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The natural triterpene 3β,6β,16β-trihydroxy-lup-20(29)-ene obtained from the flowers of Combretum leprosum induces apoptosis in MCF-7 breast cancer cells.

Viau CM, Moura DJ, Facundo VA, Saffi J - BMC Complement Altern Med (2014)

Bottom Line: We found that the treatment with IC50 and IC80 TTHL for 24 h induced apoptosis in 14% (IC50) and 52% (IC80) of MCF-7 cells.In order to further understand the biological mechanism of TTHL-induced cytotoxicity, we have also investigated its effect on different Saccharomyces cerevisiae yeast strains.Taken together, the results suggest that TTHL forms covalent adducts with cellular macromolecules, potentially disrupting cellular function and triggering apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Basic Health Sciences, Laboratory of Genetic Toxicology - UFCSPA, Porto Alegre, RS, Brazil. jenifers@ufcspa.edu.br.

ABSTRACT

Background: The 3β, 6β, 16β-trihydroxylup-20(29)-ene (TTHL) is a pentacyclic triterpene obtained from the medicinal plant Combretum leprosum Mart. In folk medicine, this plant is popularly known as mofumbo, cipoaba or mufumbo, and is used to treat several diseases associated with inflammation and pain.

Methods: We investigated the antitumor efficacy of TTHL isolated from C. leprosum. The TTHL cytotoxic effect was investigated in MRC5, MCF-7, HepG2, T24, HCT116, HT29, and CACO-2 cells after 24, 48, 72 and 120 h of treatment. The mechanisms of cell death and DNA damage induction were investigated by flow cytometry and comet assay, respectively.

Results: The results indicated that TTHL induced a time- and concentration-dependent growth inhibition in all human cancer cell lines. The cytotoxicity was more pronounced in MCF-7 breast cancer cells, with an IC50 of 0.30 μg/mL at 120 h. We therefore evaluated the cell death mechanism induced by TTHL (IC20, IC50, and IC80) in MCF-7 cells at 24 h. We found that the treatment with IC50 and IC80 TTHL for 24 h induced apoptosis in 14% (IC50) and 52% (IC80) of MCF-7 cells. The apoptosis induced by TTHL was accompanied by increased levels of both cleaved caspase-9 and intracellular ROS. In order to further understand the biological mechanism of TTHL-induced cytotoxicity, we have also investigated its effect on different Saccharomyces cerevisiae yeast strains. The mutant strains sod1Δ, sod2Δ, and sod1Δsod2Δ, which are deficient in superoxide dismutase antioxidant defenses, were hypersensitive to TTHL, suggesting that its capacity to disturb cellular redox balance plays a role in drug toxicity. Moreover, TTHL induced mutagenicity in the yeast strain XV185-14c.

Conclusions: Taken together, the results suggest that TTHL forms covalent adducts with cellular macromolecules, potentially disrupting cellular function and triggering apoptosis.

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Flow cytometric analysis of cleaved caspase 9 activity. MCF-7 cells were treated with TTHL (IC20 = 0.50 μg/mL, IC50 = 1.36 μg/mL, and IC80 = 3.70 μg/mL), fixed, permeabilized, and stained with anti-active caspase 9. Results are expressed as means ± standard deviation (SD). Data were analyzed by one-way analysis of variance (ANOVA), and means were compared using Tukey test, with P ≤ 0.05 considered as statistically significant.
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Fig4: Flow cytometric analysis of cleaved caspase 9 activity. MCF-7 cells were treated with TTHL (IC20 = 0.50 μg/mL, IC50 = 1.36 μg/mL, and IC80 = 3.70 μg/mL), fixed, permeabilized, and stained with anti-active caspase 9. Results are expressed as means ± standard deviation (SD). Data were analyzed by one-way analysis of variance (ANOVA), and means were compared using Tukey test, with P ≤ 0.05 considered as statistically significant.

Mentions: In order to characterize the apoptosis induced in MCF-7 cell line, we evaluated the activation of apoptosis mediators by different TTHL concentrations. We analyzed the cleavage and consequent activation of caspase-9 by flow cytometry using specific antibodies that recognize only the intact (inactive) forms of the enzyme. The results, summarized in Figure 4, indicate that continuous treatment of cells with TTHL concentrations (IC50 and IC80) leads to increased induction of cleaved caspase-9-labeled cells. There is a clear difference on the induction of cleaved caspase-9 when the IC50 and IC80 values are compare (Figure 4), although the increased induction of cell death (sub-G1) in treated MCF-7 cells is confirmed (Figure 2, and Additional file1: Figure S8). Moreover, TTHL (at IC20) caused no significant changes in the levels of cleaved caspase-9 in MCF-7 cells (Figure 4). These results were consistent with the data from Annexin V-PE positive FACS analysis; in fact, the treatment with TTHL at IC50 and IC80 induced a stronger apoptotic effect on MCF-7 cells when compared to IC20 and control (Figures 2,3 and4).Figure 4


The natural triterpene 3β,6β,16β-trihydroxy-lup-20(29)-ene obtained from the flowers of Combretum leprosum induces apoptosis in MCF-7 breast cancer cells.

Viau CM, Moura DJ, Facundo VA, Saffi J - BMC Complement Altern Med (2014)

Flow cytometric analysis of cleaved caspase 9 activity. MCF-7 cells were treated with TTHL (IC20 = 0.50 μg/mL, IC50 = 1.36 μg/mL, and IC80 = 3.70 μg/mL), fixed, permeabilized, and stained with anti-active caspase 9. Results are expressed as means ± standard deviation (SD). Data were analyzed by one-way analysis of variance (ANOVA), and means were compared using Tukey test, with P ≤ 0.05 considered as statistically significant.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4129108&req=5

Fig4: Flow cytometric analysis of cleaved caspase 9 activity. MCF-7 cells were treated with TTHL (IC20 = 0.50 μg/mL, IC50 = 1.36 μg/mL, and IC80 = 3.70 μg/mL), fixed, permeabilized, and stained with anti-active caspase 9. Results are expressed as means ± standard deviation (SD). Data were analyzed by one-way analysis of variance (ANOVA), and means were compared using Tukey test, with P ≤ 0.05 considered as statistically significant.
Mentions: In order to characterize the apoptosis induced in MCF-7 cell line, we evaluated the activation of apoptosis mediators by different TTHL concentrations. We analyzed the cleavage and consequent activation of caspase-9 by flow cytometry using specific antibodies that recognize only the intact (inactive) forms of the enzyme. The results, summarized in Figure 4, indicate that continuous treatment of cells with TTHL concentrations (IC50 and IC80) leads to increased induction of cleaved caspase-9-labeled cells. There is a clear difference on the induction of cleaved caspase-9 when the IC50 and IC80 values are compare (Figure 4), although the increased induction of cell death (sub-G1) in treated MCF-7 cells is confirmed (Figure 2, and Additional file1: Figure S8). Moreover, TTHL (at IC20) caused no significant changes in the levels of cleaved caspase-9 in MCF-7 cells (Figure 4). These results were consistent with the data from Annexin V-PE positive FACS analysis; in fact, the treatment with TTHL at IC50 and IC80 induced a stronger apoptotic effect on MCF-7 cells when compared to IC20 and control (Figures 2,3 and4).Figure 4

Bottom Line: We found that the treatment with IC50 and IC80 TTHL for 24 h induced apoptosis in 14% (IC50) and 52% (IC80) of MCF-7 cells.In order to further understand the biological mechanism of TTHL-induced cytotoxicity, we have also investigated its effect on different Saccharomyces cerevisiae yeast strains.Taken together, the results suggest that TTHL forms covalent adducts with cellular macromolecules, potentially disrupting cellular function and triggering apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Basic Health Sciences, Laboratory of Genetic Toxicology - UFCSPA, Porto Alegre, RS, Brazil. jenifers@ufcspa.edu.br.

ABSTRACT

Background: The 3β, 6β, 16β-trihydroxylup-20(29)-ene (TTHL) is a pentacyclic triterpene obtained from the medicinal plant Combretum leprosum Mart. In folk medicine, this plant is popularly known as mofumbo, cipoaba or mufumbo, and is used to treat several diseases associated with inflammation and pain.

Methods: We investigated the antitumor efficacy of TTHL isolated from C. leprosum. The TTHL cytotoxic effect was investigated in MRC5, MCF-7, HepG2, T24, HCT116, HT29, and CACO-2 cells after 24, 48, 72 and 120 h of treatment. The mechanisms of cell death and DNA damage induction were investigated by flow cytometry and comet assay, respectively.

Results: The results indicated that TTHL induced a time- and concentration-dependent growth inhibition in all human cancer cell lines. The cytotoxicity was more pronounced in MCF-7 breast cancer cells, with an IC50 of 0.30 μg/mL at 120 h. We therefore evaluated the cell death mechanism induced by TTHL (IC20, IC50, and IC80) in MCF-7 cells at 24 h. We found that the treatment with IC50 and IC80 TTHL for 24 h induced apoptosis in 14% (IC50) and 52% (IC80) of MCF-7 cells. The apoptosis induced by TTHL was accompanied by increased levels of both cleaved caspase-9 and intracellular ROS. In order to further understand the biological mechanism of TTHL-induced cytotoxicity, we have also investigated its effect on different Saccharomyces cerevisiae yeast strains. The mutant strains sod1Δ, sod2Δ, and sod1Δsod2Δ, which are deficient in superoxide dismutase antioxidant defenses, were hypersensitive to TTHL, suggesting that its capacity to disturb cellular redox balance plays a role in drug toxicity. Moreover, TTHL induced mutagenicity in the yeast strain XV185-14c.

Conclusions: Taken together, the results suggest that TTHL forms covalent adducts with cellular macromolecules, potentially disrupting cellular function and triggering apoptosis.

Show MeSH
Related in: MedlinePlus