Babu E et al. (2011), Role of SLC5A8, a plasma membrane transporter a...

XB-ART-44324

Oncogene 2011 Sep 22;3038:4026-37. doi: 10.1038/onc.2011.113.

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Role of SLC5A8, a plasma membrane transporter and a tumor suppressor, in the antitumor activity of dichloroacetate.

Babu E , Ramachandran S , CoothanKandaswamy V , Elangovan S , Prasad PD , Ganapathy V , Thangaraju M .

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There has been growing interest among the public and scientists in dichloroacetate (DCA) as a potential anticancer drug. Credible evidence exists for the antitumor activity of this compound, but high concentrations are needed for significant therapeutic effect. Unfortunately, these high concentrations produce detrimental side effects involving the nervous system, thereby precluding its use for cancer treatment. The mechanistic basis of the compound's antitumor activity is its ability to activate the pyruvate dehydrogenase complex through inhibition of pyruvate dehydrogenase kinase. As the compound inhibits the kinase at micromolar concentrations, it is not known why therapeutically prohibitive high doses are needed for suppression of tumor growth. We hypothesized that lack of effective mechanisms for the entry of DCA into tumor cells may underlie this phenomenon. Here we show that SLC5A8 transports DCA very effectively with high affinity. This transporter is expressed in normal cells, but expression is silenced in tumor cells by epigenetic mechanisms. The lack of the transporter makes tumor cells resistant to the antitumor activity of DCA. However, if the transporter is expressed in tumor cells ectopically, the cells become sensitive to the drug at low concentrations. This is evident in breast cancer cells, colon cancer cells and prostate cancer cells. Normal cells, which constitutively express the transporter, are however not affected by the compound, indicating tumor cell-selective therapeutic activity. The mechanism of the compound's antitumor activity still remains its ability to inhibit pyruvate dehydrogenase kinase and force mitochondrial oxidation of pyruvate. As silencing of SLC5A8 in tumors involves DNA methylation and its expression can be induced by treatment with DNA methylation inhibitors, our findings suggest that combining DCA with a DNA methylation inhibitor would offer a means to reduce the doses of DCA to avoid detrimental effects associated with high doses but without compromising antitumor activity.

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Species referenced: Xenopus laevis
Genes referenced: hdac3 slc5a8

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	Fig. 2. Characteristics of dichloroacetate (DCA) transport via SLC5A8Human SLC5A8 was expressed in X. laevis oocytes and the transport function was monitored electrophysiologically using the two-microelectrode voltage-clamp technique. (A) Inward currents induced by DCA (0.25 mM) in the presence (NaCl) or absence (NMDG chloride) of Na+. (B) Saturation kinetics for DCA-induced currents. (C) Na+-activation kinetics for DCA (0.25 mM)-induced currents. (D) Competition between [14C]-nicotinate and DCA for uptake into oocytes via SLC5A8. Uptake of [14C]-nicotinate (50 μM) in water-injected and SLC5A8 cRNA-injected oocytes was measured for 1 h in the absence and presence of acetate (0.25 mM) or DCA (0.25 mM).
	Fig. 3. Transport of monochloroacetate (MCA) and trichloroacetate via SLC5A8Human SLC5A8 was expressed in X. laevis oocytes and the transport function was monitored electrophysiologically using the two-microelectrode voltage-clamp technique. (A) Inward currents induced by MCA (1 mM) in the presence (NaCl) or absence (NMDG chloride) of Na+. (B) Saturation kinetics for monochloroacetate-induced currents. (C) Saturation kinetics for trichloroacetate-induced currents.
	Fig. 4. Induction of apoptosis in colon cancer cell lines by DCA in an SLC5A8-dependent mannerSLC5A8 protein expression was analyzed in human normal colon epithelial cell lines and colon tumor cell lines as well as human normal mammary epithelial cell lines and breast tumor cell lines by immunoblot analysis (A, B). Human colon cancer cell lines (HT29, SW620, and HCT116) were transfected with either vector alone or human SLC5A8 cDNA, and then cultured in the absence or presence of acetate, monochloroacetate (MCA), dichloroacetate (DCA), trichloroacetate (TCA), or butyrate for 48 h. Concentration of the fatty acids was 1 mM. Cells were then used for isolation of RNA, protein and analysis of apoptosis. (C) RT-PCR and immunoblot analyses of SLC5A8 expression in vector-transfected and SLC5A8 cDNA-transfected cells. (D–F) Apoptosis was quantified in these cells by FACS analysis.
	Fig. 5. Induction of apoptosis in breast and prostate cancer cell lines by DCA in an SLC5A8-dependent mannerHuman breast cancer cell lines (MCF7, an ER-positive cancer cell lines, and MB231, an ER-negative cancer cell line) and human prostate cancer cell lines (DU145, a hormone-resistant cancer cell line, and LNCaP, a hormone-sensitive cancer cell line) were transfected with either vector alone or human SLC5A8 cDNA, and then cultured in the absence or presence of dichloroacetate (DCA, 1 mM) for 48 h. Two normal cell lines, one representing colonic epithelium (CCD841) and another representing mammary epithelium (MCF10A) were also used in a similar manner. Cells were then used for isolation of RNA and protein as well as for analysis of apoptosis. (A) RT-PCR and immunoblot analyses of SLC5A8 expression in vector-transfected and SLC5A8 cDNA-transfected cells. (B) Apoptosis was quantified in these cells by FACS analysis. (C) Tet-On system-regulated lenti virus-mediated SLC5A8 and pLVX stable cell lines were generated in MCF7 and MB231 cells. RT-PCR and immunoblot analyses of SLC5A8 expression in the presence and absence of doxycylin (Dox) in pLVX and SLC5A8 stable cell lines. (D) Cell cycle analysis for pLVX and SLC5A8 stable cell lines in the presence and absence of Dox and with and without DCA. (E) Human normal colon and mammary epithelial cells as well as colon and breast tumor cells were treated with 5′-Azadc for 72 h and the re-activation of SLC5A8 expression was analyzed by immunoblot analysis. (F) Control and SLC5A8-reactivated cells were treated with DCA (1 mM) following which the extent of apoptosis was monitored by FACS analysis.
	Fig. 6. Differential effect of dichloroacetate (DCA) on intracellular levels of pyruvate in colon cancer cells and its dependence on SLC5A8Three human colon cancer cell lines (HT29, SW620, and HCT116) were transfected with either vector alone or SLC5A8 cDNA, and then cultured in the absence or presence of acetate, monocholoroacetate (MCA), dichloroacetate (DCA), or trichloroacetate (TCA) for 48 h. Concentration of the fatty acids was 1 mM. Cells were then lysed and used for determination of pyruvate and protein. Data for pyruvate were normalized with protein levels. (A) HT29; (B) SW620; (C) HCT116. , p < 0.05, , p < 0.01 and **, p < 0.001.
	Fig. 7. Lack of inhibitory effect on histone deacetylase (HDAC) activity by dichloroacetate (DCA)(A) SW620 cell lysates were used as the source of HDAC activity. Assays were done in the absence or presence of acetate, monochloroacetate (MCA), dichloroacetate (DCA), or butyrate at indicated concentrations. (B-E) Inhibition of human recombinant HDAC isoforms by butyrate, dichloroacetate, acetate, and monochloroacetate. Concentration of the fatty acids was 1 mM. Data are presented as percent of control activity measured in the absence of the fatty acids. , p < 0.05; , p < 0.01; **, p < 0.001.

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