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Autophagy inhibition enhances isorhamnetin‑induced mitochondria‑dependent apoptosis in non‑small cell lung cancer cells.

Ruan Y, Hu K, Chen H - Mol Med Rep (2015)

Bottom Line: ISO treatment also resulted in a significant increase in apoptotic cell death of A549 cells in a time- and dose-dependent manner.Furthermore, co‑treatment with autophagy inhibitors 3‑methyladenine and hydroxychloroquine significantly inhibited the ISO‑induced autophagy and enhanced the ISO‑induced apoptotic cell death in vitro as well as in vivo.In addition, the results indicated that the inhibition of autophagy may be a useful strategy for enhancing the chemotherapeutic effect of ISO on lung cancer cells.

View Article: PubMed Central - PubMed

Affiliation: Division of Respiratory Disease, Renmin Hospital of Wuhan University, Wuhan, Hubei 430060, P.R. China.

ABSTRACT
Isorhamnetin (ISO) is a flavonoid from plants of the Polygonaceae family and is also an immediate metabolite of quercetin in mammals. To date, the anti‑tumor effects of ISO and the underlying mechanisms have not been elucidated in lung cancer cells. The present study investigated the inhibitory effects of ISO on the growth of human lung cancer A549 cells. Treatment of the lung cancer cells with ISO significantly suppressed cell proliferation and colony formation. ISO treatment also resulted in a significant increase in apoptotic cell death of A549 cells in a time- and dose-dependent manner. Further investigation showed that the apoptosis proceeded via the mitochondria‑dependent pathway as indicated by alteration of the mitochondrial membrane potential, the release of cytochrome C and caspase activation. Of note, treatment with ISO also induced the formation of autophagosomes and light chain 3‑II protein in A549 cells. Furthermore, co‑treatment with autophagy inhibitors 3‑methyladenine and hydroxychloroquine significantly inhibited the ISO‑induced autophagy and enhanced the ISO‑induced apoptotic cell death in vitro as well as in vivo. Thus, the results of the present study suggested that ISO is a potential anti‑lung cancer agent. In addition, the results indicated that the inhibition of autophagy may be a useful strategy for enhancing the chemotherapeutic effect of ISO on lung cancer cells.

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ISO induces autophagy in A549 cells. (A) pEGFP-LC3-transfected A549 cells were treated with different concentrations of ISO for 24 h and the GFP-LC3-II translocation to autophagosomes was observed using confocal microscopy (scale bar, 10 μm). (B) The percentages of cells with LC3 translocation, as indicated by formation of dots, were counted (n=250 cells/sample). Cells were treated wth various concentrations of ISO for 24 h. Values are expressed the mean ± standard deviation of three independent experiments (**P<0.01). (C) A549 cells were treated with indicated concentrations of ISO for 24 h and endogenous LC3-II levels were detected by western blotting using LC3B antibody. (D) A549 cells were treated with indicated concentrations of ISO for 24 h and autophagy was detected by monodansylcadaverine staining (scale bar, 10 μm). EGFP, enhanced green fluorescence protein; ISO, isorhamnetin; DMSO, dimethyl sulfoxide; LC3, microtubule-associated protein 1 light chain 3.
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f4-mmr-12-04-5796: ISO induces autophagy in A549 cells. (A) pEGFP-LC3-transfected A549 cells were treated with different concentrations of ISO for 24 h and the GFP-LC3-II translocation to autophagosomes was observed using confocal microscopy (scale bar, 10 μm). (B) The percentages of cells with LC3 translocation, as indicated by formation of dots, were counted (n=250 cells/sample). Cells were treated wth various concentrations of ISO for 24 h. Values are expressed the mean ± standard deviation of three independent experiments (**P<0.01). (C) A549 cells were treated with indicated concentrations of ISO for 24 h and endogenous LC3-II levels were detected by western blotting using LC3B antibody. (D) A549 cells were treated with indicated concentrations of ISO for 24 h and autophagy was detected by monodansylcadaverine staining (scale bar, 10 μm). EGFP, enhanced green fluorescence protein; ISO, isorhamnetin; DMSO, dimethyl sulfoxide; LC3, microtubule-associated protein 1 light chain 3.

Mentions: Numerous chemotherapeutic agents designed to kill cancer cells are also known to induce autophagy (18). When autophagy is initiated, microtubule-associated protein 1 light chain 3 (LC3) is cut on the C-terminal to produce LC3-II protein. The resulting LC3-II is then preferentially translocated to the membranes of autophagosomes and shows a punctate staining pattern in the cytosol (33). In order to detect autophagy, EGFP-LC3 was overexpressed in A549 cells. As shown in Fig. 4A and B, the number of autophagosomes with a punctate staining pattern was significantly increased in ISO-treated cells compared with that in untreated cells. Accordingly, the protein levels of LC3-II were also significantly increased in A549 cells treated with ISO in a dose-dependent manner (Fig. 4C). Beclin1 has been shown to be the activator of the Class III phosphoinositide 3-kinase (PI3K) complex that has an important role in the regulation of autophagy induction (34). In the present study, it was found that the expression of Beclin1 was upregulated following ISO treatment (Fig. 4C).


Autophagy inhibition enhances isorhamnetin‑induced mitochondria‑dependent apoptosis in non‑small cell lung cancer cells.

Ruan Y, Hu K, Chen H - Mol Med Rep (2015)

ISO induces autophagy in A549 cells. (A) pEGFP-LC3-transfected A549 cells were treated with different concentrations of ISO for 24 h and the GFP-LC3-II translocation to autophagosomes was observed using confocal microscopy (scale bar, 10 μm). (B) The percentages of cells with LC3 translocation, as indicated by formation of dots, were counted (n=250 cells/sample). Cells were treated wth various concentrations of ISO for 24 h. Values are expressed the mean ± standard deviation of three independent experiments (**P<0.01). (C) A549 cells were treated with indicated concentrations of ISO for 24 h and endogenous LC3-II levels were detected by western blotting using LC3B antibody. (D) A549 cells were treated with indicated concentrations of ISO for 24 h and autophagy was detected by monodansylcadaverine staining (scale bar, 10 μm). EGFP, enhanced green fluorescence protein; ISO, isorhamnetin; DMSO, dimethyl sulfoxide; LC3, microtubule-associated protein 1 light chain 3.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4581743&req=5

f4-mmr-12-04-5796: ISO induces autophagy in A549 cells. (A) pEGFP-LC3-transfected A549 cells were treated with different concentrations of ISO for 24 h and the GFP-LC3-II translocation to autophagosomes was observed using confocal microscopy (scale bar, 10 μm). (B) The percentages of cells with LC3 translocation, as indicated by formation of dots, were counted (n=250 cells/sample). Cells were treated wth various concentrations of ISO for 24 h. Values are expressed the mean ± standard deviation of three independent experiments (**P<0.01). (C) A549 cells were treated with indicated concentrations of ISO for 24 h and endogenous LC3-II levels were detected by western blotting using LC3B antibody. (D) A549 cells were treated with indicated concentrations of ISO for 24 h and autophagy was detected by monodansylcadaverine staining (scale bar, 10 μm). EGFP, enhanced green fluorescence protein; ISO, isorhamnetin; DMSO, dimethyl sulfoxide; LC3, microtubule-associated protein 1 light chain 3.
Mentions: Numerous chemotherapeutic agents designed to kill cancer cells are also known to induce autophagy (18). When autophagy is initiated, microtubule-associated protein 1 light chain 3 (LC3) is cut on the C-terminal to produce LC3-II protein. The resulting LC3-II is then preferentially translocated to the membranes of autophagosomes and shows a punctate staining pattern in the cytosol (33). In order to detect autophagy, EGFP-LC3 was overexpressed in A549 cells. As shown in Fig. 4A and B, the number of autophagosomes with a punctate staining pattern was significantly increased in ISO-treated cells compared with that in untreated cells. Accordingly, the protein levels of LC3-II were also significantly increased in A549 cells treated with ISO in a dose-dependent manner (Fig. 4C). Beclin1 has been shown to be the activator of the Class III phosphoinositide 3-kinase (PI3K) complex that has an important role in the regulation of autophagy induction (34). In the present study, it was found that the expression of Beclin1 was upregulated following ISO treatment (Fig. 4C).

Bottom Line: ISO treatment also resulted in a significant increase in apoptotic cell death of A549 cells in a time- and dose-dependent manner.Furthermore, co‑treatment with autophagy inhibitors 3‑methyladenine and hydroxychloroquine significantly inhibited the ISO‑induced autophagy and enhanced the ISO‑induced apoptotic cell death in vitro as well as in vivo.In addition, the results indicated that the inhibition of autophagy may be a useful strategy for enhancing the chemotherapeutic effect of ISO on lung cancer cells.

View Article: PubMed Central - PubMed

Affiliation: Division of Respiratory Disease, Renmin Hospital of Wuhan University, Wuhan, Hubei 430060, P.R. China.

ABSTRACT
Isorhamnetin (ISO) is a flavonoid from plants of the Polygonaceae family and is also an immediate metabolite of quercetin in mammals. To date, the anti‑tumor effects of ISO and the underlying mechanisms have not been elucidated in lung cancer cells. The present study investigated the inhibitory effects of ISO on the growth of human lung cancer A549 cells. Treatment of the lung cancer cells with ISO significantly suppressed cell proliferation and colony formation. ISO treatment also resulted in a significant increase in apoptotic cell death of A549 cells in a time- and dose-dependent manner. Further investigation showed that the apoptosis proceeded via the mitochondria‑dependent pathway as indicated by alteration of the mitochondrial membrane potential, the release of cytochrome C and caspase activation. Of note, treatment with ISO also induced the formation of autophagosomes and light chain 3‑II protein in A549 cells. Furthermore, co‑treatment with autophagy inhibitors 3‑methyladenine and hydroxychloroquine significantly inhibited the ISO‑induced autophagy and enhanced the ISO‑induced apoptotic cell death in vitro as well as in vivo. Thus, the results of the present study suggested that ISO is a potential anti‑lung cancer agent. In addition, the results indicated that the inhibition of autophagy may be a useful strategy for enhancing the chemotherapeutic effect of ISO on lung cancer cells.

Show MeSH
Related in: MedlinePlus