Fig 1.
GalSph- and GlcSph-induced death of MCF7 cells is independent of apoptosis, necroptosis and ferroptosis.
(A) Death of MCF7 cells treated for 48 hours with indicated concentrations of GalSph, GlcSph, ebastine or siramesine was determined by propidium iodine and Hoechst-33342 staining employing Celigo Imaging Cytometer. Connecting lines represent non-linear fit with R2 > 0.99 for each treatment. (B, C) Death of MCF7 cells treated for 48 hours with indicated concentrations of GalSph (B) or GlcSph (C) after 1 hour pre-treatment with indicated concentrations of Z-VAD-FMK was determined as in (A). Staurosporine served as a positive control for apoptotic cell death. (D) Representative Western blots of PARP, caspase-7, and alpha tubulin (loading control) in lysates of MCF7 cells treated for indicated times with GalSph or GlcSph at concentrations corresponding to their LC90 values. Staurosporine served as a positive control for apoptotic cell death. (E-G) Death of MCF7 cells treated for 48 hours with indicated concentrations of GalSph (D), GlcSph (E), or ebastine (F) after 1 hour pre-treatment with indicated concentrations of necrostatin-1 or ferrostatin-1 was determined as in (A). Error bars, SD of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 as analyzed by two-way ANOVA followed by Dunnett’s (B-C) or Sidak’s (E-G) multiple comparisons tests.
Fig 2.
CAD-resistant MCF7 cells are resistant against GalSph- and GlcSph-induced cell death but not to growth inhibition.
(A-D) Death of parental and CAD-resistant MCF7 cells treated for 48 hours with indicated concentrations of GalSph (A), GlcSph (B), siramesine (C, positive control), or ebastine (D, positive control) was determined as in in Fig 1A. (E-H) Total cell numbers of parental and CAD-resistant MCF7 cells treated with indicated concentrations of GalSph (E), GlcSph (F), siramesine (positive control) (G), or ebastine (positive control) (H) for 48 hours were determined by Hoechst-33342 staining employing Celigo Imaging Cytometer. Error bars, SD of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 as analyzed by two-way ANOVA followed by Sidak’s multiple comparisons tests.
Fig 3.
GalSph and GlcSph induce lysosomal membrane permeabilization in MCF7 galectin-3-eGFP cells.
(A) Representative images of MCF7 galectin-3-eGFP cells treated with indicated concentration of GalSph, GlcSph, or ebastine for indicated times (hour:min) were acquired using the ImageXpress (FITC channel). Scale bars, 10 μm. (B) Percentage of galectin-3 puncta positive MCF7 galectin-3-eGFP cells treated as indicated with GalSph, GlcSph, or ebastine and visualized as in (A). (C) Average numbers of galectin-3 puncta in puncta-positive MCF7 galectin-3-eGFP cells treated as indicated with GalSph, GlcSph, or ebastine and visualized as in (A). (D) Death of MCF7 cells treated with GalSph, GlcSph, or ebastine for indicated times (n = 3). Connecting lines present the non-linear fit calculated by GraphPad Prism. Error bars, SD of three independent experiments with >100 randomly chosen cells analyzed per sample. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 as analyzed by unpaired t-test of the area under the curve (AUC) values with Welsh’s correction.
Fig 4.
Extracellular GalSph and GlcSph enter the lysosomes of MCF7 cells.
(A, B) Quantitites of HexSph (GalSph and GlcSph) in cell lysates (cells) and cell culture medium (media) of MCF7 cells treated with 45 μM GalSph (A) or 32 μM GlcSph (B) for indicated times were analyzed by shotgun lipidomics. (C) The molar percentages (mol%) of HexSph in MCF7 cells treated with 45 μM GalSph or 32 μM GlcSph for indicated times were analyzed as in (A). (D-G) The molar percentages of HexSph (D), BMP/PG (E), CL (F), and PC (G) lipid classes in total cell lysates (cells) and in purified lysosomes of MCF7 cells treated as indicated for 3 hours were analyzed as in (A). Error bars, SD of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001, ****; P < 0.0001 comparing the treated values to the first measurement at 0.02 hours (A, B) or to the control appropriate sample (D-G) and analyzed by unpaired t-test (A, B, E-G) or two-way ANOVA followed by Sidak’s multiple comparisons test (D). BMP, bis(monoacylglycero)phosphate; PG, phosphatidylglycerol; CL, cardiolipin; PC, phosphatidylcholine.
Fig 5.
GalSph and GlcSph increase cAMP levels in MCF7 cells.
(A) Representative Western blots of P-CREB and histone H2B (loading control) in lysates of MCF7 cells treated as indicated (three top panels), and densitometric quantification of P-CREB/H2B ratios normalized to untreated control samples (bottom). (B-E) Death of MCF7 cells treated with indicated concentrations of GalSph (B), GlcSph (C), siramesine (D, positive control), or ebastine (E, positive control) for 48 hours was determined as in Fig 1A. When indicated, 2–10 μM forskolin was added 1 hour before the addition of lysosphingolipids or CADs. (F) Representative Western blots of P2RX4 and histone H2B (loading control) in lysates of MCF7 cells transfected with 20 nM non-targeting control (siControl) or 20 nM siP2RX4 siRNAs for 72 hours. (G-I) Death of MCF7 cells transfected as in (F) and treated with indicated concentrations of GalSph (G), GlcSph (H) or siramesine (I, positive control) for the last 48 hours was determined as in Fig 1A. Error bars, SD of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 as analyzed by two-way ANOVA followed by Dunnett’s (A-E) or Sidak’s (G-I) multiple comparisons tests.
Fig 6.
Cholesterol inhibits cell death induced by GalSph and GlcSph in MCF7 cells.
(A-C) Death of MCF7 cells treated for 48 hours with indicated concentrations of GalSph (A), GlcSph (B), or ebastine (C) was determined as in Fig 1A. When indicated, 60 μM cholesterol was added 24 hours before the addition of lysosphingolipids or CADs. (D) Percentage of galectin-3 puncta positive MCF7 galectin-3-eGFP cells treated as indicated with GlcSph and visualized as in (E). When indicated, 60 μM cholesterol was added 24 hours before the addition of lysosphingolipids (n = 2). (E) Representative images of MCF7 galectin-3-eGFP cells treated with indicated concentrations of GlcSph, for 17.5 hours were acquired using the ImageXpress (FITC channel). When indicated, 60 μM cholesterol was added 24 hours before the addition of GlcSph. Scale bars, 10 μm (F-G) Quantities of HexSph (GalSph and GlcSph) in lysates of MCF7 cells treated with 45 μM GalSph (F) or 32 μM GlcSph (G). When indicated, 60 μM cholesterol was added 24 hours before the addition of lysosphingolipids. Error bars, SD of three independent experiments. *, P < 0.05; **, P < 0.01; ****, P < 0.0001 as analyzed by two-way ANOVA followed by Sidak’s multiple comparisons tests.
Fig 7.
Niemann-Pick type C fibroblasts are resistant against GalSph- and GlcSph-induced cell death.
(A-C) Death of fibroblasts from healthy controls or patients with Niemann-Pick type C disease (NPC) treated with indicated concentrations of GalSph (A), GlcSph (B), or ebastine (C) for 48 hours was determined as in Fig 1A. (D-E) Percentage of galectin-3 puncta positive fibroblasts from healthy controls or from patients with Niemann-Pick type C disease after 6 hours (D) or 8 hours (E) of indicated treatments was visualized by immunocytochemistry. A minimum of hundred randomly chosen cells was analyzed per sample. (F) Representative images of fibroblasts from healthy controls or from patients with Niemann-Pick type C disease treated with indicated concentration of GalSph, or GlcSph, or ebastine for 6 hours were acquired using the ImageXpress (DAPI, Cy5 and FITC channels). Scale bars, 10 μm. Error bars, SD of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 as analyzed by two-way ANOVA followed Sidak’s multiple comparisons tests.