Background
Cancer is one of the most important causes of death worldwide. In 2012, 8.2 million cancer-related deaths were reported and the number of new cases is expected to rise by about 70% over the next two decades [
1]. Cancer is characterized by the uncontrolled and invasive growth of cells. The most peculiar feature of cancer cells is their ability to metastasize to other specific organs. For example, prostate and colon cancer metastasize to bones and liver, respectively; lung cancer cells spread to adrenal glands, liver, brain, and bones, while breast cancer cells metastasize to lungs and bones [
2]. For the most important adenocarcinomas, such as lung, breast, or colorectal cancer, the treatment is barely effective, due to metastatic disease responding only transiently to conventional treatments [
3]. Therefore, scientists are still utilizing new alternatives in attempts to find novel compounds and strategies for the treatment of this disease [
4].
Plants are a valuable source of new biologically active molecules due to the presence of hundreds of biologically active components [
5]. One of the plant families i.e., Apocynaceae, is well-known for anticancer activities, mainly in the genera of
Catharanthus,
Nerium,
Strophanthus, Apocynum and
Thevetia [
6].
Thevetia peruviana (Pers.) K. Schum, also known as
Cascabela peruviana (L.) Lippold, yellow oleander or lucky nut in the West Indies, shows a diverse array of properties ranging from being a cardiotonic to a toxin.
T. peruviana plant has been used traditionally for the treatment of gastrointestinal and inflammatory diseases, heart failures and skin tumors [
7,
8]. All parts of the plant are poisonous due to the presence of cardiac toxins, but the fruit of
T. peruviana, is the most toxic part of the plant because it has the highest and diverse content of cardiac glycosides. There are many known cases of intentional and accidental poisoning of humans through ingestion of fruits and leaves [
9]
. Whereas about 10 fruits consumed may be fatal for an adult, a single fruit may be lethal for a child. The common clinical set of symptoms resembles digitalis poisoning with marked nausea, vomiting, abdominal pain, diarrhea, dysrhythmias, and hyperkalemia [
10]. Although the anticancer potential of parts of
T. peruviana plant, such as leaves, bark and seeds has been evaluated against human gastric and pancreatic cancer cell lines [
11], the anticancer potential of
T. peruviana fruit is still unknown because only a few cardiac glycosides have been identified and examined as cytotoxic agents. Therefore, the
T. peruviana fruit extract has been investigated in this study to explore its anticancer potential against the most common cancer types (lung, breast, prostate and colorectal), in terms of morphological analysis, motility and cell adhesive properties, DNA damage and induction of apoptosis in human cancer cell lines.
Methods
A T. peruviana plant specimen was collected in San Nicolás de los Garza, N.L., México (25°43′59.57″N, 100°16′4.75″W). Botanical authentication of the material was performed by the Botany Department Staff of Facultad de Ciencias Biológicas, UANL. A specimen of T. peruviana was recently deposited in the Herbarium of the Facultad de Ciencias Biológicas, Universidad Autónoma de Nuevo León (voucher UNL-028732).
Extraction and preparation of plant samples for testing
The plant extracts were obtained from samples (20 g) of dried roots, leaves, or aerial parts through maceration with methanol (3 × 300 mL) at room temperature for 24 h and under continuous shaking. The methanolic extracts were filtered and evaporated under reduced pressure using a rotary evaporator (Yamato RE801). Samples were stored at −20 °C prior to further experiments. For the cytotoxicity assays, the dried methanol extract was dissolved in dimethylsulfoxide (DMSO) in order to obtain a final concentration of 100 mg/mL (stock) and diluted in PBS (phosphate buffer saline). DMSO concentration in the culture medium was less than 0.1%. Doxorubicin (10 μg/mL, Zytokil) treated cells and untreated cells were used as positive control and negative control, respectively. The methanolic extract of T. peruviana was fractionated by column chromatography. Briefly, 1.5 g of fruit extract was adsorbed onto Celite 545 and subjected to reverse flash chromatography on a 50 g RediSep Rf Gold HP C18 column, eluting with 10:90 MeOH-H2O for 4 CV. Then a linear gradient from 10:90 MeOH-H2O to 100% MeOH for 25 CV, holding 100% MeOH for 8 CV, at a flow rate of 40 mL/min to give 120 fractions each containing 12 mL. The resulting fractions were then pooled according to their ELSD and UV profiles, which resulted in six combined fractions in total. All fractions were examined by UPLC-PDA-HRMS-MS/MS and tested for cytotoxic activity against human prostate carcinoma cell line (HTB-81) by the MTT colorimetric assay.
Cell lines and cell culture
The cancer cell lines used in this study were human colorectal adenocarcinoma (HTB-38), lung carcinoma (HTB-177), prostate adenocarcinoma (HTB-81), and breast adenocarcinoma (HTB-22), whereas the normal cell lines used were human skin fibroblast (CCL-116) and Vero cell line (CCL-81). Cell lines were obtained from the American Type Culture Collection (ATCC). Cell lines were cultured in DMEM or RPMI (only for HTB-81) supplemented with 10% (v/v) fetal bovine serum (Byproductos). All cells were cultured at 37 °C under a humidified atmosphere containing 5% CO2.
Cytotoxicity assay
For the cytotoxicity assay, 5 × 10
4-6 × 10
4 cells for colorectal, lung, breast, prostate cancer cells, and normal cells were seeded in 96-well tissue culture plates. When the cells had reached 75% confluence, they were incubated using the methanol extracts at various concentrations. After 24 h of incubation, the cytotoxicity was assessed using the MTT assay as described previously [
12]. To obtain the half maximal inhibitory concentration (IC
50)
, the percentages of cell viability and growth inhibition were calculated according to the following equations [
13]. Cell viability (%) = [(OD of treated cells-OD of blank)/ (OD of control-OD of blank)] × 100. Growth inhibition (%) = 100 – Cell viability (%). All determinations were performed five times independently with three technical replicates.
Clonogenic assay of cell in vitro
The clonogenic assay was carried out as previously described [
14]. 100–130 cells/well and incubated for 24 h at 37 °C with 5% CO
2 to lead attach. The cells were treated with the corresponding IC
50 for 24 h and were cultured for 7–14 additional days. Then, the cultures were stained with 0.5% crystal violet for 30 min. Colonies containing more than 50 cells (after 10–14 days of incubation) were counted using ImageJ software [
15]. Three independent experiments were performed with three technical replicates each one.
Wound and healing assay
Wound and healing assay was carried out as previously described [
16]. 5 × 10
4–6 × 10
5 cells were seeded and grown overnight to reach 100% confluency. The monolayer was treated with the corresponding IC
50 for 24 h. The cells were migrated into the scratched area and photographed (camera infinity 1–2, Lumenera Corp., CA) every 24 h. The migrated cells were expressed as a mean value per field. Three independent experiments were performed with three technical replicates each one.
Cell morphology and membrane permeability assays
Cells were seeded in a 96-well plate for 24 h. After attachment, the cells were treated with
T. peruviana fruit extract using the IC
50 concentration for 4, 8, 16 and 24 h. For cell morphology, the cells were observed using an inverted microscope (Olympus IX71). Trypan blue assay was used for the permeability assay (0.4% trypan blue/per well) [
17]. As a negative control, cells were cultivated in the same plate without the plant extract. Each sample was observed under the microscope and photographs were taken immediately after staining. Three independent experiments were performed with three technical replicates.
DNA fragmentation analysis
DNA fragmentation was carried out as described previously [
18]. Cells were grown in the presence or absence of
T. peruviana fruit extract using the IC
50 concentration for 24 h. Doxorubicin treated cells were used as positive control. Briefly, 5 × 10
5 cells were lysed in DNA lysis buffer [1 M Tris-HCl (pH 8.0), 0.5 M EDTA, 100% Triton X-100, 2% SDS, 0.2 M NaCl], and then, DNA was extracted. The nucleic acid concentration and purity were measured using a NanoDrop® ND-2000 spectrophotometer (Thermo Scientific). Equal amounts of DNA (10 μg/well) were electrophoresed in 1% agarose gel. DNA fragments were visualized using an UV transilluminator (MutilDoc-it Digital Imaging System UVP). Three independent experiments were performed.
Dual acridine orange/ethidium bromide (AO/EB) fluorescent staining
For the AO/EB method, 5 × 10
5 cells were seeded in a 6-well plate. Cells were treated with
T. peruviana fruit extract at the corresponding IC
50 concentration for 4 h. Then cells were subjected to AO/EB staining as described previously [
19]. Briefly, cells were tripsinized and re-suspended in cold PBS and AO/EB dye mix (100 μg/mL AO and 100 μg/mL EB; Sigma) was added. Stained cell suspensions (10 μL) were viewed and counted using a Nikon eclipse TS100 inverted microscope at 40× magnification with excitation filter 480/30 nm and barrier filter 535/40 nm. Six fields per sample were examined. The AO/EB staining method was repeated 3 times.
Liquid chromatography - Mass spectrometry analysis
HRESIMS data were collected and in positive and negative ionization modes using a Thermo QExactive Plus mass spectrometer (ThermoFisher) equipped an electrospray ionization (ESI) source and via an Acquity UPLC system (Waters Corp). The higher-energy collisional dissociation (HCD) cell used a normalized collision energy of 30 eV for all the compounds to obtain MS/MS data. The UPLC separation was performed using an Acquity BEH C
18 column equilibrated at 40 °C and a flow rate set at 0.3 mL/min. The mobile phase consisted of 15% CH
3CN–H
2O (0.1% formic acid) for 0.5 min, and then a linear gradient from 15% CH
3CN to 100% CH
3CN over 6 min, and 1 min holding 100% CH
3CN before returning to the starting conditions. Samples were dissolved in MS grade methanol and filtered through a 0.2 μm Acrodisc (Waters) filter. Tentative metabolite identification was performed by comparison of HRMS data, UV maxima and fragmentation patterns (MS/MS data) with those contained in the Dictionary of Natural Products [
20] reference compounds refined for
Thevetia plant metabolites.
Data Analysis
Statistical analysis was performed using GraphPad Prism 7 software. The
p-value was analyzed in comparison to the untreated samples using Student’s
t-Test or using one-way analysis of variance (ANOVA) followed by a Tukey test for comparison between different treatment groups. Differences were considered statistically significant at
p < 0.05. Results were expressed as mean ± SEM of data obtained from tripled or quintuplet, independent experiments. Multivariate analysis was performed using the R 3.2.5 environment. Three biological replicates for each cell line were evaluated, and the variables used were the IC
50, the clonogenic assay (CL), the membrane permeability (MP) and the wound-healing assay (WH). A standardized principal component analysis (PCA) was performed with “ade4” package [
21], followed by an independent component analysis (ICA) applied to the PCA patterns [
22].
Discussion
T. peruviana belongs to the Apocynaceae family, a plant which is native to central and southern Mexico, as well as Central America. It is a medicinal plant used to treat different diseases, including cancer [
27]. The leaves of yellow oleander had previously been studied and shown to possess antimicrobial, antifungal, antidiarrheal, insecticide, molluscacide, and rodenticide activity [
8,
28‐
30]. However, scientific evidence to demonstrate mode of action, targets and agents responsible for the bioactivity in the fruit is still needed. The purpose of the present study was to find out the cytotoxic and anti-proliferative activity of methanol extracted fruit of
T. peruviana on different types of human cancer cell lines.
Our experiments in vitro showed that
T. peruviana fruit extract exhibited strong cytotoxicity against four cancer cell lines. Among the cell lines examined, lung cells showed higher IC
50 value (12.04 μg/mL) than prostate (1.91 μg/mL), breast (5.78 μg/mL), and colorectal (6.30 μg/mL) cells. Such variation among the cell lines might be attributed in part to the fact that cancer cells possess differences in their genetic make up, morphology and doubling time, resulting in differential susceptibility to the same cytotoxic agent [
31]. Our results, together with previously reported toxic activity, suggest that
T. peruviana fruit methanol extract has an antiproliferative potential.
Cancer is a complex disease characterized by proliferation of highly resistant cells to death. An increased rate of cellular proliferation is frequent, due to the fact that most cancer cells divide more often than normal cells. The goal of targeting cell proliferation is to arrest the cell cycle or induce cancer cell death using cytotoxic compounds. We show here, that
T. peruviana fruit extract significantly reduced the cell viability and the ability of cells to form colonies in four different human cancer cell lines. The clonogenic assay has been used to detect cells that have retained their capacity to produce a large number of progeny after radiation and chemotherapy treatments [
32]. It also correlates tumorigenicity analysis in vivo and predicts the clinical response toward several agents in breast cancer patients [
33]. Hence, our results in clonogenic assay suggest that
T. peruviana fruit extract has potential anticancer activity, limiting proliferation of cancer cells after treatment. Notably, normal cells (vero and fibroblast) treated with
T. peruviana extract did not show any observable effect in clonogenic assay.
Migration is a critical step in initial progression of cancer that facilitates metastasis. Wound and healing assay is a classic and common method used for discovery and validation of molecules that affect cell migration [
16,
34] and metastasis [
35]. The methanolic
T. peruviana fruit extract inhibited and delayed the cell migration of cancer cells, but not among normal cells. These results open the door to further studies that could confirm if the cytotoxic activity of
T. peruviana fruit extract alters the regulation of the actin cytoskeleton, induces morphological changes and leads to detachment of cells, culminating in cell death. Moreover, motility and membrane permeability features were mainly affected by
T. peruviana fruit extract, according with the multivariate analysis performed on the six cell lines and the four variables evaluated, suggesting a potential antimetastatic activity in the
T. peruviana fruit extract.
One crucial and desirable mechanism by which chemotherapeutics destroy tumor cells is by inducing apoptosis. Cells undergoing apoptosis show morphological and biochemical modifications including chromatin segregation, nuclear condensation, DNA fragmentation, partition of the membrane, and vesicles formation [
36,
37]. The late-stage of apoptosis can be visualized by standard agarose gel electrophoresis as a ladder pattern due to DNA cleavage [
38], while both (the early and late stages of apoptosis) can be determined by AO/EB fluorescent staining.
Cerbera manghas, a plant belonging to the Apocynaceae family, contains a cardiac glycoside (neriifolin), which induced DNA fragmentation on hepatocellular carcinoma 48 h after treatment [
39]. Here, crude extract from
T. peruviana fruit showed death induction on cancer cell lines through early apoptosis mechanisms (AO/EB fluorescent staining) 4 h after treatment and late-stage apoptosis (DNA laddering assay) after 24 h of treatment. This result indicates that
T. peruviana could have a higher level of apoptotic activity than other members of the Apocynaceae family. In addition, it will be important to determine whether the apoptotic activity is located in the compounds present in the flesh or seeds.
Mass spectrometry analysis of active fractions from
T. peruviana fruit methanol extract indicated that one flavonoid and cardiac glycosides are secondary metabolites present in the fruit plant. Flavonoids, such as curcumin, quercentin and genistein, are known to have cell line-specific anti-proliferative and apoptosis inducing activity [
40‐
42]. It has been postulated that flavonoids possess anticancer properties manifested through several mechanisms, including decrease of reactive oxygen species, inhibition of DNA topoisomerase and downward regulation pathway of nuclear transcription factors [
43‐
46]. Further experiments are required to determine the cytotoxic effect of thevetiaflavone and individual cardiac glycosides present in the methanolic fruit extract of
T. peruviana on human cancer cell lines. Recent investigations of seeds from
T. peruviana resulted in the isolation of cardiac glycosides that had inhibitory effects against human gastric and pancreatic cancer cell lines [
11]. Cardiac glycosides are the most researched secondary metabolites in
T. peruviana due to the fact they can heal heart pathologies [
47], but are also being studied for their cytotoxic and/or apoptotic activities against myeloid leukemia (peruvoside) [
48] and hepatocellular carcinoma (neriifolin) [
39]. Thevetin and peruvoside are cardiac glycosides which are clinically important constituents due to be used in treatment of arrhythmias [
49]. One hundred nine cardenolides have been isolated and identified from members of the Apocynaceae family, and about a quarter of them are reported to have anticancer activity. Cardenolides are well known as the substrates of Na+/K + −ATPase, which is an enzyme that regulate various cell survival and death signal pathways [
6] and its relative distribution and expression are distinct in cancer cells compared with normal ones, indicating that they could serve as a novel target with great potential. This could be the reason that multivariate analysis showed an adequate separation between normal and cancer cells, revealing significant differences in their sensitivity to the toxic compounds in the
T. peruviana fruit methanolic extract, mainly due to the effect of the extract on the motility and membrane permeability on human prostate, breast, colorectal and lung cancer cell lines. The cytotoxic activity of crude extract from
T. peruviana fruit could be attributed to their phytochemical components such as thevetin A, thevetin B, peruvoside, thevenerin, and cerberin, which are toxins [
50], and also to the cardiac glycosides found in active fractions reported in this study (thevefoline, solanoside, neriifoside, peruvoside and neriifolin). Finally, the isolation of the active principles of the methanolic extract of
T. peruviana fruit is currently being undertaken to investigate their cytotoxic, molecular and genetic action mechanisms, which could provide meaningful perspectives for biomedical and biotechnological research.
Acknowledgements
We are grateful to MD Nancy B. Gordon (MD Anderson Cancer Center), for kindly providing training in tissue culture techniques, as well as Dr. Mariana Elizondo Zertuche and Álvaro Colín Oviedo for their support with the fluorescence microscope (CONACYT-INFRA 2015-251142) from Departamento de Microbiología, Facultad de Medicina, UANL. MF thank to CONACyT for the grant INFRA 252226. We thank Yesenia Cristal García Silva for her excellent technical support and express appreciation to Dr. Angel Andrade and Dr. Greoffrey Cordell for their critical examination of the manuscript.