Using Zebrafish as a Model to Study the Effect of Deguelin on COX/PGE2 Signaling Pathway
|School||South China University of Technology|
|Keywords||deguelin zebrafish COX/PGE2 signaling tumor cell|
Objective:Deguelin, a flavonoid derived from Derris elliptica within the Leguminosae family, has been shown to inhibit Tumor cell proliferation and induce several types of cancers apoptosis, including lung, stomach and breast cancer cells. Deguelin inhibited PI3K activity and reduced pAkt levels and decreased expression of cyclooxygenase 2 in vitro studies on cancer cells. However, the mechanism action of deguelin in vivo is still not clear. By using the externally developing zebrafish embryos as a model, we found that embryos treated with 0.6μM deguelin at two-cell stage exhibited gastrulation arrest at 4 hours post fertilization stage(4 hpf, it is also called sphere stage) micking phenotypes of Cox-1, Ptges, EP4-deficient and inhibition PI3K/Akt signaling embryos. Therefore, we hypothesized that deguelin may disrupt the COX/PGE2 pathway in vivo. The functional role for deguelin in vivo remains virtually unknown. We hope to analysis the alterations of COX/PGE2 signaling genes transcript level in embryos treated with deguelin compared to control using qRT-PCR and whole-mount in situ hybridization. Then we selected tumor cells associated with COX pathway to observe the proliferative inhibition process by deguelin using MTT assay. QRT-PCR assay was performed to detect the alterations of COX/PGE2 signaling genes transcript level in these tumor cells to investigate the interaction between the effect of anti-tumor of deguelin and the inhibition of COX/PGE2 signal pathway.Results:(1) Using qRT-PCR to analysis the alterations of COX/PGE2 signaling genes transcript levels in embryos treated with 0.6μM deguelin compared to control, we found that PGE2 synthetases, ptges and ptges3a, were significantly reduced, else PGE2 receptors, ep2 and ep4, were significantly reduced. Consistently the downstream genes egrl and cyclin D1 were significantly decreased.(2) Using whole-mount in situ hybridization to observe the expression of scl and gatal in embryos treated with 0.16μM deguelin from 6 hpf to 21-somite stage compared to control, we found that deguelin decreased scl and gatal expression at 10-somite and 21-somite stages. At 30 hpf stage,0.16μM deguelin inhibited the expression of runxl compared to control embryos,5μM dmPGE2 enhanced runxl expression in control embryos, while the enhancing effect of dmPGE2 on HSCs was reduced significantly by deguelin.(3) QRT-PCR was used to examine the alterations of target genes transcript levels in the 21-somite stage of embryos treated with 0.16μM deguelin from 6 hpf to 21-somite stage compared to control embryos, we found that coxl, ptges3a, ep4a, ep4b, egrl, cyclinDl, scl, gatal, runxl, cmyb were downregulated in deguelin-treated embryos. At 30 hpf stage, ep4b was found to been downregulated by deguelin, cox2b and the downstream gene egrl were significantly decreased.(4) MCF-7, Mia PaCa-2 and PC-3 cells were treated with different concentrations of deguelin for 48 hours, resulted in the inhibition of cell proliferation in a dose-dependent manner. Using qRT-PCR to analysis the alterations of COX/PGE2 signaling genes transcript levels in these three tumor cells treatment with deguelin for 48 hours, the data showed that COX1 and EP2 were downregulated at the mRNA levels in MCF-7 cell. In Mia PaCa-2 cell, PTGES was downregulated significantly by deguelin, also EP4 and CCND1 transcript levels were decreased compared to control. In PC-3 cell, COX1 was downregulated significantly by deguelin.Conclusion:(1) Deguelin downregulated the expression of COX/PGE2 signaling genes in zebrafish embryos.(2) Deguelin inhibited the proliferation of MCF-7, Mia PaCa-2 and PC-3 cells.(3) Deguelin downregulated the expression of COX/PGE2 signaling genes in MCF-7, Mia PaCa-2 and PC-3 cells.