Active fraction from clove inhibits colorectal cancer cell proliferation by downregulating M2-type pyruvate kinase to reduce aerobic glycolysis

Abstract

Background Aerobic glycolysis contributes to the high rate of metabolism in tumour cells and colorectal cancer is one of the highly metabolic malignancies. Highly expressed M2-type pyruvate kinase (PKM2) promotes tumour cell proliferation and migration as the rate-limiting enzyme of aerobic glycolysis. Previously we have shown that the active fraction from clove (AFC) can inhibit the growth of colon cancer cells in vitro. However, the mechanism of action regarding the anti-colon cancer activity of AFC, especially in aerobic glycolysis, has not been adequately investigated. Methods In vitro anti-colon cancer activity was assessed by CCK8 assay and colony formation assay, and changes in aerobic glycolytic capacity were assessed by evaluating changes in glucose consumption, lactate production, pyruvate kinase activity and pyruvate production. HCT116 cells xenograft model in nude mice was established to measure the anti-colon cancer activity of AFC in vivo. Mechanism of action studies involved western blotting, reverse transcription quantitative polymerase chain reaction (RT-qPCR), small interfering RNA (siRNA) knockdown transfection, and immunohistochemistry. Results The present study confirmed those findings by showing that the active fraction significantly inhibited the growth of five lines of colon cancer cells (HT29, SW620, Caco-2, HCT116 and LoVo). And we found that the active fraction reduced pyruvate kinase activity, glucose uptake, and production of pyruvate and lactate. It also downregulated M2-type pyruvate kinase (PKM2), c-myc and cyclin D1. Knockdown of PKM2 on its own mimicked the effects of active fraction, which could result in the ability to inhibit cell proliferation and aerobic glycolysis. Consistent with these results in cell culture, the active fraction significantly inhibited tumor growth and downregulated PKM2, c-myc and cyclin D1 in HCT116 xenografts in mice, while causing milder liver damage and weight loss than the conventional chemotherapeutic 5-fluorouracil (5-FU). Conclusion Our fndings indicate that AFC inhibits colon cancer growth by downregulating PKM2 to inhibit aerobic glycolysis and reduces tumour-specific high expression of c-myc and cyclin D1. Future work should explore how it downregulates pyruvate kinase in the first place, and the intrinsic mechanism between the down-regulation of PKM2 and the down-regulation of c-myc and cyclin D1.