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Withaferin A inhibits lysosomal activity to block autophagic flux and induces apoptosis via energetic impairment in breast cancer cells.

ABSTRACT: 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.

SUBMITTER: Muniraj N

PROVIDER: S-EPMC10893887 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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Withaferin A inhibits lysosomal activity to block autophagic flux and induces apoptosis via energetic impairment in breast cancer cells.

Muniraj Nethaji N Siddharth Sumit S Nagalingam Arumugam A Walker Alyssa A Woo Juhyung J Győrffy Balázs B Gabrielson Ed E Saxena Neeraj K NK Sharma Dipali D

Carcinogenesis 20190901 9

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 degradatio ...[more]

PMID: 30698683

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Project description:BackgroundGraphite carbon nanofibers (GCNF) have emerged as a potential alternative of carbon nanotubes (CNT) for various biomedical applications due to their superior physico-chemical properties. Therefore in-depth understanding of the GCNF induced toxic effects and underlying mechanisms in biological systems is of great interest. Currently, autophagy activation by nanomaterials is recognized as an emerging toxicity mechanism. However, the association of GCNF induced toxicity with this form of cell death is largely unknown. In this study, we have assessed the possible mechanism; especially the role of autophagy, underlying the GCNF induced toxicity.MethodsHuman lung adenocarcinoma (A549) cells were exposed to a range of GCNF concentrations and various cellular parameters were analyzed (up to 48 h). Transmission electron microscopy, immunofluorescent staining, western blot and quantitative real time PCR were performed to detect apoptosis, autophagy induction, lysosomal destabilization and cytoskeleton disruption in GCNF exposed cells. DCFDA assay was used to evaluate the reactive oxygen species (ROS) production. Experiments with N-acetyl-L-cysteine (NAC), 3-methyladenine (3-MA) and LC3 siRNA was carried out to confirm the involvement of oxidative stress and autophagy in GCNF induced cell death. Comet assay and micronucleus (MN) assay was performed to assess the genotoxicity potential.ResultsIn the present study, GCNF was found to induce nanotoxicity in human lung cells through autophagosomes accumulation followed by apoptosis via intracellular ROS generation. Mechanistically, impaired lysosomal function and cytoskeleton disruption mediated autophagic flux blockade was found to be the major cause of accumulation rather than autophagy induction which further activates apoptosis. The whole process was in line with the increased ROS level and their pharmacological inhibition leads to mitigation of GCNF induced cell death. Moreover the inhibition of autophagy attenuates apoptosis indicating the role of autophagy as cell death process. GCNF was also found to induce genomic instability.ConclusionOur present study demonstrates that GCNF perturbs various interrelated signaling pathway and unveils the potential nanotoxicity mechanism of GCNF through targeting ROS-autophagy-apoptosis axis. The current study is significant to evaluate the safety and risk assessment of fibrous carbon nanomaterials prior to their potential use and suggests caution on their utilization for biomedical research.

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Withaferin A inhibits lysosomal activity to block autophagic flux and induces apoptosis via energetic impairment in breast cancer cells.

Publications

Withaferin A inhibits lysosomal activity to block autophagic flux and induces apoptosis via energetic impairment in breast cancer cells.

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