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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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Mitochondrial target of VCD.The activities of specific complexes of the electron transport chain were analyzed in intact mitochondria in the presence and absence of drug treatment. (A) Complex I activity was recorded as absorbance (600 nm) of the electron acceptor DCIP over time. Note that inhibition of activity by nanomolar concentrations of VCD or rotenone (Rot) resulted in prominently lower absorbance. (B) Complex II activity was recorded as absorbance (490 nm) of formazan formed after reduction of electron acceptor INT. Note that neither VCD nor rotenone affected complex II activity (none of the minor variations was statistically significant, i.e., the p-value was greater than 0.05 in all two-sided comparisons). The known complex II inhibitor TTFA served as the positive control and significantly (p<0.01; asterisks) inhibited complex II activity. Ctrl: control, i.e., value in the absence of drug treatment. (C) Mitochondrial ATP synthase (complex V) activity was recorded as a decrease in absorbance (340 nm) over time. Note that ATP synthase inhibitor oligomycin inhibits complex V activity (used as a positive control; right panel), but neither VCD nor rotenone show inhibitory effect. All assays A-C were repeated at least once and yielded essentially the same outcomes.
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pone-0065695-g009: Mitochondrial target of VCD.The activities of specific complexes of the electron transport chain were analyzed in intact mitochondria in the presence and absence of drug treatment. (A) Complex I activity was recorded as absorbance (600 nm) of the electron acceptor DCIP over time. Note that inhibition of activity by nanomolar concentrations of VCD or rotenone (Rot) resulted in prominently lower absorbance. (B) Complex II activity was recorded as absorbance (490 nm) of formazan formed after reduction of electron acceptor INT. Note that neither VCD nor rotenone affected complex II activity (none of the minor variations was statistically significant, i.e., the p-value was greater than 0.05 in all two-sided comparisons). The known complex II inhibitor TTFA served as the positive control and significantly (p<0.01; asterisks) inhibited complex II activity. Ctrl: control, i.e., value in the absence of drug treatment. (C) Mitochondrial ATP synthase (complex V) activity was recorded as a decrease in absorbance (340 nm) over time. Note that ATP synthase inhibitor oligomycin inhibits complex V activity (used as a positive control; right panel), but neither VCD nor rotenone show inhibitory effect. All assays A-C were repeated at least once and yielded essentially the same outcomes.

Mentions: Because of VCD’s impairment of mitochondrial respiration, we next set out to measure the activity of specific complexes within the electron transport chain. In particular, we analyzed the effects of VCD on complex I, complex II, and complex V (ATP synthase). As summarized in Fig. 9, VCD potently blocked complex I, but not complex II or complex V. Inhibition of complex I appeared to be maximal at 100 nM, and further increased concentrations of VCD (300 and 1,000 nM) exerted only marginally stronger effects. Intriguingly, blockage of complex I activity by VCD was as effective as blockage achieved by the classic complex I inhibitor rotenone, which was used as a positive control (Fig. 9A).


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)

Mitochondrial target of VCD.The activities of specific complexes of the electron transport chain were analyzed in intact mitochondria in the presence and absence of drug treatment. (A) Complex I activity was recorded as absorbance (600 nm) of the electron acceptor DCIP over time. Note that inhibition of activity by nanomolar concentrations of VCD or rotenone (Rot) resulted in prominently lower absorbance. (B) Complex II activity was recorded as absorbance (490 nm) of formazan formed after reduction of electron acceptor INT. Note that neither VCD nor rotenone affected complex II activity (none of the minor variations was statistically significant, i.e., the p-value was greater than 0.05 in all two-sided comparisons). The known complex II inhibitor TTFA served as the positive control and significantly (p<0.01; asterisks) inhibited complex II activity. Ctrl: control, i.e., value in the absence of drug treatment. (C) Mitochondrial ATP synthase (complex V) activity was recorded as a decrease in absorbance (340 nm) over time. Note that ATP synthase inhibitor oligomycin inhibits complex V activity (used as a positive control; right panel), but neither VCD nor rotenone show inhibitory effect. All assays A-C were repeated at least once and yielded essentially the same outcomes.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0065695-g009: Mitochondrial target of VCD.The activities of specific complexes of the electron transport chain were analyzed in intact mitochondria in the presence and absence of drug treatment. (A) Complex I activity was recorded as absorbance (600 nm) of the electron acceptor DCIP over time. Note that inhibition of activity by nanomolar concentrations of VCD or rotenone (Rot) resulted in prominently lower absorbance. (B) Complex II activity was recorded as absorbance (490 nm) of formazan formed after reduction of electron acceptor INT. Note that neither VCD nor rotenone affected complex II activity (none of the minor variations was statistically significant, i.e., the p-value was greater than 0.05 in all two-sided comparisons). The known complex II inhibitor TTFA served as the positive control and significantly (p<0.01; asterisks) inhibited complex II activity. Ctrl: control, i.e., value in the absence of drug treatment. (C) Mitochondrial ATP synthase (complex V) activity was recorded as a decrease in absorbance (340 nm) over time. Note that ATP synthase inhibitor oligomycin inhibits complex V activity (used as a positive control; right panel), but neither VCD nor rotenone show inhibitory effect. All assays A-C were repeated at least once and yielded essentially the same outcomes.
Mentions: Because of VCD’s impairment of mitochondrial respiration, we next set out to measure the activity of specific complexes within the electron transport chain. In particular, we analyzed the effects of VCD on complex I, complex II, and complex V (ATP synthase). As summarized in Fig. 9, VCD potently blocked complex I, but not complex II or complex V. Inhibition of complex I appeared to be maximal at 100 nM, and further increased concentrations of VCD (300 and 1,000 nM) exerted only marginally stronger effects. Intriguingly, blockage of complex I activity by VCD was as effective as blockage achieved by the classic complex I inhibitor rotenone, which was used as a positive control (Fig. 9A).

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