Simvastatin impairs murine melanoma growth

Abstract

Background: Statins induces cell cycle arrest, apoptosis, reduction of angiogenic factors, inhibition of the endothelial growth factor, impairing tissue adhesion and attenuation of the resistance mechanisms. The aim of this study was evaluate the anti-tumoral activity of simvastatin in a B16F10 melanoma-mouse model.

Methods: Melanoma cells were treated with different concentrations of simvastatin and assessed by viability methods. Melanoma cells (5 × 10(4)) were implanted in two month old C57Bl6/J mice. Around 7 days after cells injection, the oral treatments were started with simvastatin (5 mg/kg/day, p.o.). Tumor size, hematological and biochemical analyses were evaluated.

Results: Simvastatin at a concentration of 0.8 μM, 1.2 μM and 1.6 μM had toxic effect. Concentration of 1.6 μM induced a massive death in the first 24 h of incubation. Simvastatin at 0.8 μM induces early cell cycle arrest in G0/G1, followed by increase of hypodiploidy. Tumor size were evaluated and the difference of treated group and control, after ten days, demonstrates that simvastatin inhibited the tumor expansion in 68%.

Conclusion: Simvastatin at 1.6 μM, presented cytototoxicity after 72 h of treatment, with an intense death. In vivo, simvastatin being potentially useful as an antiproliferative drug, with an impairment of growth after ten days.

Figure 1

Sinvastatin reduces the number of viable cells in cultured melanoma after 48 hours. Cells were cultured in the presence of the statin in lactone form, or with no treatment for 4 days. Three wells of each condition were subjected to the trypan blue assay. The mean values were calculated and plotted as the percentage of the vehicle-treated. The experiment was repeated three times. Sinvastatin values are indicated by p when significantly different from controls (P < 0.05). Data are expressed as mean and standard error of the mean. Statistical analysis was performed using ANOVA followed by Tukey post hoc t test. * p < 0.05 compared with the control group.

Figure 2

Flow cytometry Cell cycle analysis, using propidium iodide as a DNA intercalating agent. A) Histogram representative of Control group after 24 h of seed; B) G0/G1 cell cycle arrest induced by 0.8 μM of simvastatin. C) Percentage of G0/G1 cells in witch treatment. D) Increased cell death due to simvastatin effects evaluated by hypodiploid cells amount. Each bar represents the mean, and vertical lines are the standard error of the mean of a representative assay performed in triplicates.

Figure 3

Example of twenty days of mice bearing melanoma; A) Control group and B) Treated with high daily doses of simvastatin with notable reduction in tumor size.

Figure 4

Effect of high daily doses of simvastatin on tumor growth and survival rate of B16F10 tumor bearing mice. C57Bl6J mice were implanted with 5·104 B16 melanoma cells subcutaneously injected in dorsal region of B57CL/6 mice (8 mice per group). After the tumors had reached approximately 4 mm in diameter (10 days) the daily treatment were started for 10 days. A) Perpendicular tumor diameters were measured daily to estimate tumor volume. Controls were saline solution in the same schedule. P values were calculated by using the Student's t test and are indicated by p when significantly different from controls (P < 0.05). B) Percent B16 melanoma bearing mice survival in response to the treatment as a function of time. The survival rates were calculated daily and the experiment was terminated when all the mice of control group died (at day 28). Survival rate data were analyzed by Kaplan-Meyer curves.

Figure 5

Femoral Bone Marrow (A) and Spleen cellulartiy (B) cellularity are decreased in mice treated with simvastatin (Control-Simvastatin) but it is equal when mice bearing melanoma treated with same doses of simvastatin. Each bar represents the mean of the data from eighth animals and the vertical lines are the standard error of the mean. Statistical analysis was performed using ANOVA followed by Tukey post hoc t test. * p < 0.05 compared with the control group.