Cytotoxic effect of Erythroxylum daphnites extract is associated with G₁ cell cycle arrest and apoptosis in oral squamous cell carcinoma

ABSTRACT

Plant-derived molecules showing antineoplastic effects have recently gained increased attention as potential adjuvants to traditional therapies for various cancers. Cerrado biome in Brazil contains high floral biodiversity, but knowledge about the potential therapeutic effects of compounds derived from that flora is still limited. The present study investigated the antineoplastic activity of Erythroxylum daphnites Mart., a Brazilian native plant from Cerrado biome, in the SCC-9 oral squamous cell carcinoma cell line. Cells were treated with various concentrations of hexane extract of Erythroxylum daphnites leaves (EDH) and assessed for cytotoxicity, proliferation, and apoptosis. Thin layer chromatography was conducted to characterize the substances present in EDH. Our results showed that EDH exerted anti-proliferative effects in SCC-9 cells by stabilizing the cell cycle at G₁ phase in association with reduced intracellular levels of cyclins D and E and increased level of p21. EDH also demonstrated pro-apoptotic properties, as shown by an increased expression of caspase-3. Triterpenes were the major constituents of EDH. Our findings demonstrated a cytotoxic effect of EDH against SCC-9 cells in vitro mediated by the restraint of cellular proliferation and induction of apoptosis. Taken together, these findings support EDH constituents as potential therapeutic adjuvants for oral cancer.

KEYWORDS:

• apoptosis
• cytotoxicity
• cell cycle
• cerrado biome
• Erythroxylum daphnites
• oral squamous cell carcinoma
• triterpenes

Introduction

Head and neck squamous cell carcinoma (HNSCC) comprises tumors of the oral cavity, oropharynx, larynx, and pharynx.¹ Almost half of HNSCCs occur in the oral cavity, and the most common histopathological type is oral squamous cell carcinoma (OSCC).² Most HNSCCs develop in the upper aero-digestive epithelium after exposure to carcinogens such as tobacco and alcohol.^3-6 Moreover, human papillomavirus has also been involved as an etiologic agent in a subset of these cancers.⁷ In Brazil, there is estimated to be approximately 18,150 new cases of HNSCC in men and 4,780 cases in women every year. These findings suggest a risk of 11.54/100,000 for men and 3.92/100,000 for women of developing cancers of the oral cavity.⁸

Despite the high incidence of HNSCC, the effectiveness of the available treatments remains limited and patients with recurrent or metastatic HNSCC have a poor prognosis.⁹ The therapeutic options include surgery for resection of the tumor area and radiotherapy/chemotherapy either alone or in combination.¹⁰ For patients who may not undergo any of these treatments, the only alternative is to provide palliative support and care to improve their quality of life.^4,9 Moreover, the positive and adverse effects of these treatments should be carefully considered before recommending a specific treatment for HNSCC.¹¹

The use of plants for drug development is not a new concept. Many existing drugs are derived from plants, such as camptothecin, vincristine, vinblastine, taxol, podophyllotoxin, and combretastatin, among others.¹² Despite a large number of existing anticancer drugs of natural origin, the pharmacological potential of many plant species has not yet been fully explored. The Erythroxylaceae family comprises 4 genera containing a total of 240 different species that are found endemically in countries such as Venezuela, Brazil, and Madagascar.¹³ Brazil holds the greatest diversity of Erythroxylum species.¹⁴ We previously demonstrated that hexane extracts of Erythroxylum daphnites (EDH) show moderate cytotoxic activity against oral squamous cell carcinoma, which was supra-additive when associated with radiotherapy.¹⁵ Therefore, this species can be a strong candidate for the development of new anticancer drugs. The aims of this study were to investigate the molecular mechanisms by which hexane extract of Erythroxylum daphnites displayed antiproliferative and proapoptotic effects in oral cancer cells.

Results

EDH shows selective cytotoxicity against SCC-9 cells

To identify the cytotoxic concentration of EDH, a dose-response curve was performed. The concentration of “0 µg/mL” (addition of solvent only) was considered to have 100% viability, and the viability of other concentrations were expressed as relative values. As we can see, EDH demonstrated moderate cytotoxicity in SCC-9 cells in a dose response manner. The concentration resulting in a 50% reduction in cell viability (IC₅₀) after 24 h of treatment was calculated as 448.9 µg/mL (Fig. 1A). For comparison, HaCaT cells, an immortalized keratinocyte cell line, were treated with EDH and presented an IC₅₀ of 737.1 µg/mL (Fig. 1B). Based on these values, the tumor selective index (TSI) was calculated as 1.64, showing that EDH was more selective for OSCC cells than for keratinocytes.

Figure 1. Dose-response curves showing IC₅₀ of hexane extract of Erythroxylum daphnites (EDH) on inhibition of both SCC-9 (tongue carcinoma cells) and HaCaT (human Keratinocyte cells) cell viability. Dose-response curves were assessed by MTT assay in SCC-9 (A) and HaCat cells (B) after 24 h of treatment with increased concentration of EDH. The results represent the percentage of dead cells in the presence of different doses of EDH. They are representative of at least 3 independent experiments in triplicate and show the mean ± SEM.

EDH reduces cell proliferation by inducing G₁ cell cycle arrest in SCC-9 cells

We next evaluated the effects of EDH on cell proliferation by BrdU assay and the cell cycle distribution by flow cytometric analysis. The BrdU cell proliferation assay detects BrdU incorporated into cellular DNA during S phase using an anti-BrdU antibody. As noted, EDH significantly reduced SCC-9 cell proliferation by approximately 31% after 12 h of treatment (P < 0.01 vs control) (Fig. 2). Accordingly, when compared with the corresponding proportions in untreated control cells, EDH resulted in a significant increase in the proportions of cells in the G₀ and G₁ phases (75%) and a clear reduction in the proportion in S phase (21%) (Fig. 3A and B). To further characterize the effects of EDH on the cell cycle, proteins related to the G₁-S cell cycle phase transition were quantified by western blot analysis. As shown in Figure. 4, EDH treatment was associated with increased expression level of the CDK inhibitors p21 by 21% and decreased levels of p27 (37% reduction), cyclin D1 (88% reduction), and cyclin E (24% reduction) compared with their expression levels in untreated control cells. The modification of profile expression of proteins involved in the cell cycle induced by EDH can explain the arrest at G0/G1 phase observed by flow cytometry analysis.

Figure 2. The hexane extract of Erythroxylum daphnites extract (EDH) decreases the proliferation of SCC-9 tongue carcinoma cells. Cell proliferation was measured using BrdU incorporation assay as described in methods. The treatment with EDH significantly decreased the proliferation of SCC-9 cells compared to control. *P < 0.01 vs. control (Student's t test).

Figure 3. A – Distribution of cells in each stage of the cell cycle after 24 h of treatment with the hexane extract of Erythroxylum daphnites (EDH) (*P < 0.0001 vs. proportion of cells in S-phase). B - Distribution of events labeled with propidium iodide in the histogram, the first gray peak represents events in G₀/G₁ phase, the second gray peak (right) represents events in G₂/M phase, and the hatched region represents the number of events in S phase.

Figure 4. Expression of cell cycle proteins in SCC-9 tongue carcinoma cells assessed by Western blot after 24 hours of treatment with hexane extract of Erythroxylum daphnites (EDH). All these proteins were involved in the transition to G1/S phase of cell cycle. The reduction of expression of cyclin D, cyclin E and p27 and enhanced level of p21 suggest the arrest of cells treated with EDH at G0/G1 phase.

EDH induces apoptosis of SCC-9 cells

It was previously described that EDH induced 2 different profile of cell death, with treated cells staining positive for both annexin V (indicating apoptosis) and propidium iodide (indicating necrosis).¹⁵ To examine the role of apoptosis in EDH-induced SCC-9 cell cycle arrest, we investigated the expression of caspase-3 and pro-caspase by Western blot analysis. Our results demonstrated that the expression of pro-caspase was similar in all analyzed samples, regardless of whether they were treated or not with EDH. In contrast, EDH treatment increased the expression of cleaved caspase (17 kDa) in SCC-9 cells. Collectively, these results suggest the induction of apoptosis in SCC-9 cells treated with EDH (Fig. 5).

Figure 5. Expression of cleavage of Pro-caspase 3 and caspase in SCC-9 tongue carcinoma cells assessed by Western blot after 12 hours of treatment with hexane extract of Erythroxylum daphnites (EDH). The presence of caspase 3 band in SCC-9 cells EDH treated with EDH confirms an apoptotic effect. GAPDH expression was used as housekeeping control (Ctr). The figure represents one of 3 experiments.

Triterpenes are major components of EDH

In order to screen for substances that have cytotoxic activity against SCC-9, the EDH was fractionated into 5 fractions: hexane (EDH-FH); hexane:ethyl acetate (1:1) (EDH-FHE); ethyl acetate:methanol (1:1) (EDH-FEM); methanol (EDH-FM); and ethyl acetate (EDH-FE). Their activities were evaluated by MTT viability assay. As observed in Figure. 6A, the EDH fraction of hexane:ethyl acetate (EDH-FHE) and ethyl acetate (EDH-FE) strongly inhibit SCC-9 cell viability. After 24 hours of treatment, only 35.18% and 38.24% respectively remained viable (Fig. 6A).

Figure 6. A - Cytotoxicity of hexane extract of Erythroxylum daphnites (EDH) fractions after 24 h of treatment of SCC-9 tongue carcinoma cells. 1 - Fraction hexane:ethyl acetate (1:1) (EDH-FHE); 2 - Fraction ethyl acetate:methanol (EDH-FEM) (1:1); 3 - Fraction ethyl acetate (EDH-FE); 4 - Fraction methanol (EDH-FM); 5 - Fraction hexane (EDH-FH). *P < 0.01 vs. control. B - Chemical structure of compounds found in fraction EDH-FHE. The results show the percentage of cell viability in the presence of different extracts treatment.

The preliminary analysis of EDH-FHE and EDH-FE by thin layer chromatography showed that both fractions presented a similar chemical profile. In addition, the results suggested the presence of triterpenes as the major class of compounds. The fraction EDH-FHE was subjected to gas chromatography coupled to mass spectrometry (GC-MS) analysis and found to be constituted by the triterpenes lupenone (Fig. 6B − 1), friedelanol (Fig. 6B − 2), and friedelan-3-one (Fig. 6B − 3) and the steroidal compound β-sitosterol (Fig. 6B − 4), as shown in Table 1. Furthermore, methyl esters of fatty acids and derivatives were identified, such as methyl palmitate, methyl stearate, methyl linoleate, methyl 18-methyl nonadecanoate, methyl docosanoate, methyl lignocerate, and methyl hexacosanoate (Table 1).

Table 1. Components identified in the fraction EDH-FHE by GC-MS analysis.

UNSAPONIFIED
RT	PEAK AREA %	MOLECULAR FORMULA	CLASS OF COMPOUND	NAME
50.8	20.7	C29H50O	Steroid	β – Sitosterol
51.62	16.33	C30H48O	Triterpene	Lupenone
53.33	36.38	C29H50O2	Triterpene	Friedelanol
53.66	26.59	C30H50O	Triterpene	Friedelan-3-one
SAPONIFIED
29.27	11.96	C17H34O2	Fatty acid	Methyl palmitate
33.07	4.88	C19H38O2	Fatty acid	Methyl sterate
36.18	6.63	C19H34O2	Fatty acid	Methyl linoleate
36.54	2.61	C21H42O2	Fatty acid	Methyl 18-methylnonadecanoate
39.74	3.49	C23H46O2	Fatty acid	Methyl docosanoate
42.72	5.58	C25H50O2	Fatty acid	Methyl linocerate
45.48	3.71	C27H54O2	Fatty acid	Methyl hexacosanoate

Discussion

In recent years, plants derivatives have received much attention for their possible utility in the chemoprevention and treatment of tumors.^16-22 Among the medicinal plants used for the treatment or prevention of cancer, only 84 species were subjected to rigorous studies and only 35.71% of these species were subjected to pharmacological studies to test their activity.²⁰ The most frequently cited species are Aloe vera (Aloaceae), Euphorbia tirucalli (Euphorbiaceae), and Tabebuia impetiginosa.²⁰ Cerrado biome covers a large area of the Brazilian territory and has a great potential as a natural source of compounds for the pharmaceutical industry.^15,23-25 Many species found in this biome are popularly used for the treatment of various diseases, including cancer. Erythroxylum daphnites, from Cerrado biome, presents cytotoxic activity in OSCC cell lines, as previously described by Elias et al. in 2015.¹⁵

Furthermore, the selective cytotoxic effect of EDH is promising, with a TSI (tumor selective index) of 1.64 for SCC-9 compared to keratinocytes. This results is very interesting, since many of the adverse effects caused by chemotherapy treatment are consequences of the poor selectivity of the drugs used. Most chemotherapy drugs target rapidly dividing cells, including healthy cells undergoing mitosis as well as cancer cells.^26,27 Therefore, the identification of new agents with a higher selectivity for cancer cells is crucial. Similar results were observed in a study by Horri et al,²⁸ who found that Rhinacanthus nasutus extract was strongly selective for an OSCC cell line when compared to gingival fibroblasts, pulp cells, and cells of the periodontal ligament.

EDH also reduced the proliferation rate of SCC-9 cells to nearly 30%, triggering cell cycle arrest at the G₁ phase and reducing the proportion of cells in S phase. These effects might be involved in disrupting SCC-9 proliferation. Similar effects of Sinularia extracts were observed by Wang et al.²⁹ in the SCC-25 OSCC cell line and by Chan et al.³⁰ for the flavonoid apigenin.

Genetic alterations are primarily responsible for the development of cancer; known changes include the uncontrolled expression of oncogenes and tumor suppressor genes, and mutations in genes involved in the apoptotic pathway, which lead to genomic instability and consequently progression to a neoplasm.³¹ Many of these mutations are found in genes that regulate cell cycle G₁ phase progression.^32,33 Once the cell progresses from G₁ to S phase; it is committed to DNA replication and cell division.³³ Apoptosis is a fundamental mechanism in various stages of development and actively participates in immune system regulation and homeostasis in multicellular organisms. Failure to control apoptosis might be an underlying cause of several diseases, such as neurodegenerative diseases, autoimmune diseases, and cancer.³⁴ Since cancer development is related to dysfunctions of the apoptotic process, the primary objective of conventional chemotherapy and non-conventional antineoplastic agents, such as natural extracts, and homemade teas, is the induction of cell death in malignant cells by reactivating the apoptotic mechanism.³⁵

Cyclins and cyclin-dependent kinases (CDKs) are primarily responsible for regulating the progression of the cell cycle from G₀ to G₁ phase and subsequently through the S, G₂, and M phases. Changes in the protein levels of cyclins and CDKs mediate transitions between phases of mitosis and ultimately the generation of a daughter cell that is identical to the mother cell.³⁶ Cell cycle transitions are regulated by checkpoint signaling pathways that monitor cellular integrity and ensure the completion of each phase of the cell cycle before initiating the next phase.³⁷ With a focus on tumors that result from cell cycle deregulation, cell cycle specific antineoplastic agents have been widely used in clinical practice, including antimetabolic and hormonal agents and natural products such as vinca alkaloids and taxol.^38,39

The expression of caspase-3 in SCC-9 cells that was induced by treatment with EDH confirmed the apoptotic effect of this agent. However, EDH showed a combined profile of cell death induction, with treated cell cultures showing increased proportions of cells staining for annexin V alone, propidium iodide alone, and both markers together when analyzed by flow cytometry. These results suggested that EDH has multiple actions in oral squamous cell carcinoma, involving a rapid induction of apoptosis and progression to necrosis after a longer duration. A similar effect was observed by Chia et al.,¹⁶ who found that an extract of Toona sinensis leaves induced death either by apoptosis alone or by a combination of apoptosis and necrosis, depending on the cell type. Other studies have emphasized the importance of apoptosis induction by candidate anticancer agents and the increased use of caspase-3 labeling to establish its occurrence.^30,40-42

Once treated with EDH, SCC-9 OSCC cells presented changes in their expression of the proteins involved in cell cycle transitions. Compared with the corresponding measurements in control cells, the expression levels of cyclins D and E and p27 decreased in EDH-treated cells, while that of p21 was increased. The activity of paclitaxel, a drug widely used to treat cancer, was positively correlated with cyclin B1/CDC2 activity, prolonged mitotic arrest, and Bcl-2 phosphorylation. In contrast, carboplatin caused cell cycle arrest before mitosis in HNSCC cells.³⁷ Treatment of HNSCC cells with metformin also reduced the expression of Cdk2, Cdk4, Cdk6, cyclins D and E, and CDK inhibitors such as p15, p16, p18, and p27.⁴³ At the G₁/S phase transition, the proteins p53, pRb, and a host of CDK inhibitors (p21^Waf1/Cip1, p27^Kip1, p57^Kip2, and p16^INK4A) are necessary for checkpoint function.⁴⁴ Cyclin D, encoded by the CCND1 gene, essentially functions as a regulatory subunit of Cdk4 and Cdk6. Cyclin D activity is of fundamental importance for the transition from G₁ to S phase.³⁶ Cyclin E targets Cdk2 and the interaction between these proteins is also critical for progression from G₁ to S phase. The cyclin E/Cdk2 complex phosphorylates p27 (a cyclin D inhibitor) and induces the expression of cyclin A, which is required for entry into S phase.^36,45

Multiple compounds in EDH have shown cytotoxic activities against several cancer cell lines.^46-50 Therefore, we hypothesize that these compounds might be responsible, at least in part, for the cytotoxic effect observed in OSCC cells. Lupenone was able to increase tyrosinase mRNA expression via inhibiting the phosphorylation of extracellular signal-regulated kinases 1 and 2 by mitogen-activated protein kinase in B16 murine melanoma cells.^49,50 Additionally, lupenone exhibited weak cytotoxicity against human colorectal cancer (HT-29) and mammary breast cancer (MDA-MB) cell lines.^46,47 β-sitosterol is one of the most common phytosterols in higher plants and presents several pharmacological activities, including anti-tumor activities.^51-54 This compound showed antiproliferative effects in MCF-7 cells, mediated by caspase-induced apoptosis.⁴⁸ In addition, β-sitosterol inhibited non-metastatic 22Rv1 and metastatic DU145 human prostate cell lines but had no such effect on normal cells.⁵⁵ Fatty acids have been recognized as cytotoxic agents. The presence of fatty acids and derivatives in Erythroxylum daphnites extract and its fractions also explains the cytotoxicity of these agents against the tested OSCC cell line. Saturated fatty acids (oleic, palmitoleic, palmitic, and stearic acids) can induce hepatocyte lipo-apoptosis by activating the proapoptotic Bcl-2 proteins Bim and Bax, which trigger the mitochondrial apoptotic pathway.⁵⁶ Stearic, sterculic, and oleic acids were found to inhibit colony formation by 5 human cancer cell lines and 2 non-neoplastic cell lines in a dose-dependent manner. In that study, stearic acid was more active than oleic acid for all cell lines except HT29.⁵⁷

Our results showed that EDH induced apoptosis in the SCC-9 OSCC cell line. EDH-treated SCC-9 cells were arrested in G₁ phase and showed an altered expression of proteins involved in cell cycle progression. These effects were selective to SCC-9 cells. Taken together, EDH appears to be a promising candidate agent for the treatment of OSCC. Triterpenes and fatty acids may contribute to its cytotoxic effect in OSCC cells.

Materials and methods

Plant material

Leaves of Erythroxylum daphnites Mart. were collected from Cerrado biome in the city of Brasília (Brazil) and its surroundings, and deposited in the herbarium of the University of Brasilia [voucher number (UB) 2193]. The leaves were dried at room temperature and powdered in a knife mill. The plant material (40 g) was macerated with hexane (2 L) at room temperature for 7 days (repeated 3 times). After filtration, the solvents were removed under reduced pressure at temperatures below 40°C, furnishing hexane crude extract (EDH) (5.8% yield).

The crude extract (EDH) was subjected to filtration over silica gel 60 (Fluka) and gave 5 fractions, according to the eluent used: hexane (FH, 11.8% yield); hexane:ethyl acetate 1:1 (FHE, 51.5% yield); ethyl acetate (FE, 26.1% yield); ethyl acetate:methanol 1:1 (FEM, 9.2% yield); and methanol (FM, 1.1% yield). The crude extract and fractions were analyzed by thin layer chromatography (Alugram® Sil G, Macherey-Nagel), using a mixture of hexane and ethyl acetate (7:3) as eluent. The chromatogram was revealed by using anisaldehyde reagent.⁵⁸

The most active fraction (FHE) was subjected to saponification and transesterification reactions, according to Hartman and Lago (1973), with modifications,^59,60 resulting in saponified and unsaponified fractions. GC-MS analysis was performed on a GCMS-QP2010 Plus system (IE 70 eV) (Shimadzu) with a Rtx®-5MS column measuring 30 m × 0.32 mm × 0.25 µm, packed with 5% diphenyl and 95% dimethylpolysiloxane. The injection port was maintained at 250°C (splitless mode), and analysis was performed at a temperature gradient increasing from 100°C to 300°C at a rate of 5°C/min. The carrier gas was helium at a flow-rate of 6 mL/min.

Cell culture

SCC-9 cells, a tongue squamous carcinoma cell line, and HaCaT cells, an immortalized keratinocyte cell line, were cultured according to the recommendations of the American Type Culture Collection. SCC-9 cells were grown as monolayers in a mixture of DMEM/Ham's F12 (D2906, Sigma-Aldrich) medium and HaCaT cells were cultured in DMEM. They were supplemented with 10% (v/v) fetal bovine serum (FBS) (12657-029, Gibco, Life Technologies), 1% (v/v) antibiotics solution (P0781, Sigma-Aldrich), and 400 ng/mL hydrocortisone (H0888, Sigma-Aldrich) and maintained at 37°C in a humidified atmosphere 5% of CO₂.

Dose response curve for cell viability

Cells (5 × 10³) were seeded in 96 well plates and allowed to adhere overnight at 37°C, followed by treatments of cells with different concentrations (1050, 975, 900, 825, 750, 675, 600, 525, 450, 375, 300, 225, 150, 75, and 0 µg/mL) of EDH for 24 h. As a negative control, the solvent used for dilution of the extract was added to the culture medium at the maximum volume used for treatment. MTT reagent (M2128, Sigma-Aldrich) was added to each well and incubated for 4 h at 37°C in the dark. The media was removed and 100 µL of NHCl (1007, Vetec Quimica)/isopropanol (9084-03, J.T. Baker, Avantor Performance Materials) solution was added to each well and incubated for 15 min at room temperature under gentle agitation. Finally, absorbance was measured at 570 nm in a microplate reader. Three independent experiments were performed, each with triplicate measurements. The IC₅₀ value was calculated, and the TSI was established as the ratio of IC₅₀ in the control cells (HaCaT) to that in the neoplastic cells (SCC-9), as previously published by Horri et al.²⁸

Cell proliferation assay (BrdU)

SCC-9 cells were plated at a density of 1 × 10⁴ cells per well in 96 well plates and maintained under optimum conditions. After adherence, cells were washed with phosphate-buffered saline (PBS), and fresh serum-free medium was added to the plated cells for 24 h to induce cell synchronism. Cells were then treated with the IC₅₀ of the extract for 12 h. Finally, BrdU (11647229001, Roche) was added at a final concentration of 10 µM per well, and the cells were incubated for 2 h at 37°C. After the incubation period, medium containing BrdU was removed, and the cells were fixed for 30 min at room temperature with cold 70% (v/v) ethanol. The incorporation of BrdU into proliferating cells was quantified by ELISA as described by the manufacturer. To stop the reaction, 25 µL of a 1 M solution of H₂SO₄ (320501, Sigma-Aldrich) was added per well, and absorbance was read at a wavelength of 450 nm (reference wavelength 690 nm) using an ELISA reader (Ultramark™ Microplate Imaging System, Bio-Rad).

Cell cycle analysis

Cells were synchronized for 24 h by serum starvation and released by replacement with media containing 10% (v/v) FBS and the tested concentrations of EDH as noted above. After 24 h, cells were collected, fixed in 70% (v/v) ethanol for 30 min, treated with 10 μg/mL of RNAse (Sigma-Aldrich) and stained with 50 μg/mL of propidium iodide (Sigma-Aldrich). The distribution of cells in each cell cycle phase was analyzed with the aid of a FACSCalibur™ flow cytometer (BD Biosciences) equipped with an argon laser and ModFit LT™ software (Verity Software House).

Apoptosis analysis

The apoptosis index was determined by annexin V labeling. Briefly, cells treated for 2 and 6 h with the IC₅₀ of EDH were harvested, washed, and resuspended in binding buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 5 mM KCl, 1 mM MgCl₂, and 1.8 mM CaCl₂) containing annexin V-PE and 7-AAD dyes (BD Biosciences). Apoptosis was analyzed on a FACSCalibur™ flow cytometer equipped with an argon laser (BD Biosciences) and quantified as the number of annexin V-PE positive and 7-AAD negative cells divided by the total number of cells. A minimum of 10,000 events was analyzed for each sample.

Western blot

Cells were washed with cold PBS and lysed in a buffer containing 20 mM Tris-HCl pH 8.0, 137 mM NaCl, 10% (v/v) glycerol, 2 mM ethylenediaminetetraacetic acid, and protease inhibitors. After centrifugation, protein concentrations were measured using a protein assay according to the manufacturer's instructions (Bio-Rad Protein Assay, Bio-Rad). Four micrograms of total proteins per sample were resolved by SDS-PAGE on a 10% (v/v) polyacrylamide gel under reducing conditions and transferred to nitrocellulose membranes. The membranes were blocked with 10% (w/v) non-fat dried milk in PBS containing 0.1% (v/v) Tween® 20, rinsed in the same buffer, and incubated for 2 h with the primary antibodies (Table 2). After washing, the protein bands were detected using enhanced chemiluminescence reagent (GE Healthcare).

Table 2. List of primary antibodies.

Antibody	Clone	Supplier	Dilution
p21^WAF/Cip1	C-19	Santa Cruz	1:200
p27^Kip1	57	BD Biosciences	1:500
Ciclina E	M-20	Santa Cruz	1:1000
Ciclina D1	DSC-6	Calbiochem	1:200
Caspase-3	Sc-7148	Santa Cruz	1:500
β – actina	AC-15	Sigma-Aldrich	1:30000
GAPDH	Ab9843-200	Abcam	1:6000

Statistical analysis

Cytotoxicity results were tested by one-way ANOVA with Tukey's post-hoc multiple comparison test. For the analysis of cell proliferation (BrdU assay) and apoptosis, Student's t-test was applied. Dose-response curves were assessed by non-linear regression of inhibition vs. dose, and the IC₅₀ value was calculated by interpolation. For cell cycle analysis, 2-way ANOVA with the Bonferroni post-hoc test was performed. For all experiments, the control represents cells treated only with the solvent used to dilute EDH. All data were analyzed with Prism® software, version 5.0 (GraphPad Software). Values of p < 0.05 were considered statistically significant.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Funding

This research was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico-CNPq (MCT/CNPq/FNDCT/FAPs/MEC/CAPES/PRO-CENTRO-OESTE N. 031/2010 project 564658/2010–3 and 564208/2010-8), and by Fundação de Amparo à Pesquisa-Distrito Federal (FAP-DF) project 193.000.484/2011.