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Keywords:

  • actin filaments;
  • asteraceae;
  • cancer;
  • cell culture;
  • Spilanthes acmella

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements and funding
  7. References

Numerous natural products have pharmacological activity such that many biologically active compounds have led to the development of cancer chemotherapy drugs. Spilanthes acmella (Asteraceae) is widely cultivated in the State of Pará, Brazil, being employed in folk medicine for its anti-inflammatory, antimicrobial, antioxidant, analgesic, insecticide, and larvicidal properties. However, its cytotoxicity and influence on actin cytoskeleton organisation in tumour cell lines are practically nonexistent. We have verified the cytotoxicity of a hydroethanolic extract of the inflorescence of S. acmella, and examined its effects on the cytoskeleton of tumour cells. Decreasing concentrations of the extract (250, 500 and 1,000 µg/mL) were given to cultures of neoplastic cells (HEp-2). Cytotoxicity was assessed by the MTT test, and the influence on cytoskeleton organisation was examined by fluorescence microscopy. The IC50 of the hydroethanolic extract was 513 µg/mL, confirming the data obtained from the MTT assay that gave high cytotoxicity. The actin cytoskeleton arrangement of HEp2 cells at 500 and 1,000 µg/mL showed depolymerisation of the filaments, causing loss of morphology and consequently compromising cell adhesion.


Abbreviations
BSA

bovine serum albumin

DAPI

4′, 6-diamidino-2-phenylindole

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements and funding
  7. References

Folk knowledge and medicinal use of plants are transmitted across generations, especially in indigenous and traditional communities (Cotton, 1996). The World Health Organization reported that the majority of the populations in developed countries use traditional health practices, especially medicinal plants (WHO, 1999). Many medicinal plants have pharmacological activity and have had their active principles identified and characterised. Plants can synthesise an extensive number of secondary metabolites employed in other areas than pharmaceuticals, including biopesticides, flavourings, colourings, and food additives (Singh and Chaturvedi, 2012).

Spilanthes acmella is an herbaceous plant from the Asteraceae family (Compositae) that is widely cultivated in the State of Pará. It is popularly known as jambu, Brazil cress, jabuaçú, jaburama, buttercup, among other names (Coutinho et al., 2006). It is important in both regional cuisine and folk medicine. The Asteraceae family has about 13,000 species and 1,310 genera of flowering plants distributed throughout the world. It has a diverse morphology and is exceptionally rich in representative secondary metabolites (Heywood et al., 1977; Csupor-Löffle et al., 2009).

S. acmella is used in the treatment of severe toothache and stomatitis, but it also is effective as a larvicidal, antimicrobial, antioxidant, analgesic and insecticide. Its extract is also used as an active component in body and beauty-care products due to its fast-acting muscle relaxant property, which accelerates wrinkle repair (Perner and Rask-Madsen, 1999; Ramsewak et al., 1999; Pandey et al., 2007; Prachayasittikul et al., 2009). These activities are attributed to bioactive compounds, spilanthol and other N-isobutylamides, phenolic compounds, coumarins, and other triterpenoids (Pandey et al., 2007; Prachayasittikul et al., 2009). These compounds accumulate mainly in the inflorescence (Ratnasooriya et al., 2004; Mbeunkui et al., 2011).

Despite the wide range of biological activities and the significant cytotoxicity of similar extracts described for other genera of the Asteracea family (Réthy et al., 2007), the effect of S. acmella on tumour cell lines is little known. Cell culture testing can screen for both toxicity and basic as well as specific cell functions (Ekwall, 1983). The present study has explored the action of the hydroethanolic extract of S. acmella on HEp-2.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements and funding
  7. References

Materials

Dulbecco's minimal essential medium (DMEM) and supplements (penicillin, streptomycin, and fetal bovine serum) were purchased from Gibco Life Technologies (Gran Island, NY, USA). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide, phalloidin – TRITC, DAPI, and bovine serum albumin were purchased from Sigma Chemical Co. (St. Louis, MO, USA). The HEp-2 cell line, human laryngeal squamous cell carcinoma (ATCC CCL-23-USA) was obtained from the Cell Culture Section of Instituto Adolfo Lutz, São Paulo, SP, Brazil.

Cell culture

HEp-2 cells and L929 cells (mouse fibroblast subcutaneous connective tissue) were grown in DMEM containing penicillin and streptomycin (100 µg/mL) and 10% fetal bovine serum. The cells were maintained in humid air atmosphere containing 5% CO2 at 37 °C.

Sample collection and extract preparation

Inflorescences of S. acmella were collected from the garden of Escola São Francisco de Assis (ESFA) in Santa Teresa, in northwest Espírito Santo state. The species was identified by Ary Gomes da Silva and a voucher specimen was deposited in the herbarium of Mello Leitão Biology Museum (Voucher MBML: 22, 722). After collection, the material was dried in a forced-air oven at 40°C until constant weight. The material was milled and percolated with 96% ethanol. The drug was preswollen with a liquid extractor for 2 h and subsequently introduced into the percolator. Its extraction required a continuous flow of 1 mL/min until visible discolouration of the percolate occurred. The crude extract was concentrated on a rotary evaporator and dried in an oven at 66°C until total evaporation of the solvent and refrigerated until use. The residue obtained was analysed by thin layer chromatography (TLC) to prove the presence of spilanthol, the main metabolite associated with biological activity in this species. The procedure for spilanthol extraction and TLC analysis was done as described by Dias et al. (2012).

Treatment

Treatment was given 24 h after culture plating in 24-well plates TPP (TPP® Zellkultur-Plastik, Plastik für die Zellkultur, Switzerland) with sterile coverslips at 1 × 105 cells/well. The hydroethanolic extract of S. acmella diluted in DMEM supplemented with 5% FBS was added to the cells at varying concentrations (250, 500 and 1000 µg/mL), and the cultures incubated for 24 h. Four experiments were conducted in triplicate.

Viability

HEp-2 cells and L929 cells were treated in 24-well TPP® culture plates with hydroethanolic extract of S. acmella as above. After 24 and 48 h of incubation, 100 mL MTT solution (0.5 mg/mL) were added to each well. Absorbance was read on a plate-reader (ELISA Spectracount, Packard, USA) using a 570 nm filter. Absorbance values were converted into percentage of mitochondrial activity as compared to a control.

Actin cytoskeleton

For visualisation of the actin cytoskeleton filaments, cells grown in 24-well plates were permeabilised with 0.2% Triton X-100, PA 4% in PBS for 10 min, and incubated with TRITC-phalloidin in a 1% BSA solution for 1 h at room temperature before being treated with DAPI (300 nM). The samples were analysed with a Leica fluorescence microscope model DMLB coupled to a Leica MPS-30 photographic system.

Statistical analysis

The results were plotted in GraphPad Prism 4.01 using the means and the standard errors of means (RPM); two-way ANOVA was used for probability analysis.

Results and discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements and funding
  7. References

The S. acmella extract at 250 µg/mL had no significant effect on HEp-2 and L929 cells growth after 48 h (Figure 1), but 500 µg/mL significantly reduced cell number. At 1,000 µg/mL a highly significant reduction in cell number was recorded, indicating the dose-dependency of the response, and enabling the calculation of an IC50, which was 513 µg/mL after 24 h. The cytotoxicity of other species of the Asteraceae (Compositae) plant family has been measured by Mosaddegh et al. (2006) using the species Inula oculus Christi. Its highest activity on CACO2 cells gave an IC50 of 66 mg/mL, with other species giving IC50 values ranging from 30 to 860 µg/mL, demonstrating the significant cytotoxic activity of the S. acmella extract (Figure 1).

image

Figure 1. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay demonstrates the effect of extract of S. acmella on the growth of HEp-2 (A) and L929 (B) cells in vitro. The MTT assay was performed 24 and 48 h after treatment with decreasing concentrations (250–1,000 µg/mL). The number of cells is shown as the cell viability (%) compared to cells before treatment on the basis of OD570 value measured by the MTT assay. The results are mean values (%); bars, ±SD. *P < 0.05, **P < 0.01 and ***P < 0.001 versus nitrocellulose by two-away ANOVA (Bonferroni post-test).

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As antitumour compounds, lactones sesquiterpenes, and flavonoids have been isolated from Asteracea species. Réthy et al. (2007) assessed the antiproliferative activity of extracts of 25 species of the Asteraceae family in cell lines HeLa (cervix epithelial adenocarcinoma), A431 (skin epidermoid carcinoma), and MCF7 (breast epithelial adenocarcinoma), and found that 11 species have antiproliferative activity, four of which had not been previously investigated for the presence of active compounds with anticancer properties. This shows the importance of further studies of this family for cancer treatment.

Investigating the effects of the extract of S. acmella on actin filaments is of interest because they are considered crucial to the maintenance of cell structure and adhesion. The distribution and structure of actin filaments after treatment with the S. acmella extract at the above concentrations was assessed by fluorescence microscopy using Phalloidin-TRITC as a marker. A gradual reduction in the number of actin filaments was observed compared with controls, especially in cells incubated with 1,000 µg/mL of extract. In the control group, it was possible to verify the formation of stress fibers due to the polymerisation of the actin filaments, as well as more intense labeling in cell-to-cell interaction (Figure 2a), which was also seen at 250 µg/mL (Figure 2b). At 500 µg/mL, absence of stress fibers and a reduction in cell-to-cell interaction were seen (Figure 2c), which was aggravated at 1,000 µg (Figure 2d). The actin cytoskeleton filaments of L929 cells at 250 and 500 µg showed little change in the distribution of filaments, with small cell retraction (Figures 2f and 2g); but at 1,000 µg there was a restoration of the fibers of stress, with greater interaction between the cells (Figure 2h).

image

Figure 2. HEp-2 and L929 cells labeled with TRITC-Phalloidin, 48 h after treatment. HEp-2 cells incubated with TRITC-Phalloidin only (a). Cells incubated with 250 µg/mL extract (b). Cells incubated with 500 µg/mL extract (c). HEp-2 cells labeled with TRITC-Phalloidin. HEp-2 cells incubated with 1,000 µg/mL extract of S. acmella (d), the arrow indicates the reduction in cellular population and depolymerisation of actin filaments of the cytoskeleton. L929 cells incubated with TRITC-Phalloidin only (e). L929 cells incubated with 250 µg/mL extract (f). Cells incubated with 500 µg/mL extract (g). HEp-2 cells labeled with TRITC-Phalloidin. Cells incubated with 1,000 µg/mL extract (h), arrow indicates increased cell population, occurring maintenance of stress fibers of actin cytoskeleton.

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According to the literature, about 50% of drugs in clinical trial for anti-cancer activity are either from or correlated to natural sources (de Mesquita et al., 2009). Brazil has the largest biodiversity in the world, and plants have long been used to treat various diseases, including cancer (Brandão et al., 2008). Therefore, the existence of plants that produce antiproliferative substances capable of arresting the growth of malignant tumours is expected. According to de Mesquita et al. (2009), natural products can be advantageous to patients because they are generally less aggressive to the body compared to conventional therapies. Thus, novel extracts and their pharmacological application have been evaluated extensively and promising outcomes have been reached.

Another important aspect concerns the cell cytoskeleton, because it has an important role in cell support and structural modifications can alter adhesive interactions (Korb et al., 2004). This structural net is composed of actin filaments, microtubules, and intermediate filaments (Janmey, 1998). This complex of fibrillar elements has been recognised as an important factor in the mediation of adhesion-dependent and -independent signalling (Nojima et al., 1995; Cazaubon et al., 1997). Specifically, changes in the organisation of actin cytoskeleton result in alterations in the phosphorylation of tyrosine residues of numerous proteins located in the focal adhesion region (Thamilselvan and Basson, 2005).

Thus, based on the cytotoxic activity and changes found in the formation of actin fibers, the extract of S. acmella has proven its effectiveness in the cell adhesion process, as well as on tumour cell metabolism.

Acknowledgements and funding

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements and funding
  7. References

Financial support was received from the FAPESP (Support Research Foundation of São Paulo) Grant no. 2006/06736-5.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements and funding
  7. References
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