Figures & data
Figure 1 Autophagy inhibitors circumvent bone marrow stromal cell-mediated protection against vorinostat cytotoxicity in CLL cells.
Notes: (A) Protection of CLL cells by NKtert cells in the presence of vorinostat. The CLL cells were pre-cultured with NKtert cells for 24 hours, followed by incubation with 0, 0.5, 1, and 2 µM vorinostat for 48 hours. Cell viability was detected using annexin V/PI double staining with flow cytometry analysis. Representative dot plots of a CLL sample are shown, with numbers indicating the percentage of viable cells (annexin V/PI double negative). All experiments were performed three times. (B) Sensitization of CLL cells to vorinostat by autophagy inhibitors CQ or 3-MA. CLL and NKtert cells in co-culture were incubated with vorinostat (2 µM) or in combination with CQ (10 and 20 µM) or 3-MA (1 and 2 mM) for 24 hours and 48 hours. CLL cell viability was analyzed by annexin V/PI assay. Representative dot plots are shown with the percentage of viable cells (annexin V/PI double negative) indicated. All experiments were performed three times. (C) Effect of the combination of vorinostat and autophagy inhibitors (CQ 20 µM or 3-MA 2 mM) on CLL viability in the presence of NKtert cells. The histograms show the mean ± SD of six separate experiments using six CLL samples (*P<0.05; **P<0.01; ***P<0.001).
Abbreviations: CLL, chronic lymphocytic leukemia; Ctrl, control cells without treatment; Vor, vorinostat treatment; Single-CLL, CLL cells cultured alone; Co-CLL, CLL cells co-cultured with NKtert cells; PI, propidium iodide; CQ, chloroquine treatment; 3-MA, 3-methyladenine treatment.
Abbreviations: CLL, chronic lymphocytic leukemia; Ctrl, control cells without treatment; Vor, vorinostat treatment; Single-CLL, CLL cells cultured alone; Co-CLL, CLL cells co-cultured with NKtert cells; PI, propidium iodide; CQ, chloroquine treatment; 3-MA, 3-methyladenine treatment.
Figure 2 Vorinostat induces autophagy and mitophagy in NKtert cells co-cultured with CLL cells.
Notes: (A) Expression of LC3 in CLL cells and NKtert cells. CLL cells and NKtert cells cultured alone or in co-culture were treated with 2 µM vorinostat for 24 hours with or without CQ (20 µM), and cell lysates were assayed for LC3 using Western blot analysis. The upper panel shows the representative Western blot results, and the lower panel shows the quantification of band density of three CLL patients, with β-actin expression as an internal control (mean ± SD; n=3 different CLL samples; *P<0.05). (B) Formation of autophagosomes in NKtert cells. NKtert cells were infected with an adenovirus expressing GFP-LC3 fusion. NKtert cells were cultured alone or co-cultured with CLL cells incubated with or without 2 µM vorinostat for 24 hours, and assayed for the appearance of autophagosomes by confocal microscopy. The left panel shows the representative images of fluorescent LC3 puncta. The right panel shows the mean number of green puncta per cell (mean ± SD; n=3 different CLL samples; *P<0.05). (C) Determination of cellular mitochondrial contents in NKtert cells cultured alone or with CLL cells incubated with or without vorinostat (2 µM, 48 hours), detected by flow cytometry analysis. A representative plot is shown. All experiments were performed three times. (D) Expression of Hsp60 and COX IV in NKtert cells. NKtert cells cultured alone or with CLL cells were treated with or without vorinostat (2 µM, 24 hours and 48 hours), and cell lysates were assayed for Hsp60 and COX IV using Western blot analysis. The upper panel shows the representative Western blot results, and the lower panel shows the quantification of band density of three CLL patients, with β-actin expression as an internal control (mean ± SD; n=3 different CLL samples; *P<0.05; **P<0.01).
Abbreviations: CLL, chronic lymphocytic leukemia; LC3, light chain 3; Co-CLL, CLL cells co-cultured with NKtert cells; Single-CLL, S-CLL, CLL cells cultured alone; CQ, chloroquine treatment; Ctrl, control cells without treatment; V, Vor, vorinostat treatment; GFP, green fluorescent protein; Co-NKtert, NKtert cells co-cultured with CLL cells; Single-NKtert, S-NKtert, NKtert cells cultured alone; Hsp60, heat shock protein 60; COX IV, cytochrome c oxidase subunit IV.
Abbreviations: CLL, chronic lymphocytic leukemia; LC3, light chain 3; Co-CLL, CLL cells co-cultured with NKtert cells; Single-CLL, S-CLL, CLL cells cultured alone; CQ, chloroquine treatment; Ctrl, control cells without treatment; V, Vor, vorinostat treatment; GFP, green fluorescent protein; Co-NKtert, NKtert cells co-cultured with CLL cells; Single-NKtert, S-NKtert, NKtert cells cultured alone; Hsp60, heat shock protein 60; COX IV, cytochrome c oxidase subunit IV.
Figure 3 Release of H2O2 by CLL cells is essential to induce autophagy and mitophagy in bone marrow stromal cells.
Notes: (A) Determination of cellular ROS in NKtert cells cultured alone or with CLL cells incubated with or without 2 µM vorinostat for 30 hours, detected by flow cytometry analysis. A representative plot is shown. All experiments were performed three times. (B) Determination of cellular ROS in CLL cells cultured alone or with NKtert cells incubated with or without 2 µM vorinostat for 30 hours, detected by flow cytometry analysis. A representative plot is shown. All experiments were performed three times. (C) Comparison of H2O2 contents in culture media of CLL cells and NKtert cells. The H2O2 levels in culture media of CLL cells and NKtert cells cultured alone or in co-culture incubated with or without vorinostat (2 µM, 24 hours) were determined by amplex red assay (mean ± SD; n=3 different CLL samples; *P<0.05). (D) Expression of LC3 in NKtert cells. NKtert cells were incubated with 60 µM H2O2 for 8 hours with or without CQ (20 µM), and cell lysates were assayed for LC3 using Western blot analysis. The left panel shows the representative Western blot results, and the right panel shows the quantification of band density of three separate experiments, with β-actin expression as an internal control (mean ± SD; n=3 different CLL samples; *P<0.05). (E) Formation of autophagosomes in NKtert cells by H2O2 treatment. NKtert cells were infected with an adenovirus expressing GFP-LC3 fusion, incubated with 60 µM H2O2 for 8 hours and assayed for the appearance of autophagosomes by confocal microscopy. The left panel shows the representative images of fluorescent LC3 puncta. The right panel shows the mean number of green puncta per cell (mean ± SD; n=3 different CLL samples; **P<0.01). (F) Expression of Hsp60 and COX IV in NKtert cells. NKtert cells were incubated with 60 µM H2O2 for 8 hours and 16 hours, and cell lysates were assayed for Hsp60 and COX IV using Western blot analysis. The left panel shows the representative Western blot results, and the right two panels show the quantification of band density of three separate experiments, with β-actin expression as an internal control (mean ± SD; *P<0.05; **P<0.01). (G) Expression of LC3 in NKtert cells. NKtert cells co-cultured with CLL cells were treated with 2 µM vorinostat and with or without 500 U catalase or 1 mM GSH or 20 µM CQ for 24 hours, and cell lysates were assayed for LC3 using Western blot analysis. The left panel shows the representative Western blot results, and the right panel shows the quantification of band density of three separate experiments, with β-actin expression as an internal control (mean ± SD; **P<0.01).
Abbreviations: CLL, chronic lymphocytic leukemia; Single-NKtert, NKtert cells cultured alone; Co-NKtert, NKtert cells co-cultured with CLL cells; Co-CLL, CLL cells co-cultured with NKtert cells; Vor, vorinostat treatment; Single-CLL, CLL cells cultured alone; Ctrl, control cells without treatment; Hsp60, heat shock protein 60; COX IV, cytochrome c oxidase subunit IV; LC3, light chain 3; CQ, chloroquine treatment; GSH, glutathione.
Abbreviations: CLL, chronic lymphocytic leukemia; Single-NKtert, NKtert cells cultured alone; Co-NKtert, NKtert cells co-cultured with CLL cells; Co-CLL, CLL cells co-cultured with NKtert cells; Vor, vorinostat treatment; Single-CLL, CLL cells cultured alone; Ctrl, control cells without treatment; Hsp60, heat shock protein 60; COX IV, cytochrome c oxidase subunit IV; LC3, light chain 3; CQ, chloroquine treatment; GSH, glutathione.
Figure 4 Vorinostat activates glycolysis in stromal cells cultured with CLL cells.
Notes: (A) Oxygen consumption rate of NKtert cells. NKtert cells cultured alone or with CLL cells were treated with or without 2 µM vorinostat for 24 hours, and measured by an oxygen consumption assay. The slope of the curve reflects the respiratory activity. All experiments were performed four times. (B) Glucose uptake in NKtert cells. NKtert cells cultured alone or with CLL cells were treated with or without 2 µM vorinostat for 24 hours, and incubated with 3H-2-deoxyglucose, and glucose uptake was determined by liquid scintillation counting (mean ± SD; *P<0.05; **P<0.01). All experiments were performed four times. (C) Expression of Glut-1 in cell membrane of NKtert cells. NKtert cells cultured alone or with CLL cells were incubated with 2 µM vorinostat for 24 hours and 48 hours, and membrane fractions isolated from cell lysates were assayed for Glut-1 using Western blot analysis. The upper panel shows the representative Western blot results, and the lower panel shows the quantification of band density of three separate experiments, with Na,K-ATPase expression as an internal control (mean ± SD; *P<0.05; **P<0.01). (D) Expression of HK-II in NKtert cells. NKtert cells cultured alone or with CLL cells were incubated with 2 µM vorinostat for 24 hours and 48 hours, and cell lysates were assayed for HK-II using Western blot analysis. The upper panel shows the representative Western blot results, and the lower panel shows the quantification of band density of three separate experiments, with β-actin expression as an internal control (mean ± SD; *P<0.05; **P<0.01). (E) Lactate generation in NKtert cells. NKtert cells cultured alone or with CLL cells were incubated with 2 µM vorinostat for 24 hours, and lactate in culture media were measured at the indicated time points after changing to fresh culture medium by spectrophotometric assay. All experiments were performed three times. (F) Accumulation of ketone body in NKtert cells. NKtert cells cultured alone or with NKtert cells were incubated with 2 µM vorinostat for 24 hours, and β-hydroxybutyrate in culture media was measured by spectrophotometric assay (mean ± SD; **P<0.01). All experiments were performed three times.
Abbreviations: CLL, chronic lymphocytic leukemia; Single-NKtert, NKtert cells cultured alone; Co-NKtert, NKtert cells co-cultured with CLL cells; V, Vor, vorinostat treatment; Ctrl, control cells without treatment; Glut-1, glucose transporter-1; HK-II, hexokinase-II.
Abbreviations: CLL, chronic lymphocytic leukemia; Single-NKtert, NKtert cells cultured alone; Co-NKtert, NKtert cells co-cultured with CLL cells; V, Vor, vorinostat treatment; Ctrl, control cells without treatment; Glut-1, glucose transporter-1; HK-II, hexokinase-II.
Figure 5 Glycolytic products of stromal cells maintain mitochondrial metabolic function and survival of CLL cells.
Notes: (A) Effect of lactate, ketone and GSH on survival and drug resistance of CLL cells cultured without NKtert cells. CLL cells were pretreated with 2 mM lactate together with 2 mM β-hydroxybutyrate, or 1 mM GSH or the combination of lactate, β-hydroxybutyrate and GSH for 2 hours, or pre-cultured with NKtert cells for 24 hours, followed by 2 µM vorinostat exposure for another 48 hours. Cell viability was detected using annexin V/PI double staining with flow cytometry analysis. The histograms show the mean ± SD of three separate experiments using three CLL samples (n=3 different CLL samples; *P<0.05; **P<0.01). (B) Effect of lactate, ketone and GSH on ATP levels in CLL cells cultured with NKtert cells. CLL cells were pretreated with 2 mM lactate together with 2 mM β-hydroxybutyrate, or 1 mM GSH or the combination of lactate, β-hydroxybutyrate and GSH for 2 hours, or pre-cultured with NKtert cells for 24 hours, followed by 2 µM vorinostat exposure for another 48 hours. ATP contents were examined by luminescent assay (mean ± SD; n=3 different CLL samples; *P<0.05). (C) Effect of NKtert cells on average oxygen consumption in CLL cells. CLL cells cultured alone or with NKtert cells were treated with 2 µM vorinostat for 24 hours, and measured by an oxygen consumption assay. The histograms show the mean ± SD of four separate experiments using four CLL samples (n=4 different CLL samples; *P<0.05), where the histogram represents the slope of the curve from direct measure of total cellular oxygen consumption rate. (D) Effect of NKtert cells or the combination of lactate and ketone body on average oxygen consumption in CLL cells. CLL cells cultured alone or pre-cultured with NKtert cells for 24 hours or pretreated with 2 mM lactate and 2 mM β-hydroxybutyrate for 2 hours, followed by 2 µM vorinostat exposure for another 24 hours, and measured by an oxygen consumption assay. The histograms show the mean ± SD of four separate experiments using four CLL samples (n=4 different CLL samples; *P<0.05; **P<0.01), where the histogram represents the slope of the curve from direct measure of total cellular oxygen consumption rate. (E) Knockdown efficiency of Atg5 in NKtert cells. NKtert cells were infected with Atg5 siRNA or non-targeting siRNA control (NC), and cell lysates were assayed for Atg5 using Western blot analysis. (F) Reduced protective effect of NKtert cells on CLL cells against vorinostat treatment by knockdown of Atg5. NKtert cells infected with Atg5 siRNA or non-targeting siRNA control (NC) were cultured with CLL cells and treated with 2 µM vorinostat for 48 hours. CLL cell viability was analyzed by annexin V/PI assay. The histograms show the mean ± SD of three separate experiments using three CLL samples (n=3 different CLL samples; *P<0.05).
Abbreviations: CLL, chronic lymphocytic leukemia; Vor, vorinostat treatment; GSH, glutathione; PI, propidium iodide; Single-CLL, CLL cells cultured alone; Co-CLL, CLL cells co-cultured with NKtert cells; L, lactate; K, ketone body; NC, non-targeting siRNA control; NC-NKtert, NKtert cells infected with non-targeting siRNA control; SiAtg5-NKtert, NKtert cells infected with Atg5 siRNA.
Abbreviations: CLL, chronic lymphocytic leukemia; Vor, vorinostat treatment; GSH, glutathione; PI, propidium iodide; Single-CLL, CLL cells cultured alone; Co-CLL, CLL cells co-cultured with NKtert cells; L, lactate; K, ketone body; NC, non-targeting siRNA control; NC-NKtert, NKtert cells infected with non-targeting siRNA control; SiAtg5-NKtert, NKtert cells infected with Atg5 siRNA.
Figure 6 The combination of autophagy inhibitor and vorinostat circumvents stromal-mediated drug resistance in CLL cells.
Notes: (A) Sensitization of CLL cells to vorinostat by inhibiting autophagy in stromal cells with CQ or 3-MA. CLL and NKtert or KUSA-H1 or HS5 stromal cells in co-culture were incubated with vorinostat (2 µM) or in combination with CQ (20 µM) or 3-MA (2 mM) for 48 hours. CLL cell viability was analyzed by annexin V/PI assay. The histograms show the mean ± SD of five separate experiments using five CLL samples (n=5 different CLL samples; **P<0.01; ***P<0.001). (B) Effect of the combination of vorinostat and CQ on NKtert cells. NKtert cells were incubated with 2 µM vorinostat and 20 µM CQ for 24 hours and 48 hours. Cell viability was analyzed by annexin V/PI assay. Representative dot plots are shown with the percentage of viable cells (annexin V/PI double negative) indicated. (C) Comparison of cytotoxic effect of vorinostat and CQ or 3-MA combination in CLL cells and normal B cells in co-culture with NKtert cells. Cell viability was analyzed by annexin V/PI assay. The histograms show the mean ± SD of five separate experiments using five CLL samples and normal B cell samples (n=5 samples; *P<0.05; **P<0.01). (D) Effects of combination of vorinostat and CQ or 3-MA on CLL cells in co-culture with NKtert cells in normoxic and hypoxic conditions. CLL cells and NKtert cells in co-culture were exposed to vorinostat (2 µM) and CQ (20 µM) or 3-MA (2 mM) for 48 hours under normoxic (21%) or hypoxic (2%) conditions. Cell viability was analyzed by annexin V/PI assay. The histograms show the mean ± SD of three separate experiments using three CLL samples (n=3 different CLL samples; *P<0.05; **P<0.01).
Abbreviations: CLL, chronic lymphocytic leukemia; CQ, chloroquine; 3-MA, 3-methyladenine; Ctrl, control cells without treatment; Vor, vorinostat treatment; PI, propidium iodide.
Abbreviations: CLL, chronic lymphocytic leukemia; CQ, chloroquine; 3-MA, 3-methyladenine; Ctrl, control cells without treatment; Vor, vorinostat treatment; PI, propidium iodide.
Table S1 Clinical and biological characteristics of 101 chronic lymphocytic leukemia patients