Thelesperma megapotamicum (Asteraceae) is commonly used in Argentine to treat various diseases (renal, digestive affections, and as anaesthesia). The present study showed the mechanisms involved “ in vitro” cytotoxicity of T. megapotamicum Fractions. Five Fractions (F1 - F5) were separated by column chromatography (Silica gel) using hexane:diethyl ether as eluents. Viability was evaluated in Human breast carcinoma cell line (MCF-7) by staining with crystal violet. With respect to F1 Fraction treatment, the cell survival was 49.14% ± 8.87%, while the F2 and F3 ones exhibited a strong reduction of cell viability to only 26.35% ± 1.63% and 23.3%1 ± 0.53% of the control cell at 50 μg/ml, respectively. Apoptotic effect of these Fractions was detected using FITC-labeled Annexin V and propidium iodide binding assays and was confirmed by a higher proportion of apoptotic cells due to F2 and F3 treatments. T. megapotamicum active Fractions could facilitate the tumoral cells death by decreasing the activity of the enzyme Gamma-glutamyltranspeptidase and causing alteration in cell membrane sialoglycoconjugates and others involved anticancer mechanisms including apoptosis.
Breast cancer is the most commonly occurring non-skin malignancy among women and the second leading cause of cancer-related death. Approximately 1 in 8.2 women will receive a diagnosis of breast cancer during her lifetime, and 1 in 30 will die of the disease [
More than 80% of the world populations use the traditional medicine to satisfy their needs of primary care of the health and that great part of the traditional treatments implies the use of extracts plants or their active principles [
Argentine medicinal plants have a long-standing history in many indigenous communities and are still useful resources for treating diseases. Subsequently, pharmacological studies (activity reports) are necessary in order to extend the scarce ethnopharmaceutical data, which have given empirical support to the medicinal use of these regional plants based on traditional knowledge [
Since the native flora may contain some anticancer compounds, it has become a valuable resource looking for a chemopreventive agent. Regarding this, the medical potential of Argentine plants has been recorded based on their traditional use, with an ethnobotanical survey of central Argentina being carried out over a 26-year period (1979-2005) [
Thelesperma megapotamicum belongs to Asteraceae family, it is known as Pampa tea or Indian tea [
Accordingly, the aim of this work was to elucidate the mechanisms involved in T. megapotamicum Fractions in an “in vitro” cytotoxicity representative model and also to study the effect over some membrane components.
Plant recollection was carried out at Santa María de Punilla, “Sierras de Córdoba”. This side is located at approximately 45 km north of “Córdoba”, Argentine. Plant samples were collected during spring 2008, immediately transported to the lab and a voucher specimen was deposited in the International Herbarium of the National University of Rio Cuarto, Argentina (RIOC420).
The rhizome (12 g) was thoroughly extracted with 50 ml of hexane: ethyl ether (1:1 V/V) by maceration at room temperature for 24 hours under stirring. A supernatant was obtained by filtration, and concentrated under reduced pressure.
The hexane:diethyl ether rhizome extract was subjected to Silica gel-60 column chromatography (15.5 cm length, 0.9 cm i.d.) using hexane: ethyl ether as eluents; 50 fractions of 5 ml were checked by thin layer chromatography (TLC) and observed without chemical treatment, under UV 254 nm light. On the basis of the similar TLC profile the fractions were pooled into 5 groups: F1 - F5.
For the “in vitro” viability test, the dried Fractions were dissolved in DMSO at a concentration of 50 mg/ml.
Human breast carcinoma cell line (MCF-7, cell line ATCC HTB-22), were cultured in Dulbecco’s Modified Eagle’s medium with 10% (V/V) fetal bovine serum, 100 IU/ml penicillin G and 40 μg/ml gentamycin sulphate, incubated in a humidified atmosphere of 5% CO2 at 37˚C.
After 48 hours attachment in a 96 well-plates (2 × 104 cells/well) viable cells were incubated with 0 and 50 μg/ml of each Fraction (F1 - F5) for 6 hours. Afterwards, the medium containing extracts was removed and cell viability was determined by measuring the absorbance at 570 nm using crystal violet as followed. Viable cells were stained with 0.5% crystal violet in 50% methanol for 15 min. After washing with 50% methanol three times, the staining cells were solubilized with 20% methanol in sodium citrate solution (0.1 M, pH 5.4). Results, consistent with cellular density, were recorded by an Anthos Labtec 2010 micro ELISA reader at 570 nm. The viability percentage was defined as the relative absorbance of treated versus untreated control cells (100%). To compare the effectiveness of the extracts with a clinical drug, a concentration of 0.1 µM of Paclitaxel and 40 µM of Luteolin (Sigma Chemical Co.) were also tested as positive controls.
Apoptosis was analyzed by flow cytometry using fluorescein isothiocyanate (FITC)-labeled Annexin V and propidium iodide (PI) staining. Briefly, the cells were seeded in 6-well plates at a density of 1 × 106 cells/ml and incubated for 24 hours at 37˚C as above. The medium was then replaced with fresh medium containing the different Fractions (0, 50 µg/ml) and the cells were incubated at 37˚C for 6 hours. The cells that had become detached from the surface of the flask (floating cells), together with those which remained attached (harvested by trypsinization), were pelleted by centrifugation. Cell pellets were resuspended in complete medium (5 × 105 cells/ ml), with binding buffer and incubated with FITC-labeled Annexin V and PI (at a final concentration of 1 and 2 μg/ml, respectively) for 15 min. The stained cells were analyzed on a COULTER EPICS® flow cytometer. Cells negative for both FITC-labeled Annexin V and PI staining are live cells. Annexin V-positive and PI-negative staining cells are undergoing early stages of apoptosis. PIand Annexin V-positive staining cells are late apoptotic cells, and PIpositive and Annexin V-negative staining cells are necrotic cells [
The MCF-7 cells were seeded at a density of 1 × 106 cells/well onto 6-well culture plates and incubated for 24 hours at 37˚C, humidified air and 5% CO2. Then 50 μg/ml of the F1, F2 and F3 Fractions was added to the wells and the cells were incubated for 6 hours in the same conditions. After enzymatic harvesting with porcine trypsin, cells were homogenized at 20.000 rpm for 30 seconds in 1 ml of 10 mM HEPES buffer (pH 7.4, containing 2 μg/ml leupeptin and 1mM EDTA). They were centrifuged at 100.000 g for 1 h at 4˚C to recover the pellet, which was then resuspended in 200 μl of 10 mM HEPES buffer. The protein content was determined according to the Bradford method, and each suspension (150 μl) was mixed with 750 μl chloroform/methanol
(2:1 V/V, Fölch extraction). The mixture was centrifuged at 1.000 g for 10 min, and then the upper layer was used for sialic acid determination.
To determinate gamma glutamyltranspeptidase (γ-GT) activity the modified Szasz method was used following the γ-GT-test kinetic AA kit manufacturer’s instructions [
After Fölch extraction, the Sialic acid (SA) content (nmol/mg of protein) was measured in the upper phase at 580 nm according to Miettinen and Takki-Luukkainen [
Culture reagents and other chemicals were obtained from the Sigma-Aldrich Co. (USA). The γ-G-test kinetic AA kit™ for in vitro diagnosis was from Wiener Laboratories.
Data were expressed as mean ± standard deviation (SD) from four separate experiments performed in triplicate, unless otherwise noted. ANOVA models were used to evaluate differences of cellular viability and membrane parameters (γ-GT and SA) among treatments. The association between these variables was determined using the correlation coefficient (CC). Analytical probes were performed using the InfoStat 2007e.1 software.
In order to evaluate the Fractions cytotoxic effect, different experiments with the MCF-7 cell line were run. The survival rates in the Fractions treated MCF-7 cells, determined by Crystal violet staining, are shown in
hand, Fractions F4 and F5 had lower cytotoxic effect (77.09% ± 20.00% and 73.21% ± 7.73% survival compared to control at 50 µg/ml, respectively). With regard to 0.1 µM of Paclitaxel, this antitumoral drug was not cytotoxic towards the MCF-7 after 6 hours of incubation (data not shown), these results are in agreement with previous reports [
The apoptotic nature of T. megapotamicum Fractionsinduced cell death was confirmed using FITC-labeled Annexin V and PI binding assays as useful markers of early apoptotic and necrotic events, respectively. Stained cells were identified by flow cytometric analysis and expressed as a percentage of the total number of cells in each case.
This included cells that had become detached from the surface of the flask, as a result of poor health of the cells (including apoptotic and necrotic cells), but which had remained intact. The flow cytometric analysis excluded debris and highly condensed cells by gating on intact cells, based on light scatter. Consistent with the data shown in
The lipid sialylation and the γ-GT were studied as markers of the cell membrane status. The specific activeity of γ-GT, depicted in
p ≤ 0.02), and Fraction F3 (14.7 fold lower; p ≤ 0.02) with respect to control. MCF-7 cells had basal levels of SA in their membranes of 232.2 ± 64.3 pmole of SA/mg of proteins, being the SA content in MCF-7 incubated with Fractions F1, F2 and F3 lower than control (p ≤ 0.05): 2.7, 3.6 and 5.7 folds respectively (
Carcinogenesis involves a growing accumulation of genetic and epigenetic aberrations, leading to the deregulation of cellular homeostasis, followed by neoplastic progression [
tamicum active Fractions on cells viability, γ-GT activity and membrane sialylation in MCF-7 cell culture.
The results reported herein reveal that T. megapotamicum exerts growth inhibition in cultured human breast cancer MCF-7 cells; this cell line showed a high sensibility to T. megapotamicum active organic Fractions at a very low incubation times (6 hours). We further demonstrate that these Fractions are much more effective in decreasing the cell viability compared to the commonly used chemotherapeutic agent, Paclitaxel. Previous reports shown that Paclitaxel at 0.01 and 0.1 µM, did not induce significant death of these cancer cells, within 48 hours [
Our results suggest that F2 and F3 Fractions could facilitate the tumoral cells death by apoptosis, as indicated by the FITC-labeled Annexin and Propidium Iodide Fluorescence Assay. At the 50 μg/mL, for Fractions F2 and F3 on MCF-7 cells, FITC-labeled Annexin V fluorescence was detected substantially, as early as 6 hours post treatment. This showed that the plasma membrane of MCF-7 cells had incorporated FITC-labeled Annexin V, indicating that PS had been translocated from the inner to the outer leaflet of the plasma membrane (early step in apoptosis).
The γ-GT, a key enzyme of glutathione metabolism, can modulate crucial redox-sensitive functions, such as antioxidant/antitoxic defenses and cellular proliferative/ apoptotic balance, with potential implications in tumor progression and drug resistance [22,23].
Previous studies have shown the possible role of membrane γ-GT enzyme for evaluating anticancer drugs. This enzyme is located on the outer aspect of plasma membrane of most cell types, and is often expressed at high levels in malignant tumors and their metastases [
Gangliosides (sialylglicolipids) have previously been found to be involved in cellular surface-relates regulation and in biomembrane resistance to oxidation [27,28]. Furthermore, since some cancer cells present aberrant glycolsilation, with the SA content being a useful tumor marker [
The basal levels of SA in cells membranes incubated with different Fractions of T. megapotamicum responded decreasing its values corresponding to a decrease in the rate of cell viability.
These data suggest that the active Fractions of T. megapotamicum, apart from their effects on cellular growth, could inhibit the expression of membrane SA.
In this work, the pharmacological regulation of Fractions could be important, since ganglioside molecular interactions are involved in cancer growth due to their role in the immune response and in the metastatic process [33,34].
Taking together, T. megapotamicum active Fractions could regulate the MCF-7 cells death by decreasing the activity of the enzyme γ-GT, and causing alteration in cell membrane sialoglycoconjugates and others involved anticancer mechanisms including apoptosis.
T. megapotamicum might contain flavonoids, such as Luteolin, and phenylpropanoids [
In this work, organic “Pampa tea” Fractions have shown pronounced activity. Further evaluation will be done in the future for possible isolation of active anti-tumor compounds.
This work was partially supported by grants from the Consejo Nacional de Investigaciones científicas y Técnicas (CONICET-AVCR) No. 962/07-05-2009.
TLC: thin layer chromatography;
γ-GT: gamma glutamyltranspeptidase;
SA: sialic acid;
FITC: fluorescein isothiocyanate;
PI: propidium iodide;
PS: phosphatidylserine.