Shikonin Inhibits Tumor Growth in Mice by Suppressing Pyruvate Kinase M2-mediated Aerobic Glycolysis

Abstract

Shift metabolism profile from mitochondrial oxidative phosphorylation to aerobic glycolysis (Warburg effect) is a key for tumor cell growth and metastasis. Therefore, suppressing the tumor aerobic glycolysis shows a great promise in anti-tumor therapy. In the present study, we study the role of shikonin, a naphthoquinone isolated from the traditional Chinese medicine Lithospermum, in inhibiting tumor aerobic glycolysis and thus tumor growth. We found that shikonin dose-dependently inhibited glucose uptake and lactate production in Lewis lung carcinoma (LLC) and B16 melanoma cells, confirming the inhibitory effect of shikonin on tumor aerobic glycolysis. Treatment of shikonin also decreased tumor cell ATP production. Furthermore, pyruvate kinase M2 (PKM2) inhibitor or activator respectively altered the effect of shikonin on tumor cell aerobic glycolysis, suggesting that suppression of cell aerobic glycolysis by shikonin is through decreasing PKM2 activity. Western blot analysis confirmed that shikonin treatment reduced tumor cell PKM2 phosphorylation though did not reduce total cellular PKM2 level. In vitro assay also showed that shikonin treatment significantly promoted tumor cell apoptosis compared to untreated control cells. Finally, when mice implanted with B16 cells were administered with shikonin or control vehicle, only shikonin treatment significantly decreased B16 tumor cell growth. In conclusion, this study demonstrates that shikonin inhibits tumor growth in mice by suppressing PKM2-mediated aerobic glycolysis.

Figure 1

Shikonin inhibits tumor cell proliferation. LLC (A) and B16 (B) tumor cells were treated with various concentration of shikonin or vehicle (DMSO) for 24 h. Data were presented as a means ± SD of 3–4 individual experiments with three samples in each time point. *P < 0.05. **P < 0.01.

Figure 2

Shikonin suppresses tumor cell aerobic glycolysis. (A) Shikonin (SK) decreased relative glucose uptake in B16 cells in a dose-dependent manner. (B) Shikonin (SK) reduced relative lactate production in B16 cells in a dose-dependent manner. Data were presented as a means ± SD of 3–4 individual experiments with three samples under each condition. *P < 0.05. **P < 0.01.

Figure 3

Shikonin decreases ATP level in B16 cells in a dose-dependent manner. Data were presented as a means ± SD of three individual experiments with three samples under each condition. *P < 0.05. **P < 0.01.

Figure 4

Effect of shikonin on suppressing tumor cell aerobic glycolysis is dependent on PKM2. (A) Knockdown of PKM2 in B16 cells via PKM2 siRNA abolished the effect of shikonin on tumor cell glucose uptake. (B) Knockdown of PKM2 in B16 cells abolished the effect of shikonin on tumor cell lactate production. (C) Modulation of PKM2 activity affected the relative glucose uptake in B16 cells. (D) Modulation of PKM2 activity affected the relative lactate production in B16 cells. Data were presented as a means ± SD of three individual experiments with three samples under each condition. *P < 0.05.

Figure 5

Shikonin (SK) treatment decreases phosphorylation of PKM2 (p-PKM2). (A) Representative western blot images of p-PKM2, PKM2 and GAPDH in B16 cells treated with or without shikonin. The raw WB data of Fig. 5A were shown in Supplementary Material. (B) Analysis results of western blot images in panel A. Data were presented as a means ± SD of three individual experiments. *P < 0.05. **P < 0.01.

Figure 6

Shikonin treatment increases tumor cell apoptosis in a dose-dependent and time-dependent manner. (A) Left panel: representative flow cytometry image of B16 cell apoptosis labeled with FITC-Annexin V/PI. Right panel: analysis results of flow cytometry images. (B) Left panel: representative flow cytometry image of gastric cancer cell apoptosis labeled with FITC-Annexin V/PI. Right panel: analysis results of flow cytometry images. Data were presented as a means ± SD of three individual experiments in triplicate. *P < 0.05. **P < 0.01. ***P < 0.001.

Figure 7

Shikonin treatment inhibits the growth of implanted tumor in SCID mice. (A) Representative tumor images in SCID mice implanted with B16 melanoma. (B) B16 tumor size in mice on various days following injection with various doses of shikonin. (C) B16 tumor weight in mice on day 9 after treatment with various doses of shikonin. Data were presented as a means ± SD (n = 6). **P < 0.01.