD. b At the end of the experiment (day 14) tumors were resected and
b
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2.5
2.0
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0
518A2 Mel-JUSO 607B
weighed. Tumor weight is provided as the mean 8 SD in grams.
a
b
CCI-779+Cis CCI-779 Cis Solvent
Fig. 3. CCI-779 facilitated apoptosis and exerted antiproliferative effects in vivo. a 518A2 melanoma xenografts were analyzed by TUNEL assay for apoptotic cell death. TUNEL-positive cells increased in tumors treated with CCI-779 plus cisplatin (CCI-779+Cis) compared to CCI-779 or cisplatin (Cis) monotreatment. b Ki-67 expres- sion was evaluated by immunohistochemistry to analyze cell proliferation in 518A2 melanoma specimens. Original magnification, !200. Representative photographs of tumor sections are shown.
Table 1. Summary of the effects of the different treatment regimens on mouse body weight, tumor weight and tumor volume of Mel-JUSO, 518A2 and 607B human melanoma xenotransplants after 14 days of therapy in SCID mice (mean 8 SD)
Treatment modality (n = 6)
Mel-JUSO
CCI-779+Cis CCI-779
Cis Solvent
518A2
CCI-779+Cis CCI-779
Cis Solvent
607B
CCI-779+Cis
Tumor growth inhibition, %
76814 22816
0821 0816
9489 35817
0822 088
72813
Tumor weight, g
0.2580.15 0.7880.17 1.0780.22 1.0680.17
0.0580.07 0.4980.13 0.7580.17 0.7580.06
0.5880.26
Tumor volume, mm3
175.0898.0 6308223.0
885.08342.1 796.38312.0
14.7824.0 337.6877.3 569.08134.0 505.4893.2
363.5893.2
Body weight, g
16.881.3 19.781.7 15.481.1 19.881.0
16.481.1 19.180.7 17.281.7 18.980.7
14.081.9
CCI-779 19830 1.6880.60 1,379.08703.3 19.982.9
Cis 26813 1.5380.26 1,465.28589.5 15.582.3
Solvent 0840 2.0780.82 2,526.48868.0 19.281.4
Discussion
CCI-779 is a rapamycin analog that allows targeting of the mTOR/Akt pathway by inhibiting mTOR. It has been referred to as a compelling potential novel anticancer candidate, because of its limited toxicity and potential
additive cytotoxicity if combined with other cytotoxic agents or radiochemistry [2, 3, 20].
The main finding of our investigation was that CCI- 779 possessed the potential to increase the sensitivity to cisplatin resulting in melanoma growth inhibition. In de- tail, combined treatment with CCI-779 plus cisplatin
eradicated 4 of 6 established human 518A2 melanomas grown in SCID mice. This treatment regimen resulted also in an overt synergistic antitumor effect in Mel-JUSO and 607B melanoma xenografts, when compared to mock treated animals (p ! 0.05; fig. 2; table 1). CCI-779 itself exerted a moderate antitumor effect of approximately 20–30% compared to the solvent control group, a finding which is in excellent agreement with a recently published phase II trial in human melanoma [21]. The authors con- cluded that CCI-779 is not sufficiently active to warrant further testing as a single agent.
The ability of CCI-779 to sensitize melanoma cells to a subsequent chemotherapeutic treatment is moreover underlined by the fact that the observed antineoplastic effects were generated at 60% of the cisplatin dosage commonly used in SCID mice [17]. Notably, cisplatin monotherapy at this reduced dosage level had no effect on tumor growth, when compared to solvent control mice (p 1 0.05; fig. 2; table 1). The rationale for combin- ing CCI-779 with cisplatin was based on the fact that cisplatin is frequently administered in melanoma thera- py and additionally, can be used without further activa- tion in vitro. The in vitro data depicted in figure 1 con- firm the SCID mouse data. A synergistic antitumor ef- fect was observed for the combination treatment regimen of CCI-779 plus cisplatin compared to mock-treated cells. The antineoplastic effects generated by CCI-779 and cisplatin monotherapy were almost similar (p 1 0.05).
Next we visualized the tumor growth inhibition ob- served by quantification of apoptosis and cell prolifera- tion (fig. 3). From previous experiments there is circum- stantial evidence that, beside the direct apoptosis inhib- itory properties, rapamycin and CCI-779 are able to increase the sensitivity to apoptosis induced by chemo- therapeutic agents [22, 23]. In the present experiment we performed TUNEL staining to examine apoptotic cell death. A 5-fold increase in apoptotic cells was detected in tumor specimens treated with the combination of
CCI-779 plus cisplatin (fig. 3a). This is in accordance with recent observations demonstrating that rapamycin is able to increase the sensitivity to apoptosis induced by cisplatin in murine T cells [23] and also in multiple my- eloma cells treated with dexamethasone [7]. However, CCI-779 monotherapy exerted only a weak increase in apoptotic cells when compared to mock-treated animals. Thus, analyzing our data, it seems that CCI-779 did not induce apoptosis on its own, but influenced the suscep- tibility to apoptosis induced by cisplatin in melanoma cells.
Beside apoptosis, CCI-779 exerts antiproliferative ef- fects by inducing an arrest in the G1 phase of the cell cycle [24]. Evaluation of the antiproliferative potential of CCI-779 plus cisplatin in vivo by Ki-67 immunohisto- chemistry (fig. 3b) mirrored the in vitro data derived from a separate experiment using a 3H-thymidine incor- poration assay (fig. 1b). Ki-67 expression was inhibited in all xenografts treated with CCI-779 (fig. 3b), which cor- roborates previous findings by Frost et al. [2]. We ob- served no increase in the antiproliferative effects of CCI- 779 by addition of cisplatin, either in vitro or in vivo. These results are underlined by previously published lit- erature documenting that cisplatin mainly exerts its an- tineoplastic effect via apoptosis.
Although we found that CCI-779 increased the sensi- tivity to cisplatin in three human melanoma cell lines, a number of questions remain open. In summary, this ar- ticle reveals a good deal of experimental data that may point to a new drug combination to treat human mela- noma and to overcome chemoresistance in this tumor entity.
Acknowledgments
The work was supported by a grant from the Bürgermeister Fonds der Stadt Wien, project No. 2362, and the Hans und Blanca Moser Stiftung. CCI-779 was a gift from Wyeth Pharma (Muen- ster, Germany).
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