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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 tunicamycin-induced GRP78 expression by VCD and rotenone.Cells were exposed to hypoglycemia (0.8 mM glucose) in the presence or absence of 100 nM VCD (A) or 100 nM rotenone (B) at time 0 hours. In all cases, tunicamycin was added 8 hours later for an additional 16 hours. Thereafter, cell lysates were prepared and analyzed by Western blot for GRP78 expression levels. Actin was used as a loading control.
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pone-0065695-g011: Inhibition of tunicamycin-induced GRP78 expression by VCD and rotenone.Cells were exposed to hypoglycemia (0.8 mM glucose) in the presence or absence of 100 nM VCD (A) or 100 nM rotenone (B) at time 0 hours. In all cases, tunicamycin was added 8 hours later for an additional 16 hours. Thereafter, cell lysates were prepared and analyzed by Western blot for GRP78 expression levels. Actin was used as a loading control.

Mentions: The above results indicated that VCD (or rotenone) blocked GRP78 induction under hypglycemic conditions because combined blockage of the two cellular energy sources (glycolysis and mitochondria) precluded the availability of sufficient ATP (Fig. 7) for the synthesis of GRP78 (and many other proteins, Fig. 6). In comparison, neither VCD nor rotenone blocked GRP78 induction by tunicamycin (Fig. 3, Fig. 10), presumably because ongoing glycolysis in the presence of plentiful glucose provided sufficient energy for protein synthesis to take place. To furnish further evidence for this conjecture, we pre-treated cells with hypoglycemia+VCD (or hypoglycemia+rotenone) for 8 hours before the addition of tunicamycin. This pre-treatment, which strongly reduces cellular ATP levels (Fig. 7), would then be expected to prevent GRP78 induction by tunicamycin. As shown in Fig. 11, this was indeed the case. While pre-treatment of cells with VCD alone or hypoglycemia alone did not interfere with GRP78 induction by tunicamycin, the combination of hypoglycemia+VCD greatly minimized tunicamycin’s ability to stimulate GRP78 expression (Fig. 11A). As before, rotenone was able to faithfully mimic VCD’s effect (Fig. 11B).


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 tunicamycin-induced GRP78 expression by VCD and rotenone.Cells were exposed to hypoglycemia (0.8 mM glucose) in the presence or absence of 100 nM VCD (A) or 100 nM rotenone (B) at time 0 hours. In all cases, tunicamycin was added 8 hours later for an additional 16 hours. Thereafter, cell lysates were prepared and analyzed by Western blot for GRP78 expression levels. Actin was used as a loading control.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0065695-g011: Inhibition of tunicamycin-induced GRP78 expression by VCD and rotenone.Cells were exposed to hypoglycemia (0.8 mM glucose) in the presence or absence of 100 nM VCD (A) or 100 nM rotenone (B) at time 0 hours. In all cases, tunicamycin was added 8 hours later for an additional 16 hours. Thereafter, cell lysates were prepared and analyzed by Western blot for GRP78 expression levels. Actin was used as a loading control.
Mentions: The above results indicated that VCD (or rotenone) blocked GRP78 induction under hypglycemic conditions because combined blockage of the two cellular energy sources (glycolysis and mitochondria) precluded the availability of sufficient ATP (Fig. 7) for the synthesis of GRP78 (and many other proteins, Fig. 6). In comparison, neither VCD nor rotenone blocked GRP78 induction by tunicamycin (Fig. 3, Fig. 10), presumably because ongoing glycolysis in the presence of plentiful glucose provided sufficient energy for protein synthesis to take place. To furnish further evidence for this conjecture, we pre-treated cells with hypoglycemia+VCD (or hypoglycemia+rotenone) for 8 hours before the addition of tunicamycin. This pre-treatment, which strongly reduces cellular ATP levels (Fig. 7), would then be expected to prevent GRP78 induction by tunicamycin. As shown in Fig. 11, this was indeed the case. While pre-treatment of cells with VCD alone or hypoglycemia alone did not interfere with GRP78 induction by tunicamycin, the combination of hypoglycemia+VCD greatly minimized tunicamycin’s ability to stimulate GRP78 expression (Fig. 11A). As before, rotenone was able to faithfully mimic VCD’s effect (Fig. 11B).

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