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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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Effects of ISO on the viability and colony formation of A549 cells. (A) MTT cell viability assay. Values are expressed as the mean ± standard deviation of three independent experiments performed in duplicate. *P<0.05; **P<0.01, as compared with 0 μM isorhamnetin. (B) Colony formation assay. A549 cells were treated with indicated concentrations of ISO for 72 h and cell colonies were visualized by crystal violet staining (magnification, ×40). (C) Apoptotic DNA fragmentation was detected by agarose gel electrophoresis at the indicated time-points after exposure to 16 μM ISO. (D) Terminal deoxynucleotidyl transferase dUTP nick end labeling assay after exposure to 16 μM ISO for 48 h (apoptotic bodies in green color) (scale bars, 50 μm). (E) A549 cells were treated with indicated concentrations of ISO for 24 h, and cell apoptosis was measured by Annexin V/PI staining. ISO, isorhamnetin; FITC, fluorescein isothiocyanate; PI, propidium iodide.
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f1-mmr-12-04-5796: Effects of ISO on the viability and colony formation of A549 cells. (A) MTT cell viability assay. Values are expressed as the mean ± standard deviation of three independent experiments performed in duplicate. *P<0.05; **P<0.01, as compared with 0 μM isorhamnetin. (B) Colony formation assay. A549 cells were treated with indicated concentrations of ISO for 72 h and cell colonies were visualized by crystal violet staining (magnification, ×40). (C) Apoptotic DNA fragmentation was detected by agarose gel electrophoresis at the indicated time-points after exposure to 16 μM ISO. (D) Terminal deoxynucleotidyl transferase dUTP nick end labeling assay after exposure to 16 μM ISO for 48 h (apoptotic bodies in green color) (scale bars, 50 μm). (E) A549 cells were treated with indicated concentrations of ISO for 24 h, and cell apoptosis was measured by Annexin V/PI staining. ISO, isorhamnetin; FITC, fluorescein isothiocyanate; PI, propidium iodide.

Mentions: To investigate the proliferation inhibition effect of ISO, an MTT assay was performed. As shown in Fig. 1A, ISO treatment significantly inhibited the proliferation of A549 cells in a dose- and time-dependent manner. In addition, ISO also significantly suppressed the colony formation ability of A549 cells in a dose-dependent manner (Fig. 1B).


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)

Effects of ISO on the viability and colony formation of A549 cells. (A) MTT cell viability assay. Values are expressed as the mean ± standard deviation of three independent experiments performed in duplicate. *P<0.05; **P<0.01, as compared with 0 μM isorhamnetin. (B) Colony formation assay. A549 cells were treated with indicated concentrations of ISO for 72 h and cell colonies were visualized by crystal violet staining (magnification, ×40). (C) Apoptotic DNA fragmentation was detected by agarose gel electrophoresis at the indicated time-points after exposure to 16 μM ISO. (D) Terminal deoxynucleotidyl transferase dUTP nick end labeling assay after exposure to 16 μM ISO for 48 h (apoptotic bodies in green color) (scale bars, 50 μm). (E) A549 cells were treated with indicated concentrations of ISO for 24 h, and cell apoptosis was measured by Annexin V/PI staining. ISO, isorhamnetin; FITC, fluorescein isothiocyanate; PI, propidium iodide.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1-mmr-12-04-5796: Effects of ISO on the viability and colony formation of A549 cells. (A) MTT cell viability assay. Values are expressed as the mean ± standard deviation of three independent experiments performed in duplicate. *P<0.05; **P<0.01, as compared with 0 μM isorhamnetin. (B) Colony formation assay. A549 cells were treated with indicated concentrations of ISO for 72 h and cell colonies were visualized by crystal violet staining (magnification, ×40). (C) Apoptotic DNA fragmentation was detected by agarose gel electrophoresis at the indicated time-points after exposure to 16 μM ISO. (D) Terminal deoxynucleotidyl transferase dUTP nick end labeling assay after exposure to 16 μM ISO for 48 h (apoptotic bodies in green color) (scale bars, 50 μm). (E) A549 cells were treated with indicated concentrations of ISO for 24 h, and cell apoptosis was measured by Annexin V/PI staining. ISO, isorhamnetin; FITC, fluorescein isothiocyanate; PI, propidium iodide.
Mentions: To investigate the proliferation inhibition effect of ISO, an MTT assay was performed. As shown in Fig. 1A, ISO treatment significantly inhibited the proliferation of A549 cells in a dose- and time-dependent manner. In addition, ISO also significantly suppressed the colony formation ability of A549 cells in a dose-dependent manner (Fig. 1B).

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