In vitro and in vivo growth inhibition of human acute promyelocytic leukemia HL-60 cells by Guatteria megalophylla Diels (Annonaceae) leaf essential oil

Guatteria megalophylla Diels (Annonaceae) is an 8−10m tall tree that grows near streams and is widely spread throughout Colombian, Ecuadorian, Peruvian, Brazilian and Guianese Amazon rainforest. Herein, we investigated for the first time the chemical composition and in vitro and in vivo anti-leukemia potential of G. megalophylla leaf essential oil (EO) using human promyelocytic leukemia HL-60 cells as model. EO was obtained by a hydrodistillation clevenger-type apparatus and characterized qualiand quantitatively by GC–MS and GC–FID, respectively. In vitro cytotoxic potential of EO was evaluated in human cancer cell lines (HL-60, MCF-7 CAL27, HSC-3, HepG2 and HCT116) and in human non-cancer cell line (MRC-5) by Alamar blue method. Annexin V/propidium iodide staining, cell cycle distribution and reactive oxygen species (ROS) were assessed by flow cytometry for HL-60 cells treated with EO. In vivo efficacy of EO (50 and 100mg/kg) was evaluated in C.B17 SCID mice with HL-60 cell xenografts. Chemical composition analyses showed spathulenol, γ-muurolene, bicyclogermacrene, β-elemene and δ-elemene as main constituents of assayed sample. EO displayed in vitro cytotoxicity, including anti-leukemia effect with IC50 value of 12.51 μg/mL for HL-60 cells. EO treatment caused augment of phosphatidylserine externalization and DNA fragmentation without increasing of ROS in HL-60 cells. In vivo tumor mass inhibition rates of EO was 16.6–48.8 %. These data indicate anti-leukemia potential of G. megalophylla leaf EO.


Introduction
Cancer is a disease with high incidence and mortality worldwide. The new global cancer data indicate one-in-five men and one-in-six women in the world will develop cancer, leading to one-in-eight men and one-in-ten women deaths, respectively. In relation to leukemia, GLOBOCAN database estimated 437,033 new cases and 309,006 deaths worldwide in 2018 [1]. Therefore, researches on cancer biology, early diagnostic and new treatments are encouraged.
The genus Guatteria belongs to Annonaceae family and include about 307 species that are distributed through neotropical regions between Mexico and Brazil [2,3]. Anticancer potential of species from this genus have been reported for Guatteria friesiana (W. A. Rodrigues) megalophylla leaves and stems were assessed, but no antioxidant effect was observed at concentrations of 10 and 50 μg/mL [19]. Despite of anticancer potential of plants belonging to genus Guatteria, G. megalophylla had never been studied in relation to its cytotoxic and antitumor properties. Herein, we investigated for the first time chemical composition and in vitro and in vivo anti-leukemia potential of G. megalophylla leaf essential oil (EO) using human promyelocytic leukemia HL-60 cells as model.  G. megalophylla leaves were oven dried with air circulation at 40°C for 24 h and subjected to hydrodistillation for 4 h using a Clevenger type apparatus (Amitel, São Paulo, Brazil). EO was dried over anhydrous sodium sulphate and percentage content (w/w) was calculated based on plant material dry weight. Hydrodistillation extractions were performed in triplicate. EO was stored in freezer prior chemical and biological analyses.

GC-FID analysis
Gas chromatography coupled to flame ionization detection (GC-FID) analyses were carried out using a Shimadzu GC-2010 Plus (Shimadzu Corporation, Kyoto, Japan) fitted with a flame ionization detector and equipped with a self-injector, AOC-20i (Shimadzu Corporation). The separation of the oil constituents was achieved by employing an Rtx®-5 fused capillary column (30 m X0.25 mm X0.25 μm film thickness) coated with 5 %-diphenyl-95 %-dimethylpolysiloxane. Helium (99.99 %) was the carrier gas at a flow rate of 1.0 mL/min. The column temperature program was 40°C kept for 4 min, a heating ramp at a rate of 4°C/min up to 240°C, followed by a rate of 10°C/min until 280°C, and then280°C kept for 2 min. The injector and detector temperatures were 250°C and 280°C, respectively. Samples (10 mg/mL in CH 2 Cl 2 ) were injected with a 1:50 split ratio. Retention indexes were calculated according to Van den Dool and Kratz equation [20] in comparison with a standard solution of C 8 -C 20 n-alkanes (Sigma-Aldrich Co., Saint Louis, MO, USA). Peak areas and retention times were measured by an electronic integrator. The relative amounts of individual compounds were computed from GC peak areas without FID response factor correction.

GC-MS analysis
Gas chromatography coupled to mass spectrometry (GC-MS) analyses were also performed on a Trace gas chromatography ultra-system (Thermo-Scientific) coupled with an ISQ mass spectrometer equipped with a Tri Plus RSH auto-injector. An Rtx®-5MS fused capillary column (coated with 5 %-phenyl-95 %-methylpolysiloxane) (30 m X0.25 mm X0.25 μm film thickness) was used as stationary phase. MS data were taken at 70 eV with a scan interval of 0.5 s and mass spectra acquisition at the m/z 40−500 Da range. All injection and separation conditions were the same from GC-FID analysis. The identification of EO components was achieved based on similarity with data from Nist library [21].

Cells
HL-60 (human promyelocytic leukemia), MCF-7 (human breast adenocarcinoma), CAL27 (human oral squamous cell carcinoma), HSC-3 (human oral squamous cell carcinoma), HepG2 (human hepatocellular carcinoma), HCT116 (human colon carcinoma) and MRC-5 (human lung fibroblast) cell lines were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA), and were cultured as recommended by ATCC animal cell culture guide. All cell lines were tested for mycoplasma using a mycoplasma stain kit (Sigma-Aldrich) to validate the use of cells free from contamination.

Cytotoxicity assay
For cytotoxicity assay, cell viability was quantified by Alamar blue method as previously described [22][23][24]. For all experiments, cells were plated in 96-well plates. EO was dissolved in dimethyl sulfoxide (DMSO, Vetec Química Fina Ltda, Duque de Caxias, RJ, Brazil) and added to each well and incubated for 72 h. Doxorubicin (doxorubicin hydrochloride, purity ≥ 95 %, Laboratory IMA S.A.I.C., Buenos Aires, Argentina) and 5-fluorouracil (purity ≥ 95 %, Sigma-Aldrich Co., Saint Louis, MO, USA) were used as positive controls. At the end of treatment, 20 μL of a stock solution (0.312 mg/mL) of resazurin (Sigma-Aldrich Co.) was added to each well. Absorbances at 570 nm and 600 nm were measured using a SpectraMax 190 Microplate Reader (Molecular Data are presented as mean ± S.D. of three analyses. RI (retention indices): a calculated on RTx®-5MS column according to Van Den Dool and Kratz [20], based on a homologous series of normal alkanes; b according to Adams [21]. N.I. = Not identified.

Trypan blue exclusion assay
Trypan blue exclusion assay was used to confirm the cytotoxic effect of EO, and the number of viable cells and non-viable (stained with trypan blue) cells were counted. Cell counting was performed using a light microscope with a hemocytometer filled with a homogenized of cell suspension.

Annexin-V-FITC/propidium iodide staining assay
FITC Annexin V Apoptosis Detection Kit I (BD Biosciences, San Jose, CA, USA) was used for apoptosis quantification, and the analysis was performed according to manufacturer's instructions. At least 10 4 events were recorded per sample using a flow cytometry with a BD LSRFortessa cytometer, BD FACSDiva Software (BD Biosciences) and FlowJo Software 10 (FlowJo Lcc; Ashland, OR, USA). Cellular debris were omitted from analysis.

Internucleosomal DNA fragmentation and cell cycle distribution
Internucleosomal DNA fragmentation and cell cycle distribution were assessed by quantification of DNA content [25]. Cells were harvested in a permeabilization solution containing 0.1 % triton X-100, 2 μg/mL propidium iodide (PI), 0.1 % sodium citrate and 100 μg/mL RNAse (all from Sigma-Aldrich Co.) and incubated in dark for 15 min at room temperature. Cell fluorescence was determined by flow cytometry, as described above.

Intracellular reactive oxygen species
Intracellular reactive oxygen species (ROS) levels were measured using 2′,7′-dichlorofluorescin diacetate (DCF-DA) (Sigma-Aldrich Co.) [26]. Cells were collected, washed with saline and suspended in tubes with saline containing 5 μM DCF-DA for 30 min. Then, the cells were washed with saline, and cell fluorescence was determined by flow cytometry, as described above.

Animals
Forty-four C.B-17 severe combined immunodeficient (SCID) mice (males, six weeks old, 25-30 g) were obtained and maintained at Gonçalo Moniz Institute-FIOCRUZ animal facilities (Salvador, Bahia, Brazil). Animals were housed in cages with free access to food and water. All animals were subjected to a 12:12 h light-dark cycle (lights on at 6:00 a.m.). A local animal ethics committee approved the experimental protocol employed (number #06/2015).

Human leukemia xenograft model
HL-60 cells (1.5 × 10 7 cells per 500 μL) were implanted Data are presented as IC 50 values with their respective 95 % confidence interval obtained by nonlinear regression from three independent experiments performed in duplicate, quantified by alamar blue method after 72 h of incubation. Doxorubicin (DOX) and 5-fluorouracil (5-FU) were used as positive controls. subcutaneously into left front armpit of mice. When the tumors reached 100 to 200 mm 3 , animals were treated through intraperitoneal route (200 μL per animal) once a day for nine consecutive days. At beginning of the experiment, mice were randomly divided into four groups: group 1 animals received injections of vehicle (5 % DMSO solution) used for diluting EO (n = 11); group 2 animals received injections of doxorubicin (0.8 mg/kg, n = 11); group 3 animals received injections of EO at 50 mg/kg (n = 11); and group 4 animals received injections of EO at 100 mg/kg (n = 11). One day after the end of treatment, the animals were anesthetized, and peripheral blood samples were collected from the brachial artery. Animals were euthanized by anesthetic overdose, and tumors were excised and weighed. Inhibition ratio (%) was calculated by formula: inhibition ratio (%) = [(A-B)/A] x 100, where A is average tumor weight of negative control, and B is tumor weight of treated group.

Toxicological aspects
The mice were weighed at beginning and end of experiment to evaluation toxicological effects. Animals were observed for signs of abnormality throughout the study. A hematological analysis was performed using the Advia 60 hematology system (Bayer, Leverkusen, Germany). Livers, kidneys, lungs and hearts were removed, weighed and examined for signs of gross lesion formation, color change and/or hemorrhaging. After fixation in 4 % formaldehyde, histological analyses were performed for tumors and organs, under optical microscopy using hematoxylin-eosin and Periodic acid-Schiff (liver and kidney) staining.

Statistical analysis
Data were presented as mean ± S.E.M. or as IC 50 value with 95 % confidence intervals obtained by nonlinear regressions. Differences among experimental groups were compared through analysis of variance (ANOVA) followed by Bonferroni's Multiple Comparison Test (P < 0.05). All statistical analyses were performed using GraphPad Prism (Intuitive Software for Science; San Diego, CA, USA). . Negative control (CTL) was treated with vehicle (0.1 % DMSO) used for diluting EO, and doxorubicin (DOX, 1 μg/mL) was used as positive control. Data are presented as mean ± S.E.M. of three independent experiments performed in duplicate. Ten thousand events were evaluated per experiment, and cellular debris was omitted from analysis. * P < 0.05 compared with negative control by ANOVA, followed by Bonferroni's Multiple Comparison Test.
The presence of spathulenol along with some of major compounds were also found in other EOs from other Guatteria species [9,27,28]. In fact, spathulenol is considered a plausible chemotaxonomic marker of EOs of Guatteria [29]. On the other hand, chemical constituents of EO from this genus presented significant variations, which could be explained by climatic conditions, geographical localizations, soil characteristics and fertilization level, seasons, among other factors, which can cause such deviations.
In our cytotoxic screening program, we used a cut off limit where extracts/EOs with IC 50 values below 30 μg/mL are considered promising samples and are selected for our anticancer drug development program [15,[30][31][32]. G. megalophylla leaf EO presented IC 50 values below 30 μg/mL for most of cell lines tested and was selected for further studies.
To confirm the results obtained by Alamar blue method, we quantified the number of viable HL-60 cells by trypan blue exclusion method and annexin V-FITC/PI staining assay after 24 and 48 h incubation with EO at concentrations of 10, 20 and 40 μg/mL. EO significantly reduced the number of viable cells (Fig. 1) (internucleosomal DNA fragmentation), G 0 /G 1 , S and G 2 /M percentage distribution. Negative control (CTL) was treated with vehicle (0.1 % DMSO) used for diluting EO, and doxorubicin (DOX, 1 μg/mL) was used as positive control. Data are presented as mean ± S.E.M. of three independent experiments performed in duplicate. Ten thousand events were evaluated per experiment, and cellular debris was omitted from analysis. * P < 0.05 compared with negative control by ANOVA, followed by Bonferroni's Multiple Comparison Test. Ten thousand events were evaluated per experiment, and cellular debris was omitted from analysis. * P < 0.05 compared with negative control by ANOVA, followed by Bonferroni's Multiple Comparison Test.
66.2 % after 48 h. In addition, EO induced cell shrinkage, as observed by reduction in forward light scatter (FSC), and nuclear condensation, as observed by increasing in side scatter (SCC), both morphological alterations characteristic of apoptotic cells (Fig. 3).
Next, we examined intracellular DNA content and ROS levels in EOtreated HL-60 cells. Quantification of intracellular DNA content allowed measurement of internucleosomal DNA fragmentation and the cell cycle distribution. In this assay, all DNA that was sub-diploid in size (sub-G 1 ) was considered internucleosomal DNA fragmentation. EOtreated HL-60 cells presented an internucleosomal DNA fragmentation significantly increased after 48 h incubation (P < 0.05) (Fig. 4). At the concentrations of 10, 20 and 40 μg/mL, the sample increased DNA fragmentation to 31.5, 54.7 and 58.7 %, against 11.6 % observed for negative control, respectively. Doxorubicin (1 μg/mL) increased DNA fragmentation up to 68.1 % at same incubation time. The cell cycle phases, G 1 , S and G 2 /M, were reduced proportionally. Furthermore, the effect of EO in ROS levels was investigated in HL-60 cells after 1 and 3 h incubation. However, EO did not induce a significant increase in ROS levels (Fig. 5).
Corroborating with these data, Ferraz et al. [16] observed cell morphology consistent with cell death by apoptosis, increased internucleosomal DNA fragmentations and activation of caspase-3 in HepG2 cells treated with G. blepharophylla and G. hispida leaf EOs.

In vivo anti-leukemia effect of Guatteria megalophylla leaf essential oil
In vivo anti-leukemia effect of G. megalophylla leaf EO was evaluated in C.B-17 SCID mice with HL-60 cell xenografts. When the tumors reached 100 to 200 mm 3 , animals were treated with EO at doses of 50 and 100 mg/kg through intraperitoneal route once a day for nine consecutive days. EO significantly reduced tumor development at highest dose. One day after the end of treatment, the mean tumor weight of negative control group was 2.48 ± 0.40 g (Fig. 6A). In EO-treated groups, the mean tumor mass weights were 2.07 ± 0.19 g and 1.27 ± 0.24 g at lowest and highest doses, respectively. Tumor mass inhibition rates of EO was 16.6-48.8 % (Fig. 6B). Doxorubicin, at dose of 0.8 mg/kg, was used as positive control and reduced tumor weight by 49.4 %. Histology analysis of xenograft tumors showed a proliferation of malignant cells with abundant and granular cytoplasm, with two-or more distinct nucleoli, with characteristics similar to myeloid cells (Fig. 6C). In negative control and EO (50 mg/kg) groups, these malignant cells were often organized into small clusters of cells with scarce extracellular matrix. Instead, comparing with doxorubicin group, extensive necrosis areas were more pronounced in both EO (50 and 100 mg/kg) groups, but more extensive in EO (100 mg/kg) group.
Some toxicological aspects were also investigated in C.B-17 SCID mice with HL-60 cell xenografts treated with EO. After treatment, 100 % survival (11/11) rates have been observed for all groups with exception for doxorubicin-treated mice that showed survival rate of ∼ 73 % (8/11). No significant changes on body and organs (liver, kidney, lung and heart) weights were seen on EO-treated groups (P > 0.05) (Fig. 7). A decrease of body weight was found in doxorubicin-treated group (P < 0.05). In hematological analysis, all parameters remained unchanged after treatment with G. megalophylla leaf EO (P > 0.05) (Fig. 8).
In vivo antitumor effect of G. friesiana and G. pogonopus leaves EOs was previously investigated in murine model using Sarcoma 180-bearing mice [4,9]. The first presented tumor growth inhibition rates of 43.4-54.2 % and 6.6-42.8 %, when administrated by intraperitoneal (50 and 100 mg/kg) and oral (100 and 200 mg/kg) routes, respectively [4]. The second displayed tumor growth inhibition rate of 25.3-42.6 %, when administrated by intraperitoneal route at 50 and 100 mg/kg, respectively [9]. Herein, G. megalophylla leaf EO inhibited leukemia cells development as observed in a xenograft model in mice. Tumor growth inhibition rate was 16.6-48.8%, when administrated by intraperitoneal route at 50 and 100 mg/kg doses, respectively. Moreover, although low selectively of EO against cancer cells versus non-cancer cells was found in vitro experiments, no significant side effect was observed in vivo model, suggesting safe anticancer potential of G. megalophylla leaf EO.
Morphological analyses of lungs, liver, kidneys and heart were  performed for all experimental groups. In liver, acinar architecture and centrilobular vein were preserved in all groups. Histopathological changes included congestion, hydropic degeneration, chronic inflammation in liver portal space and focal areas of coagulation necrosis, ranging from mild to moderate. It is important to note that these histopathological characteristics were more pronounced in EO (100 mg/ kg) group and doxorubicin than in other groups (negative control and EO group -50 mg/kg). In lungs, architecture of parenchyma was partially maintained in all groups. Histopathological changes ranged from mild to severe and, frequently, focal inflammation, edema, congestion, hemorrhage, and increased airspace were observed in all experimental groups. In kidneys, tissue architecture was maintained; however, some histopathological changes were observed in all experimental groups, such as moderate vascular congestion and thickening of basal membrane of renal glomerulus, ranging from mild to moderate, with decreased urinary space. Importantly, focal areas of coagulation necrosis were observed in some animals treated with EO (100 mg/kg). The hearts of animals did not show alterations in any group.
In conclusion, G. megalophylla leaf EO has anti-leukemia potential, in which the main constituents spathulenol, γ-muurolene, bicyclogermacrene, β-elemene and δ-elemene may play a central for the recorded activities.

Data availability
The datasets used or analyzed during the current study are available from the corresponding author on reasonable request.

Ethical approval
The study was done after agreement from the local ethics committee.

Declaration of Competing Interest
The authors declare that they have no conflicts of interest regarding the publication of this paper.