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AMPK-mediated autophagy inhibits apoptosis in cisplatin-treated tumour cells.

Harhaji-Trajkovic L, Vilimanovich U, Kravic-Stevovic T, Bumbasirevic V, Trajkovic V - J. Cell. Mol. Med. (2009)

Bottom Line: The mechanisms underlying the protective effect of autophagy apparently involved the interference with cisplatin-induced modulation of Bcl-2 family proteins, as inhibition of autophagy potentiated cisplatin-mediated up-regulation of proapoptotic Bax and down-regulation of anti-apoptotic Bcl-2.The ability of cisplatin to trigger autophagy was reduced by small interfering RNA (siRNA)-mediated AMPK silencing, while transfection with mTOR siRNA was sufficient to trigger autophagy in tumour cells.Taken together, these data suggest that cisplatin-triggered activation of AMPK and subsequent suppression of mTOR activity can induce an autophagic response that protects tumour cells from cisplatin-mediated apoptotic death.

View Article: PubMed Central - PubMed

Affiliation: Institute for Biological Research, Belgrade, Serbia. vtrajkovic@eunet.rs

ABSTRACT
The role of autophagy in cisplatin anticancer action was investigated using human U251 glioma, rat C6 glioma and mouse L929 fibrosarcoma cell lines. A dose- and time-dependent induction of autophagy was observed in tumour cells following cisplatin treatment, as demonstrated by up-regulation of autophagy-inducing protein beclin-1 and subsequent appearance of acridine orange-stained acidic autophagic vesicles. The presence of autophagosomes in cisplatin-treated cells was also confirmed by electron microscopy. Inhibition of autophagy with lysosomal inhibitors bafilomycin A1 and chloroquine, or a PI3 kinase inhibitor wortmannin, markedly augmented cisplatin-triggered oxidative stress and caspase activation, leading to an increase in DNA fragmentation and apoptotic cell death. The mechanisms underlying the protective effect of autophagy apparently involved the interference with cisplatin-induced modulation of Bcl-2 family proteins, as inhibition of autophagy potentiated cisplatin-mediated up-regulation of proapoptotic Bax and down-regulation of anti-apoptotic Bcl-2. Autophagy induction in cisplatin-treated cells was preceded by activation of adenosine monophosphate-activated protein kinase (AMPK) and concomitant down-regulation of mammalian target of rapamycin (mTOR)-mediated phosphorylation of p70S6 kinase. The ability of cisplatin to trigger autophagy was reduced by small interfering RNA (siRNA)-mediated AMPK silencing, while transfection with mTOR siRNA was sufficient to trigger autophagy in tumour cells. Finally, siRNA-mediated AMPK down-regulation and AMPK inhibitor compound C increased cisplatin-induced tumour cell death, while mTOR siRNA and AMPK activator metformin protected tumour cells from cisplatin. Taken together, these data suggest that cisplatin-triggered activation of AMPK and subsequent suppression of mTOR activity can induce an autophagic response that protects tumour cells from cisplatin-mediated apoptotic death.

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The effects of autophagy inhibitors on cisplatin-induced apoptotic cell death. U251 cells (A–E), C6 or L929 cells (A) were incubated with cisplatin (cisPt; 50 μM) in the absence or presence of bafilomycin (Baf; 100 ng/ml), wortmannin (Wm; 100 nm) or chloroquine (Cq; 20 μM). LDH release (A), MTT reduction (B) and DNA fragmentation (E) were determined after 24 hrs, while ROS production (C) and caspase activation (D) were assessed after 16 hrs. (A, B) Data are mean + S.D. values of triplicates from a representative of three experiments. (C, D, E) Representative flow cytometry histograms are presented, while the accompanying graphs contain mean + S.D. values from three different experiments (#P < 0.05 and *P < 0.05 refer to untreated and cisplatin-treated cells, respectively).
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fig02: The effects of autophagy inhibitors on cisplatin-induced apoptotic cell death. U251 cells (A–E), C6 or L929 cells (A) were incubated with cisplatin (cisPt; 50 μM) in the absence or presence of bafilomycin (Baf; 100 ng/ml), wortmannin (Wm; 100 nm) or chloroquine (Cq; 20 μM). LDH release (A), MTT reduction (B) and DNA fragmentation (E) were determined after 24 hrs, while ROS production (C) and caspase activation (D) were assessed after 16 hrs. (A, B) Data are mean + S.D. values of triplicates from a representative of three experiments. (C, D, E) Representative flow cytometry histograms are presented, while the accompanying graphs contain mean + S.D. values from three different experiments (#P < 0.05 and *P < 0.05 refer to untreated and cisplatin-treated cells, respectively).

Mentions: To assess the role of autophagy in cisplatin-induced death of tumour cells, we tested if cisplatin toxicity could be modulated with different autophagy-blocking agents – lysosomal inhibitors bafilomycin and chloroquine, and the PI3 class III inhibitor wortmannin. Each autophagy blocker significantly increased LDH release when administered simultaneously with cisplatin (Fig. 2A), without affecting LDH release in untreated cells (data not shown). These results were confirmed using MTT assay, which demonstrated that cisplatin-mediated decrease in mitochondrial dehydrogenase activity of U251 cells was potentiated by autophagy inhibitors (Fig. 2B). None of the autophagy inhibitors exerted a significant effect on tumour cell mitochondrial respiration in the absence of cisplatin (Fig. 2B; similar results were obtained with C6 and L929 cells – not shown). To get some insight into the mechanisms underlying the protective effect of autophagy, we assessed the effects of autophagy inhibitors on oxidative stress, caspase activation and DNA fragmentation, the hallmarks of apoptotic cell death. Expectedly, cisplatin treatment caused an increase in ROS production (Fig. 2C), caspase activation (Fig. 2D) and DNA fragmentation (Fig. 2E) in U251 glioma cells, as demonstrated by flow cytometric analysis of the cells stained with the appropriate reporter fluorochromes that react with ROS (DHR), activated caspases (ApoStat) or DNA (PI). Each of the three autophagy inhibitors markedly increased cisplatin-induced oxidative stress, caspase activation and DNA fragmentation (Fig. 3C–E), thus further corroborating that autophagy was involved in tumour cell protection from cisplatin-induced apoptosis. Similar results were obtained with C6 glioma and L929 fibrosarcoma cells (data not shown).


AMPK-mediated autophagy inhibits apoptosis in cisplatin-treated tumour cells.

Harhaji-Trajkovic L, Vilimanovich U, Kravic-Stevovic T, Bumbasirevic V, Trajkovic V - J. Cell. Mol. Med. (2009)

The effects of autophagy inhibitors on cisplatin-induced apoptotic cell death. U251 cells (A–E), C6 or L929 cells (A) were incubated with cisplatin (cisPt; 50 μM) in the absence or presence of bafilomycin (Baf; 100 ng/ml), wortmannin (Wm; 100 nm) or chloroquine (Cq; 20 μM). LDH release (A), MTT reduction (B) and DNA fragmentation (E) were determined after 24 hrs, while ROS production (C) and caspase activation (D) were assessed after 16 hrs. (A, B) Data are mean + S.D. values of triplicates from a representative of three experiments. (C, D, E) Representative flow cytometry histograms are presented, while the accompanying graphs contain mean + S.D. values from three different experiments (#P < 0.05 and *P < 0.05 refer to untreated and cisplatin-treated cells, respectively).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4516513&req=5

fig02: The effects of autophagy inhibitors on cisplatin-induced apoptotic cell death. U251 cells (A–E), C6 or L929 cells (A) were incubated with cisplatin (cisPt; 50 μM) in the absence or presence of bafilomycin (Baf; 100 ng/ml), wortmannin (Wm; 100 nm) or chloroquine (Cq; 20 μM). LDH release (A), MTT reduction (B) and DNA fragmentation (E) were determined after 24 hrs, while ROS production (C) and caspase activation (D) were assessed after 16 hrs. (A, B) Data are mean + S.D. values of triplicates from a representative of three experiments. (C, D, E) Representative flow cytometry histograms are presented, while the accompanying graphs contain mean + S.D. values from three different experiments (#P < 0.05 and *P < 0.05 refer to untreated and cisplatin-treated cells, respectively).
Mentions: To assess the role of autophagy in cisplatin-induced death of tumour cells, we tested if cisplatin toxicity could be modulated with different autophagy-blocking agents – lysosomal inhibitors bafilomycin and chloroquine, and the PI3 class III inhibitor wortmannin. Each autophagy blocker significantly increased LDH release when administered simultaneously with cisplatin (Fig. 2A), without affecting LDH release in untreated cells (data not shown). These results were confirmed using MTT assay, which demonstrated that cisplatin-mediated decrease in mitochondrial dehydrogenase activity of U251 cells was potentiated by autophagy inhibitors (Fig. 2B). None of the autophagy inhibitors exerted a significant effect on tumour cell mitochondrial respiration in the absence of cisplatin (Fig. 2B; similar results were obtained with C6 and L929 cells – not shown). To get some insight into the mechanisms underlying the protective effect of autophagy, we assessed the effects of autophagy inhibitors on oxidative stress, caspase activation and DNA fragmentation, the hallmarks of apoptotic cell death. Expectedly, cisplatin treatment caused an increase in ROS production (Fig. 2C), caspase activation (Fig. 2D) and DNA fragmentation (Fig. 2E) in U251 glioma cells, as demonstrated by flow cytometric analysis of the cells stained with the appropriate reporter fluorochromes that react with ROS (DHR), activated caspases (ApoStat) or DNA (PI). Each of the three autophagy inhibitors markedly increased cisplatin-induced oxidative stress, caspase activation and DNA fragmentation (Fig. 3C–E), thus further corroborating that autophagy was involved in tumour cell protection from cisplatin-induced apoptosis. Similar results were obtained with C6 glioma and L929 fibrosarcoma cells (data not shown).

Bottom Line: The mechanisms underlying the protective effect of autophagy apparently involved the interference with cisplatin-induced modulation of Bcl-2 family proteins, as inhibition of autophagy potentiated cisplatin-mediated up-regulation of proapoptotic Bax and down-regulation of anti-apoptotic Bcl-2.The ability of cisplatin to trigger autophagy was reduced by small interfering RNA (siRNA)-mediated AMPK silencing, while transfection with mTOR siRNA was sufficient to trigger autophagy in tumour cells.Taken together, these data suggest that cisplatin-triggered activation of AMPK and subsequent suppression of mTOR activity can induce an autophagic response that protects tumour cells from cisplatin-mediated apoptotic death.

View Article: PubMed Central - PubMed

Affiliation: Institute for Biological Research, Belgrade, Serbia. vtrajkovic@eunet.rs

ABSTRACT
The role of autophagy in cisplatin anticancer action was investigated using human U251 glioma, rat C6 glioma and mouse L929 fibrosarcoma cell lines. A dose- and time-dependent induction of autophagy was observed in tumour cells following cisplatin treatment, as demonstrated by up-regulation of autophagy-inducing protein beclin-1 and subsequent appearance of acridine orange-stained acidic autophagic vesicles. The presence of autophagosomes in cisplatin-treated cells was also confirmed by electron microscopy. Inhibition of autophagy with lysosomal inhibitors bafilomycin A1 and chloroquine, or a PI3 kinase inhibitor wortmannin, markedly augmented cisplatin-triggered oxidative stress and caspase activation, leading to an increase in DNA fragmentation and apoptotic cell death. The mechanisms underlying the protective effect of autophagy apparently involved the interference with cisplatin-induced modulation of Bcl-2 family proteins, as inhibition of autophagy potentiated cisplatin-mediated up-regulation of proapoptotic Bax and down-regulation of anti-apoptotic Bcl-2. Autophagy induction in cisplatin-treated cells was preceded by activation of adenosine monophosphate-activated protein kinase (AMPK) and concomitant down-regulation of mammalian target of rapamycin (mTOR)-mediated phosphorylation of p70S6 kinase. The ability of cisplatin to trigger autophagy was reduced by small interfering RNA (siRNA)-mediated AMPK silencing, while transfection with mTOR siRNA was sufficient to trigger autophagy in tumour cells. Finally, siRNA-mediated AMPK down-regulation and AMPK inhibitor compound C increased cisplatin-induced tumour cell death, while mTOR siRNA and AMPK activator metformin protected tumour cells from cisplatin. Taken together, these data suggest that cisplatin-triggered activation of AMPK and subsequent suppression of mTOR activity can induce an autophagic response that protects tumour cells from cisplatin-mediated apoptotic death.

Show MeSH
Related in: MedlinePlus