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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 apoptosis in A549 cells. (A) A549 cells were treated with indicated concentrations of ISO for 24 h, and mitochondrial membrane potential was detected using JC-1 staining method (scale bars, 10 μm). (B) Cells were treated with 16 μM ISO for the indicated times, harvested and stained with tetramethylrhodamine ethyl ester to determine mitochondrial membrane potential by flow cytometry. ISO, isorhamnetin; DMSO, dimethyl sulfoxide.
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f2-mmr-12-04-5796: ISO induces apoptosis in A549 cells. (A) A549 cells were treated with indicated concentrations of ISO for 24 h, and mitochondrial membrane potential was detected using JC-1 staining method (scale bars, 10 μm). (B) Cells were treated with 16 μM ISO for the indicated times, harvested and stained with tetramethylrhodamine ethyl ester to determine mitochondrial membrane potential by flow cytometry. ISO, isorhamnetin; DMSO, dimethyl sulfoxide.

Mentions: With regard to apoptosis signaling, mitochondria are a central sensor and integration point for diverse apoptotic signals; furthermore, they function as the storehouse of cytochrome C and Smac/Diablo, which binds and disables inhibitors of apoptosis-associated proteins (IAPs) (28,29). The 'apoptosome' cascade or intrinsic pathway involves activation of pro-caspase-9 by cytochrome C released from the mitochondria, leading to the activation of the executioner pro-caspases (caspase-3, -6 and -7) that cleave poly (adenosine diphosphate ribose) polymerase (PARP) and other apoptotic protein substrates (30). To investigate whether ISO-induced apoptosis was mitochondrial-dependent, mitochondrial membrane potential and caspase assays were performed. The permeabilization of mitochondria is one of the most important events during apoptosis (31,32). Mitochondrial de-polarization in apoptotic cells can be detected by a decrease in the red/green fluorescence intensity ratio of the dye JC-1 as a result of its disaggregation into monomers. As shown in Fig. 2A, a significantly higher red/green fluorescence rate was observed in cells treated with DMSO only compared with that in ISO-treated cells, suggesting that ISO treatment resulted in the de-polarization and permeabilization of mitochondria of A549 cells. To further verify the depolarization of the mitochondrial membrane potential after ISO treatment (16 μM), A549 cells were stained with TMRE followed by flow cytometric analysis at the indicated times. As shown in Fig. 2B, ISO treatment resulted in a left-shift of the TMRE fluorescence at as early as 12 h of incubation with ISO. The ratio of cells with intact mitochondrial membrane potential decreased from 90.2% in the control cells to 80.6, 65.5, and 34.2% at 12, 24, and 48 h of ISO treatment, respectively (Fig. 2B).


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 apoptosis in A549 cells. (A) A549 cells were treated with indicated concentrations of ISO for 24 h, and mitochondrial membrane potential was detected using JC-1 staining method (scale bars, 10 μm). (B) Cells were treated with 16 μM ISO for the indicated times, harvested and stained with tetramethylrhodamine ethyl ester to determine mitochondrial membrane potential by flow cytometry. ISO, isorhamnetin; DMSO, dimethyl sulfoxide.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2-mmr-12-04-5796: ISO induces apoptosis in A549 cells. (A) A549 cells were treated with indicated concentrations of ISO for 24 h, and mitochondrial membrane potential was detected using JC-1 staining method (scale bars, 10 μm). (B) Cells were treated with 16 μM ISO for the indicated times, harvested and stained with tetramethylrhodamine ethyl ester to determine mitochondrial membrane potential by flow cytometry. ISO, isorhamnetin; DMSO, dimethyl sulfoxide.
Mentions: With regard to apoptosis signaling, mitochondria are a central sensor and integration point for diverse apoptotic signals; furthermore, they function as the storehouse of cytochrome C and Smac/Diablo, which binds and disables inhibitors of apoptosis-associated proteins (IAPs) (28,29). The 'apoptosome' cascade or intrinsic pathway involves activation of pro-caspase-9 by cytochrome C released from the mitochondria, leading to the activation of the executioner pro-caspases (caspase-3, -6 and -7) that cleave poly (adenosine diphosphate ribose) polymerase (PARP) and other apoptotic protein substrates (30). To investigate whether ISO-induced apoptosis was mitochondrial-dependent, mitochondrial membrane potential and caspase assays were performed. The permeabilization of mitochondria is one of the most important events during apoptosis (31,32). Mitochondrial de-polarization in apoptotic cells can be detected by a decrease in the red/green fluorescence intensity ratio of the dye JC-1 as a result of its disaggregation into monomers. As shown in Fig. 2A, a significantly higher red/green fluorescence rate was observed in cells treated with DMSO only compared with that in ISO-treated cells, suggesting that ISO treatment resulted in the de-polarization and permeabilization of mitochondria of A549 cells. To further verify the depolarization of the mitochondrial membrane potential after ISO treatment (16 μM), A549 cells were stained with TMRE followed by flow cytometric analysis at the indicated times. As shown in Fig. 2B, ISO treatment resulted in a left-shift of the TMRE fluorescence at as early as 12 h of incubation with ISO. The ratio of cells with intact mitochondrial membrane potential decreased from 90.2% in the control cells to 80.6, 65.5, and 34.2% at 12, 24, and 48 h of ISO treatment, respectively (Fig. 2B).

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