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

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.

Introduction

Kaurene diterpenes are considered to be important bioactive compounds,[12] with potential biological activity to be used in the development of anti-HIV-1, antispasmodic, anti-Alzheimer, and antioxidant drugs and have even been suggested in preventing osteoporosis.[34] A number of reports have shown that kaurene diterpenes may induce growth arrest in several tumor cell lines,[15-7] demonstrating that some kaurane compounds may contribute to the development of new effective chemotherapeutic agents.

In previous reports, we described kaurenic acid (KA) as a bioactive compound with anticonvulsant, sedative, antipyretic, and anti-inflammatory properties.[89] Moreover, natural diterpenoid KA have been evaluated for the development of new anticancer agents.[56] However, only a few studies have shown the in vivo anticancer activity of these compounds.

Continuing our studies to determine the biologic properties of KA, in this work we investigated the in vivo and in vitro antitumor activity of this molecule against murine melanoma, comparing it with the antineoplastic effect of taxol (Tx). Furthermore, we carried out a preliminary analysis to determine the possible influence of KA and Tx in apoptotic gene expression profile in melanoma cells.

Materials and Methods

Drugs and Reagents

KA, an ent-kaurene diterpene, was isolated from Espeletia semiglobulata as previously described[10] and provided by Dr. Alfredo Usubillaga (Research Institute, ULA, Mérida, Venezuela) as sodium kaurenate [C20H29Na02], which was dissolved in deionized distilled water before its use. Tx (Clitaxel®, Nolver, C.A., Venezuela), which was supplied by BADAN-Lara, was used for comparison in all analysis. The rest of the reagents used in antitumor assays were purchased from Sigma (St. Louis, USA).

Animals

C57BL/6 transgenic male mice, 7–8 week old were supplied by School of Veterinary Science (UCLA, Venezuela). Animals were kept under controlled conditions of temperature and light, from 6.00 am to 6.00 pm, with ad libitum access to food and water. Mice were weighted at least twice per week and its physical activity was monitored daily. Animal procedures were conducted according to the Guide for the Care and Use of Laboratory Animals, Canadian Council on Animal Care (1984), and the Guide to the Use of Laboratory Animal, National Institutes of Health, Bethesda, USA (Publication 86-23, 1986).

Tumor Cells

A transplantable tumor, B16F1 melanoma cell line, was originally provided by the Venezuelan Institute of Scientific Research (IVIC), Caracas-Venezuela. Cells were maintained by passages into the posterior left limb of C57BL/6 male mice according to modified standard inoculation.[11] Melanoma cells stained with trypan blue were counted under microscope by using a Neubauer-counting chamber. Suspensions of B16F1 tumor cells in phosphate buffered saline solution (pH 7.4) (70000 viable cells/100μL/mouse) were injected intramuscularly into the posterior left limb. The day of cell transplantation was designated as day 0.

Selection of Effective Antitumor Dose

Animals were randomly distributed into three groups; then, they were pretreated (i.p.) in different dose ranges (n = 10) starting four days before tumor engraft as follows: Experimental group, received KA in a dose range between 0.1 and 160 mg/kg/day; Standard treatment group, treated with Tx in a range from 7 to 58 mg/kg/week, each dose was fractioned in over a 3 hours lapse; Control group, received 0.9% sodium chloride (0.1 ml daily dose). The tumor inhibition rate in KA and Tx groups was calculated on the 21st post-inoculation day by comparison with values obtained from control group. Mean lethal and effective doses (ED50 - LD50) were estimated and their values were used to calculate its therapeutic index.

Survival Test

Mice bearing B16F1 tumor (n = 100) were treated daily up to day 40 following the selected schedule. The death of each animal was recorded starting from the first day of tumor engraft. The percentage of surviving mice was determined at the designated times. Median survival was calculated as control/drug ratio and analyzed by Kaplan-Meier survival curves.

Determination of Tumor Growth In Situ

The B16F1 tumor size was monitored daily after transplantation by measuring the linear diameter using callipers. Tumor growth in treated groups (n = 10) was estimated by comparison with the average growth in the control group.

Dissected Tumor Measurement

Treated and control animals (n = 10) were euthanatized by cervical dislocation in compliance with the National Institute of Health Guide for the Care and Use of Laboratory Animals and the 2007 Report of the American Veterinary Medical Association, Panel on Euthanasia. Dimensions of the dissected tumors were evaluated on experimental days 7th, 14th, and 21st by measuring the weight, volume, and liquid displacement. Tumor volume was calculated using the standard formula.[12] The weight of tumors (in g) was determined with an electronic scale (Ohaus Corp. NJ, USA). The liquid displacement (in ml) was measured to calculate the volume occupied by dissected tumor using a LE 7500 plethysmometer (LETICA, España).[13]

Histopathologic Changes

Tumors and iliac lymphatic nodes obtained from each group were fixed in formaldehyde solution (10%); then, paraffin-embedded sections were stained with H and E. Necrotic cells and hemorrhagic areas (≥0.5 mm) as well as metastatic nodes surface (≤0.5 mm throughout the area of lymphatic nodes) were visualized using an optic microscope (CH20 series, Olympus®, USA). Tissue area occupied by metastases and changes in tumor cells were quantified using an ocular micrometer (10 μm appreciation with X10). The average of the histopathology changes was compared between groups.

Cytotoxicity Assay

Two hundred thousand B16F1 melanoma cells obtained from each experiment were cultured in minimum essential medium (MEM) with 5% fetal bovine serum (Sigma®) and incubated for 24 h at 37°C in 5% CO2 humidified atmosphere. Cell suspensions were cultured without (negative control cultures) or with different concentrations of KA (0.1, 0.5, 1, and 2 μM) or Tx (1, 2, 10, 20, and 100 μM). The cytotoxic effect was considered as the relationship between dead/viable cells using the trypan blue dye exclusion method under inverted light microscopy (Nikon, Japan). The compound concentrations that produced 75% of cell death were considered as cytotoxic. The growth inhibitory effect was determined by EC50 (concentration that inhibits 50% of B16F1 melanoma cells growth). All experiments were done by triplicate (n = 6).

RT-PCR Analysis

Tumor melanoma fragments from mice treated as scheduled (n = 15) were obtained at 21st day and suspended on saline solution. Total ARN was isolated from 105 melanoma cells according Trizol method.[14] First-strand cDNA was synthesized at 37° C for 40 min in 20 μl reaction mixture containing: 1 mM dNTPs, 50 pmols of random primers, 10 mM dithiothreitol, 30 units of ribonuclease inhibitor (RNAsin®), 1x buffer, and 200 units of SuperScript® II reverse transcriptase (Invitrogen). The remaining enzyme was heat-inactivated at 65°C for 5 min. Gene expression on mRNA level was assessed using Multiplex PCR Kits for Mouse Apoptotic Genes® (Maxim Biotech Inc., USA) according to the instructions. Kits include primers for Bcl-2, Bax-alpha, Bcl-xL, c-Myc, P53, ICE, ICH1, CPP-32, ApaF1, TNF-a, Flice, MCH6, GAPDH, and 18S genes, the last two as internal control. PCR reaction was repeated twice for each sample. The PCR products were electrophoresed on agarose gel and visualized by ethidium bromide staining. Bands densities were quantified with Molecular Imaging Software, V 4.0.1 (Kodak).

Statistical Analysis

Data for early-stage studies were analyzed with GraphPad Prism Version 4.0, (GraphPad Software Inc, USA) using one-way and two-way ANOVA with repeated measurements followed by Bonferroni post-test to evaluate differences between values; Kaplan-Meyer was used for survival analysis; Log-Rank test to compare different Kaplan-Meyer curves; and Wilcoxon test for tumor growth pattern. Differences between groups were considered statistically significant at P < 0.05. Results were obtained from at least three independent experiments and expressed as mean ± SD.

Results

Antitumor Effective Response

The antitumor effective doses of KA and Tx are given in Table 1. KA (1–80 mg/kg) revealed in situ its capacity to dose-dependent decrease tumor development. Tumor inhibition rate for KA was 69.23 and 76.92% at 1 and 20 mg/kg, respectively, which showed a significant difference (P < 0.01) when compared with Tx at 14.5 mg/kg (53.84%). However, mortality increased in a dose dependent manner (20–160 mg/kg). Therefore, a dose of 1 mg/kg of KA during 25 days of treatment was selected for subsequent experiments. Tx maximum effect was observed at dose of 14.5 mg/kg administered once a week. The LD50/ED50 ratio was used to calculate the therapeutic index, which was 53.33 and 2.01 for KA and Tx, respectively.

Table 1

Lethal and effective response in B16F1 melanoma bearing C57BL/6 mice (n = 10). Therapeutic index value is expressed for each group.

In vivo Effects

The survival time of the tumor-bearing mice treated with KA was significantly prolonged compared with those shown by the Tx and control groups (P < 0.001), which evidence the beneficial effects of KA in the mouse melanoma model [Figure 1A]. In fact, two animals from KA group remained alive even at day 35; contrasting with Tx group who showed no survivor beyond day 31 (P < 0.001). The mean survival time was significantly increased (P < 0.001) for KA (28.30 ± 0.3189 days) and Tx (29.00 ± 0.43 days) groups compared with control group (23.23 ± 0.3074 days). The control/KA ratio at 50% survival was 0.82 (95% CI: 0.064–1.578), while for control/Tx was 0.79 (95% CI: 0.082–1.504). Compared with control, KA significantly reduced primary tumor growth rate similar to Tx (P < 0.001) [Figure 1B]. This effect was appreciated from day 14, being more noticeable on day 21.

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

Effects on Primary Tumors

The average weight of dissected tumors in control group gradually increased during the study to 87.5% [Figure 2A]. By contrast, the evaluation at day 21 showed that pretreatment with KA and Tx resulted in a decrease of the mean tumor weight by 49.51 and 54.16%, respectively. This finding is significant for both groups when compared with controls (P < 0.001). Figure 2B shows that KA and Tx administered to melanoma-bearing mice decreased liquid displacement of ex vivo tumor in 43.49 and 51.55%, respectively when compared to control group (P < 0.001). Figure 2C presents representative images of excised tumor treated with KA or Tx. Similarly, compared with control, the KA group showed a 35% decrease in tumor volume for the last day of the study (P < 0.01) [Figure 2D].

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.

Histological Findings of Tumor and Nodes

Primary tumors showed at day 21 extensive areas of necrosis and hemorrhage after administration of KA representing an increase of 43% with respect to Tx and control groups (P < 0.001) [Figure 3A]. Tumor histology at day 7 showed small foci of melanoma cells intermixed with connective and muscular tissues. Even at day 14, these changes were not significantly different for either group.

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

Iliac lymph node metastases were detected mainly in subcapsular and trabecular sinuses. At days 7 and 14, the lymph nodes of KA and Tx groups showed no significant changes in the metastatic area in comparison with control group. However, at day 21, metastasis cells in KA and Tx groups exhibited a lower tendency to invade compared with control group, with a 35.7% (P < 0.001) and 15.1% (P < 0.05) of reduction of the metastatic area, respectively [Figure 3B] (see supplemental material).

Cytotoxic Action in Cultured B16F1 Cells

As shown in Figure 4, the dose-response curve confirmed that KA inhibited significantly the growth of B16F1 melanoma cells in culture (P < 0.001 vs. Tx and control groups). Furthermore, compared with Tx, the inhibitory effect for KA was approximately 10-fold higher, while its concentration to achieve maximal response was lower (P < 0.001). Noninoculated control cultures were always viable (see supplemental material). The IC50 value cytotoxicity obtained for KA was 0.79 μM (95% CI: 0.6283–0.9441), while for Tx was 18.94 μM (95% CI: 6.271–23.94).

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

mRNA Expression of Genes Involved in Apoptosis in B16F1 Melanoma Cells

Results revealed that PCR fragment of 371 bp corresponding to Bcl-xL showed a decrease in intensity in samples from animals treated with KA and Tx compared with controls (P < 0.001) [Figure 5]. Moreover, Bcl-xL mRNA levels were significantly lower in samples from mice treated with KA than those receiving Tx (P < 0.001). No differences were observed in levels of mRNA expression of other analyzed genes between groups. Results were consistent in each of the replicas for each sample analyzed.

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

Discussion

The inhibition of cancer cell proliferation by ent-kaurenes, such as diterpene derivatives, has been validated in some animal models as well as on human cancer cell lines, suggesting great potential for possible treatment of cancer.[571516] In recent years, relatively few new chemotherapeutic agents have emerged with proved highly effective activity against melanoma cell lines. Some of these reports have shown cytotoxic activity of ent-kaurane diterpenoids in melanoma; however, its mechanism of action in this type of cancer has not been determined yet.[517] Furthermore, to our knowledge, this is the first study assessing the antimelanoma effect of KA in animal models.

Our results revealed that KA has inhibitory effect on B16F1 melanoma tumor in vitro and in vivo. Likewise, dissected tumor measurements were coincident with morphologic findings in this study. Moreover, KA resulted to be more active than Tx, an alternative drug used in treatment of malignant melanoma,[18] in vitro against cultured B16F1 cells, as well as in vivo against primary tumor and metastatic cells in iliac lymphatic nodes. These observations are consistent with previous reports that described the cytotoxicity of some kaurane diterpenes against solid tumors.[5715-17]

Previous studies have demonstrated the cytotoxic properties of kaurene diterpenoids, including necrosis and/or apoptosis promoting through different pathways on distinct tumor cell lines.[219-22] Moreover, some reports have shown induction of apoptosis by down-regulation of antiapoptotic Bcl-xL protein expression after exposing cancer cell lines to ent-kauranes.[2324]

Although the analysis of molecular mechanism of action of KA was not the main aim of this work, we carried out a preliminary screening of changes in mRNA expression level of genes involved in apoptotic pathways to establish possible differences with the antimelanoma effect displayed by Tx. Our results show a significant reduction of Bcl-xL mRNA levels in tumors treated with both analyzed drugs. Although no specific evaluations were performed to determine apoptosis, these results suggest that a proapoptotic mechanism could to be involved in KA-induced B16F1 melanoma cell death. Furthermore, according to our results, the molecular mechanism involved in the antimelanoma effects of KA and Tx in B16F1 cells share some aspects in common.

Several members of Bcl-2 family, including Bcl-xL protein, prevent cells from entering apoptosis.[25] Recently, it has been reported that some treatments can induce apoptosis in melanoma cells through changes in gene expression, which include down-regulation of Bcl-2 and Bcl-xL.[26] However, each tumor may behave in a particular way after antineoplastic treatments.[27] Similar to our work, a reduction of Bcl-xL expression has been shown in primary melanoma cells, but without effect on Bcl-2 expression, in UVB-induced apoptosis.[28] However, we cannot rule out post-translational changes in any protein involved in apoptosis pathways.

In summary, we have described the in vivo and in vitro antitumor activity of KA in B16F1 murine melanoma. Moreover, under assayed conditions, the cytotoxic potency of KA against melanoma cells was higher, while its IC50 was lower than that exhibited by Tx. The alteration in the expression of Bcl-xL gene could be involved in the antitumor mechanism of action of KA in B16F1 melanoma cells. Further studies are needed to elucidate and evaluate both its molecular mechanism of action and potential usefulness of KA as an agent for therapy of this cancer.