Antiproliferative Effects of Pterodon pubescens Extract and Isolated Diterpenes in HaCaT Cells

• Abstract
• Introduction
• Results and Discussion
• Materials and Methods
• Material
• Plant extraction
• Voucapan (VDFI mixture) isolation
• Voucapan compounds HPLC analysis
• Cell line
• Viability Assay
• Cell proliferation assay
• Wound-scratch assay (cell migration assay)
• Measurement of cytokine secretion
• Statistical analysis
• Contributorsʼ Statement
• References

Abstract

Pterodon pubescens fruits are popularly used because of their analgesic and anti-inflammatory actions, which are attributed to the isolated compounds with a vouacapan skeleton. This work aimed to evaluate the antiproliferative and anti-inflammatory effects of a P. pubescens fruit dichloromethane extract and the vouacapan diterpene furan isomerʼs mixture (1 : 1) (6α-hydroxy-7β-acetoxy-vouacapan-17β-oate methyl ester and 6α-acetoxy-7β-hydroxy-vouacapan-17β-oate methyl ester isomers) in HaCaT cells using the cell migration and the BrDU incorporation assay. Levels of IL-8 were measured by ELISA after TNF-α stimulation. HPLC/DAD analysis of the extract revealed the expressive presence of vouacapan diterpene furan isomerʼs mixture. P. pubescens extract (1.5625 – 25 µg/mL) and vouacapan diterpene furan isomerʼs mixture (3.125 – 50 µM) inhibited cell proliferation as indicated by a decreased BrdU-incorporation. For the evaluation of cell migration, time-lapse microscopy was used. P. pubescens presented inhibition on cell migration at all concentrations tested (3.125 – 12.5 µg/mL), whereas for the VDFI mixture, the inhibition was only observed at the highest concentrations (12.5 and 25 µM) tested. Furthermore P. pubescens extract and vouacapan diterpene furan isomerʼs mixture significantly decreased IL-8 levels. Our results showed antiproliferative and anti-inflammatory effects on HaCaT cells treated with the extract and the vouacapan isomerʼs mixture, without affecting cell viability. These activities could be attributed to the voucapan molecular structures. In conclusion, topical products developed of P. pubescens extract or the voucapan isomerʼs mixture should be further studied as a potential product for local treatment against hyperproliferative lesions as in psoriasis vulgaris, representing an alternative treatment approach.

Introduction

Pterodon pubescens Benth. fruits (Fabaceae), known in Brazil as sucupira, is used in folk medicine for their analgesic and anti-inflammatory properties as well as for the treatment of rheumatism [1]. The anti-inflammatory and anti-edematogenic pharmacological activities are reported [2], [3]. Furthermore, compounds isolated from Pterodon extract with a vouacapan moiety have been reported as a source with pharmacological activity against inflammatory and nociceptive processes and antiproliferative activities in tumor cells [4], [5]. Two vouacapan diterpenoids were isolated and identified as isomers of position 6α-hydroxy-7β-acetoxy-vouacapan-17β-oate methyl ester and 6α-acetoxy-7β-hydroxy-vouacapan-17β-oate methyl ester [6], with m/z 404 ([Fig. 1 a] and [b]). From hereafter, the vouacapan diterpene furan isomerʼs mixture (1 : 1) will be denominated VDFI mixture.

Given the extractʼs and the isolated vouacapan isomersʼ attested anti-inflammatory and antiproliferative activities on tumor cells, the antiproliferative effects of Pp and VDFI mixture on immortalized human nontumorigenic keratinocytes (HaCaT), as well as the anti-inflammatory activities on TNF-α-stimulated cells, were investigated.

Results and Discussion

The voucapan diterpenoids content in Pp extract was determined by HPLC/DAD chromatographic method. The content of compound VDFI mixture and another voucapan diterpenoid, 6α,7β-dihydroxyvouacapan-17β-oate methyl ester, with m/z 362 was expressed in mg/g of dry mass preparation ([Table 1]). The detection limit and quantification limit obtained for VDFI mixture were 2.24 µg/mL and 7.47 µg/mL, and for 6α,7β-dihydroxyvouacapan-17β-oate methyl were 0.42 µg/mL and 1.27 µg/mL, respectively. To verify cell viability, an ATP assay was chosen. The quantification of ATP levels is useful to predict cytotoxic effects on cells with different treatments [7]. The cells treated with Pp extract and VDFI mixture did not present alteration in cell viability in comparison to the untreated group ([Fig. 2 a] and [b]).

Thereafter, cell proliferation was evaluated applying the BrdU incorporation assay. This assay can determine the rate of cell proliferation activity and DNA synthesis [8]. Proliferative effects were analyzed in HaCaT cells at different concentrations. Pp extract was tested in concentrations of 1.5625, 3.125, 6.25, 12.5, and 25 µg/mL, and the VDFI mixture was tested at 3.125, 6.25, 12.5, 25, and, 50 µM, respectively. Proliferation in HaCaT cells decreased with all Pp extract concentrations tested, whereas for VDFI mixture only the highest concentrations (50 – 12.5 µM) showed that BrdU incorporation was inhibited by approximately 40%, whereas VDFI mixture in concentrations of 3.125 and 6.25 µM, inhibited BrdU incorporation by approximately 50% ([Fig. 3 a] and [b]).

Our results showed a significant antiproliferative effect in HaCaT cells treated with the extract and the isomers mixture, without affecting cell viability. A previous study reported the antiproliferative effects of P. pubescens crude ethanolic extract and an isolated vouacapan compound (vouacapan-6α, 7β, 14β, 19-tetraol) in SK MEL 37 (melanoma) cells [9]. Another study demonstrated an antiproliferative effect for 6α-acetoxy-7β-hydroxy-vouacapan, with a similar molecular structure as VDFI mixture, with high selectivity for PC-3 cell line (prostate cancer cell) [4]. Pereira et al. [10] suggested a mechanism of action in the apoptosis process by genetic regulation involving proteins of mitochondrial pathways in leukemia cells when treated with a terpene fraction from Pp extract with a furane diterpene as major component (methyl-6-acetoxy-7β-hydroxyvouacapan-17β-oate). This finding suggests that this structure is involved with the antiproliferative activity.

The effects of Pp extract and VDFI mixture on the migration of keratinocyte cells were further analyzed by a wound scratch assay. After treatment, cell migration was observed for 24 h using time-lapse microscopy ([Fig. 4]). This period was divided into 5 sections (0, 6, 12, 18, and 24 h) [11].

The untreated control group, with nonsupplemented medium (0.1% DMSO), presented the following gap closures in percentage: at t = 6 h, 8.5 ± 1.2%; at t = 12 h, 12.6 ± 6.3%; at t = 18 h, 28.4 ± 1.38; and for t = 24 h, 43.8 ± 3.5% ([Fig. 5]).

The positive control, medium with supplementation of 2% of FBS, presented the following gap closures in percentage: at t = 6 h, 28.9 ± 7.3%; at t = 12, h 55.9 ± 1.8%; at t = 18 h, 83.4 ± 4.0%; and for t = 24 h 98.7 ± 1.0% ([Fig. 5]), presenting significant difference to the untreated control (0.1% DMSO) at all times analyzed. This group showed an acceleration in gap closure as expected, since the addition of FBS in the culture medium provides lipids, growth factors, and hormones for cell maintenance and proliferation [12].

As a negative control, LY294002 (phosphatidylinositol-3-kinase inhibitor) was used. At t = 24 h, cells migrated 5.9%. In a previous study, LY294002 demonstrated an important inhibition of cell migration compared to the untreated group [13].

Pp extract presented inhibitory effects on keratinocyte migration. After 24 h of treatment, the following results for gap closure were found: for 3.125 µg/mL, 25.2%; for 6.25 µg/mL, 21.4%; and for 12.5 µg/mL, 41.3% compared to untreated control (0.1% DMSO) ([Fig. 5]).

VDFI mixture also presented inhibitory effects on cell migration, presenting the following gap closure after 24 h of treatment: for 12.5 µM, 11.6% and for 25 µM, 14.7% in comparison to untreated control (0.1% DMSO) ([Fig. 6]). For 6.25 µM concentration, however, HaCaT migration was statistically higher, with 71.8% of gap closure after 24 h of treatment ([Fig. 6]).

The effects of Pp extract on HaCaT migration were similar to the untreated control group (0.1% DMSO). As presented in [Figs. 4] and [5], the concentrations of the extract tested did not affect cell migration. For VDFI mixture, the cell migration was significantly higher after 12, 18, and 24 h of treatment ([Fig. 6]) only with the lowest concentration tested (6.25 µM).

In a recent study, the effects of fractions from Pterodon polygalaeflorus (a species belonging to the same genus of P. pubescens) on RAW 264.7 macrophage cell migration was described [14]. Both extract and fractions inhibited cell migration in the wound scratch assay. The fractions contained compounds with the vouacapan moiety (methyl 6α-hydroxy-7β-acetoxyvouacapan-17β-oate; methyl 6α,7β-dihydroxyvouacapan-17β-oate; methyl 6α-acetoxy-7β-hydroxyvouacapan-17β-oate), similar to compound VDFI mixture. These results are in accordance with our data. This chemical structure is suggested to have inhibitory effects on cell migration, in addition to antiproliferative properties.

To assess the anti-inflammatory potential of Pp extract and VDFI mixture, HaCaT cells were stimulated with TNF-α, and the pro-inflammatory cytokine IL-8 was analyzed by ELISA. Hence, keratinocytes were treated simultaneously with Pp extract (3.25 – 12.5 µg/mL) or VDFI mixture (6.25 – 25 µM) and TNF-α (20 ng/mL) for 24 h. TNF-α-stimulated group was used as a parameter group. Pp extract ([Fig. 7 a]) and VDFI mixture ([Fig. 7 b]) significantly reduced IL-8 expression in all tested concentrations. TNF-α stimulated group significantly increased IL-8 production (27 pg/mL) whereas control (0.1% DMSO) presented low levels of IL-8 (0.6 pg/mL). The positive control hydrocortisone (3.6 µg/mL corresponding to 10 µM) significantly reduced IL-8 levels (13 pg/mL) when compared to the TNF-α-stimulated group ([Fig. 7]).

IL-8 is an important chemoattractant for neutrophils, T lymphocytes, and basophils, being a key regulator in acute inflammation. This cytokine is also involved in several inflammatory and autoimmune disorders, such as psoriasis [15], [16]. Psoriasis is a chronic, autoimmune and inflammatory skin disease marked by increased keratinocyte hyperproliferation and chronic inflammation, angiogenesis, and inflammatory cell infiltration [17]. This disease can be classified according to the clinical manifestation as psoriasis vulgaris or plaque psoriasis, pustular psoriasis, among others [18]. Psoriasis vulgaris is the common form of manifestation corresponding to 90% of psoriasis cases [19].

In psoriasis vulgaris, activated keratinocytes can release several cytokines and chemokines that stimulate inflammatory cells to sustain the epidermis disorder. The network of cytokines in the epidermis stimulates the hyperproliferation of these abnormal keratinocytes, promoting the development and maturation of psoriatic lesion [20], [21].

One hallmark cytokine in the psoriatic lesion is IL-8 with local levels in skin lesions having direct relationship to disease severity [22]. IL-8 is linked to being involved in the uncontrolled growth of abnormal keratinocytes, neutrophils infiltrations, and angiogenesis and is hypothesized to mediate epidermal hyperproliferation [23], suggesting participation as a psoriasis-promoting factor [24].

In our results, Pp extract and VDFI mixture significantly decreased IL-8 levels in all tested concentrations ([Fig. 7]), suggesting an anti-inflammatory response.

Previous studies also reported the anti-inflammatory activity of P. pubescens. The ethanolic extract decreased IL-1β and TNF-α levels in experimental in vivo model [3]. More recently, a diterpenes fraction from P. polygalaeflorus was reported to inhibit the production of IL-10, IL-1β, NO, and TNF-α in macrophages [14].

Herbal products are increasingly being used in the treatment of psoriasis, aiming to specifically inhibit epidermal hyperproliferation and/or inflammation, and antiproliferative agents are valuable tools for new treatments for this disease [25].

In this context, our results presented interesting effects on reducing cell migration, the ability to prevent cell proliferation, and reducing the release of the proinflammatory cytokine IL-8 when tested in vitro with human keratinocytes, without presenting toxic effects on cell viability. These findings could be attributed to the voucapan molecular structures. Further studies are necessary to clarify the mechanism of action of antiproliferative effects in epidermal cells with supplementary evaluation of apoptotic effects.

Therefore, topical products developed with Pp extract or the VDFI mixture should be further studied as a potential product for local treatment against hyperproliferative lesions as in psoriasis vulgaris, representing an alternative treatment approach.

Materials and Methods

Material

Dichloromethane, ethyl acetate, and hexane were purchased from Merck, Brazil. Human HaCaT keratinocytes were purchased from Cell Line Services. DMSO (purity > 99.5%) and hydrocortisone (purity ≥ 90%) were obtained from Sigma-Aldrich. Ibidi provided the culture inserts for cell migration assays. DMEM with high glucose, L-glutamine and without sodium pyruvate; 0.5% trypsin/4.8 mM EDTA; and FBS were provided by Gibco. 5-Bromo-2′-deoxyuridine (BrdU) and LY294002 (purity > 99%) were obtained by LubioScience. TNF-α (purity > 97%) was obtained from R&D Systems. CellTiter-Glo Luminescent Cell Viability Assay was provided by Promega. IL-8 human ELISA kit was purchased from PeproTech.

Plant extraction

P. pubescens fruits were collected in Minas Gerais State, Brazil (Ponto Chique: 16°38′ 23.3″S, 45°03′ 54.0″W). The voucher (number 179739) was deposited in the Herbarium of Unicamp, and the identification was made by Prof. Dr. Jorge Yoshio Tamashiro (IB-Unicamp). This study is registered at SisGen-Brazil (A960ACE). The extraction process was carried out using dichloromethane as previously described by authors [26]. Ground fruits in a ratio of 1 : 3 of organic solvent were processed in 3 extractions (1.5 h each) at room temperature. The extracts were filtered, and the solvent was eliminated by rotary evaporation system under vacuum.

Voucapan (VDFI mixture) isolation

The isomers voucapan 6α-hydroxy-7β-acetoxy-vouacapan-17β-oate methyl ester and 6α-acetoxy-7β-hydroxyvouacapan-17β-oate methyl ester (VDFI mixture) from crude dichloromethane extract from P. pubescens fruit dichloromethane extract were isolated in a chromatography column (25 × 500 mm), with ethyl acetate and hexane gradients as previously published [4], [26]. The spectral data (FTIR, ¹H, and ¹³C NMR data) of the VDFI mixture were in accordance with that previously reported [4], [5].

Voucapan compounds HPLC analysis

Precisely weighted samples (Pp extract) 25 mg were solubilized with 10 mL acetonitrile and filtered in a 0.45 mm filter. Samples (10 µL) were injected into the HPLC column (Luna CN Phenomenex column, 4.6 × 250 mm, 5 mm particle size) and elution with acetonitrile and water (70 : 30) (with 1% acetic acid v : v) at a flow rate of 1 mL/min, monitored at 220 nm with column temperature kept at 35 °C. The procedure was done in triplicate. The peak area calculated from HPLC chromatograms was correlated linearly with VDFI mixture concentrations in the range of 17 – 2180 µg/mL (R² = 0.999) and compound m/z 362 range of 8.7 – 1117 µg/mL (R² = 0.999). The calculated concentrations of voucapan (in extract) were expressed in terms of mean ± SD (mg/g) [26].

Cell line

Human HaCaT keratinocytes were conditioned in DMEM with supplementation of 10% (v/v) of heat-inactivated FBS, 1% of (v/v) penicillin 10.000 U/mL, and streptomycin 10.000 µg/mL in a humidified atmosphere at 37 °C with 5% CO₂. For the accomplishment of the experiments, the cellular confluence was 80 – 90%, and the passages between 43 and 52; 2.5 × 10⁵ cells/mL was used in all experiments. All samples were homogenized in DMEM medium with 0.1% DMSO.

Viability Assay

The CellTiter-Glo Luminescent Cell Viability was performed with some modifications, as previously described [13], [27]. Cells were seeded, and after 24 h of incubation in 37 °C, 5% CO₂ in a humidified atmosphere, the medium supplemented (10% FBS) was removed, and wells were washed with PBS. The concentrations of Pp extract (50, 25, 15, 10, 5, 1, and 0.5 µg/mL) and VDFI mixture (50, 25, 12.5, 6.25, 3.125, 1.5625, and 0.78125 µM) were tested. DMEM without supplementation and with 0.1% of DMSO was used as a control. After the experimental protocol, 100 µL samples were transferred to an opaque sterile and white 96-well plate, promptly measuring luminescence (SpectraMAX L, Molecular Devices).

Cell proliferation assay

The assay was performed following the producerʼs protocol. Cells were seeded, and after 24 h of incubation in 37 °C, 5% CO₂ in a humidified atmosphere, the medium supplemented (10% FBS) was removed, and the wells were washed with PBS. Pp extract was tested in 25, 12.5, 6.25, 3.125, and 1.5625 µg/mL concentrations, and VDFI mixture was tested in 50, 25, 12.5, 6.25, and 3.125 µM concentrations, respectively. DMEM without supplementation and with 0.1% of DMSO was used as a control group. After the experimental protocol, the plate was read at 450 nm (SpectraMAX L, Molecular Devices).

Wound-scratch assay (cell migration assay)

The protocol was similar to that described previously with some modifications [11], [13], [27]. The inserts for cell culture were positioned in 12-well plates. HaCaT cells were seeded, 15 µl in each side of the insert. After 24 h of incubation in 37 °C, 5% CO₂ in a humidified atmosphere, the inserts were withdrawn, generating a 450 µm gap. Wells were washed with PBS and charged with test samples. Pp extract was tested in 12.5, 6.25, and 3.125 µg/mL concentrations, and VDFI mixture was tested in 25, 12.5, and 6.25 µM concentrations, respectively. DMEM with 2% FBS as supplementation was used as a positive control, and DMEM without supplementation and 0.1% of DMSO was used as a control group. As negative control, LY294002 (10 µM) was used. For live-cell migration, an automated inverted microscope, with an image adjusted to 10 × was used (Olympus IX83). Pictures were taken at 5 different sections: 0, 6, 12, 18, and 24 h after the addition of the extract and VDFI, with Olympus software package cellSens Dimension 1.81. For gap calculation, pictures were interpreted in Image J software (1.53e version) with wound healing tool macro, and the cell-free gap area was analyzed based on pixelʼs number. Finally, the recognized spaces were transformed into percentages: 0% represents no migration of cells and 100% represents a full gap closure. The figures represent images from only a single experiment. The graphics represent mean ± SD of triplicates.

Measurement of cytokine secretion

The protocol was according to Park et al. [28], with some alterations as previously described [18], [27]. Cells were seeded, and after 24 h of incubation in 37 °C, 5% CO₂ in humidified atmosphere, the medium supplemented (10% FBS) was removed, and wells were washed with PBS and charged with test samples. Pp extract was tested in 12.5, 6.25, and 3.125 µg/mL concentrations, and VDFI mixture were tested in 25, 12.5, and 6.25 µM concentrations. As control, hydrocortisone (10 µM) was used. For cell stimulation, wells received 20 ng/mL of TNF-α and samples tested simultaneously and incubated for 24 h. DMEM with 0% FBS group was used to verify basal levels of IL-8 in cells. After the experimental time, supernatants were stored at − 20 °C until analyses. Absorbance was quantified at 450 nm (SpectraMAX L, Molecular Devices).

Statistical analysis

Data were expressed as the mean ± SD (standard deviation). Data analysis was performed by 1-way analysis of variance (ANOVA) followed by Dunnettʼs multiple comparison test (GraphPad Prism version 6.01). All experiments were performed in triplicate with significant difference considered if p < 0.05.

Contributorsʼ Statement

Data Collection: R. T. Basting, I. M. O. Sousa; design of the study: R. T. Basting, I. M. O. Sousa, V. Butterweck, M. A. Foglio; statistical analysis: R. T. Basting, I. M. O. Sousa, V. Butterweck, M. A. Foglio; analysis and interpretation of the data: R. T. Basting, I. M. O. Sousa, V. Butterweck, M. A. Foglio; drafting the manuscript: R. T. Basting; critical revision of the manuscript: V. Butterweck, M. A. Foglio.

Conflict of Interest

The authors declare that they have no conflict of interest.

Acknowledgements

This research was funded by Fundação de Amparo à Pesquisa do Estado de São Paulo [FAPESP] grant number [2015/08600-2 and 2016/23816-4].

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Correspondence

Publication History

Received: 01 August 2020

Accepted after revision: 28 September 2020

Article published online:
03 November 2020

© 2020. Thieme. All rights reserved.

Georg Thieme Verlag KG
Rüdigerstraße 14, 70469 Stuttgart, Germany

• References

• 1 Dutra RC, Campos MM, Santos ARS, Calixto JB. Medicinal plants in Brazil: pharmacological studies, drug discovery, challenges and perspectives. Pharmacol Res 2016; 112: 4-29 doi:10.1016/j.phrs.2016.01.021
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