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Activation of p53 with ilimaquinone and ethylsmenoquinone, marine sponge metabolites, induces apoptosis and autophagy in colon cancer cells.

Lee HY, Chung KJ, Hwang IH, Gwak J, Park S, Ju BG, Yun E, Kim DE, Chung YH, Na M, Song GY, Oh S - Mar Drugs (2015)

Bottom Line: Here, we used a cell-based reporter system for the detection of p53 response transcription to identify the marine sponge metabolites, ilimaquinone and ethylsmenoquinone, as activators of the p53 pathway.In addition, ilimaquinone and ethylsmenoquinone induced G2/M cell cycle arrest and increased caspase-3 cleavage and the population of cells that positively stained with Annexin V-FITC, both of which are typical biochemical markers of apoptosis.Furthermore, autophagy was elicited by both compounds, as indicated by microtubule-associated protein 1 light chain 3 (LC3) puncta formations and LC3-II turnover in HCT116 cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Bio and Fermentation Convergence Technology, Kookmin University, Seoul 136-702, Korea. IHyunYoungg@gmail.com.

ABSTRACT
The tumor suppressor, p53, plays an essential role in the cellular response to stress through regulating the expression of genes involved in cell cycle arrest, apoptosis and autophagy. Here, we used a cell-based reporter system for the detection of p53 response transcription to identify the marine sponge metabolites, ilimaquinone and ethylsmenoquinone, as activators of the p53 pathway. We demonstrated that ilimaquinone and ethylsmenoquinone efficiently stabilize the p53 protein through promotion of p53 phosphorylation at Ser15 in both HCT116 and RKO colon cancer cells. Moreover, both compounds upregulate the expression of p21WAF1/CIP1, a p53-dependent gene, and suppress proliferation of colon cancer cells. In addition, ilimaquinone and ethylsmenoquinone induced G2/M cell cycle arrest and increased caspase-3 cleavage and the population of cells that positively stained with Annexin V-FITC, both of which are typical biochemical markers of apoptosis. Furthermore, autophagy was elicited by both compounds, as indicated by microtubule-associated protein 1 light chain 3 (LC3) puncta formations and LC3-II turnover in HCT116 cells. Our findings suggest that ilimaquinone and ethylsmenoquinone exert their anti-cancer activity by activation of the p53 pathway and may have significant potential as chemo-preventive and therapeutic agents for human colon cancer.

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Ilimaquinone and ethylsmenoquinone induce autophagy in HCT116 cells. (A) Representative images of the punctate distribution of GFP-LC3-labeled in HCT-116 cells 24 h after exposure to IQ and ESQ; (B) HCT116 cells were treated with IQ and ESQ for 24 h or DMSO for 24 h. Cell lysates were separated on 15% SDS-PAGE and analyzed by western blot using anti-LC3 antibody. Results are representative of three independent experiments.
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marinedrugs-13-00543-f006: Ilimaquinone and ethylsmenoquinone induce autophagy in HCT116 cells. (A) Representative images of the punctate distribution of GFP-LC3-labeled in HCT-116 cells 24 h after exposure to IQ and ESQ; (B) HCT116 cells were treated with IQ and ESQ for 24 h or DMSO for 24 h. Cell lysates were separated on 15% SDS-PAGE and analyzed by western blot using anti-LC3 antibody. Results are representative of three independent experiments.

Mentions: Recent studies have demonstrated that nuclear p53 negatively regulates the process of autophagy, which is a conserved catabolic pathway, by mediating the turnover of intracellular organelles and protein complexes [19]. Given that ilimaquinone and ethylsmenoquinone activated p53 responsive transcription in the nucleus, we next investigated whether these compounds induced autophagic cell death in colon cancer cells. For this purpose, we measured the conversion of the cytosolic microtubule-associated protein 1 light chain 3-1 (LC3-1) to the autophagosome-associated LC-3II form, which is widely used as an indication of autophagy [20]. HCT116 colon cancer cells were transfected with a green fluorescent protein tag of LC3 (GFP-LC3), and then, the cells were treated with ilimaquinone and ethylsmenoquinone. As depicted in Figure 6A and Supplementary Figure S3, these sesquiterpene quinone compounds stimulated the redistribution of GFP-LC3 from a diffuse pattern to punctuate dots. Furthermore, western blot analysis of the cell lysates revealed that ilimaquinone and ethylsmenoquinone promoted the formation of lipidated LC-3II from LC3-I (Figure 6B). These results indicate that autophagy may be a mechanism by which ilimaquinone and ethylsmenoquinone induce cell death in colon cancer cells.


Activation of p53 with ilimaquinone and ethylsmenoquinone, marine sponge metabolites, induces apoptosis and autophagy in colon cancer cells.

Lee HY, Chung KJ, Hwang IH, Gwak J, Park S, Ju BG, Yun E, Kim DE, Chung YH, Na M, Song GY, Oh S - Mar Drugs (2015)

Ilimaquinone and ethylsmenoquinone induce autophagy in HCT116 cells. (A) Representative images of the punctate distribution of GFP-LC3-labeled in HCT-116 cells 24 h after exposure to IQ and ESQ; (B) HCT116 cells were treated with IQ and ESQ for 24 h or DMSO for 24 h. Cell lysates were separated on 15% SDS-PAGE and analyzed by western blot using anti-LC3 antibody. Results are representative of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4306951&req=5

marinedrugs-13-00543-f006: Ilimaquinone and ethylsmenoquinone induce autophagy in HCT116 cells. (A) Representative images of the punctate distribution of GFP-LC3-labeled in HCT-116 cells 24 h after exposure to IQ and ESQ; (B) HCT116 cells were treated with IQ and ESQ for 24 h or DMSO for 24 h. Cell lysates were separated on 15% SDS-PAGE and analyzed by western blot using anti-LC3 antibody. Results are representative of three independent experiments.
Mentions: Recent studies have demonstrated that nuclear p53 negatively regulates the process of autophagy, which is a conserved catabolic pathway, by mediating the turnover of intracellular organelles and protein complexes [19]. Given that ilimaquinone and ethylsmenoquinone activated p53 responsive transcription in the nucleus, we next investigated whether these compounds induced autophagic cell death in colon cancer cells. For this purpose, we measured the conversion of the cytosolic microtubule-associated protein 1 light chain 3-1 (LC3-1) to the autophagosome-associated LC-3II form, which is widely used as an indication of autophagy [20]. HCT116 colon cancer cells were transfected with a green fluorescent protein tag of LC3 (GFP-LC3), and then, the cells were treated with ilimaquinone and ethylsmenoquinone. As depicted in Figure 6A and Supplementary Figure S3, these sesquiterpene quinone compounds stimulated the redistribution of GFP-LC3 from a diffuse pattern to punctuate dots. Furthermore, western blot analysis of the cell lysates revealed that ilimaquinone and ethylsmenoquinone promoted the formation of lipidated LC-3II from LC3-I (Figure 6B). These results indicate that autophagy may be a mechanism by which ilimaquinone and ethylsmenoquinone induce cell death in colon cancer cells.

Bottom Line: Here, we used a cell-based reporter system for the detection of p53 response transcription to identify the marine sponge metabolites, ilimaquinone and ethylsmenoquinone, as activators of the p53 pathway.In addition, ilimaquinone and ethylsmenoquinone induced G2/M cell cycle arrest and increased caspase-3 cleavage and the population of cells that positively stained with Annexin V-FITC, both of which are typical biochemical markers of apoptosis.Furthermore, autophagy was elicited by both compounds, as indicated by microtubule-associated protein 1 light chain 3 (LC3) puncta formations and LC3-II turnover in HCT116 cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Bio and Fermentation Convergence Technology, Kookmin University, Seoul 136-702, Korea. IHyunYoungg@gmail.com.

ABSTRACT
The tumor suppressor, p53, plays an essential role in the cellular response to stress through regulating the expression of genes involved in cell cycle arrest, apoptosis and autophagy. Here, we used a cell-based reporter system for the detection of p53 response transcription to identify the marine sponge metabolites, ilimaquinone and ethylsmenoquinone, as activators of the p53 pathway. We demonstrated that ilimaquinone and ethylsmenoquinone efficiently stabilize the p53 protein through promotion of p53 phosphorylation at Ser15 in both HCT116 and RKO colon cancer cells. Moreover, both compounds upregulate the expression of p21WAF1/CIP1, a p53-dependent gene, and suppress proliferation of colon cancer cells. In addition, ilimaquinone and ethylsmenoquinone induced G2/M cell cycle arrest and increased caspase-3 cleavage and the population of cells that positively stained with Annexin V-FITC, both of which are typical biochemical markers of apoptosis. Furthermore, autophagy was elicited by both compounds, as indicated by microtubule-associated protein 1 light chain 3 (LC3) puncta formations and LC3-II turnover in HCT116 cells. Our findings suggest that ilimaquinone and ethylsmenoquinone exert their anti-cancer activity by activation of the p53 pathway and may have significant potential as chemo-preventive and therapeutic agents for human colon cancer.

Show MeSH
Related in: MedlinePlus