Investigation of Epothilone B-Induced Cell Death Mechanisms in Human Epithelial Cancer Cells –in Consideration of Combined Treatment With Ionizing Radiation

Abstract

Epothilone B was shown to have promising chemo- and radiosensitizing effects on cells, but the mechanisms underlying cell death remain ambiguous. The aim of the study was to examine selected cell death pathways on the basis of FaDu and A549 cells. Western blot analyses were used for investigation of specific apoptotic markers. Immunofluorescence imaging and flow cytometry were utilized for examination of cell death mechanisms. DNA-staining was used for studying influence of epothilone B on micronucleus rate. We showed that epothilone B can initiate cell death via apoptosis and mitotic catastrophe, but induction of cell death was cell type specific.

Keywords: :

• Epothilone B
• Irradiation
• Apoptosis
• Mitotic catastrophe
• Micronucleus

Supplemental Material Available Online

Supplemental file 1: HPLC measurement to study the stability of epothilone B molecules in the medium. (A) Representative HPLC-spectrum of 10 nM epothilone B in the media. (B) Examination of the peak area of the measured HPLC-spectra of different samples. (n = 1).

Supplemental file 2: Protein expression after 10 nM epothilone B treatment by western blot analysis. A variety of tested cell death-related antibodies and the densitometric analysis of the protein-levels in the cell lysates after 48 hours of drug exposure are presented. A densitometric analysis of the protein-levels, normalized to the control bands of beta-actin, was performed. (n = 1) Horizontal lines in the table mean “no protein detectable.”

Supplemental file 3: Immunofluorescence imaging of: (A) Cytochrome C stained cells after incubation with 10 nM epothilone B for 27 hours. DMSO-treated control cells (above) and epothilone B-incubated cells (below) stained with cytochrome C antibody. (B) MitoTracker Green FM stained cells after incubation of 48 hours with 10 nM epothilone B. DMSO-treated control cells (above) and epothilone B-incubated cells (below) stained with MitoTracker Green FM.