Mitochondrial and plasma membrane lactate transporter and lactate dehydrogenase isoform expression in breast cancer cell lines - PubMed (original) (raw)
Mitochondrial and plasma membrane lactate transporter and lactate dehydrogenase isoform expression in breast cancer cell lines
Rajaa Hussien et al. Physiol Genomics. 2011.
Abstract
We hypothesized that dysregulation of lactate/pyruvate (monocarboxylate) transporters (MCT) and lactate dehydrogenase (LDH) isoforms contribute to the Warburg effect in cancer. Therefore, we assayed for the expression levels and the localizations of MCT (1, 2, and 4), and LDH (A and B) isoforms in breast cancer cell lines MCF-7 and MDA-MB-231 and compared results with those from a control, untransformed primary breast cell line, HMEC 184. Remarkably, MCT1 is not expressed in MDA-MB-231, but MCT1 is expressed in MCF-7 cells, where its abundance is less than in control HMEC 184 cells. When present in HMEC 184 and MCF-7 cells, MCT1 is localized to the plasma membrane. MCT2 and MCT4 were expressed in all the cell lines studied. MCT4 expression was higher in MDA-MB-231 compared with MCF-7 and HMEC 184 cells, whereas MCT2 abundance was higher in MCF-7 compared with MDA-MB-231 and HMEC 184 cells. Unlike MCT1, MCT2 and MCT4 were localized in mitochondria in addition to the plasma membrane. LDHA and LDHB were expressed in all the cell-lines, but abundances were higher in the two cancer cell lines than in the control cells. MCF-7 cells expressed mainly LDHB, while MDA-MB-231 and control cells expressed mainly LDHA. LDH isoforms were localized in mitochondria in addition to the cytosol. These localization patterns were the same in cancerous and control cell lines. In conclusion, MCT and LDH isoforms have distinct expression patterns in two breast cancer cell lines. These differences may contribute to divergent lactate dynamics and oxidative capacities in these cells, and offer possibilities for targeting cancer cells.
Figures
Fig. 1.
Expression of CD147 and monocarboxylate transporter and lactate dehydrogenase (LDH) isoforms detected by immunoblotting. To show relative abundances left- and right-hand plates show unsaturated and saturated autoradiograms, respectively. The expression of MCT1 (A), MCT2 (B), MCT4 (C), CD147 (D), total LDH (E), LDHB (F), and LDHA (G) in whole homogenate (WH), cytosolic fraction (Cyto), and mitochondrial fraction (MI) from 1 control (HMEC 184) and 2 cancerous breast cell lines (MCF-7, MDA-MB-231). All cell lines expressed lactate transporters (MCT1, 2, 4) in WH and MI fractions except MDA-MB-231, which did not express MCT1. An LDH antibody that reacts with all LDH isoforms was used in E. LDH was found in both Cyto and MI fractions of all cell lines. The same amount of total protein was loaded (30 μg) in each well. The fold changes in the expression levels of lactate shuttle proteins in whole homogenate fraction and mitochondrial fraction in the 2 cancerous cell lines were compared with the control cell line (H). The band of interest that was used for densitometer quantification was marked by underlining the molecular weight standard that corresponded to its size in A, B, C, D, E, F, and G and was reported in H. Data are derived from the average of 4 different experiments ± SE.
Fig. 2.
Agarose gel electrophoresis analysis of LDH isoforms. LDH isoforms from Cyto and MI in control primary breast cell line (HMEC 184), and breast cancer cell lines (MCF-7, MDA-MB-231) were separated by agarose gel electrophoresis. Total protein of 12 μg was loaded in wells (1, 2, 3, 4, 5, and 6), and 48 μg in wells (7, 8, and 9). The MCF-7 cell line expressed mainly LDHB, an LDH isoform found in oxidative cell lines. The HMEC 184 and MDA-MB-231 cell lines expressed mainly LDHA, an LDH isoform found in glycolytic cell lines. Data are from 2 different experiments.
Fig. 3.
Assessment of subcellular contamination. Representative immunoblots showing expressions of COx (A), GAPDH (B), Na+-K+-ATPase-α (C), TGFβ-R2 (D), and GLUT1 (E) in WH, Cyto, and MI from normal primary breast cell line (HMEC 184) and breast cancer cell lines (MCF-7, MDA-MB-231). MI were rich with the mitochondrial marker COx-IV and showed the presence of plasma membrane marker Na+-K+-ATPase-α in all mitochondrial fractions and the presence of plasma membrane marker GLUT1 in the mitochondrial fraction of MDA-MB-231 cells, but no signal of plasma membrane marker TGFβ-R2. There were undetectable amounts of the cytosolic marker GAPDH in mitochondrial fractions.
Fig. 4.
Immunohistochemical detection of MCT, LDH isoforms, and COx in control breast cell line HMEC 184. LDH isoforms, MCT2, and MCT4 were colocalized with mitochondrial protein marker COx (A, C, D), but MCT1 was not and was localized mainly in the plasma membrane (B). The thickness of the optical section ∼1 μm, scale bar = 10 μm.
Fig. 5.
Immunohistochemical detection of MCT, LDH isoforms, and COx in breast cancer cell line MCF-7. LDH isoforms, MCT2, and MCT4 were colocalized with mitochondrial protein marker COx (A, C, D), but MCT1 was not and was localized to the plasma membrane (B). The thickness of the optical section ∼1 μm, scale bar = 10 μm.
Fig. 6.
Immunohistochemical detection of MCT, LDH isoforms, and COx in breast cancer cell line MDA-MB-231. LDH isoforms, MCT2, and MCT4 were colocalized with mitochondrial protein marker COx (A, B, C). MCT 1 was not expressed in MDA-MB-231 cells. The thickness of the optical section ∼1 μm, scale bar = 10 μm.
Fig. 7.
Lactate production is higher in cancer cells, and the oxygen consumption is higher in control cells. Lactate production (μM) per hour in HMEC 184 (A), MCF-7 (B), and MDA-MB-231 (C) cell lines was normalized to total protein concentration (μg). Lactate production rates are represented by histogram bars and diagonal slope. Cells were incubated with media with and without 40 mM oxamate (Oxa), an LDH inhibitor, and 50 μM iodoacetate (IAA), a glycolysis blocker. Lactate production was higher in control, IAA, and Oxa dishes of cancerous cell lines compared with the control cell line. The respiration in control and cancerous cell lines were measured (A, B, C). The endogenous respiration (D), CCCP (uncoupler)-treated respiration (E), and CCCP-treated respiration to endogenous respiration ratio (F) were calculated by measuring the oxygen consumption in HMEC 184 (G), MCF-7 (H), and MDA-MB-231 (I) cell lines. The MCF-7 cell line had higher endogenous respiration rate than did MDA-MB-231. The HMEC 184 cell line has the highest endogenous and max respiration rates. Data are means ± SE. Significantly different between groups: *P < 0.05, **P < 0.001.
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