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Cytotoxicity of Eupatorium cannabinum L. ethanolic extract against colon cancer cells and interactions with Bisphenol A and Doxorubicin
© Ribeiro-Varandas et al.; licensee BioMed Central Ltd. 2014
Received: 18 February 2014
Accepted: 10 July 2014
Published: 24 July 2014
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.
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.
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.
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.
Eupatorium cannabinum L., commonly known as hemp-agrimony is a robust perennial herbaceous plant of the Asteraceae family and the only species of the Eupatorium genus found in Europe occurring also throughout North Africa and Asia . E. cannabinum has long been used for medicinal purposes being referred to by Greeks and Romans as well by the medieval Persian physician Aviccena, for what is also known as Eupatorium of Aviccena, and later by the Portuguese Renaissance pioneer in tropical medicine, Garcia da Orta (1563) . Presently, hemp-agrimnony is used in both Chinese  and Indian  traditional medicine as well as in natural medicine in western countries  with very diverse therapeutic indications including influenza-like illnesses , hypertension [3, 4, 6] and as an anti-tumour agent . E. cannabinum extracts has been previously characterized and reveal the presence of sesquiterpenes , pyrrolizidine alkaloids [3, 8] as well as several phenolic compounds [9, 10].
Sesquiterpenes were found to be a major fraction (43.3%) of essential oil from E. cannabinum aerial parts , being eupatoriopicrin the main component . Eupatoriopicrin has been associated with induction DNA damage in Ehrlich ascites tumour  as well as with cytostatic activity and both in vitro and in vivo tumour growth inhibition properties in Lewis lung carcinoma and FIG 26 fibrosarcoma .
Pyrrolizidine alkaloids are generally associated with genotoxicity and tumourigenic activities , however the isomers intermedine and lycopsamine indentified in E. cannabinum have low genotoxic potency  and lycopsamine was shown to be non-tumourigenic in rats . Additionally the phenolic compounds identified in this plant have been described to have anti-inflammatory , anti-parasitary , as well as anti-proliferative effects in several cell lines . In particular, jaceosidin cytotoxic effects have been demonstrated in normal and cancer endometrial cells  and hispidulin was shown to efficiently inhibit growth of gastric cancer cells  and liver carcinoma cells without significant toxic effect in normal liver cells .
Although the effects of specific components of Eupatorium cannabinum L. extracts have been described, the cellular effects of the full extracts have not, until now, been investigated. Thus, here different concentrations of Eupatorium cannabinum L. ethanolic extract (EcEE) were evaluated on the colon cancer cell line HT29. Moreover we also analyzed its interactions with the synthetic phenolic compound bisphenol A (BPA) as well as with the chemotherapeutic agent Doxorubicin (DOX). Human exposure to BPA is considered generalized in the common population and its adverse health effects are the focus of intense investigation [22, 23]. On the other hand, DOX is a commonly used chemotherapeutic agent to which cell resistance can emerge [24, 25]. Plant constituents are a major source of bioactive compounds and several plants have been investigated aiming to identify potential synergistic effects with DOX (reviewed in ).
Eupatorium cannabinum L. ethanolic extract
Eupatorium cannabinum L. (Asteraceae) aerial parts were collected in the Rossas fields of Arouca village, Portugal, in August during mass flowering. Formal identification of plant material was performed by A.P. Paes from “João de Carvalho e Vasconcellos Herbarium” at Instituto Superior de Agronomia (Lisboa, Portugal). A voucher specimen was deposited in the same herbarium under the number LISI 1503/2013. Plant material was dried and powdered using a grinder and ethanolic extract (EcEE) was obtained by soaking the material in absolute ethanol for 48 h at room temperature with gentle shaking. The extract were filtered and concentrated under vacuum on a rotary evaporator at 40°C and stored at -20°C for further use.
Cellular cultures, reagents and treatments
HT29 cells were purchased from European Collection of Cell Cultures (ECACC, UK) and cultivated in RPMI medium under standard conditions as previously described . Before treatments and experiments HT29, cells were allowed to stabilize for 24 h in standard medium and further cultivated in EcEE supplemented media for 24 h, 48 h or 96 h. Crude ethanolic extract was dissolved in ethanol to a final work concentration of 50 mg/ml before use and added to the culture media at four different final concentrations (0.5 μg/ml, 5 μg/ml, 25 μg/ml and 50 μg/ml). Bisphenol A (Sigma-Aldrich) was freshly diluted in ethanol and added to the culture media to the final concentration of 1 μg/ml (4.4 μM) that corresponds to the established Tolerable Daily Intake (TDI) level of 50 ug/kg BW/day [28, 29] considering an average body weight of 70 Kg and daily consumption of 3 litres of preformed water. Doxorubicin (DOX) (AppliChem) was dissolved in water at stock concentration of 1 mg/ml and added to the culture media to final concentration of 2.5 μg/ml (4 μM) which corresponds to a therapeutic dosage . For the combined EcEE/BPA or EcEE/DOX exposures, cells were pre-exposed to EcEE for 24 h followed by additional 24 h of simultaneous exposure to EcEE and BPA or EcEE and DOX. Single 24 h BPA or DOX exposure was carried-out in equivalent cell cultures. For evaluation of cell recovery capacity after treatments cells were cultivated for additional 72 h in standard culture medium. Negative controls were performed for all experiments using cells grown in standard culture medium as well as cells grown in medium supplemented with ethanol at final concentration of 170 μM, corresponding to the final concentration of ethanol used as vehicle for all EcEE concentrations as well as for BPA.
Cell viability was evaluated by CellTiter-Blue assay (Promega) following manufacturer’s instructions. Cells were plated on 96-well plates at a density of 3.2 × 104 cells/well and after treatments were incubated for 4 h with CellTiter-Blue Reagent. Additional negative controls were performed in the absence of cells to guarantee that the utilized media did not interfere with fluorescence readings. Experiments were repeated at least three times with a minimum of three replicates per experiment.
DAPI staining, TUNEL assay and immunodetection
For cytological analysis cells were grown over glass coverslips coated with 0.2% (v/v) gelatin (Sigma-Aldrich) and after treatments fixed in 4% (p/v) formaldehyde in PBS. For evaluation of colony morphology, mitotic index as well as mitotic and nuclear anomalies cells were DAPI stained and mounted on glass slides with antifade AF1 (Citifluor). DNA damage assessment with TUNEL assay (Roche) was performed accordingly to manufacturers’ instructions. Immunodetection of H3K9ac and α-tubulin was performed in fixed cells as previously described  using the primary antidodies anti-acetyl-histone H3(Lys 9) (ab10812, Abcam) and anti-α-Tubulin (T9026, Sigma-Aldrich) detected with FITC or Cy3 conjugated secondary antibodies. Images were captured using the appropriate excitation and emission filters and recorded using an epifluorescence microscope Zeiss Axioskop2 equipped with a Zeiss AxioCam MRc5 digital camera. ImageJ software (http://rsbweb.nih.gov/ij/) was used for nuclear area measurements. The analysis was performed in the pooled results of at least two independent experiments with at least two replicates.
cDNA isolation and real-time quantitative PCR
Primers used for qRT-PCR
Forward primer (5’ → 3’)
Reverse primer (5´ → 3´)
Student’s t test was used for statistical analysis of gene transcription, cell viability, nuclear area and nuclear fragmentation. No significant differences were detected between control and vehicle for all parameters analysed, and results are shown in relation to control. GraphPad Prism 6 software was used for determination of IC50 values.
E. cannabinum ethanolic extract decreases HT29 cell viability
To better understand the effects of EcEE immediately after 48 h exposure, gene transcription analysis was carried out for three proliferation-associated genes, namely nucleolin (NCL), p21 and FOS (Figure 1-C). Similarly to the cell viability results, no significant differences in transcription levels were detected after 48 h exposure to EcEE concentrations equal to or lower than 5 μg/ml. Conversely, 25 μg/ml EcEE exposure resulted in significant differences in mRNA levels of all three genes, corresponding to down regulation of both NCL and FOS (Log2 fold change = -0.813 ± 0.248 and -0.741 ± 0.078, respectively), and up regulation of p21 (Log2 fold change = 1.393 ± 0.128). Evaluation of colony morphology was performed immediately after EcEE treatments by DAPI staining. Again, significant alterations in colony morphology were detected after exposure to 25 μg/ml EcEE for 48 h, evident as cells being more dispersed and showing a flattening of cellular aggregates in comparison to controls with no detectable effect for 5 μg/ml EcEE (Figure 1-D) or 0.5 μg/ml EcEE (not shown).
E. cannabinum ethanolic extract induces alterations in nuclear structure and mitotic disruption
E. cannabinum ethanolic extract increases Bisphenol A induced mitotic disruption
Cytotoxic effects of Doxorubicin are enhanced by E. cannabinum
Eupatorium cannabinum L. is a commonly utilized plant for alternative and/or complementary medicine treatments  including as an anticancer agent . Although cellular effects of particular phytochemicals known to be present in E. cannabinum have been previously described, to our knowledge this is the first study that evaluates the cytotoxic potential of E. cannabinum extracts on human cancer cells. Here we demonstrated that E. cannabinum ethanolic extract (EcEE) has cytotoxic effects on HT29 colon cancer cells in a time and dose dependent manner. IC50 were similar after 24 and 48 h (46.75 and 44.65 μg/ml, respectively) but considerably lower (13.38 μg/ml) after 96 h of exposure. Cytotoxic activity has also been demonstrated for extracts from other Eupatorium species. For E. perfoliatum ethanolic extract, IC50 values between 12 and 14 μg/ml were obtained after 24 h exposure in three distinct mammalian cell lines . In MCF7 breast cancer cells a time dependent effect was also observed for E. odoratum ethyl acetate extract (IC50 of 65.72, 83.88 μg/ml and 92.84 μg/ml for 24, 48 and 72 h, respectively) while for acetone extract higher IC50 values were obtained but without a direct correlation with exposure time (133.9, 163.0 and 147.8 μg/ml for 24, 48, and 72 h respectively) . The immediate cytotoxicity observed here for EcEE is lower than that obtained for E. perfoliatum ethanolic extract and higher than that of ethyl acetate or acetone extracts from E. odoratum. Interestingly the time dependent increase in cytotoxicity of EcEE was only detected for the longer exposure time (96 h). Moreover, a deferred effect on cell viability was detected after 48 h exposure to EcEE at 25 μg/ml. This was also associated with disruption of cell colony three-dimensional arrangement, a generalized increase in nuclear area and H3K9 hyperacetylation. Relevantly, gene transcription analysis revealed a significant reduction in the mRNA levels of FOS, which encodes for a nuclear protein from AP-1 transcription factor complex, and nucleolin (NCL) the most profuse non-ribosomal protein of the nucleolus. Both FOS and nucleolin are involved in the regulation of cell proliferation [34, 35] as their decreased expression has been related with reduced proliferation capacity of cancer cells including colon cancer cell lines [36, 37]. On the other hand, exposure to EcEE (25 μg/ml, 48 h) also resulted in the up regulation of p21, a cyclin-dependent kinase inhibitor which is a major regulator of the cell cycle . It was previously shown that histone hyperacetylation induces p21 over expression . In colon cancer cells inhibition of histone deacetylation results in both up regulation of p21, and induction of G2/M cell cycle arrest . Relevantly, cell reduction capacity depends on the cell cycle being higher at G2/M . Considering that the cell viability assay used is based on the resazurin reduction and that overall our results were incompatible with EcEE induction of cell proliferation, the slight and transient augment of fluorescence detected after 24 h and 48 h of exposure to 25 μg/ml EcEE was also suggestive of cell arrest at G2 or M. Moreover, the increase of abnormal mitotic cells after exposure to EcEE is also suggestive of a mitotic block. This phenotype was accompanied by a significant down regulation of Aurora A transcription, which is consistent with previous results showing that decreased Aurora A levels are associated with mitotic catastrophe and consequent cell death . Induction of cell death after 48 h exposure to 25 μg/ml was evident by the prominent occurrence of pyknotic and fragmented nuclei, characteristic of both apoptotic as well as necrotic cells, and supports the marked loss in cell viability observed after recovery. This was moreover associated with transcriptional up regulation of the anti-apoptotic gene bcl-xL suggesting a non-apoptotic cell death pathway  which is also supported by limited occurrence of DNA breaks. These observations together with the increase in cell size is compatible with a necrotic cell death or necroptosis, a process which acts as backup death-inducing mechanism when apoptosis is inhibited .
Cytostatic activity was previously described for compounds identified in E. cannabinum extracts, namely the sesquiterpene eupatoriopicrin  and the flavonoids centaureidin, jaceosidin and hispidulin . Severe decrease of tumour cell survival in vitro was associated with eupatoriopicrin concentrations ranging from 1–10 μg/ml [12, 13] which was correlated with induction of DNA damage . Also, anti-proliferative effects on distinct cancer cell lines have been described for centaureidin concentrations below 1 μg/ml  as well as for jaceosidin in the concentration range of 20–50 μg/ml  and hispidulin for 4–30 μg/ml . Relevantly, both jaceosidin  and hispidulin  effects were associated with increased p21 expression. The results obtained here indicate that the anti-proliferative potency of EcEE is similar to that observed for some of its individual constituents such as eupatoriopicrin, jaceosidin and hispidulin, albeit without marked induction of DNA damage and therefore suggesting a combined action of distinct compounds.
Importantly, EcEE combined exposures with DOX at therapeutic concentration resulted in a clear enhancement of cytotoxic effects, evident as combined treatments significantly decreasing HT29 cell viability immediately after exposure, even for the lower EcEE concentration that per se did not affect cell viability. This was accompanied by increased nuclear fragmentation and reduced cell survival after recovery resulting in almost total loss of cell viability. DOX is a commonly utilized antineoplastic drug that acts in tumour cells by induction of apoptosis . Nevertheless different types of cell death can occur simultaneously, independently or through partially common pathways (reviewed in ). The severe decrease in cell viability observed after combined exposure to DOX and EcEE can thus result from induction of distinct cell death mechanisms. On the other hand therapeutic concentrations of DOX induces cell arrest at G2/M and/or G1/S checkpoints [47, 48]. The results obtained show that EcEE does not counteract DOX-induced cell cycle arrest. Considering that DOX acts by induction of apoptosis  to which cell resistance can emerge [24, 25] our data substantiates potential adjuvant EcEE properties in chemotherapeutic approaches .
On the other hand, no immediate effect on cell viability was associated with co-exposure to EcEE and the synthetic phenolic compound BPA. However, cell recovery capacity after 48 h exposure to 25 μg/ml EcEE decreased by the presence of BPA. Additionally, EcEE/BPA combined exposures resulted in increased mitotic anomalies in relation to either BPA or EcEE alone for 25 μg/ml EcEE but also for 0.5 μg/ml EcEE. BPA is characterized as an aneugenic chemical  capable of interfering with cell division mechanisms even at very low concentrations . Nonetheless BPA is widely used in a variety of consumer products leading to a generalized human exposure although its risks remain highly controversial . The present results raise the possibility that adverse BPA effects could be enhanced by interactions with other chemicals, an aspect that remains largely unknown and has barely been addressed.
E. cannabinum has been utilized as a medicinal plant for alternative and/or complementary medicine, however the effects or the mode of action of full extracts have not been evaluated at the cellular level. The present work demonstrates that E. cannabinum ethanolic extract has potent cytotoxic activity against HT29 colon cancer cells associated with mitotic disruption and cell death without marked evidences of DNA damage. Relevantly E. cannabinum extract exhibits synergistic effects with doxorubicin in the induction of HT29 cell death indicating its potential use in alternative or complementary therapeutic strategies. On the other hand, the results show also that E. cannabinum can increase aneugenic effects of the environmental pollutant BPA, drawing attention to the possibility that BPA adverse effects may be potentiated by interaction with other chemicals.
This work was funded by Fundação para a Ciência e Tecnologia (Portugal), PTDC/AACAMB/103968/2008, Pest-OE/AGR/UI0240/2014 and grant SFRH/BD/44277/2008 to E. Ribeiro-Varandas.
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