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Resveratrol and STAT inhibitor enhance autophagy in ovarian cancer cells

View Article: PubMed Central - PubMed

ABSTRACT

Autophagic activity reflects cellular response to drug treatment and can be regulated by STAT3 signaling. Resveratrol inhibits STAT3 activation and causes remarkable growth arrest and cell death of ovarian cancer (OC) cells. However, the autophagic status and its relevance with resveratrol’s anti-OC effects remain unclear. We analyzed the states of autophagic activities, the nature of autophagosomes and the levels of autophagy-related proteins (LC-3, Beclin 1 and STAT3) in resveratrol-treated CAOV-3 and OVCAR-3 OC cells using multiple approaches. We elucidated the correlation of STAT3 inhibition with autophagic activity by treating OC cells with an upstream inhibitor of STAT proteins, AG490. Resveratrol efficiently suppressed growth, induced apoptosis and inactivated STAT3 signaling of the two OC cell lines. We found enhanced autophagic activity accompanied with Beclin-1 upregulation and LC3 enzymatic cleavage in resveratrol-treated OC cells. Immunofluorescent (IF) microscopic and IF-based confocal examinations demonstrated the accumulation of cytoplasmic granules co-labeled with LC3 and cytochrome C in resveratrol- or AG490-treated OC cells. Using electron microscopy, we confirmed an increase in autophagosomes and mitochondrial spheroids in either resveratrol- or AG490-treated OC cells. This study demonstrates the abilities of resveratrol to enhance apoptotic and autophagic activities in OC cells, presumably via inactivating STAT3 signaling. Resveratrol or the selective JAK2 inhibitor also leads to mitochondrial turnover, which would be unfavorable for OC cell survival and sensitize OC cells to resveratrol.

No MeSH data available.


Double LC3 (green) and cytochrome C (red) immunofluorescent labeling of CAOV-3 cells treated by resveratrol at different time points. IF, the images taken under conventional IF microscope. Confocal, the images taken under confocal laser scanning microscope.
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fig4: Double LC3 (green) and cytochrome C (red) immunofluorescent labeling of CAOV-3 cells treated by resveratrol at different time points. IF, the images taken under conventional IF microscope. Confocal, the images taken under confocal laser scanning microscope.

Mentions: LC3 is a commonly used marker to monitor autophagosomes and cytochrome C proteins are preferably localized in mitochondria.24 To identify the feature(s) of autophagic bodies, LC3 and cytochrome C double IF staining combined with confocal laser scanning microscope observation were conducted on the OC cells without and with resveratrol treatment. The results demonstrate that the conventional IF image of normally cultured OC cells was weakly green in the cytoplasm and the confocal one showed the predominant distribution of granules labeled with LC3 (green) or cytochrome C (red; Figure 4a). The IF staining patterns were apparently altered in resveratrol-treated cells by showing the color transition of IF images from weakly green to strong orange and yellow at 24 h to green and yellow at 48 h and to yellow and green at 72 h time points. In agreement with the IF microscopic findings, the confocal images demonstrated the appearance of LC3+/cytochrome C+ granules in the cytoplasm of resveratrol-treated cells, especially those treated for 24 h (Figure 4b).


Resveratrol and STAT inhibitor enhance autophagy in ovarian cancer cells
Double LC3 (green) and cytochrome C (red) immunofluorescent labeling of CAOV-3 cells treated by resveratrol at different time points. IF, the images taken under conventional IF microscope. Confocal, the images taken under confocal laser scanning microscope.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4979504&req=5

fig4: Double LC3 (green) and cytochrome C (red) immunofluorescent labeling of CAOV-3 cells treated by resveratrol at different time points. IF, the images taken under conventional IF microscope. Confocal, the images taken under confocal laser scanning microscope.
Mentions: LC3 is a commonly used marker to monitor autophagosomes and cytochrome C proteins are preferably localized in mitochondria.24 To identify the feature(s) of autophagic bodies, LC3 and cytochrome C double IF staining combined with confocal laser scanning microscope observation were conducted on the OC cells without and with resveratrol treatment. The results demonstrate that the conventional IF image of normally cultured OC cells was weakly green in the cytoplasm and the confocal one showed the predominant distribution of granules labeled with LC3 (green) or cytochrome C (red; Figure 4a). The IF staining patterns were apparently altered in resveratrol-treated cells by showing the color transition of IF images from weakly green to strong orange and yellow at 24 h to green and yellow at 48 h and to yellow and green at 72 h time points. In agreement with the IF microscopic findings, the confocal images demonstrated the appearance of LC3+/cytochrome C+ granules in the cytoplasm of resveratrol-treated cells, especially those treated for 24 h (Figure 4b).

View Article: PubMed Central - PubMed

ABSTRACT

Autophagic activity reflects cellular response to drug treatment and can be regulated by STAT3 signaling. Resveratrol inhibits STAT3 activation and causes remarkable growth arrest and cell death of ovarian cancer (OC) cells. However, the autophagic status and its relevance with resveratrol’s anti-OC effects remain unclear. We analyzed the states of autophagic activities, the nature of autophagosomes and the levels of autophagy-related proteins (LC-3, Beclin 1 and STAT3) in resveratrol-treated CAOV-3 and OVCAR-3 OC cells using multiple approaches. We elucidated the correlation of STAT3 inhibition with autophagic activity by treating OC cells with an upstream inhibitor of STAT proteins, AG490. Resveratrol efficiently suppressed growth, induced apoptosis and inactivated STAT3 signaling of the two OC cell lines. We found enhanced autophagic activity accompanied with Beclin-1 upregulation and LC3 enzymatic cleavage in resveratrol-treated OC cells. Immunofluorescent (IF) microscopic and IF-based confocal examinations demonstrated the accumulation of cytoplasmic granules co-labeled with LC3 and cytochrome C in resveratrol- or AG490-treated OC cells. Using electron microscopy, we confirmed an increase in autophagosomes and mitochondrial spheroids in either resveratrol- or AG490-treated OC cells. This study demonstrates the abilities of resveratrol to enhance apoptotic and autophagic activities in OC cells, presumably via inactivating STAT3 signaling. Resveratrol or the selective JAK2 inhibitor also leads to mitochondrial turnover, which would be unfavorable for OC cell survival and sensitize OC cells to resveratrol.

No MeSH data available.