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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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Differential inhibition of GRP78 induction by VCD.Cells were exposed to a variety of known inducers of the ER stress response in the absence or presence of VCD for 24 hours. 2-Deoxyglucose was used at 20 mM; thapsigargin and tunicamycin were used at 200 nM. GRP78 protein expression was analyzed by Western blot analysis. In all cases, blots were re-probed with an antibody against actin (as a loading control). Ctrl: untreated cells kept under normal culture conditions. Ctrl: cells were kept in normal (25 mM glucose) culture conditions without drug treatment.
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pone-0065695-g003: Differential inhibition of GRP78 induction by VCD.Cells were exposed to a variety of known inducers of the ER stress response in the absence or presence of VCD for 24 hours. 2-Deoxyglucose was used at 20 mM; thapsigargin and tunicamycin were used at 200 nM. GRP78 protein expression was analyzed by Western blot analysis. In all cases, blots were re-probed with an antibody against actin (as a loading control). Ctrl: untreated cells kept under normal culture conditions. Ctrl: cells were kept in normal (25 mM glucose) culture conditions without drug treatment.

Mentions: As it is known that GRP78 protein expression is strongly increased not only in response to lower glucose levels, but more generally also by a great variety of conditions that cause endoplasmic reticulum (ER) stress, we next investigated additional triggers of ER stress. Cells were exposed to hypoxia, 2-deoxyglucose (2-DG, an inhibitor of hexokinase), thapsigargin (an inhibitor of SERCA, i.e., sarcoplasmic/endoplasmic reticulum calcium ATPase), and tunicamycin (an inhibitor of protein glycosylation) in the presence or absence of VCD. As shown in Fig. 3, VCD did not block GRP78 induction in response to hypoxia, thapsigargin, or tunicamycin; however, it did block GRP78 induction in response to 2-DG. Thus, altogether VCD blocked GRP78 induction under conditions that affected glycolysis, namely hypoglycemia and hexokinase inhibition by 2-DG, but it had no effect on GRP78 induction when other ER stressors were used.


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)

Differential inhibition of GRP78 induction by VCD.Cells were exposed to a variety of known inducers of the ER stress response in the absence or presence of VCD for 24 hours. 2-Deoxyglucose was used at 20 mM; thapsigargin and tunicamycin were used at 200 nM. GRP78 protein expression was analyzed by Western blot analysis. In all cases, blots were re-probed with an antibody against actin (as a loading control). Ctrl: untreated cells kept under normal culture conditions. Ctrl: cells were kept in normal (25 mM glucose) culture conditions without drug treatment.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0065695-g003: Differential inhibition of GRP78 induction by VCD.Cells were exposed to a variety of known inducers of the ER stress response in the absence or presence of VCD for 24 hours. 2-Deoxyglucose was used at 20 mM; thapsigargin and tunicamycin were used at 200 nM. GRP78 protein expression was analyzed by Western blot analysis. In all cases, blots were re-probed with an antibody against actin (as a loading control). Ctrl: untreated cells kept under normal culture conditions. Ctrl: cells were kept in normal (25 mM glucose) culture conditions without drug treatment.
Mentions: As it is known that GRP78 protein expression is strongly increased not only in response to lower glucose levels, but more generally also by a great variety of conditions that cause endoplasmic reticulum (ER) stress, we next investigated additional triggers of ER stress. Cells were exposed to hypoxia, 2-deoxyglucose (2-DG, an inhibitor of hexokinase), thapsigargin (an inhibitor of SERCA, i.e., sarcoplasmic/endoplasmic reticulum calcium ATPase), and tunicamycin (an inhibitor of protein glycosylation) in the presence or absence of VCD. As shown in Fig. 3, VCD did not block GRP78 induction in response to hypoxia, thapsigargin, or tunicamycin; however, it did block GRP78 induction in response to 2-DG. Thus, altogether VCD blocked GRP78 induction under conditions that affected glycolysis, namely hypoglycemia and hexokinase inhibition by 2-DG, but it had no effect on GRP78 induction when other ER stressors were used.

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