primary cells human aml cell lines  (ATCC)

Journal: Cancer Management and Research

Article Title:

Synergistic cytotoxicity of homoharringtonine and etoposide in acute myeloid leukemia cells involves disrupted antioxidant defense

doi: 10.2147/cmar.s187597

Figure Lengend Snippet: Figure 1 Synergistic cytotoxicity of HHT and ETP in AML cells. Notes: (A) THP1 and HL60 cells were treated with vehicle control (Ctrl), HHT, ETP, or in combination as indicated for 48 hours. Cell viability was determined via trypan blue exclusion assay. Results relative to Ctrl are shown (%). (B, C) HL60 cells were treated as in (A). (B) The percentage of apoptotic cells was determined by flow cytometry analysis using annexin-V/propidium iodide double staining. The statistical analysis is shown. (C) The protein expressions of cleaved caspase-9 and cleaved caspase-3 were measured by Western blot analysis. PARP was used as a loading control. (D) Primary AML cells from three patients (AML-1, AML-2, and AML-3) were treated as indicated for 48 hours. Cell viability was determined via trypan blue exclusion assay. Results relative to Ctrl are shown (%). (E, F) Primary AML cells (AML-2) were treated as in (D). (E) The percentage of apoptotic cells was determined by flow cytometry analysis using Annexin-V/propidium iodide double staining. The statistical analysis is shown. (F) The protein expressions of cleaved caspase-9 and cleaved caspase-3 were measured by Western blot analysis. PARP was used as a loading control. Data were obtained from at least three independent experiments and analyzed by Student’s t-test. Data are expressed as mean ± SD. **P<0.01. Abbreviations: AML, acute myeloid leukemia; ETP, etoposide; HHT, homoharringtonine.

Article Snippet: Materials and methods antibodies and reagents The antibodies and reagents used in this study were purchased from the following sources: cleaved caspase-9 (ab25758; abcam, Cambridge, MA, USA), cleaved caspase-3 (C8487; Sigma-Aldrich Co., St Louis, MO, USA), PARP (9542; Cell Signaling, Danvers, MA, USA), thioredoxin (abcam, ab26320), GAPDH (10494–1-AP; Proteintech Group, Inc., Rosemont, IL, USA), goat anti-rabbit IgG-horseradish peroxidase (HRP) (ab6721; abcam), goat anti-mouse IgG-HRP (abcam, ab6789), HHT (H0635; Sigma-Aldrich Co.), ETP (E1383; Sigma-Aldrich Co.), 2′,7′-dichlorofluorescein diacetate (DCFH-DA) (Sigma-Aldrich Co., D6883), N-acetyl-lcysteine (NAC) (Sigma-Aldrich Co.; A7250). aMl cell lines and clinical primary aMl cells Human AML cell lines HL60 and THP1 were purchased from American Type Culture Collection (ATCC) (Manassas, VA, USA) and maintained in RPMI-1640 medium supplemented with 10% FBS at 37°C in a humidified incubator containing 5% CO 2 .

Techniques: Control, Trypan Blue Exclusion Assay, Flow Cytometry, Double Staining, Western Blot

Journal: Cancer Management and Research

Article Title:

Synergistic cytotoxicity of homoharringtonine and etoposide in acute myeloid leukemia cells involves disrupted antioxidant defense

doi: 10.2147/cmar.s187597

Figure Lengend Snippet: Figure 3 ROS scavenge diminishes HHT-augmented ETP cytotoxicity. Notes: (A–C) HL60 cells were treated with vehicle control (Ctrl), HHT, ETP, or in combination in the presence or absence of NAC as indicated for 48 hours. (A) The intracellular ROS level was detected by flow cytometry analysis using DCFH-DA. Results relative to Ctrl are shown. (B) Cell viability was determined via trypan blue exclusion assay. Results relative to Ctrl are shown (%). (C) The protein expressions of cleaved caspase-9 and cleaved caspase-3 were measured by Western blot analysis. PARP was used as a loading control. (D–F) Primary AML cells (AML-2) were treated as in (A). The intracellular ROS level (D), cell viability (E), and protein expressions of cleaved caspase-9 and cleaved caspase-3 (F) were determined as in (A–C). Data were obtained from at least three independent experiments and analyzed by Student’s t-test. Data are expressed as mean ± SD. **P<0.01. Abbreviations: AML, acute myeloid leukemia; DCFH-DA, dichlorofluorescein diacetate; ETP, etoposide; HHT, homoharringtonine; NAC, N-acetyl-l-cysteine; NS, not significant; ROS, reactive oxygen species.

Article Snippet: Materials and methods antibodies and reagents The antibodies and reagents used in this study were purchased from the following sources: cleaved caspase-9 (ab25758; abcam, Cambridge, MA, USA), cleaved caspase-3 (C8487; Sigma-Aldrich Co., St Louis, MO, USA), PARP (9542; Cell Signaling, Danvers, MA, USA), thioredoxin (abcam, ab26320), GAPDH (10494–1-AP; Proteintech Group, Inc., Rosemont, IL, USA), goat anti-rabbit IgG-horseradish peroxidase (HRP) (ab6721; abcam), goat anti-mouse IgG-HRP (abcam, ab6789), HHT (H0635; Sigma-Aldrich Co.), ETP (E1383; Sigma-Aldrich Co.), 2′,7′-dichlorofluorescein diacetate (DCFH-DA) (Sigma-Aldrich Co., D6883), N-acetyl-lcysteine (NAC) (Sigma-Aldrich Co.; A7250). aMl cell lines and clinical primary aMl cells Human AML cell lines HL60 and THP1 were purchased from American Type Culture Collection (ATCC) (Manassas, VA, USA) and maintained in RPMI-1640 medium supplemented with 10% FBS at 37°C in a humidified incubator containing 5% CO 2 .

Techniques: Control, Flow Cytometry, Trypan Blue Exclusion Assay, Western Blot

Journal: Cancer Management and Research

Article Title:

Synergistic cytotoxicity of homoharringtonine and etoposide in acute myeloid leukemia cells involves disrupted antioxidant defense

doi: 10.2147/cmar.s187597

Figure Lengend Snippet: Figure 2 HHT causes elevated ROS generation in AML cells treated with ETP. Notes: HL60 cells (A) and THP1 cells (B) were treated with vehicle control (Ctrl), HHT, ETP, or in combination as indicated for 48 hours. The intracellular ROS level was detected by flow cytometry analysis using DCFH-DA. Results relative to Ctrl are shown. (C) The primary AML cells (AML-2) were treated as indicated for 48 hours. The intracellular ROS level was detected by flow cytometry analysis using DCFH-DA. Results relative to Ctrl are shown. Data were obtained from at least three independent experiments and analyzed by Student’s t-test. Data are expressed as mean ± SD. **P<0.01. Abbreviations: AML, acute myeloid leukemia; DCFH-DA, dichlorofluorescein diacetate; ETP, etoposide; HHT, homoharringtonine; NS, not significant; ROS, reactive oxygen species.

Article Snippet: Materials and methods antibodies and reagents The antibodies and reagents used in this study were purchased from the following sources: cleaved caspase-9 (ab25758; abcam, Cambridge, MA, USA), cleaved caspase-3 (C8487; Sigma-Aldrich Co., St Louis, MO, USA), PARP (9542; Cell Signaling, Danvers, MA, USA), thioredoxin (abcam, ab26320), GAPDH (10494–1-AP; Proteintech Group, Inc., Rosemont, IL, USA), goat anti-rabbit IgG-horseradish peroxidase (HRP) (ab6721; abcam), goat anti-mouse IgG-HRP (abcam, ab6789), HHT (H0635; Sigma-Aldrich Co.), ETP (E1383; Sigma-Aldrich Co.), 2′,7′-dichlorofluorescein diacetate (DCFH-DA) (Sigma-Aldrich Co., D6883), N-acetyl-lcysteine (NAC) (Sigma-Aldrich Co.; A7250). aMl cell lines and clinical primary aMl cells Human AML cell lines HL60 and THP1 were purchased from American Type Culture Collection (ATCC) (Manassas, VA, USA) and maintained in RPMI-1640 medium supplemented with 10% FBS at 37°C in a humidified incubator containing 5% CO 2 .

Techniques: Control, Flow Cytometry

Journal: Cancer Management and Research

Article Title:

Synergistic cytotoxicity of homoharringtonine and etoposide in acute myeloid leukemia cells involves disrupted antioxidant defense

doi: 10.2147/cmar.s187597

Figure Lengend Snippet: Figure 4 HHT causes elevated ROS generation by disabling thioredoxin-mediated antioxidant defense. Notes: (A) HL60, THP1, and primary AML cells (AML-2) were treated with vehicle control (Ctrl), HHT, ETP, or in combination as indicated for 48 hours. The protein expression of Trx1 was determined by Western blot. GAPDH was used as a loading control. The representative images (left) and the quantification of band intensity (right) are shown. (B–D) HL60 cells stably overexpressing vector or Trx1 were treated as in (A). (B) The protein expression of Trx1 was determined by Western blot. GAPDH was used as a loading control. The representative images (upper) and the quantification of band intensity (lower) are shown. (C) The intracellular ROS level was detected by flow cytometry analysis using DCFH-DA. Results relative to Ctrl are shown. (D) Cell viability was determined via trypan blue exclusion assay. Results relative to Ctrl are shown (%). Data were obtained from at least three independent experiments and analyzed by Student’s t-test. Data are expressed as mean ± SD. **P<0.01. Abbreviations: AML, acute myeloid leukemia; DCFH-DA, dichlorofluorescein diacetate; ETP, etoposide; HHT, homoharringtonine; NS, not significant; ROS, reactive oxygen species.

Article Snippet: Materials and methods antibodies and reagents The antibodies and reagents used in this study were purchased from the following sources: cleaved caspase-9 (ab25758; abcam, Cambridge, MA, USA), cleaved caspase-3 (C8487; Sigma-Aldrich Co., St Louis, MO, USA), PARP (9542; Cell Signaling, Danvers, MA, USA), thioredoxin (abcam, ab26320), GAPDH (10494–1-AP; Proteintech Group, Inc., Rosemont, IL, USA), goat anti-rabbit IgG-horseradish peroxidase (HRP) (ab6721; abcam), goat anti-mouse IgG-HRP (abcam, ab6789), HHT (H0635; Sigma-Aldrich Co.), ETP (E1383; Sigma-Aldrich Co.), 2′,7′-dichlorofluorescein diacetate (DCFH-DA) (Sigma-Aldrich Co., D6883), N-acetyl-lcysteine (NAC) (Sigma-Aldrich Co.; A7250). aMl cell lines and clinical primary aMl cells Human AML cell lines HL60 and THP1 were purchased from American Type Culture Collection (ATCC) (Manassas, VA, USA) and maintained in RPMI-1640 medium supplemented with 10% FBS at 37°C in a humidified incubator containing 5% CO 2 .

Techniques: Control, Expressing, Western Blot, Stable Transfection, Plasmid Preparation, Flow Cytometry, Trypan Blue Exclusion Assay

Journal: Cancer Management and Research

Article Title:

Synergistic cytotoxicity of homoharringtonine and etoposide in acute myeloid leukemia cells involves disrupted antioxidant defense

doi: 10.2147/cmar.s187597

Figure Lengend Snippet: Figure 5 Depletion of thioredoxin sensitizes AML to ETP treatment. Notes: (A–C) HL60 cells transfected with siCtrl or siTrx1 were treated with vehicle control (Ctrl) or 2 µM ETP for 48 hours. (A) The protein expression of Trx1 was determined by Western blot. GAPDH was used as a loading control. The representative images (left) and the quantification of band intensity (right) are shown. (B) The intracellular ROS level was detected by flow cytometry analysis using DCFH-DA. Results relative to Ctrl are shown. (C) Cell viability was determined via trypan blue exclusion assay. Results relative to Ctrl are shown (%). Data were obtained from at least three independent experiments and analyzed by Student’s t-test. Data are presented as mean ± SD. **P<0.01. (D) Brief schematic graph of a model for the role of HHT in augmenting ETP cytotoxicity in AML cells through targeting thioredoxin-mediated ROS elimination. Abbreviations: AML, acute myeloid leukemia; DCFH-DA, dichlorofluorescein diacetate; ETP, etoposide; HHT, homoharringtonine; NS, not significant; ROS, reactive oxygen species.

Article Snippet: Materials and methods antibodies and reagents The antibodies and reagents used in this study were purchased from the following sources: cleaved caspase-9 (ab25758; abcam, Cambridge, MA, USA), cleaved caspase-3 (C8487; Sigma-Aldrich Co., St Louis, MO, USA), PARP (9542; Cell Signaling, Danvers, MA, USA), thioredoxin (abcam, ab26320), GAPDH (10494–1-AP; Proteintech Group, Inc., Rosemont, IL, USA), goat anti-rabbit IgG-horseradish peroxidase (HRP) (ab6721; abcam), goat anti-mouse IgG-HRP (abcam, ab6789), HHT (H0635; Sigma-Aldrich Co.), ETP (E1383; Sigma-Aldrich Co.), 2′,7′-dichlorofluorescein diacetate (DCFH-DA) (Sigma-Aldrich Co., D6883), N-acetyl-lcysteine (NAC) (Sigma-Aldrich Co.; A7250). aMl cell lines and clinical primary aMl cells Human AML cell lines HL60 and THP1 were purchased from American Type Culture Collection (ATCC) (Manassas, VA, USA) and maintained in RPMI-1640 medium supplemented with 10% FBS at 37°C in a humidified incubator containing 5% CO 2 .

Techniques: Transfection, Control, Expressing, Western Blot, Flow Cytometry, Trypan Blue Exclusion Assay