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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 mitochondria-dependent caspase activation (A) Treatment with 8 μM ISO for 12 h induced alterations in the mRNA expression of marker genes associated with apoptosis in A549 cells. (B) Cleaved-caspase-3, cleaved-PARP and pro-caspase-9 were detected after treatment with the indicated concentrations of ISO for 24 h. (C) A significant increase of cytochrome C release was detected at 12 h after 16-μM ISO treatment. (D and E) Caspase-9 inhibitor Z-LEHD-FMK or caspase-3/7 inhibitor Z-DEVD-FMK significantly blocked the ISO-induced sub-G1 peaks. Cells were treated with 8 μM ISO for 48 h. Values are expressed the mean ± standard deviation (n=4). **P<0.01. C-cytochrome C, cytosolic cytochrome C; M-cytochrome C, mitochondrial cytochrome C; ISO, isorhamnetin; casp, caspase; Bcl-2, B-cell lymphoma 2; Bax, Bcl-2-associated X protein; Bclw, Bcl-2-like protein 2; Mcl1, myeloid cell leukemia 1; bid, BH3 interacting-domain death agonist; BNIP3, Bcl-2/adenovirus E1B 19 kDa protein-interacting protein 3; Bak, Bcl-2 homologous antagonist/killer; Puma, p53-upregulated modulator of apoptosis; Con, control; PARP, poly(adenosine diphosphate ribose) polymerase.
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f3-mmr-12-04-5796: ISO induces mitochondria-dependent caspase activation (A) Treatment with 8 μM ISO for 12 h induced alterations in the mRNA expression of marker genes associated with apoptosis in A549 cells. (B) Cleaved-caspase-3, cleaved-PARP and pro-caspase-9 were detected after treatment with the indicated concentrations of ISO for 24 h. (C) A significant increase of cytochrome C release was detected at 12 h after 16-μM ISO treatment. (D and E) Caspase-9 inhibitor Z-LEHD-FMK or caspase-3/7 inhibitor Z-DEVD-FMK significantly blocked the ISO-induced sub-G1 peaks. Cells were treated with 8 μM ISO for 48 h. Values are expressed the mean ± standard deviation (n=4). **P<0.01. C-cytochrome C, cytosolic cytochrome C; M-cytochrome C, mitochondrial cytochrome C; ISO, isorhamnetin; casp, caspase; Bcl-2, B-cell lymphoma 2; Bax, Bcl-2-associated X protein; Bclw, Bcl-2-like protein 2; Mcl1, myeloid cell leukemia 1; bid, BH3 interacting-domain death agonist; BNIP3, Bcl-2/adenovirus E1B 19 kDa protein-interacting protein 3; Bak, Bcl-2 homologous antagonist/killer; Puma, p53-upregulated modulator of apoptosis; Con, control; PARP, poly(adenosine diphosphate ribose) polymerase.

Mentions: Furthermore, the ISO-induced alterations in the mRNA expression of apoptosis marker genes in A549 cells were examined. RT-qPCR analysis showed a significant (P<0.01) upregulation in the expression of caspase-3 (9.6±0.53-fold), caspase-9 (9.4±0.65-fold), Bax (1.6±0.19-fold), p53 (5.89±0.21-fold), p21 (2.7±0.33-fold) and Puma (2.22±0.23-fold) at 12 h of treatment with 8 μM ISO (Fig. 3A). In addition, the protein expression of cleaved-caspase-3, cleaved-PARP and pro-caspase-9 were detected by western blotting. As shown in Fig. 3B, the expression levels of cleaved-caspase-3 and cleaved-PARP were significantly increased following ISO treatment in a dose-dependent manner, whereas the levels of pro-caspase-9 were obviously decreased, indicating that ISO treatment resulted in the activation of the caspase-dependent apoptotic pathway. As it is known that caspase activation involves changes in mitochondrial permeability and the release of cytochrome C, the levels of cytochrome C in the cytosolic fraction were then examined. As shown in Fig. 3C, a signifi-cant increase of released cytochrome C was detected at 12 h after treatment with 16 μM ISO. To further determine whether the ISO-induced apoptosis of NSCLC cells was caspase-mediated, A549 cells were incubated with caspase-9 inhibitor Z-LEHD-FMK or caspase-3/7 inhibitor Z-DEVD-FMK for 2 h followed by treatment of the cells with ISO for 48 h (Fig. 3D and E). These caspase inhibitors completely blocked the ISO-induced sub-G1 fractions in the cell cycle distribution. These results therefore suggested that ISO-induced apoptosis was mediated by mitochondria-dependent caspase activation.


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 mitochondria-dependent caspase activation (A) Treatment with 8 μM ISO for 12 h induced alterations in the mRNA expression of marker genes associated with apoptosis in A549 cells. (B) Cleaved-caspase-3, cleaved-PARP and pro-caspase-9 were detected after treatment with the indicated concentrations of ISO for 24 h. (C) A significant increase of cytochrome C release was detected at 12 h after 16-μM ISO treatment. (D and E) Caspase-9 inhibitor Z-LEHD-FMK or caspase-3/7 inhibitor Z-DEVD-FMK significantly blocked the ISO-induced sub-G1 peaks. Cells were treated with 8 μM ISO for 48 h. Values are expressed the mean ± standard deviation (n=4). **P<0.01. C-cytochrome C, cytosolic cytochrome C; M-cytochrome C, mitochondrial cytochrome C; ISO, isorhamnetin; casp, caspase; Bcl-2, B-cell lymphoma 2; Bax, Bcl-2-associated X protein; Bclw, Bcl-2-like protein 2; Mcl1, myeloid cell leukemia 1; bid, BH3 interacting-domain death agonist; BNIP3, Bcl-2/adenovirus E1B 19 kDa protein-interacting protein 3; Bak, Bcl-2 homologous antagonist/killer; Puma, p53-upregulated modulator of apoptosis; Con, control; PARP, poly(adenosine diphosphate ribose) polymerase.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3-mmr-12-04-5796: ISO induces mitochondria-dependent caspase activation (A) Treatment with 8 μM ISO for 12 h induced alterations in the mRNA expression of marker genes associated with apoptosis in A549 cells. (B) Cleaved-caspase-3, cleaved-PARP and pro-caspase-9 were detected after treatment with the indicated concentrations of ISO for 24 h. (C) A significant increase of cytochrome C release was detected at 12 h after 16-μM ISO treatment. (D and E) Caspase-9 inhibitor Z-LEHD-FMK or caspase-3/7 inhibitor Z-DEVD-FMK significantly blocked the ISO-induced sub-G1 peaks. Cells were treated with 8 μM ISO for 48 h. Values are expressed the mean ± standard deviation (n=4). **P<0.01. C-cytochrome C, cytosolic cytochrome C; M-cytochrome C, mitochondrial cytochrome C; ISO, isorhamnetin; casp, caspase; Bcl-2, B-cell lymphoma 2; Bax, Bcl-2-associated X protein; Bclw, Bcl-2-like protein 2; Mcl1, myeloid cell leukemia 1; bid, BH3 interacting-domain death agonist; BNIP3, Bcl-2/adenovirus E1B 19 kDa protein-interacting protein 3; Bak, Bcl-2 homologous antagonist/killer; Puma, p53-upregulated modulator of apoptosis; Con, control; PARP, poly(adenosine diphosphate ribose) polymerase.
Mentions: Furthermore, the ISO-induced alterations in the mRNA expression of apoptosis marker genes in A549 cells were examined. RT-qPCR analysis showed a significant (P<0.01) upregulation in the expression of caspase-3 (9.6±0.53-fold), caspase-9 (9.4±0.65-fold), Bax (1.6±0.19-fold), p53 (5.89±0.21-fold), p21 (2.7±0.33-fold) and Puma (2.22±0.23-fold) at 12 h of treatment with 8 μM ISO (Fig. 3A). In addition, the protein expression of cleaved-caspase-3, cleaved-PARP and pro-caspase-9 were detected by western blotting. As shown in Fig. 3B, the expression levels of cleaved-caspase-3 and cleaved-PARP were significantly increased following ISO treatment in a dose-dependent manner, whereas the levels of pro-caspase-9 were obviously decreased, indicating that ISO treatment resulted in the activation of the caspase-dependent apoptotic pathway. As it is known that caspase activation involves changes in mitochondrial permeability and the release of cytochrome C, the levels of cytochrome C in the cytosolic fraction were then examined. As shown in Fig. 3C, a signifi-cant increase of released cytochrome C was detected at 12 h after treatment with 16 μM ISO. To further determine whether the ISO-induced apoptosis of NSCLC cells was caspase-mediated, A549 cells were incubated with caspase-9 inhibitor Z-LEHD-FMK or caspase-3/7 inhibitor Z-DEVD-FMK for 2 h followed by treatment of the cells with ISO for 48 h (Fig. 3D and E). These caspase inhibitors completely blocked the ISO-induced sub-G1 fractions in the cell cycle distribution. These results therefore suggested that ISO-induced apoptosis was mediated by mitochondria-dependent caspase activation.

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