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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.


Flow cytometry analysis of the events linked to curcumin-induced cell death. (a and b) Flow cytometry analysis of the mitochondrial membrane potential (ΔΨm) and membrane integrity by double staining with 20 nM 3,3’-dihexyloxacarbocyanine iodide (from a 10 μM stock solution in ethanol) and 1 μg/ml PI (from a 1 mg/ml stock solution). (c and d) Percentage of cells producing ROS after treatment with various concentrations of curcumin (0–80 μM). Superoxide anion production (c) as measured by the hydroethidine staining, and hydrogen peroxide production (d) as measured by the dichlorofluorescein fluorescence of the DCFH-DA (dichlorofluorescein diacetate). All measurements were made in presence of PI to discriminate between live and dead cells. (e). A 24 h time course of ΔΨm, calcium content (with Fluo3-AM) and superoxide anion and hydroperoxide production in cells treated with 25 μM of curcumin. (f). A 8-h time course showing the rapid uptake of curcumin followed by an immediate increase in calcium levels. In all figures, when a single experiment is shown as an example, the data are expressed as a percentage of the whole population, whereas in concentration or time scale curves, the data are either expressed as mean fluorescence (in arbitrary units)±S.D. or as a percentage (%)±S.D. with n=9.
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fig2: Flow cytometry analysis of the events linked to curcumin-induced cell death. (a and b) Flow cytometry analysis of the mitochondrial membrane potential (ΔΨm) and membrane integrity by double staining with 20 nM 3,3’-dihexyloxacarbocyanine iodide (from a 10 μM stock solution in ethanol) and 1 μg/ml PI (from a 1 mg/ml stock solution). (c and d) Percentage of cells producing ROS after treatment with various concentrations of curcumin (0–80 μM). Superoxide anion production (c) as measured by the hydroethidine staining, and hydrogen peroxide production (d) as measured by the dichlorofluorescein fluorescence of the DCFH-DA (dichlorofluorescein diacetate). All measurements were made in presence of PI to discriminate between live and dead cells. (e). A 24 h time course of ΔΨm, calcium content (with Fluo3-AM) and superoxide anion and hydroperoxide production in cells treated with 25 μM of curcumin. (f). A 8-h time course showing the rapid uptake of curcumin followed by an immediate increase in calcium levels. In all figures, when a single experiment is shown as an example, the data are expressed as a percentage of the whole population, whereas in concentration or time scale curves, the data are either expressed as mean fluorescence (in arbitrary units)±S.D. or as a percentage (%)±S.D. with n=9.

Mentions: We next measured intracellular curcumin concentration that was much lower than the extracellular curcumin, although the two measures were linearly related (Figure 1b). For external curcumin at 25 μM, the intracellular curcumin is of 1.25 μM (ratio 20/1). Curcumin penetrated the cells rapidly: within 5 min a significant amount of curcumin was found within the cells (at 5 μM; Figure 1a upper left panel). Intracellular curcumin levels were maximal within 10 min and decreased slowly over the following hours (Figure 2f).


Curcumin induces crosstalk between autophagy and apoptosis mediated by calcium release from the endoplasmic reticulum, lysosomal destabilization and mitochondrial events
Flow cytometry analysis of the events linked to curcumin-induced cell death. (a and b) Flow cytometry analysis of the mitochondrial membrane potential (ΔΨm) and membrane integrity by double staining with 20 nM 3,3’-dihexyloxacarbocyanine iodide (from a 10 μM stock solution in ethanol) and 1 μg/ml PI (from a 1 mg/ml stock solution). (c and d) Percentage of cells producing ROS after treatment with various concentrations of curcumin (0–80 μM). Superoxide anion production (c) as measured by the hydroethidine staining, and hydrogen peroxide production (d) as measured by the dichlorofluorescein fluorescence of the DCFH-DA (dichlorofluorescein diacetate). All measurements were made in presence of PI to discriminate between live and dead cells. (e). A 24 h time course of ΔΨm, calcium content (with Fluo3-AM) and superoxide anion and hydroperoxide production in cells treated with 25 μM of curcumin. (f). A 8-h time course showing the rapid uptake of curcumin followed by an immediate increase in calcium levels. In all figures, when a single experiment is shown as an example, the data are expressed as a percentage of the whole population, whereas in concentration or time scale curves, the data are either expressed as mean fluorescence (in arbitrary units)±S.D. or as a percentage (%)±S.D. with n=9.
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Related In: Results  -  Collection

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fig2: Flow cytometry analysis of the events linked to curcumin-induced cell death. (a and b) Flow cytometry analysis of the mitochondrial membrane potential (ΔΨm) and membrane integrity by double staining with 20 nM 3,3’-dihexyloxacarbocyanine iodide (from a 10 μM stock solution in ethanol) and 1 μg/ml PI (from a 1 mg/ml stock solution). (c and d) Percentage of cells producing ROS after treatment with various concentrations of curcumin (0–80 μM). Superoxide anion production (c) as measured by the hydroethidine staining, and hydrogen peroxide production (d) as measured by the dichlorofluorescein fluorescence of the DCFH-DA (dichlorofluorescein diacetate). All measurements were made in presence of PI to discriminate between live and dead cells. (e). A 24 h time course of ΔΨm, calcium content (with Fluo3-AM) and superoxide anion and hydroperoxide production in cells treated with 25 μM of curcumin. (f). A 8-h time course showing the rapid uptake of curcumin followed by an immediate increase in calcium levels. In all figures, when a single experiment is shown as an example, the data are expressed as a percentage of the whole population, whereas in concentration or time scale curves, the data are either expressed as mean fluorescence (in arbitrary units)±S.D. or as a percentage (%)±S.D. with n=9.
Mentions: We next measured intracellular curcumin concentration that was much lower than the extracellular curcumin, although the two measures were linearly related (Figure 1b). For external curcumin at 25 μM, the intracellular curcumin is of 1.25 μM (ratio 20/1). Curcumin penetrated the cells rapidly: within 5 min a significant amount of curcumin was found within the cells (at 5 μM; Figure 1a upper left panel). Intracellular curcumin levels were maximal within 10 min and decreased slowly over the following hours (Figure 2f).

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.