Abstract
Background: Hypoxia-inducible factor-1α (HIF-1α) induces the expression of glycolysis-related genes, which plays a direct and key role in Warburg effect. In a recent study, honokiol (HNK) was identified as one of the potential agents that inhibited the HIF-1α signaling pathway. Because the HIF- 1α pathway is closely associated with glycolysis, we investigated whether HNK inhibited HIF-1α-mediated glycolysis. Methods: The effects of HNK on HIF-1α-mediated glycolysis and other glycolysis-related genes’ expressions, cancer cells apoptosis and tumor growth were studied in various human breast cancer models in vitro and in vivo. We performed the following tests: extracellular acidification and oxygen consumption rate assays, glucose uptake, lactate, and ATP assays for testing glycolysis; WST-1 assay for investigating cell viability; colony formation assay for determining clonogenicity; flow cytometry for assessing cell apoptosis; qPCR and Western blot for determining the expression of HIF-1α, GLUT1, HK2 and PDK1. The mechanisms of which HNK functions as a direct inhibitor of HIF-1α were verified through the ubiquitination assay, the Co-IP assay, and the cycloheximide (CHX) pulse-chase assay. Results: HNK increased the oxygen consumption rate while decreased the extracellular acidification rate in breast cancer cells; it further reduced glucose uptake, lactic acid production and ATP production in cancer cells. The inhibitory effect of HNK on glycolysis is HIF-1α-dependent. HNK also downregulated the expression of HIF-1α and its downstream regulators, including GLUT1, HK2 and PDK1. A mechanistic study demonstrated that HNK enhanced the self-ubiquitination of HIF-1α by recruiting two E3 ubiquitin ligases (UFL1 and BRE1B). In vitro, HNK inhibited cell proliferation and clonogenicity, as well as induced apoptosis of cancer cells. These effects were also HIF1α-dependent. In vivo, HNK inhibited tumor growth and HIF-1α-mediated glycolysis. Conclusion: HNK has an inhibitory effect on HIF-1α-mediated glycolysis in human breast cancer. Our research revealed a new mechanism of HNK as an anti-cancer drug, thus representing a novel strategy to improve the prognosis of cancer.
Highlights
To explore whether HNK can inhibit glycolysis in breast cancer cells, we detected the changes in Oxygen Consumption Rate (OCR), Extracellular Acidification (ECAR), glucose consumption, lactic acid production and ATP production in two human breast cancer cell lines (MCF-7 and MDA-MB-231) after HNK intervention
The results showed that HNK downregulated the protein expression of Hypoxia-inducible factor-1α (HIF-1α), glucose transporter-1 (GLUT1), HK2 and pyruvate dehydrogenase kinase-1 (PDK1) in a dose-and time-dependent manner (Figure 1B)
The results showed that HNK downregulated the level of HIF- 1α protein in both cells, which resulted in an increase in OCR and a decrease in ECAR, glucose uptake, lactic acid production and ATP production
Summary
Most eukaryotic cells can cope with environmental changes by changing their metabolic strategies (Seyfried et al, 2014). Glycolysis allows cancer cells to have a faster production rate, meet their enhanced metabolic needs, and reduce the effect of hypoxia on ATP synthesis (Dias et al, 2019; Abbaszadeh et al, 2020). It can inhibit mitochondrial function, reduce the production of reactive oxygen species, and promote the survival of cancer cells by increasing the threshold of apoptosis. HIF-1α accumulated in tumor cells is dimerized with HIF-1β, which binds to the hypoxia response element (HRE) in the transcription regulation region of the glucose transporter or other metabolic rate-limiting enzyme genes to promote their expression, resulting in reprogramming of cancer cell metabolism (Semenza, 2011a; Semenza, 2011b). Because the HIF- 1α pathway is closely associated with glycolysis, we investigated whether HNK inhibited HIF-1α-mediated glycolysis
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