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.

Figure 1.

WFA induces autophagosome accumulation, LC3B conversion and other autophagy related proteins. (A) MCF7 and MDA-MB-231 cells were treated with 5 µM WFA for 3 and 6 h as indicated and visualized under an electron microscope. Scale bars: 2 µm. Pictures are shown with ~×7400 magnifications. Double-membrane autophagosomes were counted in randomly selected ~100 cells. The number of autophagosomes was counted from randomly selected fields (shown in bar graphs). *P < 0.005, compared with vehicle-treated controls (C). (B) Breast cancer cells were treated with 5 µM WFA and subjected to immunocytochemistry using an LC3B antibody. Scale bars: 20 µm. Representative immunofluorescence images are shown. Bar diagram shows number of LC3B puncta per cell. *P < 0.001, compared with vehicle-treated controls (C). (C) Breast cancer cells were treated with 5 µM WFA, and total cell lysates were immunoblotted for LC3B (14, 16 kDa) expression. ACTB (45 kDa) was used as a loading control. (D) Total protein lysates from MDA-MB-231-derived tumors from vehicle-treated and WFA-treated mice were examined for the expression of LC3B (14, 16 kDa). ACTB (45 kDa) was used as a loading control. (E) Immunoblot analysis of ATG5 (55 kDa), ATG7 (78 kDa), and BECN1 (60 kDa) in breast cancer cells treated with 5 µM WFA as indicated. ACTB (45 kDa) was used as a loading control. (F) Tumors from vehicle and WFA-treated mice were subjected to immunohistochemical (IHC) analysis using LC3B antibodies. Scale bars: 100 µm. Bar diagrams show quantification of IHC analysis. *P< 0.01, compared with control. (G) Breast cancer cells were treated with 5 µM WFA, and total RNA were examined for the expression of ATG5, ATG7 and BECN1. ACTB was used as a loading control. (H) Schematic diagram of the tfLC3 plasmid (upper panel). MDA-MB-231 and MCF7 cells were transfected with tfLC3 followed by treatment with 5 µM WFA or 200 nM rapamycin or Earle’s balanced salt solution. Earle’s balanced salt solution and rapamycin were used as positive controls for autophagic induction. Representative fluorescent images are shown. Scale bar: 20 μm.

Figure 2.

WFA induces the fusion of autophagosomes and lysosomes. (A) MCF7 and MDA-MB-231 cells were transfected with GFP-LC3B followed by treatment with 5 µM WFA or Earle’s balanced salt solution (2 h) and staining with LysoTracker Red. Cells were fixed and subjected to confocal microscopy. Representative fluorescent images are shown. Scale bar: 10 μm. Earle’s balanced salt solution was used as positive control for autophagic induction. (B) MCF7 and MDA-MB-231 cells were cotransfected with GFP-LC3B and RFP-RAB7 followed by treatment with 5 µM WFA or Earle’s balanced salt solution (2 h). Fixed cells were subjected to confocal microscopy. Representative fluorescent images are shown. Scale bars: 10 μM. (C) MCF7 and MDA-MB-231 cells were treated with 2.5 and 5 µM WFA followed by acridine orange staining. Representative images of MCF7 cells are shown. Scale bar: 10 μm. (D) Bar graph shows acridine orange punta per cell in MCF7 and MDA-MB-231 cells. *P< 0.01, compared with control.

Figure 3.

Inhibition of autophagy does not impact WFA-mediated inhibition of cell survival and apoptosis induction in breast cancer cells. (A) MCF7 and MDA-MB-231 cells were treated with 5 µM WFA alone or in combination with 200 nM Baf, 25 μM CQ and 4 mM 3-MA as indicated and subjected to MTT assay. *P< 0.001, compared with control. (B) Breast cancer cells were treated with 5 µM WFA alone or in combination with 25 μM CQ and 4 mM 3-MA as indicated and subjected to DNA fragmentation assay. *P< 0.01, compared with control. (C) MCF7 and MDA-MB-231 cells were treated with 5 µM WFA and 4 mM 3-MA and total cell lysates were immunoblotted for cleaved PARP1 (cPARP1, 89 kDa), PARP1 (116 kDa) and ACTB (45 kDa) as indicated. (D) CRISPR/Cas9 was used to knockout BECN1 and ATG7 in MCF7 cells and total cell lysates were immunoblotted for BECN1 (60 kDa) and ATG7 (78 kDa). ACTB (45 kDa) was used as loading control. (E) Vector-control, BECN1-KO and ATG7-KO MCF7 cells were treated with 5 µM WFA for indicated time intervals and total cell lysates were immunoblotted for cleaved-PARP1 (89 kDa) and total-PARP1 (116 kDa) expression levels. ACTB (45 kDa) was used as loading control. (F) Clonogenicity of control, BECN1-KO and ATG7-KO MCF7 cells treated with 5 µM WFA as indicated. (G) Cell viability of control, BECN1-KO and ATG7-KO MCF7 cells treated with 5 µM WFA was examined using MTT assay. Representative pictures of cells are shown.

Figure 4.

WFA inhibits protein degradation in lysosomes. (A, B) MCF7 and MDA-MB-231 cells were incubated with 10 μg/ml DQ-BSA for 2 h followed by washing with medium and treatment with 5 µM WFA or 200 nM rapamycin or starving in Earle’s balanced salt solution. The cells were fixed and stained with LysoTracker Red followed by confocal microscopy. Earle’s balanced salt solution and rapamycin were used as positive controls for autophagic induction. Representative fluorescent images are shown. Scale bars: 10 μM. (C) Breast cancer cells were treated with 5 µM WFA for indicated time intervals and total lysates were immunoblotted for SQSTM1 (62 kDa) expression levels. ACTB (45 kDa) was used as loading control. (D) Total protein lysates from MDA-MB-231-derived xenograft tumors from vehicle-treated and WFA-treated mice were examined for the expression of SQSTM1 (62 kDa). ACTB (45 kDa) was used as a loading control. (E) Tumors from vehicle and WFA-treated mice were subjected to immunohistochemical (IHC) analysis using SQSTM1 antibodies. Scale bars: 100 µm. Bar diagrams show quantification of IHC analysis. *P < 0.01, compared with control. (F) MDA-MB-231 and SUM159 breast cancer cells were treated with 5 µM WFA followed by lysosome extraction. Total lysates and lysosomes were immunoblotted for cathepsin D (CTSD). LAMP2 (130 kDa) was used as control. (G) Immunocytochemical analysis of cathepsin D in breast cancer cells treated with WFA.

Figure 5.

WFA induces energy impairment with AMPK activation. (A) Breast cancer cells were treated with 5 µM WFA for the indicated time intervals and intracellular ATP production was measured. Relative ATP levels are expressed with respect to the control. *P< 0.005, compared with control. (B) Breast cancer cells were treated with 5 µM WFA for indicated time intervals and total lysates were immunoblotted for phospho-PRKAA1 (p-PRKAA1) and total PRKAA1 (62 kDa) expression levels. ACTB (45 kDa) was used as loading control. (C) SUM149 and SUM159 cells were treated with 5 µM WFA alone or in combination with 2-DG as indicated. Intracellular ATP production was measured. Relative ATP levels are expressed with respect to the control. *P< 0.005, compared with control; **P< 0.01, compared with WFA alone. (D) SUM149 and SUM159 cells were treated with 5 µM WFA alone or in combination with 2-DG as indicated followed by Hoechst 33342 staining apoptosis detection. Mean number of apoptotic cells are presented in bar graphs. *P< 0.01, compared with control; **P< 0.01, compared with WFA alone. (E) Breast cancer cells were treated with 5 µM WFA alone or in combination with 2-DG as indicated. Total lysates were immunoblotted for the expression of cPARP1 (89 kDa) and PARP1 (116 kDa). ACTB (45 kDa) was used as loading control. (F, G) SUM149 and SUM159 cells were treated with 5 µM WFA alone or in combination with 2-DG as indicated and subjected to Trypan Blue exclusion assay. Bar graph shows percentage of alive cells. *P< 0.01, compared with control; **P< 0.005, compared with WFA alone.

Figure 6.

Combined treatment with WFA and 2-DG synergistically inhibits breast cancer cells. WFA induces energy impairment with reducing LDHA and higher expression of AMPK and low expression of LDHA correlates with increased overall survival. (A, B) HCC1569, HCC1806, HS578t, MDA-MB-231 and MDA-MB-468 breast cancer cells were treated with various concentrations of WFA (1.0, 2.5, 5.0. 7.5, 10.0 µM) in combination with 4.0 mM of 2-DG. MCF7 cells were treated with various concentrations of WFA in combination with 2.0 mM 2-DG. Cells were subjected to XTT assay and combination index values were calculated using CompuSyn software. CI < 1 represents synergism. Table shows combination index of different concentrations of WFA and 2-DG. (C) MCF7 cells were treated with 5 µM WFA or 10 mM MP as indicated and intracellular ATP production was measured. Relative ATP levels are expressed with respect to the control. *P< 0.01, compared with control; **P< 0.005, compared with WFA alone. (D) MCF7 cells were treated with 5 µM WFA or 10 mM MP as indicated and subjected to Trypan Blue exclusion assay. Bar graph shows fold change of alive cells. *P< 0.01, compared with control; **P< 0.001, compared with WFA alone. (E) MCF7 cells were treated with 5 µM WFA or 10 mM MP as indicated followed by Hoechst 33342 staining apoptosis detection. Mean number of apoptotic cells are presented as fold change in bar graphs. *P < 0.01, compared with control; **P< 0.01, compared with WFA alone. (F) Total lysates were immunoblotted for the expression of cPARP1 (89 kDa) and PARP1 (116 kDa). ACTB (45 kDa) was used as loading control. (G) Schematic representation of TCA cycle and aerobic glycolysis in proliferating cancer cells. Role of LDHA is noted. (H) Breast cancer cells were treated with 5 µM WFA for various time intervals as indicated. Total RNA was examined for the expression of LDHA using RT-PCR. ACTB was used as loading control. (I) Breast cancer cells were treated with 5 µM WFA for various time intervals as indicated. Total lysates were immunoblotted for the expression of LDHA (37 kDa) and phospho-LDHA (p-LDHA). ACTB (45 kDa) was used as loading control. (J) In breast cancer patients, higher expression of AMPK and lower expression of LDHA correlate to better prognosis (n = 4,374, HR = 0.75, P = 1.3e-05 and HR = 1.59, P = 1.6e-16, respectively). When investigating ER negative HER2 negative patients, the correlation to better survival was more prominent in LDHA (HR = 1.64, P = 4.7e-05) but was smaller for AMPK (HR = 0.81, P = 0.1). The plots display relapse free-survival, ER and HER2 status were determined using gene expression.