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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

Confocal microscopy analysis of curcumin autofluorescence and mitochondrial staining with the cationic and lipophilic dye deep red. (A–C) The mitochondrial compartment (A) was stained with 40 nM deep red which fluoresces at 633 nm, whereas curcumin (B) fluoresces at 530±30 nm after excitation at 488 nm. (A and B) Panels (10 μM scale) show enlarged portions of A and B, respectively. Curcumin spots (b) in green are surrounded by lines, which are clearly empty in (a) which shows the deep red staining of the mitochondrial compartment. (C) A plot of fluorescence along the yellow line shown in A and B consisting of 413 pixels. The main peaks corresponding to deep red staining do not co-localize with those corresponding to curcumin fluorescence. In D, the ER compartment was stained with ER tracker red and the picture was taken at 585±20 nm after excitation at 488 nm. (E) Curcumin fluorescence was measured at 530±30 nm after excitation at 488 nm. (F) A plot of fluorescence along the yellow line of 596 pixels shown in the picture; curcumin fluorescence is shown in black and ER-red tracker in red.
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fig5: Confocal microscopy analysis of curcumin autofluorescence and mitochondrial staining with the cationic and lipophilic dye deep red. (A–C) The mitochondrial compartment (A) was stained with 40 nM deep red which fluoresces at 633 nm, whereas curcumin (B) fluoresces at 530±30 nm after excitation at 488 nm. (A and B) Panels (10 μM scale) show enlarged portions of A and B, respectively. Curcumin spots (b) in green are surrounded by lines, which are clearly empty in (a) which shows the deep red staining of the mitochondrial compartment. (C) A plot of fluorescence along the yellow line shown in A and B consisting of 413 pixels. The main peaks corresponding to deep red staining do not co-localize with those corresponding to curcumin fluorescence. In D, the ER compartment was stained with ER tracker red and the picture was taken at 585±20 nm after excitation at 488 nm. (E) Curcumin fluorescence was measured at 530±30 nm after excitation at 488 nm. (F) A plot of fluorescence along the yellow line of 596 pixels shown in the picture; curcumin fluorescence is shown in black and ER-red tracker in red.

Mentions: We therefore used confocal microscopy and the mitochondrial dye, deep red, to analyze simultaneously curcumin fluorescence and the mitochondrial compartment (Figures 5A–D). The mitochondrial network was clearly visible around nuclei and within the cytoplasm (Figure 5A), and this analysis confirmed the punctated pattern of curcumin fluorescence (Figure 5B). However, the curcumin signal did not colocalize with the mitochondria: curcumin fluorescent spots were close to, but did not overlap with, mitochondria (Figure 5C). Transected images confirmed that the distribution of curcumin and mitochondria was different (Figure 5C).


Curcumin induces crosstalk between autophagy and apoptosis mediated by calcium release from the endoplasmic reticulum, lysosomal destabilization and mitochondrial events
Confocal microscopy analysis of curcumin autofluorescence and mitochondrial staining with the cationic and lipophilic dye deep red. (A–C) The mitochondrial compartment (A) was stained with 40 nM deep red which fluoresces at 633 nm, whereas curcumin (B) fluoresces at 530±30 nm after excitation at 488 nm. (A and B) Panels (10 μM scale) show enlarged portions of A and B, respectively. Curcumin spots (b) in green are surrounded by lines, which are clearly empty in (a) which shows the deep red staining of the mitochondrial compartment. (C) A plot of fluorescence along the yellow line shown in A and B consisting of 413 pixels. The main peaks corresponding to deep red staining do not co-localize with those corresponding to curcumin fluorescence. In D, the ER compartment was stained with ER tracker red and the picture was taken at 585±20 nm after excitation at 488 nm. (E) Curcumin fluorescence was measured at 530±30 nm after excitation at 488 nm. (F) A plot of fluorescence along the yellow line of 596 pixels shown in the picture; curcumin fluorescence is shown in black and ER-red tracker in red.
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fig5: Confocal microscopy analysis of curcumin autofluorescence and mitochondrial staining with the cationic and lipophilic dye deep red. (A–C) The mitochondrial compartment (A) was stained with 40 nM deep red which fluoresces at 633 nm, whereas curcumin (B) fluoresces at 530±30 nm after excitation at 488 nm. (A and B) Panels (10 μM scale) show enlarged portions of A and B, respectively. Curcumin spots (b) in green are surrounded by lines, which are clearly empty in (a) which shows the deep red staining of the mitochondrial compartment. (C) A plot of fluorescence along the yellow line shown in A and B consisting of 413 pixels. The main peaks corresponding to deep red staining do not co-localize with those corresponding to curcumin fluorescence. In D, the ER compartment was stained with ER tracker red and the picture was taken at 585±20 nm after excitation at 488 nm. (E) Curcumin fluorescence was measured at 530±30 nm after excitation at 488 nm. (F) A plot of fluorescence along the yellow line of 596 pixels shown in the picture; curcumin fluorescence is shown in black and ER-red tracker in red.
Mentions: We therefore used confocal microscopy and the mitochondrial dye, deep red, to analyze simultaneously curcumin fluorescence and the mitochondrial compartment (Figures 5A–D). The mitochondrial network was clearly visible around nuclei and within the cytoplasm (Figure 5A), and this analysis confirmed the punctated pattern of curcumin fluorescence (Figure 5B). However, the curcumin signal did not colocalize with the mitochondria: curcumin fluorescent spots were close to, but did not overlap with, mitochondria (Figure 5C). Transected images confirmed that the distribution of curcumin and mitochondria was different (Figure 5C).

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