Abstract
BackgroundEupatorium 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.MethodsHT29 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.ResultsSevere 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.ConclusionsEcEE 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.
Highlights
Eupatorium cannabinum L. has long been utilized in traditional medicine, no information is available regarding cellular effects of full extracts
No decrease in cell viability was detected after 24 h or 48 h for the lower E. cannabinum ethanolic extract (EcEE) concentrations, and a slight increase in fluorescence was observed after 24 h and 48 h for 25 μg/ml EcEE
Significant decreases in cell viability were detected for the three lower EcEE concentrations after 96 h of exposure, and for 25 μg/ml EcEE (−18.89%, −14.55% and −73.25% for 0.5 μg/ml, 5 μg/ml and 25 μg/ml, respectively)
Summary
Eupatorium cannabinum L. has long been utilized in traditional medicine, no information is available regarding cellular effects of full extracts. We assessed the effects of E. cannabinum ethanolic extract (EcEE) on the colon cancer line HT29. E. cannabinum has long been presence of sesquiterpenes [7], pyrrolizidine alkaloids [3,8] as well as several phenolic compounds [9,10]. Pyrrolizidine alkaloids are generally associated with genotoxicity and tumourigenic activities [14], the isomers intermedine and lycopsamine indentified in E. cannabinum have low genotoxic potency [15] and lycopsamine was shown to be non-tumourigenic in rats [16]. The phenolic compounds identified in this plant have been described to have anti-inflammatory [9], anti-parasitary [17], as well as anti-proliferative effects in several cell lines [18]. Jaceosidin cytotoxic effects have been demonstrated in normal and cancer endometrial cells [19] and hispidulin was shown to efficiently inhibit growth of gastric cancer cells [20] and liver carcinoma cells without significant toxic effect in normal liver cells [21]
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