A rapid method for the evaluation of compounds with mitochondria-protective properties

Nuydens, Rony; Novalbos, Jesús; Dispersyn, Gwenda; Weber, Claudia; Borgers, M.; Geerts, Hugo

doi:10.1016/s0165-0270(99)00107-7

Cited by 55 publications

(31 citation statements)

References 11 publications

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“…[15][16][17]22] The direct relationship between mitochondrial respiratory metabolism and cell physiology has been largely employed in studies of cell damage caused by toxic agents. [16,18,19,26,27] Pentoxifylline, a nonspecific phosphodiesterase inhibitor, has been shown to improve tissue oxygenation and endothelial function and inhibit proinflammatory cytokine production.…”

Section: Discussionmentioning

confidence: 99%

Pentoxifylline in Ischemia-Induced Acute Kidney Injury in Rats

2009

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Ischemia is an important cause of acute kidney injury (AKI). Pentoxifylline has been shown to improve tissue oxygenation and endothelial function and inhibit proinflammatory cytokine production. The aim of this study was to evaluate a possible renal protective effect of pentoxifylline against ischemia by measuring mitochondrial respiratory metabolism as an index of cell damage. Rats were submitted to right nephrectomy. The left kidney was submitted to ischemia by clamping the renal artery for 45 minutes. Immediately after release of the clamp, 1 mL of a solution containing 20 mg of pentoxifylline/mL was injected intravenously, while a control group received 1 mL of normal saline intravenously. Five minutes after the injection, the left kidney was removed, homogenized, and subjected to refrigerated differential centrifugation. Mitochondrial respiratory metabolism was measured polarographically. The mitochondria isolated from the kidneys of saline-treated rats had an endogenous respiration of 9.20 ± 1.0 hmol O 2 /mg protein/min compared to 8.9 ± 1.4 hmol O 2 /mg protein/min in the pentoxifylline-treated rats (p > 0.05). When stimulated by sodium succinate, the respiratory metabolism increased in a similar fashion in both groups of animals: 17.9 ± 2.3 and 18.1 ± 2.1hmol O 2 /mg protein/min in the untreated and pentoxifylline-treated groups, respectively (p > 0.05). In the present study, pentoxifylline was not found to exert any protective effect on the kidney. It is possible that at the time of pentoxifylline administration, the mitochondria had already been damaged by the process of ischemia, and its effect may have been insufficient to reverse cell damage.

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Section: Discussionmentioning

confidence: 99%

Pentoxifylline in Ischemia-Induced Acute Kidney Injury in Rats

2009

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“…Measurement of Mitochondrial Membrane Potential-Mitochondrial membrane potential (⌬⌿ m ) was measured by the incorporation of cationic fluorescent dye JC-1 (5,5Ј,6,6Ј-tetracholoro-1,1Ј,3,3Ј-tetraethyl benzimidazolylcarbo cyanine iodide) into the mitochondria as described (22). MCF-7 cells were transfected with mCherry or RGS6L-mCherry and grown for 36 h. Cells were harvested as above for ROS studies and incubated with shaking for 30 min at 37°C in PBS containing JC-1 (2.5 g/ml).…”

Section: Measurement Of Intracellular Ros Levels-intracellularmentioning

confidence: 99%

Regulator of G Protein Signaling 6 (RGS6) Induces Apoptosis via a Mitochondrial-dependent Pathway Not Involving Its GTPase-activating Protein Activity

Maity¹,

Yang²,

Huang³

et al. 2011

Journal of Biological Chemistry

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Regulator of G protein signaling 6 (RGS6) is a member of a family of proteins called RGS proteins, which function as GTPase-activating proteins (GAPs) for G␣ subunits. Given the role of RGS6 as a G protein GAP, the link between G protein activation and cancer, and a reduction of cancer risk in humans expressing a RGS6 SNP leading to its increased translation, we hypothesized that RGS6 might function to inhibit growth of cancer cells. Here, we show a marked down-regulation of RGS6 in human mammary ductal epithelial cells that correlates with the progression of their transformation. RGS6 exhibited impressive antiproliferative actions in breast cancer cells, including inhibition of cell growth and colony formation and induction of cell cycle arrest and apoptosis by mechanisms independent of p53. RGS6 activated the intrinsic pathway of apoptosis involving regulation of Bax/Bcl-2, mitochondrial outer membrane permeabilization (MOMP), cytochrome c release, activation of caspases-3 and -9, and poly(ADP-ribose) polymerase cleavage. RGS6 promoted loss of mitochondrial membrane potential (⌬⌿ m ) and increases in reactive oxygen species (ROS). RGS6-induced caspase activation and loss of ⌬⌿ m was mediated by ROS, suggesting an amplification loop in which ROS provided a feed forward signal to induce MOMP, caspase activation, and cell death. Loss of RGS6 in mouse embryonic fibroblasts dramatically impaired doxorubicin-induced growth suppression and apoptosis. Surprisingly, RGS6-induced apoptosis in both breast cancer cells and mouse embryonic fibroblasts does not require its GAP activity toward G proteins. This work demonstrates a novel signaling action of RGS6 in cell death pathways and identifies it as a possible therapeutic target for treatment of breast cancer. Regulator of G protein signaling (RGS)2 proteins comprise a family of proteins, defined by the presence of a semiconserved region called the RGS domain, which negatively regulate heterotrimeric G protein signaling (1-3). These proteins were discovered as essential negative regulators of heterotrimeric G protein signaling by genetic studies in yeast followed by similar studies in Caenorhabditis elegans (4 -6). RGS proteins function as GTPase-activating proteins (GAPs) for heterotrimeric G protein subunits (7,8), an activity bestowed by their RGS domain (9) thereby enhancing the shut-off mechanism for G protein signaling. Thirty mammalian genes encode proteins with the hallmark RGS domain or less closely related versions of this domain, and RGS proteins have been classified into subfamilies based upon sequence similarities within the RGS domain that extend to sequences outside of this domain (2, 8). RGS6 is a member of the R7 subfamily of proteins that possess, in addition to their RGS domain, a GGL (G ␥ subunit-like) domain that allows for association with G␤ 5 and stabilization of RGS6 and a Drosophila/EGL10/Pleckstrin homology/DEP helical extension (DEP/DHEX) domain that allows for association with one of two additional proteins, R7BP or R9AP, and is thought to pr...

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“…[18][19][20] JC-1 can accumulate and aggregate in normal mitochondria, resulting in J-aggregate formation, although JC-1 is not capable of forming J-aggregate in membrane potential lost mitochondria, resulting in JC-1 monomer formation in cytosol. U266 cells (1ϫ10 6 cells/10 ml) were treated with 0, 50 and 100 mM Phx-3 in 25 cm 2 cell culture flasks.…”

Section: Mitochondrial Membrane Potential Analysismentioning

confidence: 99%

Apoptosis Induction Preceded by Mitochondrial Depolarization in Multiple Myeloma Cell Line U266 by 2-Aminophenoxazine-3-one

Shirato

Imaizumi

Miyazawa

et al. 2008

Biological & Pharmaceutical Bulletin

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The aim of the present study was to investigate the mechanism of apoptosis in human multiple myeloma cell line, U266, caused by 2-aminophenoxazine-3-one (Phx-3). Flow-cytometrical and morphological analyses showed that Phx-3 increased the population of annexin V-positive cells including early stage apoptotic cells and late stage apoptotic cells and induced DNA fragmentation or apoptotic body formation in U266 cells, indicating that Phx-3 induced the apoptosis of U266 cells. Activity of caspase-3 was extensively increased in U266 cells treated with Phx-3 time-dependently within 24 h, but this Phx-3-stimulated activity of the enzyme in the cells was completely cancelled by the addition of N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD-fmk), a pan-caspase inhibitor. The addition of z-VAD-fmk almost blocked the apoptotic effect of Phx-3 against U266 cells, indicating that Phx-3-induced apoptosis of U266 cells was dependent on a caspase signaling pathway. Moreover, the apoptosis of U266 cells occurred after the induction of cell cycle arrest of the cells in the S and G 2 /M phase, the loss of mitochondrial membrane potential, and activation of caspase-3 reached maximum, which were caused by Phx-3 within 24 h. These results support the views that the apoptosis of U266 cells caused by Phx-3 may be preceded by the cell cycle arrest, depolarization of mitochondria and activation of caspase-3. These results support the view that Phx-3 may be utilized in future as chemotherapeutic agent against multiple myeloma which is extremely refractory to chemotherapy.

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A rapid method for the evaluation of compounds with mitochondria-protective properties

Cited by 55 publications

References 11 publications

Pentoxifylline in Ischemia-Induced Acute Kidney Injury in Rats

Pentoxifylline in Ischemia-Induced Acute Kidney Injury in Rats

Regulator of G Protein Signaling 6 (RGS6) Induces Apoptosis via a Mitochondrial-dependent Pathway Not Involving Its GTPase-activating Protein Activity

Apoptosis Induction Preceded by Mitochondrial Depolarization in Multiple Myeloma Cell Line U266 by 2-Aminophenoxazine-3-one

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