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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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Modulation of cisplatin-induced tumour cell death by AMPK and mTOR. (A) U251 cells transfected with control, AMPK or mTOR siRNA were incubated in the absence or presence of cisplatin (cisPt; 50 μM) and the LDH release was measured after 24 hrs. (B, C) The levels of Bax and Bcl-2 mRNA were determined by real-time RT-PCR in untreated and cisplatin (50 μM)-treated cells (8 hrs) transfected with control or AMPK siRNA (B), and Bax/Bcl-2 mRNA ratio was calculated (C). (D–F) U251 (D), C6 (E) and L929 cells (F) were incubated with cisplatin (50 μM) in the absence or presence of metformin (Met; 1 mM) or compound C (compC; 1 μM) and the LDH release was determined after 24 hrs. (A–D, F) Data are mean + S.D. values of triplicate observations from a representative of three (A) or two (B–F) experiments (#P < 0.05 and *P < 0.05 refer to untreated and cisplatin-treated cells, respectively).
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fig05: Modulation of cisplatin-induced tumour cell death by AMPK and mTOR. (A) U251 cells transfected with control, AMPK or mTOR siRNA were incubated in the absence or presence of cisplatin (cisPt; 50 μM) and the LDH release was measured after 24 hrs. (B, C) The levels of Bax and Bcl-2 mRNA were determined by real-time RT-PCR in untreated and cisplatin (50 μM)-treated cells (8 hrs) transfected with control or AMPK siRNA (B), and Bax/Bcl-2 mRNA ratio was calculated (C). (D–F) U251 (D), C6 (E) and L929 cells (F) were incubated with cisplatin (50 μM) in the absence or presence of metformin (Met; 1 mM) or compound C (compC; 1 μM) and the LDH release was determined after 24 hrs. (A–D, F) Data are mean + S.D. values of triplicate observations from a representative of three (A) or two (B–F) experiments (#P < 0.05 and *P < 0.05 refer to untreated and cisplatin-treated cells, respectively).

Mentions: To further confirm the involvement of AMPK in cisplatin-induced autophagy, we tested whether siRNA-mediated AMPK silencing could mimic the stimulatory effect of autophagy inhibition on cisplatin toxicity. Indeed, siRNA-mediated down-regulation of AMPK activity in U251 cells augmented the cisplatin toxicity, as demonstrated by an increase in cisplatin-induced LDH release in AMPK siRNA-transfected, compared to control cell cultures (Fig. 5A). Accordingly, AMPK silencing also mimicked bafilomycin-mediated increase in Bax/Bcl-2 mRNA ratio in cisplatin-treated U251 cells (Fig. 5B and C). On the other hand, transfection with mTOR siRNA inhibited cisplatin-mediated tumour cell killing (Fig. 5A), thus indicating that mTOR down-regulation might be involved in the cell protection by AMPK. The protective role of AMPK in cisplatin-mediated tumour cell killing was confirmed by using compound C, an AMPK inhibitor, and metformin, an AMPK activator, which respectively enhanced and reduced cisplatin-triggered LDH release in C6, U251 and L929 cell cultures (Fig. 5D–F).


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)

Modulation of cisplatin-induced tumour cell death by AMPK and mTOR. (A) U251 cells transfected with control, AMPK or mTOR siRNA were incubated in the absence or presence of cisplatin (cisPt; 50 μM) and the LDH release was measured after 24 hrs. (B, C) The levels of Bax and Bcl-2 mRNA were determined by real-time RT-PCR in untreated and cisplatin (50 μM)-treated cells (8 hrs) transfected with control or AMPK siRNA (B), and Bax/Bcl-2 mRNA ratio was calculated (C). (D–F) U251 (D), C6 (E) and L929 cells (F) were incubated with cisplatin (50 μM) in the absence or presence of metformin (Met; 1 mM) or compound C (compC; 1 μM) and the LDH release was determined after 24 hrs. (A–D, F) Data are mean + S.D. values of triplicate observations from a representative of three (A) or two (B–F) experiments (#P < 0.05 and *P < 0.05 refer to untreated and cisplatin-treated cells, respectively).
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fig05: Modulation of cisplatin-induced tumour cell death by AMPK and mTOR. (A) U251 cells transfected with control, AMPK or mTOR siRNA were incubated in the absence or presence of cisplatin (cisPt; 50 μM) and the LDH release was measured after 24 hrs. (B, C) The levels of Bax and Bcl-2 mRNA were determined by real-time RT-PCR in untreated and cisplatin (50 μM)-treated cells (8 hrs) transfected with control or AMPK siRNA (B), and Bax/Bcl-2 mRNA ratio was calculated (C). (D–F) U251 (D), C6 (E) and L929 cells (F) were incubated with cisplatin (50 μM) in the absence or presence of metformin (Met; 1 mM) or compound C (compC; 1 μM) and the LDH release was determined after 24 hrs. (A–D, F) Data are mean + S.D. values of triplicate observations from a representative of three (A) or two (B–F) experiments (#P < 0.05 and *P < 0.05 refer to untreated and cisplatin-treated cells, respectively).
Mentions: To further confirm the involvement of AMPK in cisplatin-induced autophagy, we tested whether siRNA-mediated AMPK silencing could mimic the stimulatory effect of autophagy inhibition on cisplatin toxicity. Indeed, siRNA-mediated down-regulation of AMPK activity in U251 cells augmented the cisplatin toxicity, as demonstrated by an increase in cisplatin-induced LDH release in AMPK siRNA-transfected, compared to control cell cultures (Fig. 5A). Accordingly, AMPK silencing also mimicked bafilomycin-mediated increase in Bax/Bcl-2 mRNA ratio in cisplatin-treated U251 cells (Fig. 5B and C). On the other hand, transfection with mTOR siRNA inhibited cisplatin-mediated tumour cell killing (Fig. 5A), thus indicating that mTOR down-regulation might be involved in the cell protection by AMPK. The protective role of AMPK in cisplatin-mediated tumour cell killing was confirmed by using compound C, an AMPK inhibitor, and metformin, an AMPK activator, which respectively enhanced and reduced cisplatin-triggered LDH release in C6, U251 and L929 cell cultures (Fig. 5D–F).

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