Kaurenic acid: Evaluation of the in vivo and in vitro antitumor activity on murine melanoma

Abstract

Objective: The in vivo and in vitro antitumor activity of kaurenic acid [kaur 16-en-19 oic acid] (KA) in melanoma was evaluated in a murine model in comparison with taxol (Tx).

Materials and methods: B16F1 melanoma was developed in C57BL/6 mice and cell cultures. Survival test, tumor growth, dissected-tumor measurements, histology, cytoxicity assay on cultured cells, and changes of apoptotic gene expression at mRNA level were analyzed.

Results: KA showed antitumor effect in vivo and in vitro and compared with Tx, its antimelanoma activity was greater (P < 0.001). These results were confirmed by morphological analysis (P < 0.001). In melanoma cell cultures, KA IC(50) was 0.79 μM vs. 18.94 μM for Tx (P < 0.001). RT-PCR analysis demonstrated that Bcl-xL mRNA expression was altered in B16F1 mouse melanoma cells obtained from mice treated with either KA or Tx.

Conclusion: The data suggest that KA is active in animal melanoma models, both in vitro and in vivo, being its cytotoxic effects stronger than those exhibited by Tx. Further trials should be conducted to elucidate its mechanism of action in melanoma with respect to necrosis or apoptotic processes. Our results support other evidences indicating that KA is a potential chemotherapeutic agent against cancer that has to be widely explored.

Keywords: Antitumor activity; B16F1 melanoma; C57BL/6 mice; kaurenic acid.

Figure 1

In vivo effects of KA. (A) Survival time (n = 100), each point represents the mean of survival mice (%) up to day 40 after tumor grafting. Differences between groups became significant on day 25 **P< 0.01, reaching to ***P< 0.001 at 35 day. (B) Tumor growth (n = 10) ***P< 0.001 vs control. Values were obtained from three separated experiments performed in triplicate

Figure 2

Effect of KA on dissected tumor growth. (A) Weight (g). (B) Liquid displacement (ml). (C) At day 21, representative tumors of (a) control, (b) KA, and (c) Tx groups were removed and photographed. (D) Tumoral volume (mm³). Values are mean ± SD (n = 10) of triplicate determinations from three different experiments. *P< 0.05, **P< 0.01, and ***P< 0.001 vs. control.

Figure 3

Histology of primary and metastatic tumor in control, KA, and Tx groups (n = 10). (A) Necrotic and hemorrhagic areas in tumors after treatment. (B) Metastasis in Iliac lymph nodes. Each bar represents mean ± SD. ***P < 0.001 vs. control

Figure 4

Cytotoxicity by trypan blue dye exclusion. Drug [μM] vs. cytotoxic response (%) curve; each point represents the mean ± SD from three separated experiments. ***P < 0.001 vs. Tx

Figure 5

Bcl-xL mRNA gene expression in B16F1 cells after treatment with KA and Tx. (A) Bcl-xLmRNA levels. (B) Percentage of variation of Bcl-xL mRNA expression. Results were normalized and plotted with respect to GAPDH expression, considering the density of bands of control group as 100 % of expression. ***P < 0.001 vs. control