A bis-sulphamoylated estradiol derivative induces ROS-dependent cell cycle abnormalities and subsequent apoptosis

Visagie, M.H. (Michelle Helen); Van den Bout, Jan Iman; Joubert, Annie M.

A bis-sulphamoylated estradiol derivative induces ROS-dependent cell cycle abnormalities and subsequent apoptosis

Visagie, M.H. (Michelle Helen); Van den Bout, Jan Iman; Joubert, Annie M.

Date: 2017-04-14

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.

Description:

S1 File. Raw data and statistical analysis in excel.

S1 Fig. Thymidine does not influence hydrogen peroxide generation. Hydrogen peroxide was quantified of EMBS-treated cells in the presence or absence of 2 mM thymidine. The graph represents the average fold change between EMBS-treated- and vehicle-treated cells (3 independent experiments with error bars representing s.e.m).

S2 Fig. EMBS induces a biphasic hydrogen peroxide response. MDA-MB-231 cells were exposed to 0.4 μMEMBS at the indicated timepoints. Hydrogen peroxide was measured in the presence or absence of NAC. Histograms are representatives of 3 repeats.

S3 Fig. EMBS induces a biphasic hydrogen peroxide response. MDA-MB-231 cells were exposed to 0.4 μMEMBS at the indicated timepoints. Superoxide was measured in the presence or absence of NAC. Histograms are representatives of 3 repeats.

S4 Fig. EMBS induces mitochondrial membrane depolarisation. MDA-MB-231 cells were exposed to 0.4 μMEMBS at the indicated timepoints. Mitochondrial membrane potential of EMBS-treated cells were analysed using Mitotracker in the presence or absence of 20 mM NAC. Histograms are representatives of 3 repeats.

S5 Fig. EMBS induces cell cycle abnormalities, endoreduplication and apoptosis. Cell cycle progression was analysed using PI in cells treated with EMBS alone, EMBS together with NAC or EMBS together with the JNK inhibitor, SP600125. Histograms are representatives of 3 repeats.

S6 Fig. EMBS induces apoptosis. MDA-MB-231 cells were exposed to 0.4 μMEMBS at the indicated timepoints. Representative repeat of apoptosis induction demonstrated using Annexin V-FITC and propidium iodide.

S7 Fig. Lactate dehydrogenase release: Lactate dehydrogenase levels MCF-7-, MDA-MB- 231- and MCF-12A cells exposed to 0.4 μMEMBS-treated for 24 h were compared to vehicletreated cells. Controls included medium only as background, cells propagated in medium as the low control and cells propagated in medium containing cell lysis solution as the high control. An demonstrates a statistically significant P value <0.05 when compared to vehicletreated cells.

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