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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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Related in: MedlinePlus

EcEE exposure induces mitotic disruption. (A) DAPI stained abnormal mitotic cells after EcEE exposure showing (i) defective chromosome congression, (ii) tripolar and (iii) tetrapolar metaphases and (iv) tripolar anaphase with chromosome bridges, bar = 5 μm. (B) Percentage of abnormal mitosis after 24 h and 48 h exposure to distinct concentrations of EcEE; the total number of mitotic cells scored and utilized to calculate the percentage of abnormal mitosis is shown in brackets. (C)AURKA differential transcription after exposure to 48 h of EcEE. Results are shown as the mean log2 fold change (2-ΔΔCt) ± standard deviation in relation control, *p < 0.0001.
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Fig3: EcEE exposure induces mitotic disruption. (A) DAPI stained abnormal mitotic cells after EcEE exposure showing (i) defective chromosome congression, (ii) tripolar and (iii) tetrapolar metaphases and (iv) tripolar anaphase with chromosome bridges, bar = 5 μm. (B) Percentage of abnormal mitosis after 24 h and 48 h exposure to distinct concentrations of EcEE; the total number of mitotic cells scored and utilized to calculate the percentage of abnormal mitosis is shown in brackets. (C)AURKA differential transcription after exposure to 48 h of EcEE. Results are shown as the mean log2 fold change (2-ΔΔCt) ± standard deviation in relation control, *p < 0.0001.

Mentions: The effects of exposure to EcEE were further evaluated on mitotic cells after DAPI staining. No significant variation was observed in the mitotic index between control, vehicle and EcEE independently of the concentration assayed (varying between 4.57 and 5.94). On the other hand, although mitotic anomalies, particularly multipolar metaphases and anaphases, are a common feature of HT29 cells and therefore observed both in control and vehicle (6.67% and 11.76% after 24 h; 6.38% and 10.67% after 48 h, for control and vehicle respectively), the percentage of abnormal mitosis increased after exposure to all EcEE concentrations (Figures 3-A and B). Although a slight increase of abnormal mitosis was already detectable for 0.5 μg/ml EcEE, this effect was greater for 5 μg/ml EcEE (41% and 44% after 24 h or 48 h, respectively). After exposure to 25 μg/ml EcEE, most mitotic cells presented abnormalities (80% and 63% after 24 and 48 h, respectively). Although the frequency of abnormal mitosis was greater after 24 h at the higher EcEE concentration, these results clearly indicate that EcEE induces mitotic disruption in a dose dependent manner. Interestingly, qRT-PCR analysis revealed a significant down regulation of AURKA (Log2 fold change = -0.938 ± 0.146), a gene that encodes a key protein for mitotic chromosome segregation (Figure 3-C).Figure 3


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 exposure induces mitotic disruption. (A) DAPI stained abnormal mitotic cells after EcEE exposure showing (i) defective chromosome congression, (ii) tripolar and (iii) tetrapolar metaphases and (iv) tripolar anaphase with chromosome bridges, bar = 5 μm. (B) Percentage of abnormal mitosis after 24 h and 48 h exposure to distinct concentrations of EcEE; the total number of mitotic cells scored and utilized to calculate the percentage of abnormal mitosis is shown in brackets. (C)AURKA differential transcription after exposure to 48 h of EcEE. Results are shown as the mean log2 fold change (2-ΔΔCt) ± standard deviation in relation control, *p < 0.0001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4117973&req=5

Fig3: EcEE exposure induces mitotic disruption. (A) DAPI stained abnormal mitotic cells after EcEE exposure showing (i) defective chromosome congression, (ii) tripolar and (iii) tetrapolar metaphases and (iv) tripolar anaphase with chromosome bridges, bar = 5 μm. (B) Percentage of abnormal mitosis after 24 h and 48 h exposure to distinct concentrations of EcEE; the total number of mitotic cells scored and utilized to calculate the percentage of abnormal mitosis is shown in brackets. (C)AURKA differential transcription after exposure to 48 h of EcEE. Results are shown as the mean log2 fold change (2-ΔΔCt) ± standard deviation in relation control, *p < 0.0001.
Mentions: The effects of exposure to EcEE were further evaluated on mitotic cells after DAPI staining. No significant variation was observed in the mitotic index between control, vehicle and EcEE independently of the concentration assayed (varying between 4.57 and 5.94). On the other hand, although mitotic anomalies, particularly multipolar metaphases and anaphases, are a common feature of HT29 cells and therefore observed both in control and vehicle (6.67% and 11.76% after 24 h; 6.38% and 10.67% after 48 h, for control and vehicle respectively), the percentage of abnormal mitosis increased after exposure to all EcEE concentrations (Figures 3-A and B). Although a slight increase of abnormal mitosis was already detectable for 0.5 μg/ml EcEE, this effect was greater for 5 μg/ml EcEE (41% and 44% after 24 h or 48 h, respectively). After exposure to 25 μg/ml EcEE, most mitotic cells presented abnormalities (80% and 63% after 24 and 48 h, respectively). Although the frequency of abnormal mitosis was greater after 24 h at the higher EcEE concentration, these results clearly indicate that EcEE induces mitotic disruption in a dose dependent manner. Interestingly, qRT-PCR analysis revealed a significant down regulation of AURKA (Log2 fold change = -0.938 ± 0.146), a gene that encodes a key protein for mitotic chromosome segregation (Figure 3-C).Figure 3

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