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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

Cathepsin and calpain induction by curcumin. (a) Flow cytometry analysis of the cathepsin activity following the incubation of cells with 25 μM curcumin for 24 h in the presence or absence of a cocktail of cathepsin inhibitors. (b) Cathepsin activity in Huh-7 cells treated with various concentrations of curcumin for 24 h. The effects of z-LEHD, an inhibitor of caspase-9, were tested in samples incubated with 25 μM curcumin. (c) Caspase-8 activity in cells treated with 25 μM curcumin and one of several cathepsin inhibitors (pepstatin A, cathepsin G inhibitor and Z-FF-fmk) or pore opening inhibitors (BA and CsA). (d) Curcumin-induced calpain activity in the presence of a calcium chelator, BAPTA-AM, a mitochondrially targeted antioxidant, an inhibitor of capase-9, Z-LEHDfmk, or a calpain inhibitor.
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fig8: Cathepsin and calpain induction by curcumin. (a) Flow cytometry analysis of the cathepsin activity following the incubation of cells with 25 μM curcumin for 24 h in the presence or absence of a cocktail of cathepsin inhibitors. (b) Cathepsin activity in Huh-7 cells treated with various concentrations of curcumin for 24 h. The effects of z-LEHD, an inhibitor of caspase-9, were tested in samples incubated with 25 μM curcumin. (c) Caspase-8 activity in cells treated with 25 μM curcumin and one of several cathepsin inhibitors (pepstatin A, cathepsin G inhibitor and Z-FF-fmk) or pore opening inhibitors (BA and CsA). (d) Curcumin-induced calpain activity in the presence of a calcium chelator, BAPTA-AM, a mitochondrially targeted antioxidant, an inhibitor of capase-9, Z-LEHDfmk, or a calpain inhibitor.

Mentions: Since curcumin is taken up by a subpopulation of lysosomes (Supplementary Figures S1a and b), we examined cathepsin activity by analyzing the cleavage of Rh110-bis-CBZ-Lphe-l-Arg-amide using flow cytometry because lysosomal membrane permeabilization (LMP) is usually associated with cathepsin release (Figure 8a). Cathepsin activity was higher in curcumin-treated Huh-7 cells than in control cells and was inhibited by a cathepsin inhibitor cocktail (Figure 8a). Cathepsin activity increased with increasing curcumin concentrations (Figure 8b). We also tested whether cathepsin activity was affected by inhibition of the mitochondrial pathway. In cells treated with 25 μM curcumin, cathepsin activity was significantly higher in than presence of the caspase-9 inhibitor Z-LEHD-fmk than in control conditions (P<0.05; Figure 8b).


Curcumin induces crosstalk between autophagy and apoptosis mediated by calcium release from the endoplasmic reticulum, lysosomal destabilization and mitochondrial events
Cathepsin and calpain induction by curcumin. (a) Flow cytometry analysis of the cathepsin activity following the incubation of cells with 25 μM curcumin for 24 h in the presence or absence of a cocktail of cathepsin inhibitors. (b) Cathepsin activity in Huh-7 cells treated with various concentrations of curcumin for 24 h. The effects of z-LEHD, an inhibitor of caspase-9, were tested in samples incubated with 25 μM curcumin. (c) Caspase-8 activity in cells treated with 25 μM curcumin and one of several cathepsin inhibitors (pepstatin A, cathepsin G inhibitor and Z-FF-fmk) or pore opening inhibitors (BA and CsA). (d) Curcumin-induced calpain activity in the presence of a calcium chelator, BAPTA-AM, a mitochondrially targeted antioxidant, an inhibitor of capase-9, Z-LEHDfmk, or a calpain inhibitor.
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Related In: Results  -  Collection

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fig8: Cathepsin and calpain induction by curcumin. (a) Flow cytometry analysis of the cathepsin activity following the incubation of cells with 25 μM curcumin for 24 h in the presence or absence of a cocktail of cathepsin inhibitors. (b) Cathepsin activity in Huh-7 cells treated with various concentrations of curcumin for 24 h. The effects of z-LEHD, an inhibitor of caspase-9, were tested in samples incubated with 25 μM curcumin. (c) Caspase-8 activity in cells treated with 25 μM curcumin and one of several cathepsin inhibitors (pepstatin A, cathepsin G inhibitor and Z-FF-fmk) or pore opening inhibitors (BA and CsA). (d) Curcumin-induced calpain activity in the presence of a calcium chelator, BAPTA-AM, a mitochondrially targeted antioxidant, an inhibitor of capase-9, Z-LEHDfmk, or a calpain inhibitor.
Mentions: Since curcumin is taken up by a subpopulation of lysosomes (Supplementary Figures S1a and b), we examined cathepsin activity by analyzing the cleavage of Rh110-bis-CBZ-Lphe-l-Arg-amide using flow cytometry because lysosomal membrane permeabilization (LMP) is usually associated with cathepsin release (Figure 8a). Cathepsin activity was higher in curcumin-treated Huh-7 cells than in control cells and was inhibited by a cathepsin inhibitor cocktail (Figure 8a). Cathepsin activity increased with increasing curcumin concentrations (Figure 8b). We also tested whether cathepsin activity was affected by inhibition of the mitochondrial pathway. In cells treated with 25 μM curcumin, cathepsin activity was significantly higher in than presence of the caspase-9 inhibitor Z-LEHD-fmk than in control conditions (P<0.05; Figure 8b).

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