Figure 1.
Zymography of human breast cell lines.
Note the absent or decreased expression of LDH isoenzymes 1–4 in T-47D and MCF7 cells. In contrast, expression of LDH isoenzymes 1–4, can be seen in MDA-MB-436 and MDA-MB-231 cells. Expression of LDH isoenzymes 2-5, are predominant in the MCF10AT, MCF10DCIS.com and MCF10A cells. The expression of all 5 LDH isoenzymes can be seen in LNCaP cells, while absent or decreased expression of LDH isoenzymes 1–4, can be seen in the LNCaP-LN3 cells used as controls. In MCF10AT, MDA-MB-436, MDA-MB-231, MCF10DCIS.com and MCF10A cells, a weak band (arrow head), can be seen under the LDH-4 isoenzyme band, and may represent a posttranslationally modified form of LDH [28].
Figure 2.
Sequencing chromatograms of sodium bisulfite modified DNA
Methylation at CpG sites (marked by arrowheads), can be seen in (a) T-47D cells and in (b) a case of human breast cancer tissue by the presence of unmodified CpG residues (arrowheads). Identical sites can be seen to be modified and therefore unmethylated in (c) normal human breast tissue and (d) peripheral blood lymphocyte DNA from a patient with breast cancer.
Figure 3.
Immunoexpression of LDH-B in human breast tissues.
(a) Strong cytoplasmic expression of LDH-B protein can be seen in the glands of non-malignant epithelium (x20 magnification), and (b) in a case of pre-invasive DCIS lesion (x10). (c) A DCIS lesion (marked by asterix) showing absent expression of LDH-B, while strong expression can be seen in the adjacent and surrounding non-malignant glands (x10). (d) A breast cancer case showing absent LDH-B expression, while expression can be seen in non-malignant glands at the top left of the image (x10).
Figure 4.
Reverse-transcription PCR for LDH-B mRNA.
Untreated (U) T-47D cells showed an absence of LDH-B mRNA expression. Treatment (T) of cells with 1 µM 5'-Azacytidine for 5 days, was able to restore mRNA expression. GAPDH was used as a loading and amplification control.
Figure 5.
Lactate levels in conditioned medium from breast cell lines under normoxia and hypoxia.
Following hypoxic growth, mean lactate levels in the conditioned medium from MCF10AT cells increased from 27.9 µM to 33.6 µM per 1×106 cells (1.2 fold change), but this difference was not statistically significant (p = 0.09, Mann Whitney test). In MDA-MB-436 cells the mean lactate levels decreased slightly from 38.5 µM to 34.5 µM per 1×106 cells (-0.9 fold change), but was not statistically significant (p = 0.229). However, following hypoxic growth of MCF7 cells, mean lactate levels increased significantly (p = 0.002), from 2.9 µM to 40.7 µM per 1×106 cells (14.0 fold change). Similarly, in T-47D cells the mean lactate levels were found to increase significantly (p = 0.009), from 15.2 µM to 43.5 µM per 1×106 cells (2.9 fold change), following hypoxic growth. * indicate statistically significant differences (p<0.05).
Figure 6.
Zymography of T-47D cancer cells following hypoxic treatment.
Note the relative increased activity of LDH-5 following hypoxic growth.