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Isoginkgetin, a Natural Biflavonoid Proteasome Inhibitor, Sensitizes Cancer Cells to Apoptosis via Disruption of Lysosomal Homeostasis and Impaired Protein Clearance

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Isoginkgetin, a Natural Biflavonoid Proteasome Inhibitor, Sensitizes Cancer Cells to Apoptosis via Disruption of Lysosomal Homeostasis and Impaired Protein Clearance

Jessica Tsalikis et al. Mol Cell Biol.

Abstract

Protein degradation pathways are critical for maintaining proper protein dynamics within the cell, and considerable efforts have been made toward the development of therapeutics targeting these catabolic processes. We report here that isoginkgetin, a naturally derived biflavonoid, sensitized cells undergoing nutrient starvation to apoptosis, induced lysosomal stress, and activated the lysosome biogenesis gene TFEB Isoginkgetin treatment led to the accumulation of aggregates of polyubiquitinated proteins that colocalized strongly with the adaptor protein p62, the 20S proteasome, and the endoplasmic reticulum-associated degradation (ERAD) protein UFD1L. Isoginkgetin directly inhibited the chymotrypsin-like, trypsin-like, and caspase-like activities of the 20S proteasome and impaired NF-κB signaling, suggesting that the molecule may display its biological activity in part through proteasome inhibition. Importantly, isoginkgetin was effective at killing multiple myeloma (MM) cell lines in vitro and displayed a higher rate of cell death induction than the clinically approved proteasome inhibitor bortezomib. We propose that isoginkgetin disturbs protein homeostasis, leading to an excess of protein cargo that places a burden on the lysosomes/autophagic machinery, eventually leading to cancer cell death.

Keywords: TFEB; autophagy; biflavonoid; drug discovery; proteasome.

Figures

FIG 1
FIG 1
Isoginkgetin sensitizes nutrient-starved cancer cells to apoptotic cell death. (A) Quantification of cell death measured via DAPI stain in HeLa cells treated with 10 μM isoginkgetin (Iso) for 24 h, measured as a percentage of the total number of cells (three experiments [n = 3]; roughly 200 cells quantified per condition). The value for cells treated with isoginkgetin for 24 h was significantly different (P < 0.001) from the control (CTL) value as indicated by the bar and asterisks. (B) Western blot analysis of cleaved PARP-1, cleaved caspase-3 (Casp-3), and tubulin loading control upon treatment with 10 μM isoginkgetin for 0 to 24 h. (C) Quantification of cell death measured via DAPI stain in HeLa cells treated with 10 μM isoginkgetin either alone or in combination with KRB or EBSS for 6 h, measured as a percentage of the total number of cells (n = 3; roughly 200 cells quantified per condition; ***, P < 0.001). (D) Western blot analysis of the levels of tubulin (loading control), cleaved PARP-1 and caspase-3 in HeLa cells treated for 6 h with KRB and 10 μM isoginkgetin, either alone or in combination, or 20 μM staurosporine (Stauro.). (E) HeLa cells were stimulated with KRB or 10 μM isoginkgetin for 6 h, followed by staining with annexin V-FITC and propidium iodide and analyzed via flow cytometry. (F) Soft-agar colony-forming assay of HeLa and MDA-MD-468 breast cancer cells, treated with either DMSO or 10 μM isoginkgetin for 72 h. (G and H) Quantification of cell death measured via DAPI stain (G) and Western blot analysis of cleaved PARP-1, cleaved caspase-3, and tubulin (H) in HeLa cells incubated in either DMEM with serum, DMEM without serum, or KRB, with or without the addition of 10 μM isoginkgetin for 6 h, measured as a percentage of the total number of cells (n = 3; roughly 200 cells quantified per condition; ***, P < 0.001). (I) Western blot analysis of cleaved PARP-1, cleaved caspase-3, and tubulin in HeLa cells incubated in either nutrient-rich DMEM or low-glucose (gluc.) DMEM for 6 h with or without 10 μM isoginkgetin.
FIG 2
FIG 2
Isoginkgetin induces the perinuclear aggregation of p62/ubiquitin-positive protein cargo. (A) Confocal immunofluorescence analysis of HeLa cells treated with 10 μM isoginkgetin for 24 h, stained with ubiquitin (Ub) and p62 antibodies and DAPI nuclear stain. (B) Quantification of the percentage of HeLa cells with p62-positive aggregates upon treatment with isoginkgetin for 8 or 24 h (n = 3; approximately 100 cells per condition). Values are significantly different (P < 0.001) from the no-isoginkgetin value by one-way analysis of variance (ANOVA) as indicated by asterisks. (C) Graph showing the colocalization between ubiquitin and p62 using Pearson’s coefficient obtained via the Zen Blue software (n = 3; approximately 100 cells per conditions). Values are significantly different (P < 0.01) by Student’s t test as indicated by the asterisks. (D) Confocal immunofluorescence analysis of HeLa cells treated with 10 μM isoginkgetin, KRB or 10 μM bafilomycin A1 (Baf A1) for 6 h, stained with p62 antibody and DAPI nuclear stain. (E) Zoomed-in images from the dashed boxes in panel D. (F) Quantification of the percentage of HeLa cells with p62-positive aggregates upon treatment with isoginkgetin for 6 h in either complete DMEM or KRB (n = 3; approximately 100 cells per condition). Values are significantly different by two-way ANOVA as indicated by asterisks as follows: ***, P < 0.001; **, P < 0.01. (G) Confocal immunofluorescence analysis of HeLa cells treated with isoginkgetin for 24 h and stained with p62 and LAMP2. N, nucleus.
FIG 3
FIG 3
Isoginkgetin induces ER stress, impairs the unfolded protein response, and results in accumulation of ERAD substrates. (A) Confocal immunofluorescence analysis of HeLa cells treated with 10 μM isoginkgetin stained with antibodies against ubiquitin, 20S proteasome. or UFD1l. (B) Confocal immunofluorescence analysis of HeLa cells treated with isoginkgetin for 24 h, stained with antibodies against Ub and calreticulin (CRT). (C) Quantitative PCR analysis of the relative mRNA expression levels of ER stress genes BiP, CHOP, GADD34, and ATF3 upon treatment with 10 μM isoginkgetin or 10 μM thapsigargin (Thapsi) for 6 h (n = 3). Values that are significantly different by one-way ANOVA test are indicated by asterisks as follows: *, P < 0.01; **, P < 0.005; ***, P < 0.001. (D) Western blot analysis of the protein levels of ATF4, ATF3, and tubulin loading control in HeLa cells upon treatment with 10 μM isoginkgetin for 0 to 6 h.
FIG 4
FIG 4
Isoginkgetin induces lysosomal stress and activation of transcription factor TFEB. (A) Confocal immunofluorescence microscopy of HeLa cells stained with LAMP2 upon treatment with 10 μM isoginkgetin for the indicated times. Cytoplasm in each cell is outlined by a dashed line. (B) HeLa cells treated with KRB, 10 μM isoginkgetin, or 1 μM bafilomycin A1 for 6 h, followed by acridine orange (AO) staining for 30 min and visualization via immunofluorescence microscopy and quantification of the acridine orange puncta in each of the conditions. Values that are significantly different by Student’s t test are indicated by asterisks as follows: ***, P < 0.001; **, P < 0.01. (C) Confocal immunofluorescence of HeLa cells treated with either 10 μM isoginkgetin or 10 μM MG132, stained with DAPI and LAMP2, and quantification of the total lysosome cluster area per cell upon 24-h isoginkgetin treatment (n = 3 with approximately 50 cells per condition; ***, P < 0.001 by Student’s t test). Bars = 5 μm. (D) Western blot analysis of the levels of LC3 I and II in HeLa cells treated with 10 μM isoginkgetin over an 8-h period or with KRB for 6 h compared to a tubulin control. (E) Confocal immunofluorescence microscopy analysis and quantification of the levels of nuclear TFEB in HeLa and HCT-116 cell lines. ***, P < 0.001. (F) Quantification of the percentage of HeLa cells with nuclear TFEB upon treatment with 10 μM isoginkgetin for 24 h. (G) Western blot analysis of TFEB expression in nuclear (N) fractions versus cytoplasmic (C) fractions in HeLa cells incubated with nutrient-rich DMEM or KRB buffer for 6 h with or without 10 μM isoginkgetin compared to fractionation controls lamin A (nuclear) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (cytoplasmic).
FIG 5
FIG 5
Isoginkgetin inhibits the proteolytic activities of purified 20S proteasome and impairs NF-κB signaling. (A) Activity of purified 20S proteasome measured by cleavage of fluorogenic substrates of the chymotrypsin-like, trypsin-like, and caspase-like enzymatic activities over a 2-h period upon treatment with 10 μM isoginkgetin or 10 μM MG132. Representative data of three independent experiments are shown. (B and C) Activity of purified 20S proteasome upon treatment with isoginkgetin, MG132, or 5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole (DRB) at various doses. Values represent measurement readings taken 2 h following addition of the drugs (chymotrypsin-like, trypsin-like, and caspase-like). (D) SUnSET assay to measure active translation in HeLa cells treated with 1 μg/ml cycloheximide (CHX) for 8 h or isoginkgetin for 0 to 8 h. Puro, puromycin. (E) Analysis of NF-κB luciferase activity in HEK cells transfected with NF-κB luciferase reporter plasmid. Cells were either left unstimulated or pretreated with 10 μM MG132 or 10 μM isoginkgetin for 6 h, followed by TNF-α stimulation for 4 h (n = 3). (F) Western blot analysis of IκB protein levels in HeLa cells treated with TNF-α for 30 min, isoginkgetin for 6 h, or isoginkgetin pretreatment for 6 h followed by a 30-min TNF-α stimulation.
FIG 6
FIG 6
Isoginkgetin induces apoptosis in human MM cell lines in vitro and displays a higher rate of cell death than bortezomib. (A) Cell viability assays and corresponding IC50 values in multiple myeloma cell lines treated with various doses of isoginkgetin or bortezomib (BTZ) for 72 h (n = 3; values graphed as a percentage compared to the values for untreated control samples). (B) Western blot analysis of PARP-1 and tubulin expression in MM cell lines treated with 30 μM isoginkgetin for 8 or 24 h. (C) Analysis of cell viability in MM cell lines treated with 30 μM isoginkgetin or 20 nM bortezomib for 24 h (n = 3) (values graphed as a percentage compared to the values for untreated control samples).
FIG 7
FIG 7
Proposed model of proteasome inhibition and disruption of lysosomal homeostasis via isoginkgetin. Isoginkgetin is a novel inhibitor of the 20S proteasome, which results in compensatory activation of autophagy and the accumulation of ubiquitinated protein aggregates stemming at the perinuclear region. The increased protein load induced by isoginkgetin treatment leads to increased lysosomal acidification, swelling, and repositioning, as well as activation of the transcription factor TFEB. Upon prolonged isoginkgetin treatment, alone or in combination with nutrient starvation, cancer cells undergo apoptotic cell death because of increased proteotoxic stress and impaired protein clearance.

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