Prodigiosin inhibits Wnt/β-catenin signaling and exerts anticancer activity in breast cancer cells PNAS 2016 113(46) 13150-13155 Speaker：Wu Po-Huei Adviser : Jung-Yie Kao Date：2017.05.02

Non-canonical Wnt pathway Wnt signaling pathway Canonical Wnt pathway Embryonic development Cancer Non-canonical Wnt pathway Cell movement Wnt-Ca2+ pathway

OFF STATE ON STATE clathrin P DVL Axin APC P P P β-catenin P P DVL P LRP= low-density-lipoprotein receptor protein GSK3= glycogen synthase kinase-3β DVL= Dishevelled clathrin P DVL Multivescular body Axin APC P GSK3 β-catenin P P P Proteasomal degradation P DVL P OFF STATE ON STATE Cyclin D1 LEF1 Fibronectin

GSK3 inhibitor Prodigiosin induces the proapoptotic gene NAG-1 via glycogen synthase kinase-3beta activity in human breast cancer cells Mol Cancer Ther 6(1):362–369

Prodigiosin inhibits the transcription activity of Wnt/β-catenin signaling pathway LRP6 TCF/LEF promoter Luciferase SuperTop Flash FI = TCF/LEF RLU β-galactose RLU WNT sequence Transfect to HEK293T Add prodigiosin Calculate FI value Plasmid

Prodigiosin inhibits the transcription activity of Wnt/β-catenin signaling pathway prodigiosin can reduces Wnt signaling by inhibiting LRP6 and DVL2 prodigiosin likely inhibits Wnt signaling upstream of β-catenin LRP6

prodigiosin does not inhibit gene expression of Wnt ligand Prodigiosin inhibits the transcription activity of Wnt/β-catenin signaling pathway WNT3A-CM=WNT3A conditioned medium TCF/LEF promoter Luciferase SuperTop Flash Transfect to HEK293T Add prodigiosin Calculate FI value WNT prodigiosin does not inhibit gene expression of Wnt ligand

AP1 promoter Luciferase AP1-Luc Ras Plasmid Prodigiosin did not influence the luciferase activity of other pathway NFAT(transcription factor) Nuclear Factor of Activated T-cells NFAT promoter Luciferase NFAT-Luc NFATc Plasmid Conculsion: Prodigiosin inhibits Wnt/β-catenin transcription activity and likely to be Wnt signaling upstream of β-catenin

Prodigiosin inhibits the Wnt/β-catenin signaling pathway in HEK293T cells WNT Transfect to HEK293T Add prodigiosin Plasmid prodigiosin may block Wnt/β-catenin signaling by inhibiting the phosphorylation of LRP6 and DVL LRP6 Prodigiosin noticeably inhibited the phosphorylation of GSK3β

Prodigiosin inhibits the Wnt/β-catenin signaling pathway in breast cancer MDA-MB-231 MDA-MB-468 Prodigiosin inhibits the phosphorylation of LRP6 DVL2 and GSK3β , and it also can inhibit total LRP6 and DVL2 in breast cancer

Prodigiosin inhibits the Wnt/β-catenin signaling pathway in breast cancer MDA-MB-231 MDA-MB-468 RT-qPCR

Prodigiosin suppresses breast cancer cell migration and invasion Serum –free medium MDA-MB-231 medium Stain cells Crystal violet

Prodigiosin suppresses breast cancer cell invasion matrigel 6hr Conculsion: prodigiosin exerts inhibitory effects on the migratory and invasive capacity of breast cancer cells in vitro

Prodigiosin inhibits tumor growth in a breast cancer xenograft mouse model MDA-MB-231 BALB/c Salin Prodigiosin 5mg/kg 50mm³ (i.p. injection 3wk)

Prodigiosin inhibits tumor growth in a breast cancer xenograft mouse model

Prodigiosin inhibits tumor growth in a breast cancer xenograft mouse model LRP6 RT-qPCR

Prodigiosin induces tumor regression in MMTV-Wnt1 transgenic mice Mouse mammary tumor virus (retrovirus) WNT MMTV-Wnt1 BALB/c

Prodigiosin induces tumor regression in MMTV-Wnt1 transgenic mice BALB/c

Prodigiosin induces tumor regression in MMTV-Wnt1 transgenic mice RT-qPCR

．Prodigiosin acts proximally in the Wnt signaling cascade by inhibiting the phosphorylation of LRP6, DVL2, and GSK3β, thereby preventing the activation of β-catenin ．Prodigiosin inhibits breast cancer cell growth, migration, and invasion 本篇研究證實靈菌紅素可藉由抑制磷酸化的LRP6 DVL2 GSK3，來抑制β-catenin，使整個wnt signaling pathway活性下降 並且在in vitro和in vivo實驗均有相同的結果 另外靈菌紅素可抑制乳癌細胞的 爬行跟轉移能力 他比起一些化學療藥物，具有較低細胞毒性，當濃度大於300nM時才具有明顯的淋巴細胞抑制作用。 靈菌紅素對於許多種癌細胞具有效果，如肺，結腸，腎和乳癌 因此，靈菌紅素在未來有潛力可開發成為癌症用藥

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