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-tolactate 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.

The Preparation of Ginsenoside Rg5, Its Antitumor Activity against Breast Cancer Cells and Its Targeting of PI3K

“…Rg5 treatment increased the level of LC3B puncta, and microscopic evaluation of tandem GFP-RFP-tagged LC3B in MCF-7 cells demonstrated that Rg5 induced the occurrence of autophagic flux. Furthermore, autophagy can be limited due to the delayed trafficking of autophagosomes to lysosomes and the fusion of autophagosomes and lysosomes [36,37]. Immunofluorescence analyses from LysoTracker Red staining with GFP-LC3B were used to establish that Rg5 treatment increased the formation of autophagolysosomes and led to the fusion of autophagosomes and lysosomes.…”

Role of autophagic response induced by major phytochemicals in cancer prevention and treatment

“…WA was shown to impair the proteolytic activity of lysosomes causing blockade of autophagic flux, a decrease in the substrates required for the TCA cycle and impaired oxidative phosphorylation, resulting in apoptosis in breast cancer cells [49] . Similarly, another study concluded that the inhibition of proteasome activity and induction of impaired autophagy constituted the main mechanism associated with the antitumor effects of WA in breast cancer cells [50] . WA was shown to induce G2/M cell cycle arrest in myelodysplasia and leukemia cells [51] .…”

“…Liver WA and WA + Chloroquine or Bafilomycin WA induced autophagy. Combination of WA with Chloroquine or Bafilomycin had a higher efficacy than monotherapy Muniraj et al [50] 2019 Cell Culture, MDA-MB-231…”

Induction of STK11-dependent cytoprotective autophagy in breast cancer cells upon honokiol treatment

“…Cells were embedded in EPON, sectioned, stained and examined with an H7600 transmission electron microscope (Hitachi, Tokyo, Japan) 45 . Breast cancer cells (5 ×10 5 cells/well) were plated in 4-well chamber slides (Nunc, Rochester, NY) followed by HNK treatment as indicated and subjected to immunofluorescence analysis 77 . Fixed and immunofluorescently stained cells were imaged using a Zeiss LSM510 Meta (Zeiss, Dublin, California, USA) laser scanning confocal system configured to a Zeiss Axioplan 2 upright microscope (Zeiss, Dublin, California, USA).…”

“…Multiple view-fields were examined and representative cells from 3 view-fields were photographed. For puncta, at least 50-100 cells/sample were counted in triplicates 45,77,78 .…”