Cytotoxicity of citral against melanoma cells: The involvement of oxidative stress generation and cell growth protein reduction

Abstract

Citral is a natural compound that has shown cytotoxic and antiproliferative effects on breast and hematopoietic cancer cells; however, there are few studies on melanoma cells. Oxidative stress is known to be involved in all stages of melanoma development and is able to modulate intracellular pathways related to cellular proliferation and death. In this study, we hypothesize that citral exerts its cytotoxic effect on melanoma cells by the modulation of cellular oxidative status and/or intracellular signaling. To test this hypothesis, we investigated the antiproliferative and cytotoxic effects of citral on B16F10 murine melanoma cells evaluating its effects on cellular oxidative stress, DNA damage, cell death, and important signaling pathways, as these pathways, namely, extracellular signal-regulated kinases 1/2 (ERK1/2), AKT, and phosphatidylinositol-3 kinase, are involved in cell proliferation and differentiation. The p53 and nuclear factor kappa B were also investigated due to their ability to respond to intracellular stress. We observed that citral exerted antiproliferative and cytotoxic effects in B16F10; induced oxidative stress, DNA lesions, and p53 nuclear translocation; and reduced nitric oxide levels and nuclear factor kappa B, ERK1/2, and AKT. To investigate citral specificity, we used non-neoplastic human and murine cells, HaCaT (human skin keratinocytes) and NIH-3T3 cells (murine fibroblasts), and observed that although citral effects were not specific for cancer cells, non-neoplastic cells were more resistant to citral than B16F10. These findings highlight the potential clinical utility of citral in melanoma, with a mechanism of action involving the oxidative stress generation, nitric oxide depletion, and interference in signaling pathways related to cell proliferation.

Keywords

Melanoma B16F10 citral apoptosis autophagy oxidative stress

Introduction

Melanoma is a malignant tumor that arises from melanocytes, dendritic cells that produce melanin, a pigment that protects the body from damaging ultraviolet (UV) radiation. A cluster of melanocytes form nevi (pigmented lesions or moles) and melanoma occurs when these melanocytes undergo a malignant transformation.^1,2 Although melanoma represents the least common form of skin cancer (accounting for only about 5% of all skin cancer cases), it is the deadliest form of skin cancer claiming about 75% of related deaths, with increasing incidence worldwide. According to an estimative from the American Cancer Society, one person dies every hour from melanoma.³

Standard treatment for patients with primary melanoma with thickness higher or equal to 2.0 mm with or without regional metastases to lymph nodes is surgery followed by adjuvant therapy; however, the surgical excision can be curative only for patients who have localized disease.⁴ In patients with disseminated disease, post-resection treatment aims prolonging survival and improving quality of life.⁵ Patients who progress to stage IV metastatic melanoma have a median survival of less than 1 year. Among available treatments, chemotherapy yields low response rates, with high rates of recurrence, and interleukin-2 (IL-2) therapy achieves durable benefits in a greater fraction, but it is accompanied by severe toxicity that requires hospitalization for support during treatment.⁴

In this context, the use of natural products has shown promising potential as a new therapeutic strategy in many diseases. Since 1961, several anticancer drugs derived from plants have been available, such as Taxol, oncovin, navelbine, and vumon.^6,7 In cancer therapy, the focus is on strategies that suppress tumor growth through cell cycle disruption and activation of apoptotic programs in cells.^8,9

Citral (3,7-dimethyl-2,6-octadienal) is a naturally occurring aliphatic aldehyde of the terpene series and is an isomeric moisture of geranial and neral; it is a key component of essential oils extracted from several herbal plants such as lemongrass (Cymbopogon citratus), Melissa (Melissa officinalis), and verbena (Verbena officinalis).¹⁰ It is used as food additive and as fragrance in the cosmetic industry.^10,11 Studies indicate that citral is devoid of major toxicity and carcinogenic potential in mice and rats.^12,13 It was also reported that citral is devoid of mutagenic effect in in vitro models.¹⁴ Moreover, substantial antibacterial, antifungal, antiparasitic, and insecticidal effects of citral on different organisms have been described.^15,16 With regard to effects of citral on cancer, studies demonstrated that this compound is cytotoxic to breast cancer and hematopoietic cancer cell lines;^11,17 however, its effects on melanoma are poorly understood.

Because oxidative stress is known to be important in all stages of melanoma development and to be able to modulate intracellular pathways related to cellular proliferation and death, we hypothesize that citral could be cytotoxic to melanoma cells by the modulation of cellular oxidative status. Furthermore, it is known that patients with the disease present systemic and tumor redox deregulation.¹⁸ With this in mind, we investigated the antiproliferative and cytotoxic effects of citral on B16F10 murine melanoma cells evaluating the participation of the compound in cellular oxidative stress, DNA damage, cell death, and several signaling pathways involved in melanoma. The extracellular signal-regulated kinases 1 and 2 (ERK1/2), AKT, and phosphatidylinositol-3 kinase (PI3K) were investigated due to its ability to interfere in cell proliferation, survival, and differentiation. The p53 and nuclear factor kappa B (NF-KB) were also investigated due to their ability to respond to intracellular stress.

Materials and methods

Cell culture and treatment

The murine melanoma cell line (B16F10) was seeded in tissue culture flasks and grown in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Thermo Fisher, USA), supplemented with 5% fetal bovine serum (FBS; Gibco, Thermo Fisher) and 1% penicillin/streptomycin mixture (Santa Cruz Biotechnology, USA). The culture was maintained in a humidified atmosphere with 5% CO₂ at 37°C (Sanyo CO₂ Incubator; Sanyo, Japan). To choose the concentration of citral, a dose–response curve with different concentrations (0.01, 0.05, 0.1, 0.5, 1.0, and 2.5 µM) was plotted using proliferation and MTT (2-(3,5-diphenyltetrazol-2-ium-2-yl)-4,5-dimethyl1-1,3-thiazole bromide) assay, after 24 and 48 h of exposure. For this, 10⁵ cells were seeded in 24-well plates and cultured with fresh culture medium for 24 h prior to citral treatment (Citral C.A.S. 5392-40-5, mixture of cis and trans, >96%; Sigma-Aldrich, USA,). Citral was diluted in dimethyl sulfoxide (DMSO; maximum 1% final concentration) and, for this reason, 1% of DMSO was added in the culture medium of the control group.

The concentrations of citral used in the other experiments were 0.1, 0.5, 1.0, and 2.5 µM and for a duration of 24 h. For analysis of genotoxicity, cell death patterns, and immunocytochemistry, 10⁵ cells were cultured on circular glass coverslips distributed in 24-well plates. To analyze oxidative stress parameters and nitric oxide (NO) levels, 10⁶ cells were seeded in 25-cm² cell culture flasks for 24 h and then exposed to citral, as described above. All the experiments were performed in triplicate and three independent repetitions.

To also verify citral cytotoxicity against human melanoma and normal cell lines, SK-MEL-147 (Homo sapiens skin melanoma, NRas mutant), UACC-257 (Homo sapiens skin melanoma, BRaf mutant), HaCaT (Homo sapiens skin keratinocyte, p53 mutant), and NIH-3T3 (murine fibroblast cells) were used. Cells were cultured and treated under the same conditions as the B16F10 cells. The cells used in this study were kindly supplied by Professors Marco Andrey Cipriani Frade (HaCaT), Glaucia Regina Martinez (B16F10), João Ernesto de Carvalho (UACC-257), Eliana Saul Furquim Werneck Abdelhay (NIH-3T3), and Silvya Stuchi Maria Engler (SK-MEL-147).

Determination of citral inhibitory concentration 50 and the MTT assay

The determination of the inhibitory concentration 50 (IC₅₀) was performed using the MTT assay, according to Mosmann.¹⁹ A regression analysis was performed on MTT assay proliferation data and the resultant equation was used to calculate the concentration required to produce 50% reduction in cell proliferation. The determination of citral IC₅₀ was done in B16F10, SK-MEL-147, UACC-257, HaCaT, and NIH-3T3 cells.

Cell proliferation assay

After 24 and 48 h of treatment, B16F10 cells were washed with phosphate-buffered saline (PBS) and trypsinized. Cells were then suspended in Trypan Blue (0.05%) and counted using a Neubauer chamber. To identify the cytostatic or cytotoxic effect of citral and determine the percentage of viable cells, the cells were classified as viable (no staining) and unviable (blue staining), according to the procedure described by Queiroz et al.²⁰

Cell death patterns

Annexin V-FITC/propidium iodide staining

The cells were washed and trypsinized after treatment. Cellular suspension was centrifuged at 600g (4°C, 3 min) and cells were stained using the Annexin V-FITC/propidium iodide (PI) kit (BD, USA), as described previously²¹ by flow cytometry. Double-negative cells were considered intact, annexin⁺/PI⁻ cells are presumably in early apoptosis, and the annexin⁻/PI⁺ were considered necrotic. Results were expressed in percentage of apoptotic and necrotic cells.

TUNEL assay

After treatment, B16F10 cells were washed and fixed with formaldehyde 10%. The apoptosis were evaluated using the DeadEnd Colorimetric Terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling (TUNEL) System (Promega, USA). The experimental procedures were carried out according to the manufacturer’s protocol and the coverslips were photographed utilizing the Olympus Fluorescence System Microscope BX3-URA (Olympus Corporation, Japan), and 20 photos were taken randomly to ensure that the obtained data were representative.

Lactate dehydrogenase release assay

The in vitro assay kit for lactic dehydrogenase quantification (Doles, BR) was used for the measurement of lactate dehydrogenase (LDH) released from the cytosol of damaged B16F10 cells into the supernatant, to estimate cellular membrane damage associated with necrosis. The assay was based on the reduction of nicotinamide adenine dinucleotide (NAD) by LDH. Absorbance was read at 490 nm using a microplate spectrophotometer (Multiscan Go; Thermo Scientific, USA).

Autophagy assay: monodansylcadaverine

After treatment, cells were washed with PBS and incubated with 0.05 mM monodansylcadaverine (MDC) in fresh culture medium at 37°C for 10 min. Cells were immediately analyzed using a fluorescence microscope (Olympus Fluorescence System Microscope BX3-URA) with 380 and 525 nm wavelength of excitation and emission filters, respectively. Autophagy was determined in B16F10, SK-MEL-147, UACC-257, HaCaT, and NIH-3T3 cells.

Comet assay

Single-cell gel electrophoresis (SCGE; Comet assay) was performed according to the procedure described by Tice et al.²² After treatment, B16F10 cells were harvested and suspended in low-melting-point agarose (0.5%) and deposited on pregelatinized slides (agarose: 1.5%). Subsequently, the slides were placed in an electrophoresis buffer (pH: 13) for 20 min for DNA denaturation. Then, the slides were subjected to alkaline electrophoresis (pH: 13.0; 25 V; 300 mA; 4°C). All steps were performed under undirected light. Finally, the slides were treated with pH-neutralizing buffer, fixed with ethanol, and stained with Gel Red (33%). A total of 100 random nucleoids from each slide were blindly examined at 400× magnification using a fluorescence microscope (excitation filter was set at 420–490 nm, while emission filter was set at 520 nm) connected to an image capture system. For the determination of tail moment of each nucleoid, Comet ScoreTM Freeware (version: 1.5; USA) was used. Apoptotic/necrotic nucleoids with extensive DNA damage were not included in the analysis. The mean value of the tail moment was considered as an index of DNA damage.²³

Determination of NO levels

The determination of NO levels was performed as previously described by Panis et al.²⁴ Aliquots cells plus supernatants (60 µL) were deproteinized by adding 50 µL of ZnSO₄ 75 mM solution (Merck, Germany), agitated and centrifuged at 10,000 r/min, 2 min, 25°C, and then 70 µL of NaOH 55 mM (Merck, Germany) was added, and agitated and centrifuged at 10,000 r/min, 5 min, 25°C. The limpid supernatant was recovered and diluted in glycine buffer solution (45 g/L pH 9.7, Merck, Germany) in a proportion of 5:1. Cadmium granules (Fluka; Sigma-Aldrich, USA) stored in H₂SO₄ 100 mM solution (Merck, Germany) were rinsed three times in distilled sterile water and added to a CuSO₄ 5 mM solution in glycine–NaOH buffer (15 g/L, pH 9.7, Merck, Germany) for 5 min and the copper-coated cadmium granules were used within 10 min. Activated granules (600–1000 mg, approximately 1–2 granules) were added to the glycine buffer–diluted supernatant and stirred for 10 min. Aliquots of 200 µL were recovered in appropriated tubes for nitrite determination and the same volume of Griess reagent was added (Reagent I: 50 mg of N-naphthylethylenediamine in 250 mL of distilled water; reagent II: 5 g of sulfanilic acid in 500 mL of 3 M HCl; Sigma-Aldrich, USA). After incubation for 10 min at room temperature, the tubes were centrifuged at 10,000 r/min, 2 min, 25°C, and 100 µL was added to 96-well microplates in triplicate. To determine sample nitrite concentration, a calibration curve was prepared by dilution of NaNO₂ (Merck, Germany) in distilled sterile water to concentrations of 125 to 0 µM, and 100 µL of the curve point + 100 µL of Griess reagent was added in triplicate to the microplate wells. The absorbance was measured at 550 nm in a microplate reader. The final results were expressed in micromolar (µM).

Oxidative stress parameters

For the evaluation of oxidative stress parameters, B16F10 cells were washed and trypsinized after treatment. Cellular suspension was centrifuged at 600g (4°C, 3 min). The cellular pellets were suspended in cold PBS buffer and frozen (−80°C) until analysis.

Determination of intracellular reduced glutathione levels

Glutathione (GSH) levels were measured as described by Locatelli et al.²⁵ The assay involved spectrophotometric (Multiscan Go; Thermo Scientific, USA) measurement of reduction of 5,5′-dithiobis-(2-nitrobenzoic acid) to 5-thio-2-nitrobenzoic acid by GSH at 412 nm, by the formation of yellow-colored 5-thio-2-nitrobenzoic acid. Results were expressed as GSH per gram (µM/g) of total protein. The total protein content was determined based on the Lowry method²⁶ modified by Miller.²⁷

Determination of intracellular malondialdehyde levels

The measurement was performed as described by Victorino et al.,²⁸ using a high-performance liquid chromatography (HPLC) system (HPLC-Shimadzu 20AT) equipped with a LC20AT pump, an absorbance detector (UV SPDM20A diode-array), and the analytical column Zorbax Eclipse XDB-C18 (4.6 × 250 mm, 5 µm; Agilent Technologies, Santa Clara, USA). Results were expressed in nanomolar malondialdehyde (MDA) per gram (nM MDA/g) of total protein.

Determination of oxidative stress participation in citral cytotoxicity to B16F10 cells

To evaluate the involvement of reactive oxygen species (ROS) in citral cytotoxicity, we performed an MTT assay (24 h) with simultaneous treatment of citral 0.5, 1.0, and 2.5 µM and different specific ROS scavengers: histidine (100 mM), Trolox (50 mM), and Tempol (50 mM); singlet oxygen and hydroxyl radicals; and peroxyl radical and superoxide anion scavengers, respectively.^29–31 These concentrations of citral were chosen because in these concentrations we observed decrease in GSH levels and predominantly apoptotic cell death.

Evaluation of direct pro-oxidant effect of citral: oxygen uptake in red blood cells exposed to tert-butyl hydroperoxide

To evaluate citral ability to exert direct pro-oxidant effects, the oxygen uptake in erythrocytes exposed to tert-butyl hydroperoxide (t-BHP) was determined. The method consists in the delay of oxygen uptake in erythrocytes exposed to t-BHP, and this delay could be reduced by antioxidants,³² or increased in pro-oxidative situations.³³ The assay was performed according to Lissi et al.³² In summary, the heparinized blood sample was obtained from a healthy donator, and blood was centrifuged and washed three times to obtain isolated red blood cells. Aliquots of erythrocytes were incubated with citral (0.1, 0.5, 1.0, or 2.5 µM), diluted in NaH₂PO₄10 mM pH 7.4 for 5 min at 37°C. The control group was incubated with NaH₂PO₄ buffer at the same conditions as citral treatment. After incubation time, the erythrocytes were transferred to the oxygen consumption chamber of an oxymeter equipped with a polarographic electrode for oxygen level measurement. Red blood cell suspensions were treated with t-BHP 2 mM, 37°C, and oxygen levels were monitored for 10 min. Data obtained from curves were converted to area under the curve graphic using numerical integration.

Immunocytochemistry labeling for p53, ERK1/2, AKT, PI3K, and NF-KB

After treatment, B16F10 cells were washed and fixed in a commercial mixture of polyethylene glycols (Citofix^®; Doles, BR). Immunocytochemistry analysis was performed on coverslip-adherent cells using the labeled streptavidin biotin method using a LSAB KIT (DAKO, Japan). The sections were incubated with 0.1% Triton X-100 solution for 1 h, washed three times in PBS, and treated for 40 min at room temperature with 10% BSA. In addition, coverslips were incubated overnight at 4°C with the primary antibodies (anti-p53 (DO-1), sc-126; anti-NF-KB p65-A, sc-109; anti-ERK1/2, 12D4, sc: 81492; anti-PI3K p110, H-239, sc:7189; and anti-AKT, B-1, sc: 5298; diluted 1:200, Santa Cruz Biotechnology, USA). After secondary antibody treatment (2 h, room temperature), horseradish peroxidase activity was visualized by treatment with H₂O₂ and 3,3′-diaminobenzidine (DAB) for 5 min. At the last step, the sections were weakly counterstained with Harry’s hematoxylin (Merck, Germany). For each case, negative controls were performed by omitting the primary antibody. Intensity and localization of immunoreactivities against primary antibody used were examined on all coverslips using a photomicroscope (Olympus BX41, Olympus Optical Co., Ltd., Japan). For the image analysis study, photomicroscopic color slides of representative areas (400× magnification) were digitally acquired. Then, positive pixels for DAB were thresholded and processed by ImageJ software. Positive immunostained area was calculated as positive labeled area. Nuclear labeling for p53, ERK1/2, and NF-KB images was also analyzed using ImageJ software; for each image, the total number of cells were counted and the number of cells with nuclear labeling were used to calculate the percentage of cells with nuclear labeling (%). For all analyses, we used at least 10 representative images per treatment.

Statistical analysis

The normality of data was investigated and all of them presented Gaussian distribution. The data were expressed as mean ± standard error of the mean and analyzed using one-way analysis of variance (ANOVA). Intergroup differences were analyzed by Tukey’s test, and p < 0.05 was considered statistically significant. Data analysis was conducted using GraphPad Prisma (version 5.0; USA). Data obtained from 24 h of treatment with citral were also used to calculate Spearman’s correlation using GraphPad Prisma, and correlations with p < 0.05 were considered significant.

Results

Cytotoxic effect of citral

Citral reduced the proliferation of B16F10 cells in concentrations equal to or higher than 0.5 µM in 24 h and 0.05 µM in 48 h (Figure 1(a) and (b), respectively; p < 0.0001). Trypan Blue exclusion demonstrated that citral decreased cell viability in concentrations equal to or higher than 0.5 µM in 24 h and 48 h (Figure 1(c) and (d), respectively; p < 0.0001). In the MTT assay, citral was able to decrease cellular viability from the concentration of 0.5 µM onward in 24 h and 0.1 µM after 48 h (Figure 1(e); p < 0.0001; and 1F: p < 0.0001, respectively). The citral IC₅₀ in B16F10 cells was estimated in 1.04 µM for 24 h (Table 1). The IC₅₀ values of the drug were 11.7 µM in SK-MEL-147, 13.4 µM in UACC-257, 50.3 µM in HaCaT, and 2.50 µM in NIH-3T3 cells (Table 1).

Figure 1.

Citral effect in proliferation and viability in metastatic B16F10 murine melanoma cells. The cells were treated for 24 h or 48 h with increasing concentrations of citral (0.01–2.5 µM). (a) Cell counting after 24 h of citral treatment. (b) Cell counting after 48 h of citral treatment. (c) Cell viability (%) obtained from Trypan Blue exclusion after 24 h of citral treatment. (d) Cell viability (%) obtained from Trypan Blue exclusion after 48 h of citral treatment. (e) Cell viability (% of control) obtained from MTT assay after 24 h of citral treatment. (f) Cell viability (% of control) obtained from MTT assay after 48 h of citral treatment.

Table 1.

Half maximal inhibitory concentration (IC₅₀) of citral after 24 h of treatment on human and murine cell lineages.

Cell lineage	Description	IC₅₀ 24 h (µM)^a
SK-MEL-147	Homo sapiens skin melanoma NRas mutant	11.7
UACC-257	Homo sapiens skin melanoma BRaf mutant	13.4
HaCaT	Homo sapiens skin keratinocyte Tp53 mutant	50.3
B16F10	Mus musculus skin melanoma PTEN and Tp53 mutant	1.04
NIH-3T3	Mus musculus embryo fibroblast Hypertriploid karyotype	2.50

IC₅₀ was obtained from nonlinear regression of MTT assay results.

Cell death pattern

The annexin V-FITC/PI staining showed that citral increased the percentage of apoptotic cells in 1.0 µM concentration and the percentage of necrotic cells in 2.5 µM concentration (Figure 2(a); p < 0.0001). The TUNEL assay demonstrated apoptosis from 0.5 µM concentration onward (Figure 2(b); p < 0.0001), and in the LDH release assay, necrosis was also observed at citral 1.0 and 2.5 µM concentrations (Figure 2(c); p < 0.0001).

Figure 2.

Analysis of citral-induced cell death patterns in metastatic B16F10 murine melanoma cells. (a) Percentage of cell death patterns obtained from annexin V-FITC/propidium iodide staining. (b) Percentage of apoptotic cells revealed by TUNEL assay, with representative photomicrographs with the nucleus of apoptotic cells labeled in dark brown. (c) Measurement of lactate dehydrogenase (UI/L) released into culture medium. (d) Percentage of cells with autophagic vacuoles revealed by monodansylcadaverine assay, with representative images.

To better characterize the cell death patterns of citral, we evaluated the autophagy, using the MDC. In this assay, autophagic vacuoles accumulate MDC emitting fluorescence. In our conditions of treatment, only citral 1.0 µM significantly induced autophagy in B16F10 cells (Figure 2(d); p < 0.0001).

The analysis of cell death in other human and murine cell lineages treated with citral demonstrated that the minimum concentration of drug capable of significantly inducing apoptosis was 5.0 µM in SK-MEL-147, UACC-257, and NIH-3T3 cells, and 25.0 µM in HaCaT cells (Table 2). In relation to necrosis, citral significantly increased its rates in concentrations equal to or higher than 20.0 µM in SK-MEL-147 and in UACC-257, 50.0 µM in HaCaT and 5.0 µM in NIH-3T3 cells (Table 2). The MDC staining revealed that citral-induced autophagy in SK-MEL-147 and in UACC-257 cells from 5.0 µM concentration onward (Table 2). Autophagy could not be observed in the other analyzed cell lines (HaCaT and NIH-3T3) even at the highest concentration (100 µM).

Table 2.

Apoptosis, necrosis, and autophagy observed in human and murine cell lineages treated (24 h) with citral.

Specie	Cell lineage	Apoptosis(citral µM)	Necrosis(citral µM)	Autophagy(citral µM)
Human	SK-MEL-147	+ (≥5.0)	+ (≥20.0)	+ (≥5.0)
	UACC-257	+ (≥5.0)	+ (≥20.0)	+ (≥5.0)
	HaCaT	+ (≥25.0)	+ (≥50.0)	–
Murine	B16F10	+ (≥0.5)	+ (≥1.0)	+ (1.0)
Murine	NIH-3T3	+ (≥5.0)	+ (≥5.0)	–

Citral induces DNA lesions and reduces NO levels in B16F10 cells

Citral increased DNA lesions observed in the comet assay from 0.5 µM concentration onward (Figure 3(a); p < 0.0001) and reduced NO levels in all concentrations used (Figure 3(b); p < 0.0001) in a dose-dependent manner.

Figure 3.

Induction of DNA damage and depletion of nitric oxide observed after 24 h of citral treatment. (a) Mean tail moment (arbitrary unities) was obtained from comet assay in metastatic B16F10 murine melanoma cells. (b) Nitric oxide levels obtained from nitrite levels measured by Griess/Cadmium assay.

Induction of oxidative stress is related to citral cytotoxicity to B16F10 cells

To investigate the involvement of oxidative stress in the citral effects in B16F10 cells, we determined the GSH and MDA levels. The drug decreased GSH levels in concentrations equal to or higher than 0.5 µM and increased MDA at 2.5 µM (Figure 4(a), p < 0.000; Figure 4(b), p = 0.0188). The use of specific ROS scavengers simultaneously with citral treatment demonstrated that the presence of all three scavengers restored cellular viability, observed in the MTT assay, to control levels at 0.5 µM, and reduced citral toxicity at 1.0 and 2.5 µM although it was not able to revert the viability to control levels (Figure 4(c)). These results demonstrated that the induction of oxidative stress is related to citral cytotoxicity to B16F10 cells. However, the evaluation of direct pro-oxidant effect of citral, by the measurement of oxygen uptake in erythrocytes exposed to t-BHP, demonstrated that the drug did not exert direct pro-oxidant action (Figure 4(d)).

Figure 4.

Oxidative stress parameters in metastatic B16F10 murine melanoma cells treated with citral. (a) Reduced glutathione (GSH) levels (µM of GSH/g of protein). (b) Malondialdehyde levels (nM). (c) Evaluation of citral cytotoxicity by the MTT assay in the presence and in the absence of specific scavengers of reactive oxygen species: L-histidine 100 mM (Hist), Trolox 50 mM (Trol), and Tempol 50 mM (Temp). (d) Area under the curve of data obtained from oxygen uptake by red blood cells treated with citral during 5 min (37°C).

Immunocytochemistry

In this work, we also investigated the interference of citral on proteins involved in cell signaling pathways. For immunocytochemistry, only the concentrations 0.1, 0.5, and 1.0 µM were used. Citral 1.0 µM decreased the ERK1/2-labeled area/cell and citral 0.5 and 1.0 µM also decreased the nuclear labeling for this protein (Figure 5(a); p < 0.0001). Citral 0.5 and 1.0 µM reduced the expression of AKT and PI3K proteins (Figure 5(b), p < 0.0001; and Figure 5(c), p < 0.0001, respectively). For p53 protein, citral at 0.5 and 1.0 µM increased the percentage of labeled cells (Figure 5(d); p < 0.0001). Citral 1.0 µM increased NF-KB-labeled area/cell, but all tested concentrations reduced the nuclear labeling of this protein (Figure 5(e); p < 0.0001).

Figure 5.

Immunocytochemical labeling in metastatic B16F10 murine melanoma cells treated with citral. (a) ERK1/2-labeled area/cell and percentage of cells with nuclear labeling for this protein. (b) PI3K-labeled area/cell. (c) AKT-labeled area/cell. (d) Percentage of cell labeled with p53 antibody. (e) NF-KB-labeled area/cell and percentage of cells with nuclear labeling for this protein.

Spearman’s correlation of the results obtained after 24 h of citral 0.1 µM treatment

Table 3 shows the correlation matrix obtained from treatment with citral 1 µM for a duration of 24 h in B16F10 cells. The most significant correlations (p < 0.05) allowed to realize that (1) reduced cell counting was associated with decreased NO levels; (2) diminished cell viability was associated with increased percentage of necrosis, increased LDH release, increased p53 labeling, and decreased PI3K and ERK1/2 labeling; (3) apoptosis percentage was correlated to increased p53 labeling; (4) increased DNA damage was associated with increased p53 labeling, decreased GSH levels, and decreased PI3K and ERK1/2 labeling; and (5) autophagy was correlated to increased p53 labeling and decreased PI3K and ERK1/2 labeling.

Table 3.

Spearman’s correlation matrix of citral 1 µM (24 h) effects against B16F10 cells.

Variables	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18
Cell counting (1)	–	0.921	0.973	−0.737	−0.894	−0.864	−0.403	−0.953	0.946	−0.847	0.988	0.881	0.891	0.859	0.968	−0.942	−0.965	0.911
Cell viability (2)	0.921	–	0.979	−0.542	−0.996	−0.988	−0.182	−0.977	0.946	−0.963	0.939	0.996	0.998	0.992	0.785	−0.987	−0.944	0.773
MTT assay (3)	0.973	0.979	–	−0.694	−0.958	−0.937	−0.358	−0.996	0.984	−0.902	0.974	0.980	0.984	0.970	0.854	−0.991	−0.967	0.827
Apoptosis (%) (4)	−0.737	−0.542	−0.694	–	0.466	0.411	0.915	0.707	−0.774	0.315	−0.684	−0.986	−0.990	−0.979	−0.833	0.991	0.960	−0.810
Necrosis (%) (5)	−0.894	−0.996	−0.958	0.466	–	0.998	0.095	0.954	−0.916	0.981	−0.922	−0.996	−0.994	−0.989	−0.779	0.993	0.956	−0.797
LDH levels (6)	−0.864	−0.988	−0.937	0.411	0.998	–	0.038	0.934	−0.892	0.986	−0.898	−0.996	−0.988	−0.990	−0.717	0.983	0.937	−0.766
Autophagy (%) (7)	−0.403	−0.182	−0.358	0.915	0.095	0.038	–	0.387	−0.483	−0.081	−0.341	−1.000	−0.999	−0.997	−0.750	0.983	0.933	−0.751
Comet assay (8)	−0.953	−0.977	−0.996	0.707	0.954	0.934	0.387	–	−0.991	0.886	−0.955	−0.997	−0.999	−0.994	−0.779	0.985	0.940	−0.765
GSH levels (9)	0.946	0.946	0.984	−0.774	−0.916	−0.892	−0.483	−0.991	–	−0.829	0.951	0.996	0.991	0.989	0.761	−0.992	−0.954	0.794
MDA levels (10)	−0.847	−0.963	−0.902	0.315	0.981	0.986	−0.081	0.886	−0.829	–	−0.880	−0.910	−0.918	−0.891	−0.949	0.962	0.976	−0.905
NO levels (11)	0.988	0.939	0.974	−0.684	−0.922	−0.898	−0.341	−0.955	0.951	−0.880	–	0.877	0.876	0.848	0.942	−0.954	−0.990	0.965
ERK1/2 labeling (12)	0.881	0.996	0.980	−0.986	−0.996	−0.996	−1.000	−0.997	0.996	−0.910	0.877	–	0.998	0.998	0.733	−0.980	−0.928	0.742
ERK1/2 nuclear labeling (%) (13)	0.891	0.998	0.984	−0.990	−0.994	−0.988	−0.999	−0.999	0.991	−0.918	0.876	0.998	–	0.998	0.749	−0.976	−0.922	0.732
PI3K labeling (14)	0.859	0.992	0.970	−0.979	−0.989	−0.990	−0.997	−0.994	0.989	−0.891	0.848	0.998	0.998	–	0.703	−0.966	−0.904	0.700
AKT labeling (15)	0.968	0.785	0.854	−0.833	−0.779	−0.717	−0.750	−0.779	0.761	−0.949	0.942	0.733	0.749	0.703	–	−0.831	−0.892	0.912
p53 labeling (16)	−0.942	−0.987	−0.991	0.991	0.993	0.983	0.983	0.985	−0.992	0.962	−0.954	−0.980	−0.976	−0.966	−0.831	–	0.983	−0.860
NF-KB labeling (17)	−0.965	−0.944	−0.967	0.960	0.956	0.937	0.933	0.940	−0.954	0.976	−0.990	−0.928	−0.922	−0.904	−0.892	0.983	–	−0.938
NF-KB nuclear labeling (%) (18)	0.911	0.773	0.827	−0.810	−0.797	−0.766	−0.751	−0.765	0.794	−0.905	0.965	0.742	0.732	0.700	0.912	−0.860	−0.938	–

All variables were log (X + 1) transformed before analysis. Values shown are the correlation coefficients between variables. Bold coefficients were statistically significant (p < 0.05). Positive and negative coefficients indicate positive or negative correlations, with 1.0 or −1.0 being the strongest relationship.

Discussion

Citral is an important compound of many essential oils, especially lemongrass. It has been related to antibacterial, antifungal, antiparasitic, and insecticidal effects on different organisms.^15,16,34 Recently, citral demonstrated to have antiproliferative action against human breast cancer cells MCF-7;³⁵ however, the drug effects on melanoma cells are poorly understood. In this context, the main goal of this work was to evaluate the antiproliferative and cytotoxic effect of citral on B16F10 murine melanoma cells and to identify some mechanisms involved in this effect.

We first estimated the IC₅₀-24 h citral at 1.04 µM, revealing its cytotoxic effect on B16F10 cells. For 48 h of treatment, citral 0.05 and 0.1 µM reduced the cell counting but did not reduce cell viability, suggesting that citral also has antiproliferative activities. This result agrees with previous works revealing antiproliferative effects of citral on human leukemia cells (HL-60)^36,37 and MCF-7 cells.³⁵ Furthermore, we observed that B16F10 was more sensitive to citral than the human melanoma cell lines analyzed (SK-MEL-147 and UACC-257), presenting lower values of IC₅₀ and induction of apoptosis, necrosis, and autophagy in lower concentrations of citral. For this reason, B16F10 cells were chosen to elucidate some mechanisms of citral-induced cytotoxicity.

Regarding the mechanisms enrolled in cell death triggering, citral-induced cytotoxicity was evaluated by annexin V-FITC/PI staining, TUNEL assay, and LDH release. Citral was able to induce apoptotic and necrotic patterns of cell death according to tested concentrations. Using annexin V-FITC/PI staining, necrosis was recognized for citral 2.5 µM, and confirmed by the increase in LDH release to the culture medium. Annexin V-FITC/PI staining also revealed the induction of apoptosis for citral 1.0 µM, which was further confirmed by TUNEL assay. However, in this assay, apoptosis was observed from 0.5 µM concentration onward. Dudai et al.¹¹ observed that citral induced apoptosis in hematopoietic cancer cell lines (22.25 µM) and suggest that this proapoptotic effect depends on α,β-unsaturated aldehyde group. In our work, we also observed apoptosis in B16F10 cells in much lower concentrations of citral (1.0 µM), but we could not associate apoptosis with the increased p53 levels. Traditionally, increased p53 levels are associated with DNA damage and apoptosis induction.³⁸ However, B16F10 cells have functional mutations in the Tp53 gene that allow changes in Tp53 expression, but the produced protein is not capable of binding to the DNA and inducing p53-associated responses.³⁹ The ability of citral to increase p53 levels was previously reported by Duerksen-Hughes et al.⁴⁰ The increased p53 levels can also be associated with oxidative stress induction.⁴¹ In our work, the apoptosis induction by citral could be better related to the oxidative stress generation, which will be further discussed.

We also observed that citral was able to induce the generation of autophagic vacuoles in B16F10 cells. Autophagy is considered a non-selective process for bulk degradation, and it has been associated with physiological processes of adaptation to starvation, cell differentiation and development, the degradation of aberrant structures, among others.⁴² Increased autophagy offers a distinct advantage in various physiological and stress conditions suggesting that this process represents an adaptive mechanism to rescue cells from death.⁴³ Many anticancer treatments including novel targeted therapies stimulate autophagy, which can both lead to increased cytotoxicity and to therapeutic resistance.⁴⁴ The presence of autophagic vesicles in dying cells may reflect an adaptive response that failed to maintain cell survival under stress conditions.⁴⁵ In this study, citral 1.0 µM was able to induce autophagic vesicles, suggesting that B16F10 cells shifted cellular metabolism, trying to recycle damaged structures by oxidative stress under treatment with citral.

Many anticancer drugs (or their metabolites) are able to generate ROS usually by electron transfer processes,⁴⁶ which damage several cellular structures such as DNA, leading to cell death. For this reason, we investigated the ability of citral to alter redox balance of B16F10 cells. To reach this goal, we evaluated the antioxidant content of cells by measuring GSH content, as well as investigated the pro-oxidant profiling by analyzing MDA. GSH is a tripeptide involved in non-enzymatic antioxidant defenses, usually depleted during xenobiotic biotransformation and enhanced oxidative stress conditions. Furthermore, GSH depletion has been recently implicated in cell death.⁴⁷

MDA is a product of polyunsaturated fatty acid peroxidation, and it is considered more than a marker of lipid peroxidation, due to its interaction with DNA and proteins.⁴⁸ In this work, citral was able to slightly reduce GSH levels, revealing the consumption of antioxidant defenses. Only the highest tested concentration (2.5 µM) was able to increase MDA levels, revealing that citral, at highest concentrations, generates ROS that overcome antioxidant defense mechanisms in B16F10 cells and induced a significant increase in lipoperoxidation. The citral-induced oxidative stress is probably associated with citral metabolism, as we did not observe modification in oxygen uptake measurement in red blood cells treated with citral 5 min (37°C). The same result was observed in 30 min of treatment with citral (data not shown). Taking together, these results indicate that citral is able to induce oxidative stress in B16F10 cells, and the oxidative stress is probably enrolled with cell death, since ROS induces DNA damage and interacts with cellular membranes, such as mitochondrial membrane. Lesions in mitochondrial membrane are associated with apoptosis, through the release of proapoptotic proteins, such as c cytochrome, promoting the activation of effector caspases, independently of previous p53 signaling.⁴⁹ It could explain the observed apoptosis in B16F10 cells treated with citral even with the functional mutation in Tp53 in these cells.

The relation between oxidative stress induction and citral toxicity to B16F10 melanoma cells became clear when we exposed cells simultaneously to citral (0.5 µM) and to ROS scavengers (histidine, tempol, and trolox) and the cell viability restored to control levels. In the concentration of 1.0 and 2.5 µM, although scavengers were not able to revert the viability to control levels, they reduced citral toxicity at 1.0 µM, but at 2.5 µM, only Trolox reduced citral toxicity, demonstrating that the cytotoxic effect of citral on B16F10 cells can be partially explained by the generation of oxidative stress, without the involvement of particular oxidative specie. High levels of ROS can induce oxidative damage to lipids and proteins. ROS are also associated with DNA damage and subsequent activation of signal transduction pathways.⁵⁰ In addition to this, ROS can elicit autophagy as well as apoptotic cell death,⁵¹ and it is important to emphasize that autophagy and apoptosis are closely interrelated because major players of both pathways cross-talk to each other.⁵² ROS generation would thus partially explain the presence of autophagic vesicles and induction of apoptosis in B16F10 cells. Although we did not detect any alteration in MDA levels during the treatment of melanoma cells with citral 1.0 µM, it is possible that ROS enrolled in citral cytotoxicity interacted with cellular GSH, resulting in the observed GSH depletion. This hypothesis helps to understand why MDA did not increase at citral 1.0 µM. On the other hand, it is also probable that citral-generated oxidative stress also promoted DNA injury with increased DNA damage (observed in Comet assay) and induction of apoptosis and autophagy during citral exposition.

We also investigated the effects of citral in NO levels, which is synthesized endogenously by a family of enzymes called nitric oxide synthases (NOS). NO has a variety of different biological roles. In melanoma, it was demonstrated that inducible nitric oxide synthase (iNOS) is overexpressed and this enzyme and NO stimulate survival and proliferation of human melanoma cells.^53,54 Among the mechanisms that elucidate NO role in melanoma tumorigenesis is the NO (produced by iNOS) ability to inhibit the expression of CXCL10, an antitumorigenic chemokine that inhibits angiogenesis, tumor cell growth, and metastasis.⁵³ In this work, we observed a progressive decrease in NO levels induced by citral, which could represent a potential therapeutic target in melanoma therapy.

In this work, we also evaluated the ability of citral to interfere in cell signaling pathways: ERK1/2, AKT, PI3K, and NF-KB. ERK1/2 are members of the mitogen-activated protein kinase (MAPK) super-family and participate in the RAS-RAF-MEK-ERK signal transduction cascade involved in cell proliferation, survival, and differentiation.⁵⁵ Citral was able to reduce ERK1/2-labeled area/cell and nuclear labeling of this protein, demonstrating that citral reduces ERK1/2 protein and inhibits the translocation of this protein to the nucleus, blocking further cellular signaling. This result is very important, once it is well established that mutations in NRas and BRaf genes are associated with melanoma carcinogenesis, and ERK1/2 is the transcriptional factor activated in this pathway. Thus, citral could be able to reduce the intracellular consequences of these mutations, by inhibiting the last step of the MAPK cascade.

PI3K is involved in many cellular responses, including cell cycle progression and cellular growth. During its activation, PI3K recruits a subset of signaling proteins, including AKT. Once activated, AKT mediates the activation and inhibition of several targets, resulting in cellular survival, growth, and proliferation.⁵⁶ Citral 0.5 and 1.0 µM were able to reduce PI3K and AKT-labeled area/cell, revealing that citral is also able to reduce the expression of both proteins, inhibiting PI3K/AKT/mechanistic target of rapamycin (mTOR) pathway, which could also explain citral effects on cell proliferation.

We also observed that citral increased NF-KB levels in cytoplasm, but reduced nuclear labeling of this protein, suggesting that citral inhibited the translocation of this signaling molecule to the nucleus. NF-KB controls aspects of the immune and inflammatory response and it is activated by a variety of stimuli, including oxidative stress.⁵⁷ NF-KB regulates inflammatory and cell growth responses by interacting with responsive elements that include iNOS⁵⁸ and cyclin D1⁵⁹ genes, among others. Previously, Lee et al.⁶⁰ reported that citral was able to suppress the activation of NF-KB in RAW264.7 (macrophage) cells, reducing the lipopolysaccharide (LPS)-induced NF-KB DNA binding activity. In our work, citral reduced the percentage of cells with nuclear labeling, revealing that the inhibitory effect of citral includes the inhibition of NF-KB translocation into the nucleus; this effect explains the decreased NO levels, as iNOS is one of the target genes for NF-KB signaling. The citral effects on NF-KB should be highlighted because it was previously reported that this transcription factor increases the expression of CD271, a transmembrane protein associated with melanoma cell survival and resistance to vemurafenib (BRAF inhibitor).⁶¹

Another interesting result observed in this work is that citral action was not shown to be exclusive to tumor cells, but it presents a pronounced toxic effect in neoplastic cells, since HaCaT (human skin keratinocytes) and NIH-3T3 cells (murine fibroblasts) undergo apoptosis and necrosis only in high concentrations.

Taken together, all results allowed to obtain the interactome of citral effect on tested pathways (Figure 6), revealing that AKT seems to be an important key to mediate citral effects. This effect of citral is very significant since functional experiments have demonstrated important roles for the PI3K-AKT in both melanoma initiation and therapeutic resistance.⁶² Furthermore, our data reveal that citral is able to exert both antiproliferative and cytotoxic effects in B16F10 cells. It seems that the reduction of cell proliferation is related to decreased NF-KB, and consequently, NO levels, and the decreased levels of ERK1/2 and AKT signaling. The cytotoxic effect could be explained by the oxidative stress induction, which could be responsible for DNA lesions and apoptosis induction. These findings highlight the potential antineoplastic action of citral tested in vivo models of melanoma, with a tumor-driven mechanism of action enrolling the depletion of NO and interference in signaling pathways pivotal to cell proliferation and survival.

Figure 6.

(a) Overall view of citral-induced effects in B16F10 cells and (b) interactome analysis of all evaluated pathways. The protein interaction network was obtained from the Ingenuity^® Pathway Analysis (IPA^®), QIAGEN (USA).

Footnotes

Acknowledgements

We are very grateful to Glaucia Regina Martinez of Universidade Federal do Paraná (UFPR)—Curitiba, Brazil, for the supply of B16F10 cells. Authors would like to thank Pedro Sebastião Raimundo Dionízio Filho and Jesus Antônio Vargas for technical support.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The study was supported by grants from the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).

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