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. 2008 Jun;109(3):394-402.
doi: 10.1016/j.ygyno.2008.01.038. Epub 2008 Apr 18.

Dichloroacetate induces apoptosis in endometrial cancer cells

Jason Y Y Wong  1 , Gordon S Huggins, Marcella Debidda, Nikhil C Munshi, Immaculata De Vivo
Affiliations

Dichloroacetate induces apoptosis in endometrial cancer cells

Jason Y Y Wong et al. Gynecol Oncol. 2008 Jun.

Abstract

Purpose: A recent landmark study demonstrated that Dichloroacetate (DCA) treatment promoted apoptosis in lung, breast, and glioblastoma cancer cell lines by shifting metabolism from aerobic glycolysis to glucose oxidation coupled with NFAT-Kv1.5 axis remodeling. The objective of this study was to determine whether DCA induces apoptosis in endometrial cancer cells and to assess apoptotic mechanism.

Methods: A panel of endometrial cancer cell lines with varying degrees of differentiation was treated with DCA and analyzed for apoptosis via flow cytometry. Biological correlates such as gene expression, intracellular Ca(2+), and mitochondrial membrane potential were examined to assess apoptotic mechanism.

Results: Initiation of apoptosis was observed in five low to moderately invasive cancer cell lines including Ishikawa, RL95-2, KLE, AN3CA, and SKUT1B while treatment had no effect on non-cancerous 293T cells. Two highly invasive endometrial adenocarcinoma cell lines, HEC1A and HEC1B, were found to be resistant to DCA-induced apoptosis. Apoptotic responding cell lines had a significant increase in early and late apoptotis, a decrease in mitochondrial membrane potential, and decreased Survivin transcript abundance, which are consistent with a mitochondrial-regulated mechanism. DCA treatment decreased intracellular calcium levels in most apoptotic responding cell lines which suggests a contribution from the NFAT-Kv1.5-mediated pathway. DCA treatment increased p53 upregulated modulator of apoptosis (PUMA) transcripts in cell lines with an apoptotic response, suggesting involvement of a p53-PUMA-mediated mechanism.

Conclusions: Dichloroacetate effectively sensitizes most endometrial cancer cell lines to apoptosis via mitochondrial, NFAT-Kv1.5, and PUMA-mediated mechanisms. Further investigation of the cancer therapeutic potential of DCA is warranted.

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Conflict of interest statement

Conflict of Interest Statement:

The authors declare there are no conflicts of interest.

Figures

Figure 1
Figure 1. Dichloroacetate decreases cell viability through promotion of apoptosis in most endometrial cancer cell lines
A) In a dose-response experiment, the cell viability of endometrial cancer cell lines after 40 hours of treatment with 10 mM DCA was measured by reduction of Resazurin to Resorufin by metabolically healthy cells. Error bars represent standard error between 2 to 3 replicate experiments performed in triplicate wells. p < 0.01 between 0 mM and 10 mM DCA for those with decreased viability. B) FACS Traces from the Annexin-V / 7-AAD assay for apoptosis. Seven endometrial cancer cell lines and 293T were treated with 10 mM DCA for 40 hours and analyzed for apoptosis via flow cytometry. The upper bar graph is the difference in early apoptotic cells with and without DCA treatment. The lower bar graph is the difference in late apoptotic cells with and without DCA treatment. Error bars represent standard deviation between 3 replicate experiments. p ≤ 0.05 for those with increased percentage of apoptotic cells. C) Several representative cell lines were treated with 10 mM DCA for 48 hours and TUNEL-stained to confirm late apoptotic genomic fragmentation. Slides were viewed in bright field under 40X objective magnification. Arrows indicate TUNEL-positive cells.
Figure 2
Figure 2. DCA induced apoptosis consistent with mitochondrial-regulated and NFAT-Kv1.5 mechanisms
A) In a dose-response experiment, intracellular calcium levels were measured after treatment of endometrial cancer cell lines with increasing concentrations of DCA for 8 hours. Error bars represent standard error between 2 to 3 independent experiments performed in triplicate wells. p < 0.01 between 0 mM and 10 mM for those with decreased calcium levels. B) One representative apoptotic responder and non-responder were treated with 10 mM DCA for 24 hours and their mitochondrial membrane potentials measured via Mitocapture. Cells with hyperpolarized MMP stain strongly for PE (red) and apoptotic cells with decreased MMP stain for FITC (green). The experiment was formed in triplicate C) Reduction of hyperpolarized MMP is further confirmed via TMRM fluorescent staining in a DCA dose-response experiment. Error bars represent standard error from 2 independent experiments performed in triplicate wells.
Figure 3
Figure 3. Dichloroacetate decreases Survivin mRNA abundance and increases PUMA mRNA abundance in endometrial cancer cell lines
A) Real-time Quantitative PCR was used to evaluate Survivin transcription after 40 hours of treatment with and without 10 mM DCA. Relative Quantification (RQ) of Survivin mRNA was performed with the Comparative –dCt method against a RPLP0 housekeeping gene. B) Real-time Quantitative PCR was used to evaluate PUMA transcription after 40 hours of treatment with and without 10 mM DCA. Relative Quantification of PUMA mRNA was performed with the Comparative –dCt method against a RPLP0 housekeeping gene. Error bars represent RQ 95% Confidence Intervals of 2 replicate experiments performed in triplicate wells. p < 0.01.
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