Molecular Mechanism Of Cucurbitacin B In Hypoxia-Inducible Factor-1A Signaling Pathway

Objective:Cucurbitacin B is a triterpenoid compound isolated from Trichosanthes kirilowii Maximowicz, which has been used in oriental medicine for its antitumor activities. However, the mechanisms by which cucurbitacin B inhibits tumor growth are not folly understood. In the present paper we investigated the effects of cucurbitacin B on HIF-1activation induced by different stimulation.Methods:Activation of HIF-1α was detected by reporter gene assay. HIF-1α, HIF-1β, Topo-I, phosphorylated-Akt (p-Akt), Akt, p-mTOR, mTOR, p-ERKl/2, ERK, p-p70S6K, p70S6K, p-4E-BP1,4E-BP1, p-eIF4E, eIF4E were measured by Western blot. The expressions of VEGF, EPO, and HIF-1α mRNA were measured by quantitative real-time PCR. The VEGF protein concentration in the culture supernatant was detected by ELISA. The in vitro matrigel invasion assays used HeLa cells. Antitumor activity of cucurbitacin B was detected in a murine xenograft model.Result:Cucurbitacin B showed the potent inhibitory activity against HIF-1activation induced by hypoxia in various human cancer cell lines. This compound markedly decreased the hypoxia-induced accumulation of HIF-1α protein dose-dependently, whereas it did not affect the expressions of HIF-1β and topoisomerase-I. Further analysis revealed that cucurbitacin B strongly inhibited HIF-1α protein synthesis, without affecting the expression level of HIF-1α mRNA or degradation of HIF-1α protein. Rather, we found that suppression of HIF-1α accumulation by cucurbitacin B correlated with strong dephosphorylation of mammalian target of rapamycin (mTOR) and its effectors ribosomal protein S6kinase (p70S6K) and eukaryotic initiation factor4E-binding protein-1(4E-BP1) and extracellular signal-regulated kinase-1/2(ERK1/2), a pathway known to regulate HIF-1α expression at the translational level. Cucurbitacin B also activated Akt, a mechanistic feature exhibited by established mTOR inhibitors in many tumor cells. Furthermore, cucurbitacin B prevented hypoxia-induced expression of HIF-1target genes and suppresses the invasiveness of tumor cells. In vivo studies further confirmed the inhibitory effect of cucurbitacin B on the expression of HIF-1α proteins, leading to a decrease in tumor vascularisation and growth of the xenografts.Conclusion:1. Cucurbitacin B rapidly down-regulates not only HIF-la by decreasing its protein synthesis without affecting mRNA levels or protein degradation, but also the expression of HIF target genes such as vascular endothelial growth factor (VEGF) and erythropoietin.2. The HIF-1activation-inhibitory effects of cucurbitacin B were associated with the suppression of mTOR/p70S6k/4E-BP1and MEK/ERK signaling pathways.3. Cucurbitacin B inhibits growth of HeLa cells in a murine xenograft tumor model with no apparent toxicity to the animals.