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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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Inhibition of autophagy sensitizes A549 cells to ISO-induced growth inhibition and apoptosis. (A) pEGFP-LC3-transfected A549 cells were treated with 4 μM isorhamnetin alone or in combination with 10 mM 3-MA or 50 μM CQ for 24 h, and autophagy was observed using confocal microscopy (scale bar, 10 μm). (B) The percentages of cells with LC3 translocation as indicated by the formation of dots were counted (n=250 cells/sample). Values are expressed as the mean ± standard deviation of three independent experiments (**P<0.01). (C) Endogenous LC3-II levels in cells with the same treatment as in A were detected by western blotting using the LC3B antibody. (D) A549 cells were treated as in C, and an MTT cell viability assay was conducted. Values are expressed as the mean ± standard deviation of three independent experiments (*P<0.05; **P<0.01). (E) A549 cells were treated as in C and apoptosis was detected using Annexin V/PI staining followed by flow cytometric analysis. ISO, isorhamnetin; DMSO, dimethyl sulfoxide; LC3, microtubule-associated protein 1 light chain 3; PI, propidium iodide; MA, methyladenine; EGFP, enhanced fluorescence protein; CQ, hydroxychloroquine; FITC, fluorescein isothiocyanate.
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f5-mmr-12-04-5796: Inhibition of autophagy sensitizes A549 cells to ISO-induced growth inhibition and apoptosis. (A) pEGFP-LC3-transfected A549 cells were treated with 4 μM isorhamnetin alone or in combination with 10 mM 3-MA or 50 μM CQ for 24 h, and autophagy was observed using confocal microscopy (scale bar, 10 μm). (B) The percentages of cells with LC3 translocation as indicated by the formation of dots were counted (n=250 cells/sample). Values are expressed as the mean ± standard deviation of three independent experiments (**P<0.01). (C) Endogenous LC3-II levels in cells with the same treatment as in A were detected by western blotting using the LC3B antibody. (D) A549 cells were treated as in C, and an MTT cell viability assay was conducted. Values are expressed as the mean ± standard deviation of three independent experiments (*P<0.05; **P<0.01). (E) A549 cells were treated as in C and apoptosis was detected using Annexin V/PI staining followed by flow cytometric analysis. ISO, isorhamnetin; DMSO, dimethyl sulfoxide; LC3, microtubule-associated protein 1 light chain 3; PI, propidium iodide; MA, methyladenine; EGFP, enhanced fluorescence protein; CQ, hydroxychloroquine; FITC, fluorescein isothiocyanate.

Mentions: 3-MA is a PI3K inhibitor, which suppresses autophagy formation by blocking the activity of the the Class III PI3K complex at an early stage of autophagy (35,36). CQ inhibits autophagy by interfering with the fusion of autophagosomes and lysosomes at a late stage of autophagy (37,38). As shown in Fig. 5A, pre-treatment with 3-MA significantly inhibited the ISO-induced autophagosome formation in the A549 cells (Fig. 5A and B). By contrast, an increased amount of ISO-induced autophagosomes was accumulated in the A549 cells pre-treated with CQ compared with that in cells treated with ISO only (Fig. 5A and B). Next, the protein levels of endogenous LC3-II were detected. As expected, 3-MA pre-treatment significantly inhibited the formation of endogenous LC3-II protein in ISO-treated cells, compared to that in cells treated with ISO only. However, there was no obvious difference in endogenous LC3-II levels between cells treated with ISO only and those co-treated with CQ (Fig. 5C).


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)

Inhibition of autophagy sensitizes A549 cells to ISO-induced growth inhibition and apoptosis. (A) pEGFP-LC3-transfected A549 cells were treated with 4 μM isorhamnetin alone or in combination with 10 mM 3-MA or 50 μM CQ for 24 h, and autophagy was observed using confocal microscopy (scale bar, 10 μm). (B) The percentages of cells with LC3 translocation as indicated by the formation of dots were counted (n=250 cells/sample). Values are expressed as the mean ± standard deviation of three independent experiments (**P<0.01). (C) Endogenous LC3-II levels in cells with the same treatment as in A were detected by western blotting using the LC3B antibody. (D) A549 cells were treated as in C, and an MTT cell viability assay was conducted. Values are expressed as the mean ± standard deviation of three independent experiments (*P<0.05; **P<0.01). (E) A549 cells were treated as in C and apoptosis was detected using Annexin V/PI staining followed by flow cytometric analysis. ISO, isorhamnetin; DMSO, dimethyl sulfoxide; LC3, microtubule-associated protein 1 light chain 3; PI, propidium iodide; MA, methyladenine; EGFP, enhanced fluorescence protein; CQ, hydroxychloroquine; FITC, fluorescein isothiocyanate.
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Related In: Results  -  Collection

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f5-mmr-12-04-5796: Inhibition of autophagy sensitizes A549 cells to ISO-induced growth inhibition and apoptosis. (A) pEGFP-LC3-transfected A549 cells were treated with 4 μM isorhamnetin alone or in combination with 10 mM 3-MA or 50 μM CQ for 24 h, and autophagy was observed using confocal microscopy (scale bar, 10 μm). (B) The percentages of cells with LC3 translocation as indicated by the formation of dots were counted (n=250 cells/sample). Values are expressed as the mean ± standard deviation of three independent experiments (**P<0.01). (C) Endogenous LC3-II levels in cells with the same treatment as in A were detected by western blotting using the LC3B antibody. (D) A549 cells were treated as in C, and an MTT cell viability assay was conducted. Values are expressed as the mean ± standard deviation of three independent experiments (*P<0.05; **P<0.01). (E) A549 cells were treated as in C and apoptosis was detected using Annexin V/PI staining followed by flow cytometric analysis. ISO, isorhamnetin; DMSO, dimethyl sulfoxide; LC3, microtubule-associated protein 1 light chain 3; PI, propidium iodide; MA, methyladenine; EGFP, enhanced fluorescence protein; CQ, hydroxychloroquine; FITC, fluorescein isothiocyanate.
Mentions: 3-MA is a PI3K inhibitor, which suppresses autophagy formation by blocking the activity of the the Class III PI3K complex at an early stage of autophagy (35,36). CQ inhibits autophagy by interfering with the fusion of autophagosomes and lysosomes at a late stage of autophagy (37,38). As shown in Fig. 5A, pre-treatment with 3-MA significantly inhibited the ISO-induced autophagosome formation in the A549 cells (Fig. 5A and B). By contrast, an increased amount of ISO-induced autophagosomes was accumulated in the A549 cells pre-treated with CQ compared with that in cells treated with ISO only (Fig. 5A and B). Next, the protein levels of endogenous LC3-II were detected. As expected, 3-MA pre-treatment significantly inhibited the formation of endogenous LC3-II protein in ISO-treated cells, compared to that in cells treated with ISO only. However, there was no obvious difference in endogenous LC3-II levels between cells treated with ISO only and those co-treated with CQ (Fig. 5C).

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