Inhibition of metastatic potential of B16-F10 melanoma cell line in vivo and in vitro by biflorin

doi:10.1016/j.lfs.2013.05.018

Life Sciences

Volume 93, Issues 5–6, 14 August 2013, Pages 201-207

https://doi.org/10.1016/j.lfs.2013.05.018 Get rights and content

Abstract

Aim

The aim of this study was to determine the antimetastatic potential of biflorin using in vivo and in vitro approaches.

Main methods

Biflorin was isolated from Capraria biflora collected in Fortaleza, Ceará, Brazil. Adhesion, migration and invasion assays were performed to avail of the antimetastatic potential of this quinone. Experimental metastasis was performed to avail of the antimetastatic potential of bilflorin using in vivo assay.

Key findings

Treatment with biflorin (25 and 50 mg/kg/day) was shown to be effective in reducing B16-F10 melanoma metastasis in C57BL/6 mice. The administration of biflorin at 25 mg/kg/day intraperitoneally inhibited the formation of metastases by about 57% compared to untreated control animals. When the animals were treated with 50 mg/kg/day intraperitoneally, there was a 71% decrease in the number of lung metastases. Morphological assays showed the presence of hemosiderin and erythrocytes in the lung parenchyma, indicating the occurrence of hemorrhage, probably a side effect of biflorin. Biflorin at non-toxic concentrations (0.5, 1.0 and 1.5 g/mL) was tested directly on B16-F10 cells in vitro, and it inhibited cell adhesion to type I collagen and cell motility using the wound-healing assay.

Significance

These data suggest that biflorin has a promising antimetastatic potential, as shown by its anti-adhesion, anti-migration and anti-invasion properties against a metastatic melanoma cell line. However, further studies are essential to elucidate its mechanism of action.

Introduction

The spread of cancer cells from the primary tumor to distant locations is known as metastasis. The occurrence of metastasis is the major cause of mortality in cancer patients (Lee et al., 2003, Weiss, 1990), and the treatment of metastasis is still far from satisfactory (Han et al., 2009). Metastasis of cancer cells involves multiple processes including inhibition of cell-to-cell adhesion, enhancement of cell-to-extracellular matrix (ECM) adhesion, and invasion, which involves the degradation of the ECM (Lee et al., 2003, Lee et al., 2006, Cavallaro and Christofori, 2001). Tumor invasion of tissues by penetrating the basement membrane is also an important step, which involves the adhesion of tumor cells to ECM components followed by its degradation (Cavallaro and Christofori, 2001).

Natural products are a rich source of pharmacologically active compounds, in which plant materials hold an important position. One such plant Capraria biflora L., a perennial shrub of the family Schrophulariaceae, is used to treat various symptoms such as pain, fever, flu, vomiting, childbirth recovery, diarrhea, hemorrhoids, rheumatism, and swelling. The roots of this plant have antibacterial properties (Vasconcellos et al., 2007) and its aqueous extract has demonstrated both peripheral and central analgesic effects (Acosta et al., 2003).

Biflorin is a natural product that can be isolated from the roots of C. biflora L., a substance with an o-quinone structure. This quinone has antibiotic activity against Gram-positive bacteria, yeasts and fungi (Aquino et al., 2007). Moreover, biflorin has antifungal and antitumor effects, such as in the melanoma model (Vasconcellos et al., 2011).

Several studies have shown that some compounds isolated from plants can prevent tumor metastasis through inhibition of tumor adhesion and migration (Lee et al., 2006, Yang et al., 2007, Huey-Chun et al., 2003). Thus, the aim of this study was to determine the antimetastatic potential of biflorin using in vivo and in vitro approaches.

Section snippets

Plant material

C. biflora L. (Scrophulariaceae) was collected at a plantation located in Fortaleza, Ceará, Brazil in 2008 and identified by Dr. Edson Nunes. A voucher specimen (No. 30848) was deposited in the Herbarium Prisco Bezerra of the Biology Department of the Federal University of Ceará.

Isolation of biflorin

The isolation of biflorin was performed as described by Fonseca et al. (2003). Air-dried powdered roots (4.5 kg) was extracted with light petroleum (4 L) for 2 days and solvent was evaporated under reduce pressure to yield

Effect of biflorin on experimental lung metastasis and survival

Long-term treatment with biflorin with 25 or 50 mg/kg/day reduced by 57 and 71%, respectively, the number of lung metastases in comparison to control group (Fig. 1, Fig. 2). At these dose levels, there was no effect on body weight, nor were there any other clinical signs of toxicity (Table 1).

In addition, the life span of these animals was significantly increased by biflorin treatment (Fig. 3). The control animals survived for 22.3 ± 1.9 days after inoculation of mice with B16-F10 melanoma. Animals

Discussion

Initially, a previous study in our laboratory by Vasconcellos et al. (2011) demonstrated in vitro and in vivo anticancer activities of biflorin against a murine melanoma cell line. At 25 mg/kg/day i.p., this quinone improved the lifespan and also reduced tumor growth in animals bearing B16 melanoma tumors (Vasconcellos et al., 2011).

It is well known that B16-F10 melanoma cells are highly metastatic and form tumor nodules in the parenchyma of the lungs when administered through the tail vein (

Conclusion

We demonstrated that biflorin exhibits antimetastatic action in the mouse melanoma lung metastasis model. Furthermore, the antimetastatic effect is suggested due to the inhibition of the adhesion, migration and invasion of melanoma cells. These results indicate that biflorin is a promising candidate for an antimetastatic agent. However, more experiments are necessary to elucidate its mechanism of action.

Conflict of interest statement

The authors declare that there are no conflicts of interest.

Acknowledgments

We are grateful to the Brazilian agencies FINEP, CNPq, BNB/FUNDECI, PRONEX, and CAPES for fellowships and financial support. We thank Silvana França dos Santos for providing excellent technical assistance. Dr. A. Leyva helped with English editing of the manuscript.

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Inhibition of metastatic potential of B16-F10 melanoma cell line in vivo and in vitro by biflorin

Abstract

Aim

Main methods

Key findings

Significance

Introduction

Section snippets

Plant material

Isolation of biflorin

Effect of biflorin on experimental lung metastasis and survival

Discussion

Conclusion

Conflict of interest statement

Acknowledgments

Fitoterapia

Biochim Biophys Acta

Food Chem Toxicol

Food Chem Toxicol

Food Chem Toxicol

J Immunol Methods

Adv Cancer Res

J Ethnopharmacol

Preclinical and clinical studies of lapachol and beta-lapachone

Open Nat Prod J

Influence of biflorin on the labelling of red blood cells, plasma protein, cell protein, and lymphocytes with technetium-99 m: in vitro study

Braz J Pharmacogn