Ultrastructural Examination of Intracellular Calcium Changes in Ovarian Cancer

Abstract

In this study, following the fibronectin-coated integrin activation in ovarian cancer cells (SKOV-3), endoplasmic reticulum (ER) stress was generated by intracellular calcium increase and then possible apoptotic effects were investigated. Tunicamycin (TN) concentrations and intracellular calcium alterations in SKOV-3 cells were analyzed by flow cytometry using Fluo-3 AM ye Rhod-2 AM flou-rescent dyes. Apoptotic cell death dependent to ER stress that generated after the treatment with 18 mu M TN were investigated by Annexin V / PI protocol and the localisations of cytochrome c and EndoG proteins were also imaged by fluorescent microscope. In addition, morphological alterations were determined by Transmission electron microscope. Following the treatment with 18 mu M TN of SKOV-3 cells staining with Fluo-3 AM ye Rhod-2 AM, cytoplasmic and mitochondrial Ca2+ increases were observed, respectively. Correspondingly, early apoptosis percentage was 11.38 % and late apoptosis percentage was 10.24% in SKOV-3 cells. After 2 h, TN incubations of mitochodrial apoptotic proteins cytocrome c and EndoG, flourescent radiations were found as more intense than non-coating cells. Apoptotic changes depending on ER stress in fibronectin coated SKOV-3 cells were determined as plasma and nucleus membrane damages, nucleus condensation and intense heterochromatin structures in nucleus by fragmentation. On the other hand, autophagic findings were also obseved. As a result, calcium dependent apoptotic process in SKOV-3 cells in metastatic stage were partially shown. Electron microscopic findings showed that TN treatment effectively induced ER stress-associated apoptosis and autophagy in SKOV-3 cells that effect becomes stronger with binding of cells to fibronectin. However, the detailed mechanism underlying the crosstalk between TN-induced autophagy and apoptosis in SKOV-3 cells warrants further study.