In vivo experiments were conducted in male C57BL/6J mice (18-25 g), housed at 22 ± 2°C under a 12/12 h light/dark cycle with free access to food and water until use. The animals were acclimatized to the laboratory for at least 24 h before testing and were used only once. All animal handling and experimental procedures were conducted with prior approval of the ethics committee on animal use of the University of São Paulo Medical School Hospital Ethics Committee.

Simvastatin, in both, compressed powders and as a lactone form were a giftly provide from Prati Donaduzzi (Toledo, Brazil).

For in vitro assays, simvastatin in lactone form was dissolved in 1.8 mL of ethanol, added to 19 mL of 0.1 M NaOH and then incubated for 40 min at 40°C to yield the active form, followed by the addition of 0.1 M HCl to adjust the pH to 7,4 [11].

To mice experimentations, the simvastatin compressed powders were macerated and watery solutions were made.

B16F10 mice melanoma cell line was generously provided by Dr. Roger Chammas (USP, São Paulo, Brazil). Cells were routinely cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) at 37°C in a humidified atmosphere containing 5% CO_2.

Cells were treated with different concentrations of simvastatin (0.8; 1.6; 3.2 and 5.6 μM) and assessed by colorimetric method of MTT, based on the reduction of MTT to Formazan by living cells. The cells were incubated in plates of 96 wells, treated with simvastatin and MTT was added at times 24, 48 and 72 hours. To dissolve the crystals of Formazan was used dimethylsulfoxide (DMSO). Quantification was obtained by the absorbance reading at a wavelength of 450 nm to 690 nm. Cell viability was also evaluated by the method of Trypan Blue exclusion (Invitrogen, Carlsbad, CA), by direct counting in a hemocytometer [12].

Cell cycle analysis was determined by flow cytometry at the Federal State University of Paraná. Briefly, after the different treatments, 2 × 10⁶ cells were trypsinized, washed three times with PBS, fixed in 70% ethanol, and stained with Propidium Iodide (PI) solution. All analyses were done using a FACScalibur flow cytometer (Becton Dickinson, San Jose, CA). The red fluorescence of PI was collected through a 585/42-nm band -pass filter, and the fluorescence signals were measured in a linear scale of 1024 channels. For each sample, at least 10000 events were acquired and the data were analyzed using an appropriate software (CELLQuest, Becton Dickinson, San Jose, CA) [12].

All animal's procedures were made in Butantan Institute with supervision of Dr. Durvanei Augusto Maria, in accordance with University of Sao Paulo Medical School Hospital Ethics Committee. Briefly, 5 × 10⁴ B16F10 melanoma cells were injected subcutaneously into C57Bl/6J mice. When tumors were palpable (around 8-10 days after implanted) mice were treated with oral daily dose of 5 mg/Kg of simvastatin. Tumors were measured using a caliper and volume was estimated using the formula V = (small diameter)² × (large diameter)/¾ πR [13].

C57BL/6J mice, weighing about 20 g and 60 days older were used. After the experiments, blood was collected from the axillary plexus. The bone marrow was collected from the femur of the same animals. The spleen cellularities were also analyzed [14].

Taking account the importance of the liver in lipids and simvastatin metabolism, we evaluated a possible hepatotoxic effect, evaluating three enzymes associated with liver injury, the glutamic-oxalacetic transaminase (GOT), the glutamic pyruvic transaminase (ALT) and gamma glutamyl transferase(gamma-GT). The experimental procedure consists in a simple addition of the 50 μL of the serum sample to 1000 μL of commercial reagent for each tested enzyme [15]. Twenty days after the melanoma cells implant and hence ten days after sinvastatin treatment has started the blood was collect.

Blood samples were collected twenty days after tumor implant from the axillary plexus of the animals in all experimental groups, which were used to perform blood and reticulocyte count, according to the techniques used in the Laboratory of Experimental Hematology of FCF/USP [14].

The animals were first anesthetized, exsanguinated and sacrificed by cervical dislocation. The bone marrow was obtained by washing the cavity with the culture femoral McCoy's 5A modified (Sigma, Chemical Company, USA) ice cream [14].

The bone marrow cells suspension samples were prepared on slides of cell spin cytocentrifuge (Incibrás) and stained by the method of May-Grünwald-Giemsa modified Rosenfeld. The cells were classified as blastic and mitotic cells, neutrophil young granulocytes (promyelocytic and myelocytes) in ring stage (metamyelocytes and rod), and segmented eosinophils, monocytes and macrophages, lymphocytes and plasma cells, pro-erythroblasts and basophilic erythroblasts, polychromatic and orthochromatic [14,16].

Sleen cells were collected by the same sacrificed animals of hematological procedures. The spleen was transferred to plastic Petri dish with iced culture McCoy's 5A modified (Sigma, Chemical Company, USA) containing 10% EDTA, used to collect the cells. In the Petri dish, the spleen capsule was broken in one of its ends, with the aid of two needles bent into "L", and fixed in syringes. The cells were gently removed from the dish by the dissociation method. The suspension was homogenized in the Petri dish with standard Pasteur pipette to complete cell separation and transferred to conical plastic tube and kept in an ice bath. The cells obtained from the spleen were quantified in a Neubauer chamber hemocytometer after dilution of 1:500 liquid Turk, diluent composed of 1% solution of glacial acetic acid. For morphological evaluation, the slides were performed in cell suspension cellspin cytocentrifuge (Incibras) and subsequently submitted to the staining of May-Grunwald GIENS, modified by Rosenfeld, and peroxidase method modified by Graham Knoll, for identification and quantification of different types cell [16].

Tumor growth data were analyzed by Student's t test, survival rate data were analyzed by Kaplan-Meyer curves and hematological data were analyzed by one-way ANOVA with Tukey posttest using Graph Pad Prism Stat version 4.0 for Windows.

The Melanoma cell line B16F10 was exposed to different amounts of simvastatin, the results evidenced that at a concentration of 0.8 μM, 1.2 μM and 1.6 μM simvastatin had toxic effect. When incubated with concentration of 1.6 μM, the toxicity was present in the first 24 h of incubation, inducing a massive death (Figure 1). Cell cycle analyses (Figure 2) shows that 0.8 μM of simvastatin induces early cell cycle arrest in G0/G1, 24 hours, followed by increase of hypodiploid cells, a characteristic of apoptosis cell death.

To test the hypothesis that inhibiting endogenous cholesterol synthesis could limit melanoma growth in vivo, we administered daily oral doses of simvastatin on B16F10 melanoma bearing mice. Concentration of 5 mg/Kg/day of simvastatin were orally given to mice for 10 days, 10 days after the inoculation of the tumor cells.

The effects of simvastatin on tumor volume are demonstrated in Figure 3 and 4. The Figure 3 exemplifies the action over tumor size after high daily doses of simvastatin compared to control mouse with same tumoral implant. As can be see in Figure 4, the 10 days follow-up effects of simvastatin treatment significantly impair tumor growth (p < 0,05). The melanoma presented a 25% of final tumor sizing compared to the controls. The Figure 4B shows the Kaplan-Meyer survival curves of mice treated with simvastatin. There are no survival mice of the control group on day 24. Twenty-five percent of the simvastatin treated mice had a survival of 28 days.

Total tumoral regression was not developed in anyone of the melanoma bearing mice, and when the pharmacologic treatment was aborted the tumor re-started to grow (data not show).

The transaminases are typically intracellular enzymes. Increased plasma concentration of both leads to the predominant diagnosis of hepatocellular injury, which may be acute as in viral hepatitis, drug-induced or autoimmune, or chronic liver disease or ischemic, and others. The gamma-GT, gamma glutamyl transferase, is present in the liver (in hepatocytes and in biliary epithelium) and in other organs such as pancreas and kidneys. Quantification of Gamma-GT is useful in evaluation of acute and chronic liver diseases, with high enzyme activity in the tables of cholestasis intra or extra-hepatic. Despite being a very sensitive marker of hepatobiliary disease, is not specific, being increased in other diseases such as diabetes mellitus and renal failure. In addition, some drugs act as inducers, especially the barbiturates, the diphenyl-hydantoin and the tricyclic antidepressants, in addition to ethanol.

Our results showed at Table 1 had a large variation being not statistically significant for GOT or Gamma-GT. The ALT showed significance between the groups treated with simvastatin and control groups. These isolated findings did not represent liver injury when analyzed globally with the other transaminase, Gamma-GT and liver histopathology (data not show).

To estimate how toxic simvastatin could be, we evaluated the cellularity of bone marrow and spleen, remembering that these are hematological producing centers in rodents. The results summarized in Figure 5 and Table 2 show that simvastatin protect or keep the myeloid tissue in a normal account on melanoma bearing mice. Interestingly, the animals without tumor that received simvastatin had a decrease on the amount of cells into the analyzed tissues.

The evaluation of white blood cell count showed no significant differences in the amount of total leukocytes. The differential leukocytes count showed significant difference only for the eosinophils percentage in melanoma bearing mice treated with simvastatin. Studies such as Cormier [17] have previously shown that the increase in the number of eosinophils in solid tumors such as melanoma, is an important factor for the demonstration of persistent inflammation and on the initial tumor [17]. There are no difference in percentage of promyelocytic, plasma, total blasts, erythroblasts and megakaryocytes in all groups (data not shown).