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A novel sLRP6E1E2 inhibits canonical Wnt signaling, epithelial-to-mesenchymal transition, and induces mitochondria-dependent apoptosis in lung cancer.

Lee JS, Hur MW, Lee SK, Choi WI, Kwon YG, Yun CO - PLoS ONE (2012)

Bottom Line: We designed a Wnt antagonist sLRP6E1E2, and generated a replication-incompetent adenovirus (Ad), dE1-k35/sLRP6E1E2, and a replication-competent oncolytic Ad, RdB-k35/sLRP6E1E2, both expressing sLRP6E1E2. sLRP6E1E2 prevented Wnt-mediated stabilization of cytoplasmic β-catenin, decreased Wnt/β-catenin signaling and cell proliferation via the mitogen-activated protein kinase, and phosphatidylinositol 3-kinase pathways. sLRP6E1E2 induced apoptosis, cytochrome c release, and increased cleavage of PARP and caspase-3. sLRP6E1E2 suppressed growth of the human lung tumor xenograft, and reduced motility and invasion of cancer cells.In addition, sLRP6E1E2 upregulated expression of epithelial marker genes, while sLRP6E1E2 downregulated mesenchymal marker genes.Taken together, sLRP6E1E2, by inhibiting interaction between Wnt and its receptor, suppressed Wnt-induced cell proliferation and epithelial-to-mesenchymal transition.

View Article: PubMed Central - PubMed

Affiliation: Brain Korea 21 Project for Medical Sciences, Yonsei Cancer Center, Yonsei University College of Medicine, Seoul, Korea.

ABSTRACT
Aberrant activation of the Wnt pathway contributes to human cancer progression. Antagonists that interfere with Wnt ligand/receptor interactions can be useful in cancer treatments. In this study, we evaluated the therapeutic potential of a soluble Wnt receptor decoy in cancer gene therapy. We designed a Wnt antagonist sLRP6E1E2, and generated a replication-incompetent adenovirus (Ad), dE1-k35/sLRP6E1E2, and a replication-competent oncolytic Ad, RdB-k35/sLRP6E1E2, both expressing sLRP6E1E2. sLRP6E1E2 prevented Wnt-mediated stabilization of cytoplasmic β-catenin, decreased Wnt/β-catenin signaling and cell proliferation via the mitogen-activated protein kinase, and phosphatidylinositol 3-kinase pathways. sLRP6E1E2 induced apoptosis, cytochrome c release, and increased cleavage of PARP and caspase-3. sLRP6E1E2 suppressed growth of the human lung tumor xenograft, and reduced motility and invasion of cancer cells. In addition, sLRP6E1E2 upregulated expression of epithelial marker genes, while sLRP6E1E2 downregulated mesenchymal marker genes. Taken together, sLRP6E1E2, by inhibiting interaction between Wnt and its receptor, suppressed Wnt-induced cell proliferation and epithelial-to-mesenchymal transition.

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Decoy Wnt receptor sLRP6E1E2 induces apoptosis in human lung cancer cells.(a) Cells were transduced with dE1-k35/LacZ or dE1-k35/sLRP6E1E2 at (20 MOI), and photographs were taken at 72 hr later. Original magnification, ×200. (b) Detection of sLRP6E1E2-induced apoptosis by TUNEL staining. Original magnification, ×400. (c) Total number of TUNEL-positive cells per fields (mean ± SEM). *P<0.05 versus PBS or dE1-k35/LacZ treated with Wnt3a; **P<0.001 versus PBS-treated or dE1-k35/LacZ-transduced controls. n.s.  =  not significant. (d) Western analysis of sLRP6E1E2-mediated apoptosis. H460 cells were transduced with dE1-k35/LacZ or dE1-k35/sLRP6E1E2 (20 MOI). The western blot using specific antibodies against uncleaved PARP, cleaved PARP, pro-caspase-3, cleaved caspase-3, and cytochrome c. (e) H460 cells were treated as indicated above (Fig. 4d). Subcellular localization of cytochrome c was determined by western blot analysis of cytosolic and microsomal fractions.
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pone-0036520-g004: Decoy Wnt receptor sLRP6E1E2 induces apoptosis in human lung cancer cells.(a) Cells were transduced with dE1-k35/LacZ or dE1-k35/sLRP6E1E2 at (20 MOI), and photographs were taken at 72 hr later. Original magnification, ×200. (b) Detection of sLRP6E1E2-induced apoptosis by TUNEL staining. Original magnification, ×400. (c) Total number of TUNEL-positive cells per fields (mean ± SEM). *P<0.05 versus PBS or dE1-k35/LacZ treated with Wnt3a; **P<0.001 versus PBS-treated or dE1-k35/LacZ-transduced controls. n.s.  =  not significant. (d) Western analysis of sLRP6E1E2-mediated apoptosis. H460 cells were transduced with dE1-k35/LacZ or dE1-k35/sLRP6E1E2 (20 MOI). The western blot using specific antibodies against uncleaved PARP, cleaved PARP, pro-caspase-3, cleaved caspase-3, and cytochrome c. (e) H460 cells were treated as indicated above (Fig. 4d). Subcellular localization of cytochrome c was determined by western blot analysis of cytosolic and microsomal fractions.

Mentions: Wnt signaling can prevent apoptosis and promote cellular proliferation and survival [26]. To characterize the molecular mechanisms by which sLRP6E1E2 inhibits non-small cell lung cancer proliferation, we evaluated the effects of sLRP6E1E2 on apoptosis. At 3 days after dE1-k35/sLRP6E1E2 transduction, we observed that A549, H1299, and H358 cells gradually detached from the culture dish and became rounder and smaller than attached cells (Fig. 4A), suggesting that sLRP6E1E2 induced apoptosis. Evidence of apoptosis was sought by looking for nuclear apoptotic bodies (data not shown), and then assessed using the TUNEL assay to detect internucleosomal DNA fragmentation [27]. As shown in Fig. 4B, more TUNEL-positive cells were observed among dE1-k35/sLRP6E1E2-transduced cells than among control cells in the presence or absence of Wnt3a. Quantitation of TUNEL staining revealed that the rate of apoptosis was approximately 1.9-fold higher (without Wnt3a) and 2.8-fold higher (with Wnt3a) in dE1-k35/sLRP6E1E2-transduced cells than in dE1-k35/LacZ-transduced controls (P<0.001) (Fig. 4C).


A novel sLRP6E1E2 inhibits canonical Wnt signaling, epithelial-to-mesenchymal transition, and induces mitochondria-dependent apoptosis in lung cancer.

Lee JS, Hur MW, Lee SK, Choi WI, Kwon YG, Yun CO - PLoS ONE (2012)

Decoy Wnt receptor sLRP6E1E2 induces apoptosis in human lung cancer cells.(a) Cells were transduced with dE1-k35/LacZ or dE1-k35/sLRP6E1E2 at (20 MOI), and photographs were taken at 72 hr later. Original magnification, ×200. (b) Detection of sLRP6E1E2-induced apoptosis by TUNEL staining. Original magnification, ×400. (c) Total number of TUNEL-positive cells per fields (mean ± SEM). *P<0.05 versus PBS or dE1-k35/LacZ treated with Wnt3a; **P<0.001 versus PBS-treated or dE1-k35/LacZ-transduced controls. n.s.  =  not significant. (d) Western analysis of sLRP6E1E2-mediated apoptosis. H460 cells were transduced with dE1-k35/LacZ or dE1-k35/sLRP6E1E2 (20 MOI). The western blot using specific antibodies against uncleaved PARP, cleaved PARP, pro-caspase-3, cleaved caspase-3, and cytochrome c. (e) H460 cells were treated as indicated above (Fig. 4d). Subcellular localization of cytochrome c was determined by western blot analysis of cytosolic and microsomal fractions.
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pone-0036520-g004: Decoy Wnt receptor sLRP6E1E2 induces apoptosis in human lung cancer cells.(a) Cells were transduced with dE1-k35/LacZ or dE1-k35/sLRP6E1E2 at (20 MOI), and photographs were taken at 72 hr later. Original magnification, ×200. (b) Detection of sLRP6E1E2-induced apoptosis by TUNEL staining. Original magnification, ×400. (c) Total number of TUNEL-positive cells per fields (mean ± SEM). *P<0.05 versus PBS or dE1-k35/LacZ treated with Wnt3a; **P<0.001 versus PBS-treated or dE1-k35/LacZ-transduced controls. n.s.  =  not significant. (d) Western analysis of sLRP6E1E2-mediated apoptosis. H460 cells were transduced with dE1-k35/LacZ or dE1-k35/sLRP6E1E2 (20 MOI). The western blot using specific antibodies against uncleaved PARP, cleaved PARP, pro-caspase-3, cleaved caspase-3, and cytochrome c. (e) H460 cells were treated as indicated above (Fig. 4d). Subcellular localization of cytochrome c was determined by western blot analysis of cytosolic and microsomal fractions.
Mentions: Wnt signaling can prevent apoptosis and promote cellular proliferation and survival [26]. To characterize the molecular mechanisms by which sLRP6E1E2 inhibits non-small cell lung cancer proliferation, we evaluated the effects of sLRP6E1E2 on apoptosis. At 3 days after dE1-k35/sLRP6E1E2 transduction, we observed that A549, H1299, and H358 cells gradually detached from the culture dish and became rounder and smaller than attached cells (Fig. 4A), suggesting that sLRP6E1E2 induced apoptosis. Evidence of apoptosis was sought by looking for nuclear apoptotic bodies (data not shown), and then assessed using the TUNEL assay to detect internucleosomal DNA fragmentation [27]. As shown in Fig. 4B, more TUNEL-positive cells were observed among dE1-k35/sLRP6E1E2-transduced cells than among control cells in the presence or absence of Wnt3a. Quantitation of TUNEL staining revealed that the rate of apoptosis was approximately 1.9-fold higher (without Wnt3a) and 2.8-fold higher (with Wnt3a) in dE1-k35/sLRP6E1E2-transduced cells than in dE1-k35/LacZ-transduced controls (P<0.001) (Fig. 4C).

Bottom Line: We designed a Wnt antagonist sLRP6E1E2, and generated a replication-incompetent adenovirus (Ad), dE1-k35/sLRP6E1E2, and a replication-competent oncolytic Ad, RdB-k35/sLRP6E1E2, both expressing sLRP6E1E2. sLRP6E1E2 prevented Wnt-mediated stabilization of cytoplasmic β-catenin, decreased Wnt/β-catenin signaling and cell proliferation via the mitogen-activated protein kinase, and phosphatidylinositol 3-kinase pathways. sLRP6E1E2 induced apoptosis, cytochrome c release, and increased cleavage of PARP and caspase-3. sLRP6E1E2 suppressed growth of the human lung tumor xenograft, and reduced motility and invasion of cancer cells.In addition, sLRP6E1E2 upregulated expression of epithelial marker genes, while sLRP6E1E2 downregulated mesenchymal marker genes.Taken together, sLRP6E1E2, by inhibiting interaction between Wnt and its receptor, suppressed Wnt-induced cell proliferation and epithelial-to-mesenchymal transition.

View Article: PubMed Central - PubMed

Affiliation: Brain Korea 21 Project for Medical Sciences, Yonsei Cancer Center, Yonsei University College of Medicine, Seoul, Korea.

ABSTRACT
Aberrant activation of the Wnt pathway contributes to human cancer progression. Antagonists that interfere with Wnt ligand/receptor interactions can be useful in cancer treatments. In this study, we evaluated the therapeutic potential of a soluble Wnt receptor decoy in cancer gene therapy. We designed a Wnt antagonist sLRP6E1E2, and generated a replication-incompetent adenovirus (Ad), dE1-k35/sLRP6E1E2, and a replication-competent oncolytic Ad, RdB-k35/sLRP6E1E2, both expressing sLRP6E1E2. sLRP6E1E2 prevented Wnt-mediated stabilization of cytoplasmic β-catenin, decreased Wnt/β-catenin signaling and cell proliferation via the mitogen-activated protein kinase, and phosphatidylinositol 3-kinase pathways. sLRP6E1E2 induced apoptosis, cytochrome c release, and increased cleavage of PARP and caspase-3. sLRP6E1E2 suppressed growth of the human lung tumor xenograft, and reduced motility and invasion of cancer cells. In addition, sLRP6E1E2 upregulated expression of epithelial marker genes, while sLRP6E1E2 downregulated mesenchymal marker genes. Taken together, sLRP6E1E2, by inhibiting interaction between Wnt and its receptor, suppressed Wnt-induced cell proliferation and epithelial-to-mesenchymal transition.

Show MeSH
Related in: MedlinePlus