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Curcumin induces crosstalk between autophagy and apoptosis mediated by calcium release from the endoplasmic reticulum, lysosomal destabilization and mitochondrial events

View Article: PubMed Central - PubMed

ABSTRACT

Curcumin, a major active component of turmeric (Curcuma longa, L.), has anticancer effects. In vitro studies suggest that curcumin inhibits cancer cell growth by activating apoptosis, but the mechanism underlying these effects is still unclear. Here, we investigated the mechanisms leading to apoptosis in curcumin-treated cells. Curcumin induced endoplasmic reticulum stress causing calcium release, with a destabilization of the mitochondrial compartment resulting in apoptosis. These events were also associated with lysosomal membrane permeabilization and of caspase-8 activation, mediated by cathepsins and calpains, leading to Bid cleavage. Truncated tBid disrupts mitochondrial homeostasis and enhance apoptosis. We followed the induction of autophagy, marked by the formation of autophagosomes, by staining with acridine orange in cells exposed curcumin. At this concentration, only the early events of apoptosis (initial mitochondrial destabilization with any other manifestations) were detectable. Western blotting demonstrated the conversion of LC3-I to LC3-II (light chain 3), a marker of active autophagosome formation. We also found that the production of reactive oxygen species and formation of autophagosomes following curcumin treatment was almost completely blocked by N-acetylcystein, the mitochondrial specific antioxidants MitoQ10 and SKQ1, the calcium chelators, EGTA-AM or BAPTA-AM, and the mitochondrial calcium uniporter inhibitor, ruthenium red. Curcumin-induced autophagy failed to rescue all cells and most cells underwent type II cell death following the initial autophagic processes. All together, these data imply a fail-secure mechanism regulated by autophagy in the action of curcumin, suggesting a therapeutic potential for curcumin. Offering a novel and effective strategy for the treatment of malignant cells.

No MeSH data available.


Related in: MedlinePlus

Effect of curcumin on mitochondrial bioenergetics and mitochondrial PTP opening. (a) Trace control. Recordings of ΔΨm (TPP+ electrode measurements; green line) and oxygen consumption (Clark electrode; black line) in purified mouse liver mitochondria. The numbers in black along the trace show nmol O2/min/mg protein and the ΔΨm is reported in mV. TPP+ was added to calibrate the electrode. Measurements were performed in the presence of succinate (1 mM) and rotenone (5 μM) together with a saturating amount of ADP (0.5 mM). The uncoupler, mClCCP (10 μM), was added at the end of the trace to uncouple fully respiration. (b) Effect of 25 μM curcumin on ΔΨm (TPP+ electrode measurements; green line) and oxygen consumption (Clark electrode; black line) in purified mouse liver mitochondria. The experiment was performed in the same conditions as for trace a. (c) Opening of the mitochondrial permeability transition pore in different conditions; comparison between PTP opening with 25 μM calcium, 5 μM ter-butylhydroperoxide or 25 μM curcumin. CsA was used at 5 μM. One representative experiment (n=6) is shown. (d) Opening of the mitochondrial permeability transition pore in response to increasing concentrations of curcumin (from 2.5 to 100 μM).
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fig3: Effect of curcumin on mitochondrial bioenergetics and mitochondrial PTP opening. (a) Trace control. Recordings of ΔΨm (TPP+ electrode measurements; green line) and oxygen consumption (Clark electrode; black line) in purified mouse liver mitochondria. The numbers in black along the trace show nmol O2/min/mg protein and the ΔΨm is reported in mV. TPP+ was added to calibrate the electrode. Measurements were performed in the presence of succinate (1 mM) and rotenone (5 μM) together with a saturating amount of ADP (0.5 mM). The uncoupler, mClCCP (10 μM), was added at the end of the trace to uncouple fully respiration. (b) Effect of 25 μM curcumin on ΔΨm (TPP+ electrode measurements; green line) and oxygen consumption (Clark electrode; black line) in purified mouse liver mitochondria. The experiment was performed in the same conditions as for trace a. (c) Opening of the mitochondrial permeability transition pore in different conditions; comparison between PTP opening with 25 μM calcium, 5 μM ter-butylhydroperoxide or 25 μM curcumin. CsA was used at 5 μM. One representative experiment (n=6) is shown. (d) Opening of the mitochondrial permeability transition pore in response to increasing concentrations of curcumin (from 2.5 to 100 μM).

Mentions: As it has been demonstrated that isolated mitochondria where sensitive to curcumin we tried to test this in our conditions. We incubated a mitochondrial suspension with 25 μM of curcumin. This inhibited state 3 respiration (Figure 3a), uncoupled state 4 respiration and led to a drop in ΔΨm in state 4 (Figure 3b). We tested permeability transition pore (PTP) opening in presence of 25 μM curcumin (Figure 3c). Calcium induces the opening of the PTP in a similar fashion as ter-butylhydroperoxide and pore opening is inhibited by cyclosporin A (Figures 3c and d). The presence of 25 μM curcumin opened the PTP, but the initial mitochondrial shrinkage associated with calcium uptake was not observed and the curve of PTP opening was identical to that in the presence of 5 μM of ter-butylhydroperoxide. The minimal curcumin concentration that caused the PTP to open in isolated mitochondria was ≥2.5 μM (Figure 3d); higher concentrations promoted PTP opening and slightly modified the kinetics of opening (Figure 3d).


Curcumin induces crosstalk between autophagy and apoptosis mediated by calcium release from the endoplasmic reticulum, lysosomal destabilization and mitochondrial events
Effect of curcumin on mitochondrial bioenergetics and mitochondrial PTP opening. (a) Trace control. Recordings of ΔΨm (TPP+ electrode measurements; green line) and oxygen consumption (Clark electrode; black line) in purified mouse liver mitochondria. The numbers in black along the trace show nmol O2/min/mg protein and the ΔΨm is reported in mV. TPP+ was added to calibrate the electrode. Measurements were performed in the presence of succinate (1 mM) and rotenone (5 μM) together with a saturating amount of ADP (0.5 mM). The uncoupler, mClCCP (10 μM), was added at the end of the trace to uncouple fully respiration. (b) Effect of 25 μM curcumin on ΔΨm (TPP+ electrode measurements; green line) and oxygen consumption (Clark electrode; black line) in purified mouse liver mitochondria. The experiment was performed in the same conditions as for trace a. (c) Opening of the mitochondrial permeability transition pore in different conditions; comparison between PTP opening with 25 μM calcium, 5 μM ter-butylhydroperoxide or 25 μM curcumin. CsA was used at 5 μM. One representative experiment (n=6) is shown. (d) Opening of the mitochondrial permeability transition pore in response to increasing concentrations of curcumin (from 2.5 to 100 μM).
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fig3: Effect of curcumin on mitochondrial bioenergetics and mitochondrial PTP opening. (a) Trace control. Recordings of ΔΨm (TPP+ electrode measurements; green line) and oxygen consumption (Clark electrode; black line) in purified mouse liver mitochondria. The numbers in black along the trace show nmol O2/min/mg protein and the ΔΨm is reported in mV. TPP+ was added to calibrate the electrode. Measurements were performed in the presence of succinate (1 mM) and rotenone (5 μM) together with a saturating amount of ADP (0.5 mM). The uncoupler, mClCCP (10 μM), was added at the end of the trace to uncouple fully respiration. (b) Effect of 25 μM curcumin on ΔΨm (TPP+ electrode measurements; green line) and oxygen consumption (Clark electrode; black line) in purified mouse liver mitochondria. The experiment was performed in the same conditions as for trace a. (c) Opening of the mitochondrial permeability transition pore in different conditions; comparison between PTP opening with 25 μM calcium, 5 μM ter-butylhydroperoxide or 25 μM curcumin. CsA was used at 5 μM. One representative experiment (n=6) is shown. (d) Opening of the mitochondrial permeability transition pore in response to increasing concentrations of curcumin (from 2.5 to 100 μM).
Mentions: As it has been demonstrated that isolated mitochondria where sensitive to curcumin we tried to test this in our conditions. We incubated a mitochondrial suspension with 25 μM of curcumin. This inhibited state 3 respiration (Figure 3a), uncoupled state 4 respiration and led to a drop in ΔΨm in state 4 (Figure 3b). We tested permeability transition pore (PTP) opening in presence of 25 μM curcumin (Figure 3c). Calcium induces the opening of the PTP in a similar fashion as ter-butylhydroperoxide and pore opening is inhibited by cyclosporin A (Figures 3c and d). The presence of 25 μM curcumin opened the PTP, but the initial mitochondrial shrinkage associated with calcium uptake was not observed and the curve of PTP opening was identical to that in the presence of 5 μM of ter-butylhydroperoxide. The minimal curcumin concentration that caused the PTP to open in isolated mitochondria was ≥2.5 μM (Figure 3d); higher concentrations promoted PTP opening and slightly modified the kinetics of opening (Figure 3d).

View Article: PubMed Central - PubMed

ABSTRACT

Curcumin, a major active component of turmeric (Curcuma longa, L.), has anticancer effects. In vitro studies suggest that curcumin inhibits cancer cell growth by activating apoptosis, but the mechanism underlying these effects is still unclear. Here, we investigated the mechanisms leading to apoptosis in curcumin-treated cells. Curcumin induced endoplasmic reticulum stress causing calcium release, with a destabilization of the mitochondrial compartment resulting in apoptosis. These events were also associated with lysosomal membrane permeabilization and of caspase-8 activation, mediated by cathepsins and calpains, leading to Bid cleavage. Truncated tBid disrupts mitochondrial homeostasis and enhance apoptosis. We followed the induction of autophagy, marked by the formation of autophagosomes, by staining with acridine orange in cells exposed curcumin. At this concentration, only the early events of apoptosis (initial mitochondrial destabilization with any other manifestations) were detectable. Western blotting demonstrated the conversion of LC3-I to LC3-II (light chain 3), a marker of active autophagosome formation. We also found that the production of reactive oxygen species and formation of autophagosomes following curcumin treatment was almost completely blocked by N-acetylcystein, the mitochondrial specific antioxidants MitoQ10 and SKQ1, the calcium chelators, EGTA-AM or BAPTA-AM, and the mitochondrial calcium uniporter inhibitor, ruthenium red. Curcumin-induced autophagy failed to rescue all cells and most cells underwent type II cell death following the initial autophagic processes. All together, these data imply a fail-secure mechanism regulated by autophagy in the action of curcumin, suggesting a therapeutic potential for curcumin. Offering a novel and effective strategy for the treatment of malignant cells.

No MeSH data available.


Related in: MedlinePlus