Molecular crosstalk between apoptosis and autophagy induced by a 2-methoxyestradiol analogue (C19) in HeLa cells

Abstract

Cervical cancer is reported by the World Health Organisation to be the second most common type of cancer to affect women in poorer socioeconomic countries. Treatment of this pathology remains sub-optimal at advanced stages and continues to be of importance on the research agenda. Previous studies have reported cytotoxic and antiproliferative effects of 2-methoxyestradiol (2-ME) in vitro on a HeLa cervical cancer cell line. These results were promising but use of 2-ME itself is limited due to pharmacodynamic constraints. In an attempt to overcome these, a sulphamoylated analogue of 2-ME, namely 2-ethyl-3-O-sulphamoyl-estra- 1,3,5(10)16-tetraene or compound 19 (C19), was synthesised. In this in vitro study, the induction of a block in mitosis with subsequent culmination of apoptosis and autophagy as types of cells death was investigated after HeLa cells were exposed for 24 hours to a 0.5 μM C19 solution. This was achieved by morphological assessment (fluorescent, Polarization-optical transmitted light differential interference contrast microscopy (PlasDIC) and transmission electron microscopy (TEM)) and flow cytometry (cell cycle progression, cyclin B1 analysis, phosphatidylserine (PS) flip and aggresome formation). Spectrophotometric quantification of the apoptotic initiator and executioner caspases 8 and 3 respectively was done to determine their involvement in the crosstalk between apoptosis and autophagy. Results included the following: (i) PlasDIC microscopy illustrated the appearance of an increased number of cells blocked in metaphase, stress signaling, premature cell shrinkage, hypercondensed chromatin and the presence of apoptotic bodies after C19 exposure. The presence of ghost cells, cell debris and decreased cell density of the treated cells correlated with the autophagy control. (ii) Fluorescence microscopy employing triple staining highlighted an increased lysosomal activity and staining of C19-exposed cells when compared to the control, as well as evidence of apoptotic and metaphase-blocked cells. This is indicative of both the autophagic and apoptotic cell death process. (iii) TEM allowed for examination of the ultrastructure of the intracellular processes, and revealed that apoptotic cells have hallmarks of both autophagy and apoptosis, confirming the results of light microscopy. (iv) Cell cycle analysis demonstrated more cells present in the sub-G1 and G2/M populations, indicating the induction of apoptosis (confirmed with PS fip flow cytometric quantification) and a metaphase block (corroborated by an increased cyclin B1 fluorescence). (v) The increase in autophagosome formation seen on fluorescence- and transmission electron microscopy was confirmed by flow cytometry demonstrating an upregulation of aggresome formation in C19-exposed cells. This investigation demonstrated induction of both types of cells death by this novel compound. (vi) The upregulation of caspases 8 and 3 was demonstrated in the C19-treated cells, indicating apoptosis induction via the extrinsic pathway. (vii) Confocal microscopy demonstrated complete microtubule disintegration in the C19-exposed HeLa cells. Both apoptotic and autophagic cell death mechanisms were induced in C19-treated HeLa cells after spindle abrogation kept the cells in metaphase block. Insight gained into the molecular effect of C19 on HeLa cells may be used as a springboard for in vivo studies, furthering the development of this promising anticancer agent toward clinical application.