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Resveratrol Specifically Kills Cancer Cells by a Devastating Increase in the Ca 2+ Coupling Between the Greatly Tethered Endoplasmic Reticulum and Mitochondria

View Article: PubMed Central - PubMed

ABSTRACT

Background/aims: Resveratrol and its derivate piceatannol are known to induce cancer cell-specific cell death. While multiple mechanisms of actions have been described including the inhibition of ATP synthase, changes in mitochondrial membrane potential and ROS levels, the exact mechanisms of cancer specificity of these polyphenols remain unclear. This paper is designed to reveal the molecular basis of the cancer-specific initiation of cell death by resveratrol and piceatannol.

Methods: The two cancer cell lines EA.hy926 and HeLa, and somatic short-term cultured HUVEC were used. Cell viability and caspase 3/7 activity were tested. Mitochondrial, cytosolic and endoplasmic reticulum Ca2+ as well as cytosolic and mitochondrial ATP levels were measured using single cell fluorescence microscopy and respective genetically-encoded sensors. Mitochondria-ER junctions were analyzed applying super-resolution SIM and ImageJ-based image analysis.

Results: Resveratrol and piceatannol selectively trigger death in cancer but not somatic cells. Hence, these polyphenols strongly enhanced mitochondrial Ca2+ uptake in cancer exclusively. Resveratrol and piceatannol predominantly affect mitochondrial but not cytosolic ATP content that yields in a reduced SERCA activity. Decreased SERCA activity and the strongly enriched tethering of the ER and mitochondria in cancer cells result in an enhanced MCU/Letm1-dependent mitochondrial Ca2+ uptake upon intracellular Ca2+ release exclusively in cancer cells. Accordingly, resveratrol/piceatannol-induced cancer cell death could be prevented by siRNA-mediated knock-down of MCU and Letm1.

Conclusions: Because their greatly enriched ER-mitochondria tethering, cancer cells are highly susceptible for resveratrol/piceatannol-induced reduction of SERCA activity to yield mitochondrial Ca2+ overload and subsequent cancer cell death.

No MeSH data available.


Representative curves (left panels) show cytATP (A) and mtATP (B) ratio signals of HeLa cells, measured by AT1.03 targeted to cytosol or mitochondria, respectively, stimulated with first 100 µM resveratrol (red curves), 100 µM piceatannol (green curves), or 10 µM oligomycin A (blue curves) and afterwards with 2.5 mM 2-DG and 10 µM oligomycin A in order to gain a complete depletion of ATP. Bars (right panels) reveal average ∆ ratio signals to resveratrol (red columns; cytATP: n=40/7, mtATP: n=41/7), piceatannol (green columns; cytATP: n=47/7, mtATP: n=31/7) or oligomycin A (blue columns; cytATP: n=48/6, mtATP: n=34/7), presented as percentage of complete ATP depletion. The cells‘ response to the combination of 2.5 mM 2-DG and 10 µM oligomycin A was set to 100 %.
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Figure 5: Representative curves (left panels) show cytATP (A) and mtATP (B) ratio signals of HeLa cells, measured by AT1.03 targeted to cytosol or mitochondria, respectively, stimulated with first 100 µM resveratrol (red curves), 100 µM piceatannol (green curves), or 10 µM oligomycin A (blue curves) and afterwards with 2.5 mM 2-DG and 10 µM oligomycin A in order to gain a complete depletion of ATP. Bars (right panels) reveal average ∆ ratio signals to resveratrol (red columns; cytATP: n=40/7, mtATP: n=41/7), piceatannol (green columns; cytATP: n=47/7, mtATP: n=31/7) or oligomycin A (blue columns; cytATP: n=48/6, mtATP: n=34/7), presented as percentage of complete ATP depletion. The cells‘ response to the combination of 2.5 mM 2-DG and 10 µM oligomycin A was set to 100 %.

Mentions: Because resveratrol, piceatannol, and oligomycin A showed similiar results in cell viability and mitochondrial Ca2+ uptake experiments, we next compared their effect on mitochondrial and cytosolic/global ATP levels. Measuring cytosolic and mitochondrial ATP by respectively-targeted fluorescence ATP probes [27, 64] revealed for all ATP-synthase inhibitors a small reduction in the cytosolic ATP level of app. 20% of the decrease observed by the combined addition of 2-deoxy-D-glucose and oligomycin A (Fig. 5A). In contrast, resveratrol, piceatannol, and oligomycin A strongly affected mitochondrial ATP levels of more than 50% of the decrease obtained by the addition of 2-deoxy-D-glucose and oligomycin A (Fig. 5B). These data indicate that three compounds, in the concentration used in this study (resveratrol 100 μM, piceatannol 100 μM, oligomycin A 10 μM), exhibited similar inhibitory potential and predominantly affected mitochondrial ATP levels while their effect for lowering global ATP was very small.


Resveratrol Specifically Kills Cancer Cells by a Devastating Increase in the Ca 2+ Coupling Between the Greatly Tethered Endoplasmic Reticulum and Mitochondria
Representative curves (left panels) show cytATP (A) and mtATP (B) ratio signals of HeLa cells, measured by AT1.03 targeted to cytosol or mitochondria, respectively, stimulated with first 100 µM resveratrol (red curves), 100 µM piceatannol (green curves), or 10 µM oligomycin A (blue curves) and afterwards with 2.5 mM 2-DG and 10 µM oligomycin A in order to gain a complete depletion of ATP. Bars (right panels) reveal average ∆ ratio signals to resveratrol (red columns; cytATP: n=40/7, mtATP: n=41/7), piceatannol (green columns; cytATP: n=47/7, mtATP: n=31/7) or oligomycin A (blue columns; cytATP: n=48/6, mtATP: n=34/7), presented as percentage of complete ATP depletion. The cells‘ response to the combination of 2.5 mM 2-DG and 10 µM oligomycin A was set to 100 %.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC5382978&req=5

Figure 5: Representative curves (left panels) show cytATP (A) and mtATP (B) ratio signals of HeLa cells, measured by AT1.03 targeted to cytosol or mitochondria, respectively, stimulated with first 100 µM resveratrol (red curves), 100 µM piceatannol (green curves), or 10 µM oligomycin A (blue curves) and afterwards with 2.5 mM 2-DG and 10 µM oligomycin A in order to gain a complete depletion of ATP. Bars (right panels) reveal average ∆ ratio signals to resveratrol (red columns; cytATP: n=40/7, mtATP: n=41/7), piceatannol (green columns; cytATP: n=47/7, mtATP: n=31/7) or oligomycin A (blue columns; cytATP: n=48/6, mtATP: n=34/7), presented as percentage of complete ATP depletion. The cells‘ response to the combination of 2.5 mM 2-DG and 10 µM oligomycin A was set to 100 %.
Mentions: Because resveratrol, piceatannol, and oligomycin A showed similiar results in cell viability and mitochondrial Ca2+ uptake experiments, we next compared their effect on mitochondrial and cytosolic/global ATP levels. Measuring cytosolic and mitochondrial ATP by respectively-targeted fluorescence ATP probes [27, 64] revealed for all ATP-synthase inhibitors a small reduction in the cytosolic ATP level of app. 20% of the decrease observed by the combined addition of 2-deoxy-D-glucose and oligomycin A (Fig. 5A). In contrast, resveratrol, piceatannol, and oligomycin A strongly affected mitochondrial ATP levels of more than 50% of the decrease obtained by the addition of 2-deoxy-D-glucose and oligomycin A (Fig. 5B). These data indicate that three compounds, in the concentration used in this study (resveratrol 100 μM, piceatannol 100 μM, oligomycin A 10 μM), exhibited similar inhibitory potential and predominantly affected mitochondrial ATP levels while their effect for lowering global ATP was very small.

View Article: PubMed Central - PubMed

ABSTRACT

Background/aims: Resveratrol and its derivate piceatannol are known to induce cancer cell-specific cell death. While multiple mechanisms of actions have been described including the inhibition of ATP synthase, changes in mitochondrial membrane potential and ROS levels, the exact mechanisms of cancer specificity of these polyphenols remain unclear. This paper is designed to reveal the molecular basis of the cancer-specific initiation of cell death by resveratrol and piceatannol.

Methods: The two cancer cell lines EA.hy926 and HeLa, and somatic short-term cultured HUVEC were used. Cell viability and caspase 3/7 activity were tested. Mitochondrial, cytosolic and endoplasmic reticulum Ca2+ as well as cytosolic and mitochondrial ATP levels were measured using single cell fluorescence microscopy and respective genetically-encoded sensors. Mitochondria-ER junctions were analyzed applying super-resolution SIM and ImageJ-based image analysis.

Results: Resveratrol and piceatannol selectively trigger death in cancer but not somatic cells. Hence, these polyphenols strongly enhanced mitochondrial Ca2+ uptake in cancer exclusively. Resveratrol and piceatannol predominantly affect mitochondrial but not cytosolic ATP content that yields in a reduced SERCA activity. Decreased SERCA activity and the strongly enriched tethering of the ER and mitochondria in cancer cells result in an enhanced MCU/Letm1-dependent mitochondrial Ca2+ uptake upon intracellular Ca2+ release exclusively in cancer cells. Accordingly, resveratrol/piceatannol-induced cancer cell death could be prevented by siRNA-mediated knock-down of MCU and Letm1.

Conclusions: Because their greatly enriched ER-mitochondria tethering, cancer cells are highly susceptible for resveratrol/piceatannol-induced reduction of SERCA activity to yield mitochondrial Ca2+ overload and subsequent cancer cell death.

No MeSH data available.