As previously stated, 2MEBM holds therapeutic potential as an anticancer agent, since it exerts antiproliferative effects in vitro and inhibits tumour growth in vivo . In this article the effects of 2MEBM on the MCF-7 cell line was investigated by demonstrating the influence of 2MEBM on cell cycle progression, membrane integrity and possible production of reactive oxygen species in order to suggests the types of cell death induced by 2MEBM.
Previous reports have indicated that 2MEBM induces a G2/M arrest followed by induction of apoptosis in cell lines including MCF-7, MCF-7 DOX40, MCF-7 MR and MDA-MB-231 [8, 17]. In addition, 2MEBM caused a significant apoptotic sub-G1 peak after 48 h. Day, et al (2009) observed similar results where 24 h 2MEBM exposure resulted in G2/M arrest; however, 48 h exposure resulted in a sub-G1 peak accompanied with no changes in the cell cycle distribution in the human 2MEBM-resistant cell line (A2780.140) derived from the human ovarian carcinoma cell line (A2780) . B-Cell Lymphoma-2 Family Members (BCL-2) phosphorylation has also been found to be induced by 2MEBM in MCF-7 cells and correlates with a G2/M arrest . Foster, et al (2008) reported similar findings pertaining to the decrease of 2MEBM-treated cells observed in the G1-phase in this study [8, 22, 23]. Induction of apoptosis by 2MEBM in breast adenocarcinoma CAL51 cell line was demonstrated by Wood, et al (2004) . In this study 2MEBM caused a reduction in the mitochondrial membrane potential, confirming the induction of apoptosis. The mitochondrial membrane potential depolarization is an early event in the intrinsic (mitochondrial) apoptotic pathway [20, 25, 26]. Our study suggests that 2-methoxyestradiol-bis-sulphamate (2MEBM) influences the mitochondrial membrane potential minimally, thus the involvement of the intrinsic pathway to a lesser extent. Furthermore, exposure to 2MEBM revealed a sub-G1 peak. This is confirmed by studies conducted in our laboratory where flow cytometry utilizing annexin V-FITC demonstrated that 2MEBM induced apoptosis in MCF-7 cells . During apoptosis, the electrochemical mitochondrial membrane gradient collapses [27–30]. The latter precipitates cell death through either the release of molecules involved in apoptosis, or the loss of mitochondrial functions essential for cell survival .
This study demonstrates the novel finding that 2MEBM exposure in the MCF-7 cell line resulted in increased ROS production. However, the source and processing of ROS and the role they play in 2MEBM-induced growth inhibition and cell death remains to be elucidated as no reports has been published on this matter. It was previously reported that the precursor molecule, 2ME2, inhibits superoxide dismutase and increased ROS production in human leukaemia cells . 2ME2 also induces ROS generation in cell lines including myeloid leukaemia U937 cells, myeloid leukaemia HL-60 cell line, human acute T cell leukaemia cells (Jurkat cells), HeLa cells and MCF-7 cells [32–35]. Azad, et al (2009) revealed that ROS can induce autophagy by means of Atg4, catalase and the mitochondrial electron transport chain leading to both cell survival and cell death and is a selective towards cancer treatment. ROS generation upregulates Beclin-1 expression by an unknown mechanism increasing the occurrence of autophagy and hydrogen peroxide is essential for starvation-induced autophagy and directly targets Atg4 for oxidation and inactivates Atg4 inducing autophagy . In addition, upregulated mitochondrial matrix calcium leads to increased reactive oxygen species production results in a reduction of the mitochondrial membrane potential (permeability transition pore is triggered). The latter leads to cytochrome c release from the mitochondria and subsequent activation of caspases leading to apoptosis [37, 38]. Ling, et al (2003) reported that upregulated reactive oxygen species was accompanied by a reduction in the mitochondrial membrane potential and subsequent apoptosis . Thus, the increased ROS production accompanied with reduced mitochondrial potential found in this study suggests that both of the above-mentioned contribute to the apoptosis induced by 2MEBM. The novel finding regarding the in vitro influence of 2MEBM on either of these reactive oxygen species has not been reported as previously.