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Cytotoxicity of Eupatorium cannabinum L. ethanolic extract against colon cancer cells and interactions with Bisphenol A and Doxorubicin.

Ribeiro-Varandas E, Ressurreição F, Viegas W, Delgado M - BMC Complement Altern Med (2014)

Bottom Line: Severe loss of HT29 cell viability was detected for 50 μg/ml EcEE immediately after 24 h exposure whereas the lower concentrations assayed (0.5, 5 and 25 μg/ml) resulted in significant viability decreases after 96 h.Exposure to 25 μg/ml EcEE for 48 h resulted in irreversible cell damage leading to a drastic decrease in cell viability after 72 h recovery in EcEE-free medium. 48 h 25 μg/ml EcEE treatment also induced alteration of colony morphology, H3K9 hyperacetylation, transcriptional up regulation of p21 and down regulation of NCL, FOS and AURKA, indicating reduced proliferation capacity.Interaction experiments showed that EcEE can increase BPA aneugenic effects and EcEE synergistic effects with DOX supporting a potential use as adjuvant in chemotherapeutic approaches.

View Article: PubMed Central - PubMed

Affiliation: Centro de Botânica Aplicada à Agricultura, Instituto Superior de Agronomia, Universidade de Lisboa, Tapada da Ajuda, 1349-017 Lisboa, Portugal. mxdelgado@isa.ulisboa.pt.

ABSTRACT

Background: Eupatorium cannabinum L. has long been utilized in traditional medicine, however no information is available regarding cellular effects of full extracts. Here we assessed the effects of E. cannabinum ethanolic extract (EcEE) on the colon cancer line HT29. Potential interactions with bisphenol A (BPA) a synthetic phenolic compound to which humans are generally exposed and a commonly used chemotherapeutic agent, doxorubicin (DOX) were also evaluated.

Methods: HT29 cells were exposed to different concentrations (0.5 to 50 μg/ml) of EcEE alone or in combination with BPA or DOX. Cell viability was analyzed through resazurin assay. Gene transcription levels for NCL, FOS, p21, AURKA and bcl-xl were determined through qRT-PCR. Cytological analysis included evaluation of nuclear and mitotic anomalies after DAPI staining, immunodetection of histone H3 lysine 9 acetylation (H3K9ac) and assessment of DNA damage by TUNEL assay.

Results: Severe loss of HT29 cell viability was detected for 50 μg/ml EcEE immediately after 24 h exposure whereas the lower concentrations assayed (0.5, 5 and 25 μg/ml) resulted in significant viability decreases after 96 h. Exposure to 25 μg/ml EcEE for 48 h resulted in irreversible cell damage leading to a drastic decrease in cell viability after 72 h recovery in EcEE-free medium. 48 h 25 μg/ml EcEE treatment also induced alteration of colony morphology, H3K9 hyperacetylation, transcriptional up regulation of p21 and down regulation of NCL, FOS and AURKA, indicating reduced proliferation capacity. This treatment also resulted in drastic mitotic and nuclear disruption accompanied by up-regulation of bcl-xl, limited TUNEL labeling and nuclear size increase, suggestive of a non-apoptocic cell death pathway. EcEE/BPA co-exposure increased mitotic anomalies particularly for the lowest EcEE concentration, although without major effects on viability. Conversely, EcEE/DOX co-exposure decreased cell viability in relation to DOX for all EcEE concentrations, without affecting the DOX-induced cell cycle arrest.

Conclusions: EcEE has cytotoxic activity on HT29 cancer cells leading to mitotic disruption and non-apoptotic cell death without severe induction of DNA damage. Interaction experiments showed that EcEE can increase BPA aneugenic effects and EcEE synergistic effects with DOX supporting a potential use as adjuvant in chemotherapeutic approaches.

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EcEE has synergistic effects with DOX at a therapeutic dose. (A) Cell viability after co-exposure to DOX (2.5 μg/ml) and distinct EcEE concentrations and subsequent 72 h recovery in standard medium. Results are presented as percentage over control, the level of significance in relation to DOX alone is indicated by horizontal brackets, **p < 0.0001 and *p < 0.01. (B) DAPI stained cell after co-exposure to EcEE 25 μg/ml/DOX illustrating the occurrence of (i) micronuclei (arrow head) and pyknotic nuclei (arrow) and (ii) fragmented nuclei (arrows), bar = 5 μm. (C) Percentage fragmented nuclei after exposure to EcEE 25 μg/ml or DOX alone and the combination of both. Total number of cells analyzed is shown in brackets, **p < 0.0001 and *p < 0.03 in relation to EcEE 25 μg/ml or DOX alone.
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Fig5: EcEE has synergistic effects with DOX at a therapeutic dose. (A) Cell viability after co-exposure to DOX (2.5 μg/ml) and distinct EcEE concentrations and subsequent 72 h recovery in standard medium. Results are presented as percentage over control, the level of significance in relation to DOX alone is indicated by horizontal brackets, **p < 0.0001 and *p < 0.01. (B) DAPI stained cell after co-exposure to EcEE 25 μg/ml/DOX illustrating the occurrence of (i) micronuclei (arrow head) and pyknotic nuclei (arrow) and (ii) fragmented nuclei (arrows), bar = 5 μm. (C) Percentage fragmented nuclei after exposure to EcEE 25 μg/ml or DOX alone and the combination of both. Total number of cells analyzed is shown in brackets, **p < 0.0001 and *p < 0.03 in relation to EcEE 25 μg/ml or DOX alone.

Mentions: Potential interactions between different concentrations of EcEE and the chemotherapeutic drug doxorubicin (DOX) at a therapeutic concentration of 2.5 μg/ml were investigated. Immediately after exposure, DOX alone resulted in a slight decrease in cell viability (-4.15%) (Figure 5-A). Interestingly, the loss of cell viability was significantly more pronounced after co-exposure to EcEE/DOX for all EcEE concentrations (-10.03%, -19.88% and -18.67% for 0.5 μg/ml, 5 μg/ml and 25 μg/ml, respectively) (Figure 5-A) contrasting with the lack of effects observed for 48 h exposure to EcEE alone (Figure 1-A). Recovery experiments showed that the effects of both DOX and 25 μg/ml EcEE/DOX have long lasting negative effects on viability, apparent as prominent decreases in cell viability after 72 h recovery in standard medium in relation to what was observed immediately after exposure (Figure 5-A). After recovery, EcEE 25 μg/ml/DOX exposure resulted in an even more pronounced loss of cell viability (-93.95%) than that observed for exposure to 25 μg/ml EcEE alone (Figure 1-B). Conversely, for the lower EcEE concentrations, no significant differences were detected between exposure to DOX alone and in combination with EcEE (Figure 1-B).


Cytotoxicity of Eupatorium cannabinum L. ethanolic extract against colon cancer cells and interactions with Bisphenol A and Doxorubicin.

Ribeiro-Varandas E, Ressurreição F, Viegas W, Delgado M - BMC Complement Altern Med (2014)

EcEE has synergistic effects with DOX at a therapeutic dose. (A) Cell viability after co-exposure to DOX (2.5 μg/ml) and distinct EcEE concentrations and subsequent 72 h recovery in standard medium. Results are presented as percentage over control, the level of significance in relation to DOX alone is indicated by horizontal brackets, **p < 0.0001 and *p < 0.01. (B) DAPI stained cell after co-exposure to EcEE 25 μg/ml/DOX illustrating the occurrence of (i) micronuclei (arrow head) and pyknotic nuclei (arrow) and (ii) fragmented nuclei (arrows), bar = 5 μm. (C) Percentage fragmented nuclei after exposure to EcEE 25 μg/ml or DOX alone and the combination of both. Total number of cells analyzed is shown in brackets, **p < 0.0001 and *p < 0.03 in relation to EcEE 25 μg/ml or DOX alone.
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Fig5: EcEE has synergistic effects with DOX at a therapeutic dose. (A) Cell viability after co-exposure to DOX (2.5 μg/ml) and distinct EcEE concentrations and subsequent 72 h recovery in standard medium. Results are presented as percentage over control, the level of significance in relation to DOX alone is indicated by horizontal brackets, **p < 0.0001 and *p < 0.01. (B) DAPI stained cell after co-exposure to EcEE 25 μg/ml/DOX illustrating the occurrence of (i) micronuclei (arrow head) and pyknotic nuclei (arrow) and (ii) fragmented nuclei (arrows), bar = 5 μm. (C) Percentage fragmented nuclei after exposure to EcEE 25 μg/ml or DOX alone and the combination of both. Total number of cells analyzed is shown in brackets, **p < 0.0001 and *p < 0.03 in relation to EcEE 25 μg/ml or DOX alone.
Mentions: Potential interactions between different concentrations of EcEE and the chemotherapeutic drug doxorubicin (DOX) at a therapeutic concentration of 2.5 μg/ml were investigated. Immediately after exposure, DOX alone resulted in a slight decrease in cell viability (-4.15%) (Figure 5-A). Interestingly, the loss of cell viability was significantly more pronounced after co-exposure to EcEE/DOX for all EcEE concentrations (-10.03%, -19.88% and -18.67% for 0.5 μg/ml, 5 μg/ml and 25 μg/ml, respectively) (Figure 5-A) contrasting with the lack of effects observed for 48 h exposure to EcEE alone (Figure 1-A). Recovery experiments showed that the effects of both DOX and 25 μg/ml EcEE/DOX have long lasting negative effects on viability, apparent as prominent decreases in cell viability after 72 h recovery in standard medium in relation to what was observed immediately after exposure (Figure 5-A). After recovery, EcEE 25 μg/ml/DOX exposure resulted in an even more pronounced loss of cell viability (-93.95%) than that observed for exposure to 25 μg/ml EcEE alone (Figure 1-B). Conversely, for the lower EcEE concentrations, no significant differences were detected between exposure to DOX alone and in combination with EcEE (Figure 1-B).

Bottom Line: Severe loss of HT29 cell viability was detected for 50 μg/ml EcEE immediately after 24 h exposure whereas the lower concentrations assayed (0.5, 5 and 25 μg/ml) resulted in significant viability decreases after 96 h.Exposure to 25 μg/ml EcEE for 48 h resulted in irreversible cell damage leading to a drastic decrease in cell viability after 72 h recovery in EcEE-free medium. 48 h 25 μg/ml EcEE treatment also induced alteration of colony morphology, H3K9 hyperacetylation, transcriptional up regulation of p21 and down regulation of NCL, FOS and AURKA, indicating reduced proliferation capacity.Interaction experiments showed that EcEE can increase BPA aneugenic effects and EcEE synergistic effects with DOX supporting a potential use as adjuvant in chemotherapeutic approaches.

View Article: PubMed Central - PubMed

Affiliation: Centro de Botânica Aplicada à Agricultura, Instituto Superior de Agronomia, Universidade de Lisboa, Tapada da Ajuda, 1349-017 Lisboa, Portugal. mxdelgado@isa.ulisboa.pt.

ABSTRACT

Background: Eupatorium cannabinum L. has long been utilized in traditional medicine, however no information is available regarding cellular effects of full extracts. Here we assessed the effects of E. cannabinum ethanolic extract (EcEE) on the colon cancer line HT29. Potential interactions with bisphenol A (BPA) a synthetic phenolic compound to which humans are generally exposed and a commonly used chemotherapeutic agent, doxorubicin (DOX) were also evaluated.

Methods: HT29 cells were exposed to different concentrations (0.5 to 50 μg/ml) of EcEE alone or in combination with BPA or DOX. Cell viability was analyzed through resazurin assay. Gene transcription levels for NCL, FOS, p21, AURKA and bcl-xl were determined through qRT-PCR. Cytological analysis included evaluation of nuclear and mitotic anomalies after DAPI staining, immunodetection of histone H3 lysine 9 acetylation (H3K9ac) and assessment of DNA damage by TUNEL assay.

Results: Severe loss of HT29 cell viability was detected for 50 μg/ml EcEE immediately after 24 h exposure whereas the lower concentrations assayed (0.5, 5 and 25 μg/ml) resulted in significant viability decreases after 96 h. Exposure to 25 μg/ml EcEE for 48 h resulted in irreversible cell damage leading to a drastic decrease in cell viability after 72 h recovery in EcEE-free medium. 48 h 25 μg/ml EcEE treatment also induced alteration of colony morphology, H3K9 hyperacetylation, transcriptional up regulation of p21 and down regulation of NCL, FOS and AURKA, indicating reduced proliferation capacity. This treatment also resulted in drastic mitotic and nuclear disruption accompanied by up-regulation of bcl-xl, limited TUNEL labeling and nuclear size increase, suggestive of a non-apoptocic cell death pathway. EcEE/BPA co-exposure increased mitotic anomalies particularly for the lowest EcEE concentration, although without major effects on viability. Conversely, EcEE/DOX co-exposure decreased cell viability in relation to DOX for all EcEE concentrations, without affecting the DOX-induced cell cycle arrest.

Conclusions: EcEE has cytotoxic activity on HT29 cancer cells leading to mitotic disruption and non-apoptotic cell death without severe induction of DNA damage. Interaction experiments showed that EcEE can increase BPA aneugenic effects and EcEE synergistic effects with DOX supporting a potential use as adjuvant in chemotherapeutic approaches.

Show MeSH
Related in: MedlinePlus