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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 - Oncogene (2011)

Bottom Line: Credible evidence exists for the antitumor activity of this compound, but high concentrations are needed for significant therapeutic effect.Here we show that SLC5A8 transports DCA very effectively with high affinity.The mechanism of the compound's antitumor activity still remains its ability to inhibit pyruvate dehydrogenase kinase and force mitochondrial oxidation of pyruvate.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Medical College of Georgia, Georgia Health Sciences University, Augusta, GA 30912, USA.

ABSTRACT
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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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.
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Figure 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.

Mentions: We then wanted to determine if dichloroacetate-induced apoptosis exhibits tumor cell selectivity and also if the effect is seen in cancer cell lines of tissue origin other than colon. For this, we selected CCD841 and MCF10A as the representative of normal cell lines (CCD841, colon; MCF10A, mammary epithelium) and four human cancer cell lines: MCF7 (an estrogen receptor-positive breast cancer cell line), MB231 (an estrogen receptor-negative breast cancer cell line), DU145 (an androgen-insensitive prostate cancer cell line) and LNCaP (an androgen-sensitive prostate cancer cell line). As reported previously (Thangaraju et al., 2006, 2008), the normal cell lines CCD841 and MCF10A expressed detectable levels of SLC5A8, at both mRNA and protein levels (Fig. 5A). In contrast, none of the four cancer cell lines examined here expressed the transporter. The expression became evident in cancer cell lines upon transient transfection of a mammalian expression construct. In normal cells, the expression levels increased upon transfection. Using these cell lines, we compared the ability of dichloroacetate to induce apoptosis between normal and cancer cell lines (Fig. 5B). We found no significant difference in apoptosis in normal cell lines with and without treatment with dichloroacetate (1 mM). The increased levels of SLC5A8 expression also did not have any effect on the extent of apoptosis. In contrast, dichloroacetate was able to induce marked apoptosis in the four cancer cell lines, but only if the cells expressed the transporter. Without the expression of the transporter, the cancer cell lines did not undergo apoptosis upon treatment with dichloroacetate. These results show that dichloroacetate is able to cause apoptosis even at concentrations as low as 1 mM in a cancer cell-selective manner, but only if SLC5A8 is expressed. These observations were also confirmed with lenti virus-mediated stable expression of SLC5A8 (doxycylin-inducible) in two breast cancer cell lines, MCF7 and MB231 (Fig. 5C, D). It has been well established that the silencing of SLC5A8 is associated with DNA methylation and that treatment of cancer cells with DNA-demethylating agents re-actives SLC5A8 expression. Therefore, we treated normal and cancer cell lines with 5′-aza-2-deoxycytidine (5-Azadc), a DNA-demethylating agent, and the re-activation of SLC5A8 was confirmed by immunoblotting analysis (Fig. 5E). 5-Azadc treatment did not alter SLC5A8 protein expression in normal colon and mammary epithelial cells while it re-activated SLC5A8 expression in all cancer cell lines. Furthermore, treatment of these cells with 5-Azadc itself induced significant apoptosis; however, treatment of these cells with dichloroacetate (1 mM) dramatically enhanced the 5-Azadc-induced apoptosis (Fig. 5F).


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 - Oncogene (2011)

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.
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Figure 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.
Mentions: We then wanted to determine if dichloroacetate-induced apoptosis exhibits tumor cell selectivity and also if the effect is seen in cancer cell lines of tissue origin other than colon. For this, we selected CCD841 and MCF10A as the representative of normal cell lines (CCD841, colon; MCF10A, mammary epithelium) and four human cancer cell lines: MCF7 (an estrogen receptor-positive breast cancer cell line), MB231 (an estrogen receptor-negative breast cancer cell line), DU145 (an androgen-insensitive prostate cancer cell line) and LNCaP (an androgen-sensitive prostate cancer cell line). As reported previously (Thangaraju et al., 2006, 2008), the normal cell lines CCD841 and MCF10A expressed detectable levels of SLC5A8, at both mRNA and protein levels (Fig. 5A). In contrast, none of the four cancer cell lines examined here expressed the transporter. The expression became evident in cancer cell lines upon transient transfection of a mammalian expression construct. In normal cells, the expression levels increased upon transfection. Using these cell lines, we compared the ability of dichloroacetate to induce apoptosis between normal and cancer cell lines (Fig. 5B). We found no significant difference in apoptosis in normal cell lines with and without treatment with dichloroacetate (1 mM). The increased levels of SLC5A8 expression also did not have any effect on the extent of apoptosis. In contrast, dichloroacetate was able to induce marked apoptosis in the four cancer cell lines, but only if the cells expressed the transporter. Without the expression of the transporter, the cancer cell lines did not undergo apoptosis upon treatment with dichloroacetate. These results show that dichloroacetate is able to cause apoptosis even at concentrations as low as 1 mM in a cancer cell-selective manner, but only if SLC5A8 is expressed. These observations were also confirmed with lenti virus-mediated stable expression of SLC5A8 (doxycylin-inducible) in two breast cancer cell lines, MCF7 and MB231 (Fig. 5C, D). It has been well established that the silencing of SLC5A8 is associated with DNA methylation and that treatment of cancer cells with DNA-demethylating agents re-actives SLC5A8 expression. Therefore, we treated normal and cancer cell lines with 5′-aza-2-deoxycytidine (5-Azadc), a DNA-demethylating agent, and the re-activation of SLC5A8 was confirmed by immunoblotting analysis (Fig. 5E). 5-Azadc treatment did not alter SLC5A8 protein expression in normal colon and mammary epithelial cells while it re-activated SLC5A8 expression in all cancer cell lines. Furthermore, treatment of these cells with 5-Azadc itself induced significant apoptosis; however, treatment of these cells with dichloroacetate (1 mM) dramatically enhanced the 5-Azadc-induced apoptosis (Fig. 5F).

Bottom Line: Credible evidence exists for the antitumor activity of this compound, but high concentrations are needed for significant therapeutic effect.Here we show that SLC5A8 transports DCA very effectively with high affinity.The mechanism of the compound's antitumor activity still remains its ability to inhibit pyruvate dehydrogenase kinase and force mitochondrial oxidation of pyruvate.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Medical College of Georgia, Georgia Health Sciences University, Augusta, GA 30912, USA.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus