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

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.

Sulphamoylated Estradiol Analogue Induces Reactive Oxygen Species Generation to Exert Its Antiproliferative Activity in Breast Cancer Cell Lines

“…Due to previous studies that demonstrated that sulphamoylated compounds induce antiproliferative effects via ROS production [ 8 ], ROS scavengers were utilized to identify the different ROS involved in the antiproliferative activity exerted by sulphamoylated compounds such as ESE-one. Three out of six scavengers (tiron, trolox, DMTU) had a rescue effect in the antiproliferative action induced by ESE-one.…”

“…The addition of N-acetyl cysteine (NAC) abrogated antiproliferative activity, cell cycle arrest and cell death suggesting that ROS production is essential for the ability of EMBS to induce cell death. However, the specific ROS-dependent signaling cascade induced by EMBS including the relevant ROS involved remains unknown [ 8 ].…”

“…ROS include, among others, singlet oxygen, superoxide radical, perhydroxyl radical, hydrogen peroxide and hydroxyl radical, and play a key role in apoptosis induction [ 10 , 11 , 12 , 13 ]. However, an excessive quantity of ROS is known to induce cell death including via the apoptotic pathways [ 8 , 9 , 10 ]. The identification of aberrant ROS modulated by sulphamoylated estradiol antimitotic compounds in cancer cells therefore contributes towards the understanding of oxidative-stress-dependent signaling and improvement of anticancer strategies.…”

Dysregulation of Catalase by a Sulphamoylated Estradiol Analogue Culminates in Antimitotic Activity and Cell Death Induction in Breast Cancer Cell Lines

“…Several previous studies have shown that sulfamoylated compounds such as 2-methoxyestradiol-bis-sulphamate and 2-ethyl-13-methyl-decahydro-6-cyclopenta[a]phenanthrane-3,17-diyl bis-sulfamate (EMBS) reduce cell growth and induce aberrant cell cycle activity in several tumorigenic cell lines including MCF-7 and MDA-MB-231 breast tumorigenic cell lines and the SNO esophageal tumorigenic cell line [ 2 , 3 , 4 , 5 , 6 , 7 , 8 ]. In addition, the antiproliferative and antimitotic activity exerted by EMBS in breast cancer cell lines was abrogated by N-acetyl cysteine (potent scavenger of reactive oxygen species) indicating that these effects induced by EMBS are dependent on increased reactive oxygen species generated due to exposure to EMBS [ 8 ]. Further studies demonstrated that exposure to sulfamoylated compounds, including ESE-one, resulted in decreased cell growth and increased quantities of hydrogen peroxide and superoxide when compared to their counterparts that was not sulfamoylated which had no significant effect.…”

2-Methyl 2-butanol suppresses human retinoblastoma cells through cell cycle arrest and autophagy

“…Autophagy is the natural, regulated, destructive mechanism of the cell that disassembles unnecessary components. Various studies highlight that the therapeutic agents to treat cancer act by inhibiting cell cycle, apoptosis, and autophagy ( 4 , 5 ). These agents affect the cell cycle and cause autophagy through multi-signaling pathways within the cells.…”