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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.


Related in: MedlinePlus

Representative curves (left panels) reflect mtCa2+ ratio signals upon 100 µM histamine (His) addition in Ca2+-free solution of HeLa cells with transient knockdown of UCP2/3 (A), Letm1 (B) or MCU (C) after treatment with 100 µM resveratrol (red curves), 100 µM piceatannol (green curves) or 10 µM oligomycin A (blue curves) as well as under control conditions with corresponding knockdown (black curves) or without knockdown (dotted lines). Bars (right panels) represent an average of maximal ∆ ratio signals of IP3 agonist stimulated response of cells without knockdown (dotted columns; n=71/13), under corresponding control conditions (white columns; siUCP2/3: n=47/12, siMCU: n =46/8, siLetm1: n=28/9) or after treatment with resveratrol (red columns; siUCP2/3: n=31/8, siMCU: n=38/9, siLetm1: n=30/8), piceatannol (green columns; siUCP2/3: n=20/6, siMCU: n=35/6, siLetm1: n=22/6) or oligomycin A (blue columns; siUCP2/3: n=21/7, siMCU: n=27/6, siLetm1: n=23/6).
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Figure 7: Representative curves (left panels) reflect mtCa2+ ratio signals upon 100 µM histamine (His) addition in Ca2+-free solution of HeLa cells with transient knockdown of UCP2/3 (A), Letm1 (B) or MCU (C) after treatment with 100 µM resveratrol (red curves), 100 µM piceatannol (green curves) or 10 µM oligomycin A (blue curves) as well as under control conditions with corresponding knockdown (black curves) or without knockdown (dotted lines). Bars (right panels) represent an average of maximal ∆ ratio signals of IP3 agonist stimulated response of cells without knockdown (dotted columns; n=71/13), under corresponding control conditions (white columns; siUCP2/3: n=47/12, siMCU: n =46/8, siLetm1: n=28/9) or after treatment with resveratrol (red columns; siUCP2/3: n=31/8, siMCU: n=38/9, siLetm1: n=30/8), piceatannol (green columns; siUCP2/3: n=20/6, siMCU: n=35/6, siLetm1: n=22/6) or oligomycin A (blue columns; siUCP2/3: n=21/7, siMCU: n=27/6, siLetm1: n=23/6).

Mentions: In HeLa cells, mitochondrial Ca2+ uptake is sensitive to a knock-down of MCU and UCP2/3 but not Letm1 (Fig. 7A). An inhibition of SERCA is known to impact mitochondrial Ca2+ uptake and to shift mitochondrial Ca2+ uptake route to become UCP2/3-independent but Letm1-dependent [66]. Accordingly, we next tested whether the redirection of the mitochondrial Ca2+ uptake route also occurs by incubation with resveratrol, piceatannol, and oligomycin A as consequence of their inhibition of SERCA due to the inhibition of ATP-synthase by these compounds. In line with the reported effect of thapsigargin, all three ATP-synthase inhibitors abolished the contribution of UCP2/3 to mitochondrial Ca2+ uptake of intracellularely released Ca2+ (Fig. 7B). Hence, under condition of an inhibition of the altered mitochondrial Ca2+ uptake route by a depletion of Letm1 with respective siRNA resveratrol, piceatannol, and oligomycin A failed to enhance mitochondrial Ca2+ sequestration (Fig. 7C) that entirely depended on the core mitochondrial Ca2+ uniporter protein MCU under all conditions (Fig. 7D). These data indicate that thapsigargin as well as the ATP-synthase inhibitors resveratrol, piceatannol, and oligomycin A shift the MCU-established mitochondrial Ca2+ uptake from UCP2-dependent to an UCP2/3-independent but Letm1-dependent route.


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) reflect mtCa2+ ratio signals upon 100 µM histamine (His) addition in Ca2+-free solution of HeLa cells with transient knockdown of UCP2/3 (A), Letm1 (B) or MCU (C) after treatment with 100 µM resveratrol (red curves), 100 µM piceatannol (green curves) or 10 µM oligomycin A (blue curves) as well as under control conditions with corresponding knockdown (black curves) or without knockdown (dotted lines). Bars (right panels) represent an average of maximal ∆ ratio signals of IP3 agonist stimulated response of cells without knockdown (dotted columns; n=71/13), under corresponding control conditions (white columns; siUCP2/3: n=47/12, siMCU: n =46/8, siLetm1: n=28/9) or after treatment with resveratrol (red columns; siUCP2/3: n=31/8, siMCU: n=38/9, siLetm1: n=30/8), piceatannol (green columns; siUCP2/3: n=20/6, siMCU: n=35/6, siLetm1: n=22/6) or oligomycin A (blue columns; siUCP2/3: n=21/7, siMCU: n=27/6, siLetm1: n=23/6).
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Figure 7: Representative curves (left panels) reflect mtCa2+ ratio signals upon 100 µM histamine (His) addition in Ca2+-free solution of HeLa cells with transient knockdown of UCP2/3 (A), Letm1 (B) or MCU (C) after treatment with 100 µM resveratrol (red curves), 100 µM piceatannol (green curves) or 10 µM oligomycin A (blue curves) as well as under control conditions with corresponding knockdown (black curves) or without knockdown (dotted lines). Bars (right panels) represent an average of maximal ∆ ratio signals of IP3 agonist stimulated response of cells without knockdown (dotted columns; n=71/13), under corresponding control conditions (white columns; siUCP2/3: n=47/12, siMCU: n =46/8, siLetm1: n=28/9) or after treatment with resveratrol (red columns; siUCP2/3: n=31/8, siMCU: n=38/9, siLetm1: n=30/8), piceatannol (green columns; siUCP2/3: n=20/6, siMCU: n=35/6, siLetm1: n=22/6) or oligomycin A (blue columns; siUCP2/3: n=21/7, siMCU: n=27/6, siLetm1: n=23/6).
Mentions: In HeLa cells, mitochondrial Ca2+ uptake is sensitive to a knock-down of MCU and UCP2/3 but not Letm1 (Fig. 7A). An inhibition of SERCA is known to impact mitochondrial Ca2+ uptake and to shift mitochondrial Ca2+ uptake route to become UCP2/3-independent but Letm1-dependent [66]. Accordingly, we next tested whether the redirection of the mitochondrial Ca2+ uptake route also occurs by incubation with resveratrol, piceatannol, and oligomycin A as consequence of their inhibition of SERCA due to the inhibition of ATP-synthase by these compounds. In line with the reported effect of thapsigargin, all three ATP-synthase inhibitors abolished the contribution of UCP2/3 to mitochondrial Ca2+ uptake of intracellularely released Ca2+ (Fig. 7B). Hence, under condition of an inhibition of the altered mitochondrial Ca2+ uptake route by a depletion of Letm1 with respective siRNA resveratrol, piceatannol, and oligomycin A failed to enhance mitochondrial Ca2+ sequestration (Fig. 7C) that entirely depended on the core mitochondrial Ca2+ uniporter protein MCU under all conditions (Fig. 7D). These data indicate that thapsigargin as well as the ATP-synthase inhibitors resveratrol, piceatannol, and oligomycin A shift the MCU-established mitochondrial Ca2+ uptake from UCP2-dependent to an UCP2/3-independent but Letm1-dependent route.

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.


Related in: MedlinePlus