content in MCF-7 cells. Cells were incubated under the following conditions: none (Control), Fractions F1-F3. Data are mean ± SD. Values significantly different from those of the control group: *p < 0.05.

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 [21].

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 [24]. The administration of the T. megapotamicum Fractions to MCF-7 cells was reflected in a decrease of γ-GT activity. Since, γ-GT expression is regulated by the cytosolic glutathione pool and steroids [25,26], we speculated that the decreased Fractions-induced activity might generate a fewer intracellular glutathione to sustain the enzymatic activity level necessary into the cells. So, a γ-GT reduction may participate in the mechanism of Fractions sensibility and increase the apoptosis on MCF-7 cells.

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 [29]. SA, the end moieties of the carbohydrate chains are biologically important and essential for functions of glycoconjugates and are reported to be altered in cancer patients. A positive correlation between serum levels of different forms of SA and extent of malignant disease was observed [30]. Glycosylation has been demonstrated to play a critical role during malignant transformation. Patients with breast cancer had significantly higher levels of different forms of SA as compared to the controls [31,32].

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 [7]. Previous investigators have shown that both family compounds have anticancer effects [12,35]. Meanwhile, we have not yet specifically looked into the active ingredient(s) of the T. megapotamicum that inhibits the growth of breast cancer.

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.

5. Acknowledgements

This work was partially supported by grants from the Consejo Nacional de Investigaciones científicas y Técnicas (CONICET-AVCR) No. 962/07-05-2009.

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Abbreviations:

TLC: thin layer chromatography;

γ-GT: gamma glutamyltranspeptidase;

SA: sialic acid;

FITC: fluorescein isothiocyanate;

PI: propidium iodide;

PS: phosphatidylserine.

NOTES

*There is no conflict of interest with regard to this manuscript.

#Corresponding author.

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