Abstract
Clinical trials have revealed that the potential anticancer agent, 2-methoxyestradiol (2ME2) has limitations due to its low bioavailability. Subsequently, 2ME2 derivatives including (8R,13S,14S,17S)-2-ethyl-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthrane-3,17-diyl bis(sulphamate) (EMBS) have shown improved efficacies in inducing apoptosis. However, no conclusive data exist to explain the mode of action exerted by these drugs. This study investigated the mode of action used by EMBS as a representative of the sulphamoylated 2ME2 derivatives. Hydrogen peroxide and superoxide production was quantified using dichlorofluorescein diacetate and hydroethidine. Cell proliferation and mitochondrial metabolism were investigated using crystal violet and Alamar Blue. Apoptosis was assessed using Annexin V-FITC while mitochondrial integrity was assessed using Mitocapture. Autophagy was visualised using LC3B II antibodies. The effects of EMBS on H2A phosphorylation and nuclei were visualised using phospho H2A antibody and 4',6-diamidino-2-phenylindole, dihydrochloride. Data showed that EMBS exposure leads to increased reactive oxygen species (ROS) production which is correlated with loss of cell proliferation, mitochondrial membrane damage, decreased metabolic activity, G2/M arrest, endoreduplication, DNA double stranded breaks, micronuclei and apoptosis induction. Treatment of EMBS-exposed cells with the ROS scavenger, N-acetyl cysteine, abrogated ROS production, cell cycle arrest and apoptosis implying an essential role for ROS production in EMBS signaling. The inhibition of c-Jun N-terminal kinase (JNK) activity also inhibited EMBS-induced apoptosis suggesting that EMBS triggers apoptosis via the JNK pathway. Lastly, evaluation of LC3IIB protein levels indicated that autophagy is not activated in EMBS-exposed cells. Our data shows that EMBS targets a pathway that leads to increased ROS production as an early event that culminates in G2/M arrest and apoptosis by means of JNK-signaling in cancer cells. This study suggests a novel oxidative stress-dependent mode of action for sulphamoylated derivatives.
Highlights
MethodsCell linesThe estrogen receptor-positive MCF-7 cell line [22] and the estrogen receptor-negative metastatic MDA-MB-231 cell line [23] were obtained from Cellonex. (Johannesburg, South Africa)
EMBS induces reactive oxygen species (ROS)-dependent apoptosis, a G2/M block and endoreduplication of Health Sciences, awarded to Dr MH Visagie
To identify the sequence of events resulting in EMBS-induced apoptosis, ROS production was measured over time
Summary
Cell linesThe estrogen receptor-positive MCF-7 cell line [22] and the estrogen receptor-negative metastatic MDA-MB-231 cell line [23] were obtained from Cellonex. (Johannesburg, South Africa). Both cell lines were propagated and maintained in a humidified atmosphere at 37 ̊C and 5% CO2 in Dulbecco’s Minimum Essential Medium Eagle (DMEM) supplemented with 10% heat-inactivated fetal calf serum (FCS), 100 U/ml penicillin G, 100 μg/ml streptomycin and fungizone (250 μg/l). MCF-12A cells were cultured in growth medium consisting of a 1:1 mixture of DMEM and Ham’s-F12 medium supplemented with 20 ng/ml epidermal growth factor, 100 ng/ml cholera toxin, 10 μg/ml insulin and 500 ng/ml hydrocortisone, 10% heat-inactivated FCS, 100 U/ml penicillin G, 100 μg/ml streptomycin and fungizone (250 μg/l). Heat-inactivated FCS, sterile cell culture flasks and plates were purchased from Sterilab Services (Kempton Park, Johannesburg, Gauteng, South Africa). DCF and the 2-hydroethidine cation fluorescent product fluorescence was measured with a FC500 System flow cytometer (Beckman Coulter South Africa (Pty) Ltd). Information generated from at least 10 000 cells were analyzed by means of Cyflogic version 1.2.1 software (Pertu Therho, Turko, Finland)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
