Oleanolic Acid Inhibits High Salt-Induced Exaggeration of Warburg-like Metabolism in Breast Cancer Cells

Abstract

Cancer cells have a proliferative advantage by utilizing intermediates of aerobic glycolysis (Warburg effect) for their macromolecule synthesis. Although the exact causes of this Warburg effect are unclear, high osmotic stress in solid tumor microenvironment is considered one of the important factors. Oleanolic acid (OA) is known to exert anti-inflammatory and anti-cancer effect. In our current studies, using breast cancer cell lines, we determined the protective role of OA in high salt-mediated osmotic stress-induced cancer growth. Hypertonic (0.16 M NaCl) culture conditions enhanced the cancer cell growth (26 %, p < 0.05) and aerobic glycolysis as marked by increased glucose consumption (34 %, p < 0.05) and lactate production (25 %, p < 0.05) over untreated cells. This effect was associated with increased expression and activity of key rate-limiting enzymes of aerobic glycolysis, namely hexokinase, pyruvate kinase type M2, and lactate dehydrogenase A. Interestingly, this high salt-mediated enhanced expression of aerobic glycolytic enzymes was efficiently reversed by OA along with the decreased cancer cell proliferation. In cancer cells, enhanced aerobic glycolysis is associated with the decreased mitochondrial activity and mitochondrial-associated caspase activity. As expected, high salt further inhibited the mitochondrial related cytochrome oxidase and caspase-3 activity. However, OA efficiently reversed the high salt-mediated inhibition of cytochrome oxidase, caspase activity, and pro-apoptotic Bax expression, thus suggesting that OA induced mitochondrial activity and enhanced apoptosis. Taken together, our data indicate that OA efficiently reverses the enhanced Warburg-like metabolism induced by high salt-mediated osmotic stress along with potential application of OA in anti-cancer therapy.

Keywords: Apoptosis; Breast cancer; Glycolysis; Oleanolic acid; Osmotic Stress; Warburg effect.

Figure 1. Oleanolic acid suppresses high salt induced aerobic glycolysis in breast cancer cells

(A) Dose titration of salt (NaCl - 0.1 to 0.26 M) on MDA-MB-231, highly invasive epithelial breast cancer cell line; (■) without oleanolic acid (5 μM), (□) with oleanolic acid (5 μM); (*) statistical significance at 0.16 M NaCl used in further experiments. (B) Dose titration of oleanolic acid (OA – 0 to 10 μM) on MDA-MB-231, highly invasive epithelial breast cancer cell line; (◇) with regular NaCl (0.1 M), (◆) with high NaCl (0.16 M); (*) statistical significance at 5 μM used in further experiments. (C) Dose titration of salt (NaCl - 0.1 to 0.26 M) on MCF10A, normalized epithelial breast cancer cell line; (●) without oleanolic acid (5 μM), (○) with oleanolic acid (5 μM); (*) statistical significance at 0.16 M NaCl used in further experiments. (D) Dose titration of oleanolic acid (OA – 0 to 10 μM) on MCF10A, highly invasive epithelial breast cancer cell line; (unfilled▼) with regular NaCl (0.1 M), (filled▼) with high NaCl (0.16 M); (*) statistical significance at 5 μM used in further experiments. Cell viability analysis using MTT assay for 12-96 hours on MDA-MB-231 (E) and MCF10A (F) cell lines; (Δ) with vehicle-control (negative control) DMSO treatment; (▲) with OA (5 μM); (□) with 0.16M NaCl; (■)with 0.16M NaCl+OA (5 μM). (G-H)Glucose consumption assay in MDA-MB-231(G) and MCF10A (H) cell lines under various culture conditions. (G-H) Lactate production analysis in MDA-MB-231(I) and MCF10A (J) cell lines under various culture conditions. All data represented as mean values ± SEM from four independent experiments. (*) p-value <0.05.

Figure 2. Oleanolic acid suppresses rate-limiting enzymes in aerobic glycolysis

(A) Western blot analysis of the expression of key rate-limiting enzymes in aerobic glycolysis, hexokinase, PKM1, PKM2, LDH-A following high salt (0.16M NaCl) and OA (5 μM) in MDA-MB-231 and MCF10A cell lines. As it can be noted PKM1 and PKM2 demonstrated an opposite expression pattern and β-actin is used as loading control. Quantitative densitometric analysis (not shown) was performed for data-analysis. (B-C) Pyruvate kinase (PK) enzymatic activity is MDA-MB-231 (B) and MCF10A (C) cell lines following various culture conditions. Enzyme activity presented as units per mg total protein. All data represented as mean values ± SEM from four independent experiments. (*) p-value <0.05.

Figure 3. Oleanolic acid induces mitochondrial activity in breast cancer cells

(A) Western blot analysis of the expression of key mitochondrial activity associated enzymes, peroxisome proliferator-activated receptor gamma co-activator-1α PGC1 and cytochrome-C (CytC) following high salt (0.16M NaCl) and OA (5 μM) in MDA-MB-231 and MCF10A cell lines. (B-C) Colorimetric-based cytochrome oxidase enzymatic activity is MDA-MB-231 (B) and MCF10A (C) cell lines following various culture conditions. (D-E) Fluorimetric-based cytochrome oxidase enzymatic activity is MDA-MB-231 (D) and MCF10A (E) cell lines following various culture conditions. All data represented as mean values ± SEM from four independent experiments. (*) p-value <0.05.

Figure 4. Oleanolic acid induces apoptosis in breast cancer cells

(A) Western blot analysis of the expression of key apoptosis associated enzymes, Bcl-2 (anti-apoptosis) and Bax (proapoptosis) following high salt (0.16M NaCl) and OA (5 μM) in MDA-MB-231 and MCF10A cell lines. (B-C) Flow cytometry based annexin-V binding assay to determine apoptosis in MDA-MB-231 (B) and MCF10(C) cell lines following various culture conditions. All data represented as mean values ± SEM from four independent experiments. (*) p-value <0.05.