Human GM3 Synthase Attenuates Taxol-Triggered Apoptosis Associated with Downregulation of Caspase-3 in Ovarian Cancer Cells

Abstract

Background: Taxol (paclitaxel) inhibits proliferation and induces apoptosis in a variety of cancer cells, but it also upregulates cytoprotective proteins and/or pathways that compromise its therapeutic efficacy.

Materials and method: The roles of GM3 synthase (α2,3-sialyltransferase, ST3Gal V) in attenuating Taxol-induced apoptosis and triggering drug resistance were determined by cloning and overexpressing this enzyme in the SKOV3 human ovarian cancer cell line, treating SKOV3 and the transfectants (SKOV3/GS) with Taxol and determining apoptosis, cell survival, clonogenic ability, and caspase-3 activation.

Results: In this report, we demonstrated that Taxol treatment resulted in apoptosis which was associated with caspase-3 activation. Taxol treatment upregulated the expression of human GM3 synthase, an enzyme that transfers a sialic acid to lactosylceramide. Moreover, we cloned the full-length GM3 synthase gene and showed for the first time that forced expression of GM3 synthase attenuated Taxol-induced apoptosis and increased resistance to Taxol in SKOV3 cells.

Conclusions: GM3 synthase overexpression inhibited Taxol-triggered caspase-3 activation, revealing that upregulation of GM3 synthase prevents apoptosis and hence reduces the efficacy of Taxol therapy.

Keywords: Apoptosis; Caspase-8; Drug Resistance; GM3 Synthase; Ovarian Cancer; Taxol.

Figure 1

Taxol-induced apoptosis and GM3 synthase expression in SKOV3 cells. (a) Cells were treated with 100 nM Taxol for 48 h, harvested, and apoptosis was determined by FACS analysis as described in Materials and Methods. Compensation was executed for each experiment using untreated cells and cells stained only with annexin V or propidium iodide, respectively. Error bars show standard deviation from triplicate measurements (*P < 0.05). (b) Total RNA from SKOV3 cells was extracted and the levels of GM3 synthase mRNA were measured by RT-PCR. β-actin was used as an internal control in the RT-PCR reactions. (c) SKOV3 and SKOV3/GS cells were treated with 100 nM of Taxol for 48 h and apoptotic cell death was determined by DAPI staining as described in Materials and Methods (c1 and c3 are control untreated SKOV3 and SKOV3/GS, respectively; c2 and c4 SKOV3 and SKOV3/GS cells treated with 100 nM Taxol, respectively). (d) Apoptotic cells were identified by condensation and fragmentation of nuclei. A minimum of 300 cells were counted for each treatment, and the percentage of apoptotic cells was calculated. Error bars show S.D. from triplicate measurements.

Figure 2

GM3 synthase prevents caspase-3 activation. Western blot analysis of GM3 synthase expression in SKOV3/GS cells using V5 antibody. SKOV3 and SKOV3/GS cells were treated with 100 nM Taxol for 48 h and GM3 synthase was detected by Western blot analysis using V5 antibody as described in the Material and Methods. Note that forced expression of GM3 synthase attenuates Taxolinduced apoptosis (Figure 1(a)) and caspase-3 activation.

Figure 3

GM3 synthase increases proliferation and decreases Taxol cytotoxicity in SKOV3/GS cells. (a) SKOV3 and SKOV3/GS cells were grown for 48 h and the number of cells was determined as described in Materials and Methods. (b) SKOV3 and SKOV3/GS cells were treated with 1 – 100 nM Taxol for 48 h and the numbers of cells which survived was determined by methylene blue cytotoxicity assay as described in Materials and Methods. (c) SKOV3 cells untreated and SKOV3 cells treated with 5 or 10 nM Taxol for 48 h; SKOV3/GS untreated cells and SKOV3/GS cells treated with 5 or 10 nM Taxol for 48 h as described in the Materials and Methods. Colonies were counted 7 days later. The values are representative of triplicate counts and error bars show standard deviations.