Oleanolic acid suppresses aerobic glycolysis in cancer cells by switching pyruvate kinase type M isoforms

Abstract

Warburg effect, one of the hallmarks for cancer cells, is characterized by metabolic switch from mitochondrial oxidative phosphorylation to aerobic glycolysis. In recent years, increased expression level of pyruvate kinase M2 (PKM2) has been found to be the culprit of enhanced aerobic glycolysis in cancer cells. However, there is no agent inhibiting aerobic glycolysis by targeting PKM2. In this study, we found that Oleanolic acid (OA) induced a switch from PKM2 to PKM1, and consistently, abrogated Warburg effect in cancer cells. Suppression of aerobic glycolysis by OA is mediated by PKM2/PKM1 switch. Furthermore, mTOR signaling was found to be inactivated in OA-treated cancer cells, and mTOR inhibition is required for the effect of OA on PKM2/PKM1 switch. Decreased expression of c-Myc-dependent hnRNPA1 and hnRNPA1 was responsible for OA-induced switch between PKM isoforms. Collectively, we identified that OA is an antitumor compound that suppresses aerobic glycolysis in cancer cells and there is potential that PKM2 may be developed as an important target in aerobic glycolysis pathway for developing novel anticancer agents.

Figure 1. OA suppresses the aerobic glycolysis in cancer cells.

Glucose consumption (A), lactic acid production (B) and oxygen consumption (C) were assessed in PC-3 and MCF-7 cells treated with 50 or 100 µg/ml OA for 6 or 12 hr respectively. The details of methodology have been described in the section of Materials and Methods. The ratio of each group to control was presented here. The bars showed the average values of three independent experiments (Mean ± SD). *, P<0.05; **, P<0.01.

Figure 2. OA treatment affects the expression profile of PKM isoforms in cancer cells in a dose- and time-dependent manner.

PKM1 and PKM2 expression level was evaluated by immunoblotting assays in PC-3 and MCF-7 cells treated with 10, 25, 50,100 µg/ml OA respectively for 12 hr (A) or 100 µg/ml OA for 0.5, 1, 2, 3, 6, 12 hr respectively (B). β-tubulin was used as endogenous references. (C) The PKM2 levels (Green) were in PC-3 and MCF-7 cells treated with 100 µg/ml OA for 12 hr respectively, as determined by immunfluorescent staining. The nuclei (Blue) were stained with DAPI. The merged pictures were shown in the bottom of the panel. The fluorescence intensity was quantified with ImageJ software. The ratio of each group to control was presented here. The bars represented the average values of 5 randomly selected experiments.

Figure 3. PKM2 overexpression abolishes the effect of OA on aerobic glycolysis in cancer cells.

(A) OA-treated PC-3 cells were transfected with pWZL Neo Myr Flag PKM2 (Flag-PKM2) or control vector (Flag-GFP). Immunoblot assays were performed to determine the expression of PKM2 protein. β-tubulin was used as endogenous references. Glucose consumption (B), lactic acid production (C) and oxygen consumption (D) were assessed in PC-3 cells treated with 100 µg/ml OA for 12 hr. The details of methodology was described in the section of Materials and Methods. The ratio of each group to control was presented here. The bars showed the average values of three independent experiments (Mean ± SD). *, P<0.05; **, P<0.01.

Figure 4. mTOR suppression is required for OA-induced metabolic switch.

(A) The levels of phosphorylated mTOR was examined in PC-3 and MCF-7 cells treated with 50 or 100 µg/ml OA for 6 or 12 hr respectively as determined by immunoblot assays. β-tubulin was used as endogenous references. (B) Immunoblot analysis of PKM1 and PKM2 expression was performed in PC-3 cells incubated with OA (100 µg/ml) or/and a mTOR activator MHY1485 (2 µM). Glucose consumption (C), lactic acid production (D) and oxygen consumption (E) were detected in PC-3 cells treated with OA (100 µg/ml) and/or MHY1485 (2 µM) for 12 hr. The ratio of each group to control was presented here. The bars showed the average values of three independent experiments (Mean ± SD). *, P<0.05; **, P<0.01.

Figure 5. The switch from PKM2 to PKM1 by OA results from the decrease in Myc, hnRNPA1 and hnRNPA2 expression.

(A) The expression of c-Myc, hnRNPA1 and hnRNPA2 was determined by immunoblot analysis in PC-3 and MCF-7 cells treated with 50 or 100 µg/ml OA for 6 or 12 hr respectively. (B) The expressionof c-Myc, hnRNPA1 and hnRNPA2 expression was determined by immunoblot analysis in PC-3 cells treated with OA (100 µg/ml) or/and MHY1485 (2 µM) respectively. (C) The level of c-Myc, hnRNPA1, hnRNPA2, PKM1 and PKM2 was determined by immunoblot assays in PC-3 cells treated with plasmids pMXs-hcMYC (Flag-cMyc) in the presence or absence of OA. β-tubulin was used as endogenous references.

Figure 6. OA suppression of aerobic glycolysis contributes to its anti-tumor activity.

(A) PC-3 cells was counted 12, 24, 36 and 48 hr after the treatment of OA (100 µg/ml) or/and MHY1485 (2 µM) using hemacytometry. The average values of three independent experiments were shown as Mean ± SD. **, P<0.01. The representative figure was shown for each group. (B) Cells were treated with OA (100 µg/ml) or/and MHY1485 (2 µM) for 10 days, the number of colonies of PC-3 cells were counted and analyzed using ImageJ software. The representative figure was shown for each group. (C) OA suppresses the activation of mTOR in cancer cells. This inhibitory effect on mTOR signaling, in turn, abrogated c-Myc/hnRNPA1/hnRNPA2-dependent PKM2 expression. Consequently, the aerobic glycolysis was inhibited in cancer cells treated with OA.