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

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.

Figure 1

EcEE affect cell viability and proliferation. (A) Cell viability after 24 h, 48 h and 96 h of exposure to distinct concentrations of EcEE and (B) after 72 h recovery in EcEE-free medium following 24 h and 48 h treatments. Results are presented as percentage over control, **p < 0.0001 and *p < 0.01. (C) NCL, FOS and p21 differential transcription after 48 h exposure to distinct EcEE concentrations. Results are shown as mean log2 fold change (2^-ΔΔCt) ± standard deviation in relation control, *p < 0.0001. (D) DAPI stained HT29 colonies after 48 h in control medium and medium supplemented with EcEE 5 μg/ml or EcEE 25 μg/ml. All images have identical magnification, bar = 50 μm.

Figure 2

Nuclear organization is disrupted after 48 h of exposure to EcEE 25 μg/ml. (A) DAPI stained HT29 interphase cells. Nuclear anomalies, namely pyknosis (arrow), micronuclei (open arrow head) and karyorrhexis (arrow head) are detectable only for 25 μg/ml EcEE. The lack of effects induced by EcEE lower concentrations is exemplified by 5 μg/ml EcEE. (B) TUNEL positive nuclei (arrow) with sparse labeling are detectable for 25 μg/ml EcEE. (C) bcl-xl differential expression after 48 h exposure to distinct EcEE concentrations. Results in are shown as the mean log2 fold change (2^-ΔΔCt) ± standard deviation in relation control, **p < 0.0001 and *p < 0.01. (D) DAPI staining (blue) and α-tubulin immunodetection (red in merged images at the bottom) of interphase cells after 48 h in control or 25 μg/ml EcEE. (E) Immunodetection of H3K9ac after 48 h in control or EcEE 25 μg/ml supplemented medium. Within each experiment all images have identical magnification, bar = 5 μm.

Figure 3

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.

Figure 4

EcEE interacts with BPA at reference level. (A) Cell viability after co-exposure to BPA (1 μg/ml) and distinct EcEE concentrations and subsequent 72 h recovery in standard medium. Results are presented as percentage over control, *p < 0.001. (B) Percentage of mitotic anomalies after 48 h culture in standard medium (control) or medium supplemented with ethanol (vehicle), BPA or EcEE at distinct concentrations in combination with BPA. The total number of mitotic cells scored and utilized to calculate the percentage of abnormal mitosis is shown in brackets.

Figure 5

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.