TY - JOUR
T1 - Withaferin A inhibits lysosomal activity to block autophagic flux and induces apoptosis via energetic impairment in breast cancer cells
AU - Muniraj, Nethaji
AU - Siddharth, Sumit
AU - Nagalingam, Arumugam
AU - Walker, Alyssa
AU - Woo, Juhyung
AU - Gyorffy, Balázs
AU - Gabrielson, Ed
AU - Saxena, Neeraj K.
AU - Sharma, DIpali
N1 - Funding Information:
This work was supported by National Institute of Health National Cancer Institute [R01CA204555], Breast Cancer Research Foundation [90047965] and The Fetting Fund (to D.S.). Conflict of Interest Statement: None declared.
Publisher Copyright:
© 2019 The Author(s) 2019. Published by Oxford University Press. All rights reserved.
PY - 2019/9/1
Y1 - 2019/9/1
N2 - Withaferin A (WFA), a steroidal lactone, negatively regulates breast cancer growth however, its mechanisms of action remain largely elusive. We found that WFA blocks autophagy flux and lysosomal proteolytic activity in breast cancer cells. WFA increases accumulation of autophagosomes, LC3B-II conversion, expression of autophagy-related proteins and autophagosome/lysosome fusion. Autolysosomes display the characteristics of acidic compartments in WFA-treated cells; however, the protein degradation activity of lysosomes is inhibited. Blockade of autophagic flux reduces the recycling of cellular fuels leading to insufficient substrates for tricarboxylic acid (TCA) cycle and impaired oxidative phosphorylation. WFA decreases expression and phosphorylation of lactate dehydrogenase, the key enzyme that catalyzes pyruvate-to-lactate conversion, reduces adenosine triphosphate levels and increases AMP-activated protein kinase (AMPK) activation. AMPK inhibition abrogates while AMPK activation potentiates WFA's effect. WFA and 2-deoxy-d-glucose combination elicits synergistic inhibition of breast cancer cells. Genetic knockout of BECN1 and ATG7 fails to rescue cells from WFA treatment; in contrast, addition of methyl pyruvate to supplement TCA cycle protects WFA-treated cells. Together, these results implicate that WFA is a potent lysosomal inhibitor; energetic impairment is required for WFA-induced apoptosis and growth inhibition and combining WFA and 2-DG is a promising therapeutic strategy for breast cancer.
AB - Withaferin A (WFA), a steroidal lactone, negatively regulates breast cancer growth however, its mechanisms of action remain largely elusive. We found that WFA blocks autophagy flux and lysosomal proteolytic activity in breast cancer cells. WFA increases accumulation of autophagosomes, LC3B-II conversion, expression of autophagy-related proteins and autophagosome/lysosome fusion. Autolysosomes display the characteristics of acidic compartments in WFA-treated cells; however, the protein degradation activity of lysosomes is inhibited. Blockade of autophagic flux reduces the recycling of cellular fuels leading to insufficient substrates for tricarboxylic acid (TCA) cycle and impaired oxidative phosphorylation. WFA decreases expression and phosphorylation of lactate dehydrogenase, the key enzyme that catalyzes pyruvate-to-lactate conversion, reduces adenosine triphosphate levels and increases AMP-activated protein kinase (AMPK) activation. AMPK inhibition abrogates while AMPK activation potentiates WFA's effect. WFA and 2-deoxy-d-glucose combination elicits synergistic inhibition of breast cancer cells. Genetic knockout of BECN1 and ATG7 fails to rescue cells from WFA treatment; in contrast, addition of methyl pyruvate to supplement TCA cycle protects WFA-treated cells. Together, these results implicate that WFA is a potent lysosomal inhibitor; energetic impairment is required for WFA-induced apoptosis and growth inhibition and combining WFA and 2-DG is a promising therapeutic strategy for breast cancer.
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U2 - 10.1093/carcin/bgz015
DO - 10.1093/carcin/bgz015
M3 - Article
C2 - 30698683
AN - SCOPUS:85064814086
VL - 40
SP - 1110
EP - 1120
JO - Carcinogenesis
JF - Carcinogenesis
SN - 0143-3334
IS - 9
ER -