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. 2019 Jan 29;40(9):1110–1120. doi: 10.1093/carcin/bgz015

Figure 6.

Figure 6.

Combined treatment with WFA and 2-DG synergistically inhibits breast cancer cells. WFA induces energy impairment with reducing LDHA and higher expression of AMPK and low expression of LDHA correlates with increased overall survival. (A, B) HCC1569, HCC1806, HS578t, MDA-MB-231 and MDA-MB-468 breast cancer cells were treated with various concentrations of WFA (1.0, 2.5, 5.0. 7.5, 10.0 µM) in combination with 4.0 mM of 2-DG. MCF7 cells were treated with various concentrations of WFA in combination with 2.0 mM 2-DG. Cells were subjected to XTT assay and combination index values were calculated using CompuSyn software. CI < 1 represents synergism. Table shows combination index of different concentrations of WFA and 2-DG. (C) MCF7 cells were treated with 5 µM WFA or 10 mM MP as indicated and intracellular ATP production was measured. Relative ATP levels are expressed with respect to the control. *P< 0.01, compared with control; **P< 0.005, compared with WFA alone. (D) MCF7 cells were treated with 5 µM WFA or 10 mM MP as indicated and subjected to Trypan Blue exclusion assay. Bar graph shows fold change of alive cells. *P< 0.01, compared with control; **P< 0.001, compared with WFA alone. (E) MCF7 cells were treated with 5 µM WFA or 10 mM MP as indicated followed by Hoechst 33342 staining apoptosis detection. Mean number of apoptotic cells are presented as fold change in bar graphs. *P < 0.01, compared with control; **P< 0.01, compared with WFA alone. (F) Total lysates were immunoblotted for the expression of cPARP1 (89 kDa) and PARP1 (116 kDa). ACTB (45 kDa) was used as loading control. (G) Schematic representation of TCA cycle and aerobic glycolysis in proliferating cancer cells. Role of LDHA is noted. (H) Breast cancer cells were treated with 5 µM WFA for various time intervals as indicated. Total RNA was examined for the expression of LDHA using RT-PCR. ACTB was used as loading control. (I) Breast cancer cells were treated with 5 µM WFA for various time intervals as indicated. Total lysates were immunoblotted for the expression of LDHA (37 kDa) and phospho-LDHA (p-LDHA). ACTB (45 kDa) was used as loading control. (J) In breast cancer patients, higher expression of AMPK and lower expression of LDHA correlate to better prognosis (n = 4,374, HR = 0.75, P = 1.3e-05 and HR = 1.59, P = 1.6e-16, respectively). When investigating ER negative HER2 negative patients, the correlation to better survival was more prominent in LDHA (HR = 1.64, P = 4.7e-05) but was smaller for AMPK (HR = 0.81, P = 0.1). The plots display relapse free-survival, ER and HER2 status were determined using gene expression.