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The cell competition-based high-throughput screening identifies small compounds that promote the elimination of RasV12-transformed cells from epithelia.

Yamauchi H, Matsumaru T, Morita T, Ishikawa S, Maenaka K, Takigawa I, Semba K, Kon S, Fujita Y - Sci Rep (2015)

Bottom Line: Among several Rebeccamycin-derivative compounds, we have found that VC1-8 has least cytotoxicity against normal cells but shows the comparable effect on the elimination of transformed cells.This cell competition-promoting activity of VC1-8 is observed both in vitro and ex vivo.These data demonstrate that the cell competition-based screening is a promising tool for the establishment of a novel type of cancer preventive medicine.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Oncology, Institute for Genetic Medicine, Hokkaido University Graduate School of Chemical Sciences and Engineering, Sapporo, Japan.

ABSTRACT
Recent studies have revealed that cell competition can occur between normal and transformed epithelial cells; normal epithelial cells recognize the presence of the neighboring transformed cells and actively eliminate them from epithelial tissues. Here, we have established a brand-new high-throughput screening platform that targets cell competition. By using this platform, we have identified Rebeccamycin as a hit compound that specifically promotes elimination of RasV12-transformed cells from the epithelium, though after longer treatment it shows substantial cytotoxic effect against normal epithelial cells. Among several Rebeccamycin-derivative compounds, we have found that VC1-8 has least cytotoxicity against normal cells but shows the comparable effect on the elimination of transformed cells. This cell competition-promoting activity of VC1-8 is observed both in vitro and ex vivo. These data demonstrate that the cell competition-based screening is a promising tool for the establishment of a novel type of cancer preventive medicine.

No MeSH data available.


Related in: MedlinePlus

VC1-8 induces cell death of transformed cells that are surrounded by normal cells.(a) Immunofluorescence images of MDCK-pTR GFP-RasV12 cells that are surrounded by MDCK cells in the absence (left) or presence (right) of 2 μM VC1-8 for 16 h. Cells are stained with Hoechst 33342 (blue) and Alexa-Fluor-568-conjuated phalloidin (red). Arrows indicate a cell with fragmentation. Scale bars: 20 μm. (b) Images of a representative time-lapse analysis of MDCK-pTR GFP-RasV12 cells that are surrounded by MDCK cells (red) in the presence of 2 μM VC1-8. Scale bar: 20 μm. (c) A graph showing the survival rate of MDCK-pTR GFP-RasV12 cells surrounded by MDCK cells in the presence of DMSO (blue) or 2 μM VC1-8 (red). n = 49 cells (DMSO) and 33 cells (VC1-8). The effect of VC1-8 was statistically significant (P < 0.0001). (d) Effect of apoptosis inhibitor (4-aminopyridine, Z-VAD-FMK) and/or necroptosis inhibitor (Necrostatin-1) on MDCK-pTR GFP-RasV12 cells mixed with MDCK cells. Data are mean ± SD from three independent experiments. Values are expressed as a ratio relative to DMSO treatment. **P < 0.01, ***P < 0.001.
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f3: VC1-8 induces cell death of transformed cells that are surrounded by normal cells.(a) Immunofluorescence images of MDCK-pTR GFP-RasV12 cells that are surrounded by MDCK cells in the absence (left) or presence (right) of 2 μM VC1-8 for 16 h. Cells are stained with Hoechst 33342 (blue) and Alexa-Fluor-568-conjuated phalloidin (red). Arrows indicate a cell with fragmentation. Scale bars: 20 μm. (b) Images of a representative time-lapse analysis of MDCK-pTR GFP-RasV12 cells that are surrounded by MDCK cells (red) in the presence of 2 μM VC1-8. Scale bar: 20 μm. (c) A graph showing the survival rate of MDCK-pTR GFP-RasV12 cells surrounded by MDCK cells in the presence of DMSO (blue) or 2 μM VC1-8 (red). n = 49 cells (DMSO) and 33 cells (VC1-8). The effect of VC1-8 was statistically significant (P < 0.0001). (d) Effect of apoptosis inhibitor (4-aminopyridine, Z-VAD-FMK) and/or necroptosis inhibitor (Necrostatin-1) on MDCK-pTR GFP-RasV12 cells mixed with MDCK cells. Data are mean ± SD from three independent experiments. Values are expressed as a ratio relative to DMSO treatment. **P < 0.01, ***P < 0.001.

Mentions: Next, we further analyzed the effect of VC1-8 on the cellular phenotype by microscopic analyses. Addition of VC1-8 induced cell death-like morphology; irregular cell shape with frequent fragmentation into small pieces (Fig. 3a). Time-lapse microscopic analyses revealed that the morphological changes initiated around 8–12 h after addition of VC1-8 (Fig. 3b), and the quantification of the time-lapse data showed that VC1-8 substantially enhanced the elimination of RasV12-transformed cells from the epithelial monolayer (Fig. 3c). Co-incubation with apoptosis inhibitor (4-aminopyridine or Z-VAD-FMK) and/or necroptosis inhibitor (Necrostatin-1) significantly diminished the effect of VC1-8, suggesting that VC1-8 mediates apoptosis and/or necroptosis of RasV12 cells surrounded by normal cells (Fig. 3d).


The cell competition-based high-throughput screening identifies small compounds that promote the elimination of RasV12-transformed cells from epithelia.

Yamauchi H, Matsumaru T, Morita T, Ishikawa S, Maenaka K, Takigawa I, Semba K, Kon S, Fujita Y - Sci Rep (2015)

VC1-8 induces cell death of transformed cells that are surrounded by normal cells.(a) Immunofluorescence images of MDCK-pTR GFP-RasV12 cells that are surrounded by MDCK cells in the absence (left) or presence (right) of 2 μM VC1-8 for 16 h. Cells are stained with Hoechst 33342 (blue) and Alexa-Fluor-568-conjuated phalloidin (red). Arrows indicate a cell with fragmentation. Scale bars: 20 μm. (b) Images of a representative time-lapse analysis of MDCK-pTR GFP-RasV12 cells that are surrounded by MDCK cells (red) in the presence of 2 μM VC1-8. Scale bar: 20 μm. (c) A graph showing the survival rate of MDCK-pTR GFP-RasV12 cells surrounded by MDCK cells in the presence of DMSO (blue) or 2 μM VC1-8 (red). n = 49 cells (DMSO) and 33 cells (VC1-8). The effect of VC1-8 was statistically significant (P < 0.0001). (d) Effect of apoptosis inhibitor (4-aminopyridine, Z-VAD-FMK) and/or necroptosis inhibitor (Necrostatin-1) on MDCK-pTR GFP-RasV12 cells mixed with MDCK cells. Data are mean ± SD from three independent experiments. Values are expressed as a ratio relative to DMSO treatment. **P < 0.01, ***P < 0.001.
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f3: VC1-8 induces cell death of transformed cells that are surrounded by normal cells.(a) Immunofluorescence images of MDCK-pTR GFP-RasV12 cells that are surrounded by MDCK cells in the absence (left) or presence (right) of 2 μM VC1-8 for 16 h. Cells are stained with Hoechst 33342 (blue) and Alexa-Fluor-568-conjuated phalloidin (red). Arrows indicate a cell with fragmentation. Scale bars: 20 μm. (b) Images of a representative time-lapse analysis of MDCK-pTR GFP-RasV12 cells that are surrounded by MDCK cells (red) in the presence of 2 μM VC1-8. Scale bar: 20 μm. (c) A graph showing the survival rate of MDCK-pTR GFP-RasV12 cells surrounded by MDCK cells in the presence of DMSO (blue) or 2 μM VC1-8 (red). n = 49 cells (DMSO) and 33 cells (VC1-8). The effect of VC1-8 was statistically significant (P < 0.0001). (d) Effect of apoptosis inhibitor (4-aminopyridine, Z-VAD-FMK) and/or necroptosis inhibitor (Necrostatin-1) on MDCK-pTR GFP-RasV12 cells mixed with MDCK cells. Data are mean ± SD from three independent experiments. Values are expressed as a ratio relative to DMSO treatment. **P < 0.01, ***P < 0.001.
Mentions: Next, we further analyzed the effect of VC1-8 on the cellular phenotype by microscopic analyses. Addition of VC1-8 induced cell death-like morphology; irregular cell shape with frequent fragmentation into small pieces (Fig. 3a). Time-lapse microscopic analyses revealed that the morphological changes initiated around 8–12 h after addition of VC1-8 (Fig. 3b), and the quantification of the time-lapse data showed that VC1-8 substantially enhanced the elimination of RasV12-transformed cells from the epithelial monolayer (Fig. 3c). Co-incubation with apoptosis inhibitor (4-aminopyridine or Z-VAD-FMK) and/or necroptosis inhibitor (Necrostatin-1) significantly diminished the effect of VC1-8, suggesting that VC1-8 mediates apoptosis and/or necroptosis of RasV12 cells surrounded by normal cells (Fig. 3d).

Bottom Line: Among several Rebeccamycin-derivative compounds, we have found that VC1-8 has least cytotoxicity against normal cells but shows the comparable effect on the elimination of transformed cells.This cell competition-promoting activity of VC1-8 is observed both in vitro and ex vivo.These data demonstrate that the cell competition-based screening is a promising tool for the establishment of a novel type of cancer preventive medicine.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Oncology, Institute for Genetic Medicine, Hokkaido University Graduate School of Chemical Sciences and Engineering, Sapporo, Japan.

ABSTRACT
Recent studies have revealed that cell competition can occur between normal and transformed epithelial cells; normal epithelial cells recognize the presence of the neighboring transformed cells and actively eliminate them from epithelial tissues. Here, we have established a brand-new high-throughput screening platform that targets cell competition. By using this platform, we have identified Rebeccamycin as a hit compound that specifically promotes elimination of RasV12-transformed cells from the epithelium, though after longer treatment it shows substantial cytotoxic effect against normal epithelial cells. Among several Rebeccamycin-derivative compounds, we have found that VC1-8 has least cytotoxicity against normal cells but shows the comparable effect on the elimination of transformed cells. This cell competition-promoting activity of VC1-8 is observed both in vitro and ex vivo. These data demonstrate that the cell competition-based screening is a promising tool for the establishment of a novel type of cancer preventive medicine.

No MeSH data available.


Related in: MedlinePlus