Instituto de Genética Experimental, Facultad de Ciencias Médicas, Universidad Nacional de Rosario, Santa Fe 3100 (2000) Rosario, Argentina
Correspondence to: O. Graciela Scharovsky, Instituto de Genética Experimental, Facultad de Ciencias Médicas, UNR, Santa Fe 3100 (2000) Rosario, Argentina. Tel: +54-341-4804559/63 ext. 244; Fax: +54-341-4804569; Email: ogs{at}citynet.net.ar
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Abstract |
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Methods: Adult inbred rats were challenged with lymphoma TACB and sarcoma E100 s.c. on day 0. Animals were divided into two groups: group I, control, injected with saline three times a week; and group II, treated with Cy 10 mg/kg three times a week, from day 10 until the tumour was non-palpable, or 5 mg/kg three times a week from day 7. Tumours were measured and animals were weighed twice weekly. Periodic blood samples were taken for determination of urea, creatinine, serum glutamic-oxaloacetic transaminase, lactate dehydrogenase and haematological parameters.
Results: The administration of low-dose Cy eradicated established rat lymphomas and sarcomas; there was neither metastatic growth nor recurrence at primary sites for 100% of the lymphomas and 83% of the sarcomas. In addition, the treatment did not cause weight loss, and was devoid of haematological, cardiac, hepatic and renal toxicity.
Conclusions: Metronomic administration of Cy at low doses on a thrice weekly schedule to already grown rat lymphomas and sarcomas demonstrated itself to be a successful antitumour therapy that did not cause weight loss and was devoid of haematological, cardiac, hepatic and renal toxicity.
Key words: cyclophosphamide, lymphoma, metronomic dosing, sarcoma, toxicity
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Introduction |
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In order to avoid the problems caused by traditional chemotherapeutic treatments, several researchers, including ourselves, recently began to search for new modalities of drug administration oriented towards a more efficient and non-toxic antitumoral and/or antimetastatic therapy. We have already demonstrated that a single low dose of cyclophosphamide (Cy), a treatment completely devoid of toxicity, inhibits the growth of spontaneous and experimental metastasis of a rat lymphoma, while it does not affect primary tumour growth [1]. Such an effect would mainly be due to modulation of the immune response [2
5
]. These results proved that in our tumour model, the targets of low-dose chemotherapy were the immune cells and that metastatic cells were affected indirectly by the drug.
On the other hand, Kerbel, Folkman and colleagues have demonstrated the efficacy of some of the most widely used chemotherapeutic drugs as antiangiogenic agents [69
]. The proposal implies that a change should be made with respect to the rationale behind chemotherapy, involving a different target. This new concept includes the possibility of treating tumours that no longer respond to traditional chemotherapy. Results obtained at the clinical level [10
] provide a point of departure for future studies in this interesting area of cancer therapy.
Our objective in this study was to investigate the possibility of obtaining an antitumoral effect by the metronomic administration of low-dose Cy. Another main focus of our work was to determine the toxicity of the treatment. We report here the effect of administration of (i) Cy 10 mg/kg on a thrice weekly schedule, from day 10 until approximately day 60, on lymphoma TACB (L-TACB) growth; and (ii) Cy 5 mg/kg on the same schedule, from day 7 until approximately day 30, on sarcoma E100 (S-E100) growth, along with its toxicity.
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Methods |
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Drugs
Cy (Endoxan-Asta, Labinca SA, Argentina) was dissolved in sterile, distilled water to a concentration of 20 mg/ml.
Tumours
L-TACB is a poorly differentiated B-cell lymphoma, which arose spontaneously in an inbred e rat [12]. When L-TACB is injected subcutaneously, lymph nodes are the exclusive site of metastatic growth.
S-E100 is a fibrosarcoma that appeared spontaneously in an outbred population of IIM rats in 1955 and is maintained by subcutaneous passage in rats of the allogeneic inbred line m, with 100% lethality [13].
Experimental models
Adult e or m rats were challenged with L-TACB or S-E100, respectively, subcutaneously by trocar, on day 0, and distributed as follows. L-TACB: group I control rats (n=7) were injected i.p. with saline three times a week, from day 10 until animals were killed; and group II rats (n=6) were injected i.p. with Cy (10 mg/kg body weight) three times a week, from day 10 until the day on which the tumour was no longer palpable. S-E100: group I control animals (n=12) were injected i.p. with saline three times a week, from day 7 until animals were killed; and group II rats (n=12) were injected with Cy 5 mg/kg i.p., three times a week, from day 7. In the case of the lymphoma tumour model, a third experimental group was added (group III), in which naïve animals (n=4) were injected with Cy, employing the same schedule and dose as in the respective group II.
Tumours were measured twice weekly using a calliper. Tumour volumes were calculated as follows: v = 0.4 (ab2), where v = volume (cm3), a = largest diameter (cm) and b = smallest diameter (cm). Animals were weighed weekly. On days 0, 20, 40 and 60, blood samples were taken and each rat was subjected to an electrocardiogram (ECG). Group II animals were clinically controlled and the appearance of tumour recurrences and metastasis was checked until the end of the experiment (day 150 for L-TACB and day 120 for S-E100).
Haematological and serological parameters
Blood and serum samples were obtained from animals belonging to all experimental groups [L-TACB: group I (n=7), group II (n=6) and group III (n=4); S-E100: groups I and II (n=4)] for the determination of haematological and serological parameters, including haemoglobinemia, haematocrit, total leukocyte count and proportion of white blood cell (WBC) types (May GrünwaldGiemsa colouration), and serum concentration of urea (direct colorimetric method), creatinine (Jafeé reaction), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH, E.C.1.1.1.27) by the optimised ultraviolet method.
Electrocardiograms
The ECG DII derivation at a velocity of 50 mm/s was registered with awake animals, employing a conventional electrocardiograph. Cardiac rate, PRi (PR interval), QTi (QT interval) and ÂQRS (main ventricular depolarisation vector) were measured, and the presence of arrhythmia or other manifestations of myocardial lesions were analysed.
Statistical analyses
Analysis of variance and the TukeyKramer Multiple Comparison tests, the MannWhitney non-parametric test and Student's t-test (InStat, GraphPad Software, Inc.) were used. Results were considered significant if P-values were <0.05.
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Results |
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Discussion |
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The experimental models were designed to resemble the clinical situation of a patient with a recently detected tumour (not too large, but large enough to be easily detected) who begins therapy immediately after being diagnosed. Metronomic therapy began after 10 and 7 days of the tumour challenge, for L-TACB and S-E100, respectively. The response of L-TACB-bearing rats to treatment was complete in 100% of the animals, achieving total regression between days 30 and 60. Importantly, neither recurrences nor metastases were detected until the end of the experiment. The behaviour of S-E100 after Cy treatment was similar to that of L-TACB until day 28, when tumour growth at the primary site resumed in two out of 12 rats. The rest of the animals remained recurrence-free until the end of the experiment. These results, obtained with tumour types and species different from those assayed up to now, provide additional experimental evidence for the antitumour effect of metronomic therapy. The demonstration that different kinds of tumours can be eliminated or significantly diminished [610
] by chronic administration of low doses of very different kinds of chemotherapeutic drugs [19
] increases the likelihood of successful treatment of human cancer. It also encourages the development of phase I and II clinical trials for different cancer types using different drugs, which, ultimately, will determine the efficacy of metronomic therapy for the treatment of human cancers.
Other authors have demonstrated clearly the antiangiogenic nature of the mechanism of action of several drugs administered by metronomic dosing. Moreover, the combination of such treatment with specific antiangiogenic reagents increased significantly the observed antitumour effect of metronomic dosing [68
, 20
, 21
]. Hence, the antitumour effect described herein is probably due to an antiangiogenic mechanism. Some recent findings by Mauceri et al. [22
] support this hypothesis. She found that the addition of Cy to angiostatin, an internal fragment of plasminogen with antiangiogenic properties that is generally secreted by tumours [23
], produced inhibition of tube formation. Moreover, recent preliminary results, from our laboratory, utilising the Matrigel plug angiogenesis assay in e rats showed an antiangiogenic effect exerted by four injections of Cy (10 mg/kg) administered over 6 days. Nevertheless, we do not dismiss the existence of other mechanisms contributing to the antitumour effect, mainly the modulation of the immune response, as we have demonstrated for the antimetastatic effect of single low-dose Cy in the L-TACB tumour model [2
5
]. The recurrence of the tumour in 17% of the Cy-treated, S-E100-bearing rats suggested that, in this case, the cell target of Cy would not be the genetically stable endothelial cells, which in all likelihood would not generate drug resistance. Future studies will address this important matter.
Determination of body weight showed no weight loss in either treatment group for each tumour model. The body weight of treated animals increased steadily, with the exception of transient, non-significant decreases. Concomitantly, animals were monitored for the appearance of any other clinical sign of drug toxicity, which never appeared.
From a haematological point of view, determinations of the haematological parameters throughout the study demonstrated the lack of toxicity of the treatment. Haemoglobin concentration and haematocrit did not suffer variation in any of the Cy-treated groups throughout the experiments, while in both control groups there were significant decreases in both values. Also, while the WBC count increased significantly with tumour growth, a significant decrease was observed in the group of naïve Cy-treated rats. These opposite effects caused by tumour growth and Cy treatment, respectively, might account for the lack of WBC changes observed in Cy-treated L-TACB- and S-E100-bearing rats. The altered WBC proportions observed in treated tumour-bearing rats, namely an increase in neutrophils and a decrease in lymphocytes, tended to return to basal values by day 60. Such changes are mainly due to tumour growth, because naïve rats treated only with Cy showed no important modifications to these proportions. Moreover, the Cy-treated tumour-bearing rats returned to normal values between days 40 and 60, despite continuation of chemotherapy.
Similarly, the evaluation of biochemical parameters showed a lack of renal, hepatic or cardiac toxicity with the treatment. Serum concentrations of urea and creatinine in L-TACB-bearing rats remained unaltered, while in the S-E100 tumour model, the significant increase in serum urea in control rats, caused by tumour growth, was moderated by the treatment. Cy-treated S-E100-bearing rats showed normalised uraemia by days 4060; therefore, there was no renal toxicity when measured using these parameters.
The growth of L-TACB or S-E100 was accompanied by a striking elevation in AST concentration. AST is an enzyme present in high concentrations in tissues with high metabolic activity. Elevated levels of AST have been correlated with certain malignancies [24]. Severely damaged or dead cells release AST into the blood in quantities directly proportional to the amount of tissue damage [25
]. Interestingly, the treatment administered herein did not modify the normal enzyme levels in S-E100-treated rats. Besides, in L-TACB Cy-treated rats there was a transient increase in AST concentration at day 20; an increase that, nevertheless, was significantly lower than that of control rats (P < 0.01), returning to normal levels by day 40.
Many tissues, including the liver, red blood cells and the brain, produce LDH, an enzyme normally found in the blood. The fact that tumour growth is frequently associated with an increase in LDH serum levels is already known, and this protein can be considered as a tumour marker [26, 27
]. Also, LDH, as well as AST, are protein markers associated with different types and degrees of cardiac damage [28
]. The significant increases in LDH serum concentration found in control rats belonging to both tumour models were moderated by the treatment at day 20 and continued to decrease towards day 60. The augmentation of serum LDH during tumour growth would probably be compensated for by the decrease observed during Cy treatment in naïve rats. The AST and LDH results obtained herein indicate a lack of hepatic and cardiac toxicity with the treatment. Moreover, there were no changes in ECG results during the treatment, a fact that confirmed the absence of gross cardiac alterations.
In summary, metronomic administration of Cy at low doses on a thrice weekly schedule to already grown rat lymphomas and sarcomas was demonstrated to be a successful antitumour therapy that, interestingly, was devoid of toxicity. Further studies will help to clarify the mechanism of action and what type of cells are being targeted by this chronic chemotherapy.
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Acknowledgements |
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Notes |
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Received for publication October 12, 2003. Revision received March 15, 2004. Accepted for publication May 12, 2004.
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References |
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