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Repositioning of Verrucosidin, a purported inhibitor of chaperone protein GRP78, as an inhibitor of mitochondrial electron transport chain complex I.

Thomas S, Sharma N, Gonzalez R, Pao PW, Hofman FM, Chen TC, Louie SG, Pirrung MC, Schönthal AH - PLoS ONE (2013)

Bottom Line: Rather, VCD blocked mitochondrial energy production via inhibition of complex I of the electron transport chain.Altogether, our study identifies mitochondria as the primary target of VCD.The possibility that other purported GRP78 inhibitors (arctigenin, biguanides, deoxyverrucosidin, efrapeptin, JBIR, piericidin, prunustatin, pyrvinium, rottlerin, valinomycin, versipelostatin) might act in a similar GRP78-independent fashion will be discussed.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Microbiology and Immunology, University of Southern California, Los Angeles, California, United States of America.

ABSTRACT
Verrucosidin (VCD) belongs to a group of fungal metabolites that were identified in screening programs to detect molecules that preferentially kill cancer cells under glucose-deprived conditions. Its mode of action was proposed to involve inhibition of increased GRP78 (glucose regulated protein 78) expression during hypoglycemia. Because GRP78 plays an important role in tumorigenesis, inhibitors such as VCD might harbor cancer therapeutic potential. We therefore sought to characterize VCD's anticancer activity in vitro. Triple-negative breast cancer cell lines MDA-MB-231 and MDA-MB-468 were treated with VCD under different conditions known to trigger increased expression of GRP78, and a variety of cellular processes were analyzed. We show that VCD was highly cytotoxic only under hypoglycemic conditions, but not in the presence of normal glucose levels, and VCD blocked GRP78 expression only when glycolysis was impaired (due to hypoglycemia or the presence of the glycolysis inhibitor 2-deoxyglucose), but not when GRP78 was induced by other means (hypoxia, thapsigargin, tunicamycin). However, VCD's strictly hypoglycemia-specific toxicity was not due to the inhibition of GRP78. Rather, VCD blocked mitochondrial energy production via inhibition of complex I of the electron transport chain. As a result, cellular ATP levels were quickly depleted under hypoglycemic conditions, and common cellular functions, including general protein synthesis, deteriorated and resulted in cell death. Altogether, our study identifies mitochondria as the primary target of VCD. The possibility that other purported GRP78 inhibitors (arctigenin, biguanides, deoxyverrucosidin, efrapeptin, JBIR, piericidin, prunustatin, pyrvinium, rottlerin, valinomycin, versipelostatin) might act in a similar GRP78-independent fashion will be discussed.

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Inhibition of ATP synthesis by VCD.Intracellular levels of ATP were determined after incubation of cells under various culture conditions. Cells were exposed to 100 nM VCD or 100 nM rotenone in the presence of 0.8 or 25 mM glucose for up to 12 h. Ctrl: control for the respective time point in the absence of drug treatment. Asterisks: p<0.05 (treated compared to untreated cells at the same time point).
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pone-0065695-g007: Inhibition of ATP synthesis by VCD.Intracellular levels of ATP were determined after incubation of cells under various culture conditions. Cells were exposed to 100 nM VCD or 100 nM rotenone in the presence of 0.8 or 25 mM glucose for up to 12 h. Ctrl: control for the respective time point in the absence of drug treatment. Asterisks: p<0.05 (treated compared to untreated cells at the same time point).

Mentions: To determine VCD’s postulated effects on energy production more directly, we measured intracellular ATP levels after treatment of cells with VCD or rotenone in the presence or absence of glucose. As shown in Fig. 7, neither VCD nor rotenone displayed pronounced effects on ATP levels when cells were kept in the presence of normal glucose levels. However, in the absence of glucose both compounds drastically lowered intracellular ATP levels. Hypoglycemia by itself also lowered ATP levels over time (to 30% at 12 hours), but this decline was substantially slower and much less severe than in the presence of VCD or rotenone (<5%). As shown by earlier studies (for examples, see [49], [50]), ATP decline to about 20% can be tolerated by cells for extended periods (weeks), especially when growth factors/serum are present; however, if levels drop substantially lower, cellular adaptation processes (inclusive of autophagy [51]) are unable to compensate and cells will die (e.g., [52], [53]). Therefore, the observed massive depletion of ATP levels by VCD under hypoglycemic condition is consistent with general shutdown of cellular functions (inclusive of global gene expression, Fig. 5) and cell death due to energy exhaustion.


Repositioning of Verrucosidin, a purported inhibitor of chaperone protein GRP78, as an inhibitor of mitochondrial electron transport chain complex I.

Thomas S, Sharma N, Gonzalez R, Pao PW, Hofman FM, Chen TC, Louie SG, Pirrung MC, Schönthal AH - PLoS ONE (2013)

Inhibition of ATP synthesis by VCD.Intracellular levels of ATP were determined after incubation of cells under various culture conditions. Cells were exposed to 100 nM VCD or 100 nM rotenone in the presence of 0.8 or 25 mM glucose for up to 12 h. Ctrl: control for the respective time point in the absence of drug treatment. Asterisks: p<0.05 (treated compared to untreated cells at the same time point).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3675020&req=5

pone-0065695-g007: Inhibition of ATP synthesis by VCD.Intracellular levels of ATP were determined after incubation of cells under various culture conditions. Cells were exposed to 100 nM VCD or 100 nM rotenone in the presence of 0.8 or 25 mM glucose for up to 12 h. Ctrl: control for the respective time point in the absence of drug treatment. Asterisks: p<0.05 (treated compared to untreated cells at the same time point).
Mentions: To determine VCD’s postulated effects on energy production more directly, we measured intracellular ATP levels after treatment of cells with VCD or rotenone in the presence or absence of glucose. As shown in Fig. 7, neither VCD nor rotenone displayed pronounced effects on ATP levels when cells were kept in the presence of normal glucose levels. However, in the absence of glucose both compounds drastically lowered intracellular ATP levels. Hypoglycemia by itself also lowered ATP levels over time (to 30% at 12 hours), but this decline was substantially slower and much less severe than in the presence of VCD or rotenone (<5%). As shown by earlier studies (for examples, see [49], [50]), ATP decline to about 20% can be tolerated by cells for extended periods (weeks), especially when growth factors/serum are present; however, if levels drop substantially lower, cellular adaptation processes (inclusive of autophagy [51]) are unable to compensate and cells will die (e.g., [52], [53]). Therefore, the observed massive depletion of ATP levels by VCD under hypoglycemic condition is consistent with general shutdown of cellular functions (inclusive of global gene expression, Fig. 5) and cell death due to energy exhaustion.

Bottom Line: Rather, VCD blocked mitochondrial energy production via inhibition of complex I of the electron transport chain.Altogether, our study identifies mitochondria as the primary target of VCD.The possibility that other purported GRP78 inhibitors (arctigenin, biguanides, deoxyverrucosidin, efrapeptin, JBIR, piericidin, prunustatin, pyrvinium, rottlerin, valinomycin, versipelostatin) might act in a similar GRP78-independent fashion will be discussed.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Microbiology and Immunology, University of Southern California, Los Angeles, California, United States of America.

ABSTRACT
Verrucosidin (VCD) belongs to a group of fungal metabolites that were identified in screening programs to detect molecules that preferentially kill cancer cells under glucose-deprived conditions. Its mode of action was proposed to involve inhibition of increased GRP78 (glucose regulated protein 78) expression during hypoglycemia. Because GRP78 plays an important role in tumorigenesis, inhibitors such as VCD might harbor cancer therapeutic potential. We therefore sought to characterize VCD's anticancer activity in vitro. Triple-negative breast cancer cell lines MDA-MB-231 and MDA-MB-468 were treated with VCD under different conditions known to trigger increased expression of GRP78, and a variety of cellular processes were analyzed. We show that VCD was highly cytotoxic only under hypoglycemic conditions, but not in the presence of normal glucose levels, and VCD blocked GRP78 expression only when glycolysis was impaired (due to hypoglycemia or the presence of the glycolysis inhibitor 2-deoxyglucose), but not when GRP78 was induced by other means (hypoxia, thapsigargin, tunicamycin). However, VCD's strictly hypoglycemia-specific toxicity was not due to the inhibition of GRP78. Rather, VCD blocked mitochondrial energy production via inhibition of complex I of the electron transport chain. As a result, cellular ATP levels were quickly depleted under hypoglycemic conditions, and common cellular functions, including general protein synthesis, deteriorated and resulted in cell death. Altogether, our study identifies mitochondria as the primary target of VCD. The possibility that other purported GRP78 inhibitors (arctigenin, biguanides, deoxyverrucosidin, efrapeptin, JBIR, piericidin, prunustatin, pyrvinium, rottlerin, valinomycin, versipelostatin) might act in a similar GRP78-independent fashion will be discussed.

Show MeSH
Related in: MedlinePlus