Synergistic apoptosis of CML cells by buthionine sulfoximine and hydroxychavicol correlates with activation of AIF and GSH-ROS-JNK-ERK-iNOS pathway

Abstract

Background: Hydroxychavicol (HCH), a constituent of Piper betle leaf has been reported to exert anti-leukemic activity through induction of reactive oxygen species (ROS). The aim of the study is to optimize the oxidative stress -induced chronic myeloid leukemic (CML) cell death by combining glutathione synthesis inhibitor, buthionine sulfoximine (BSO) with HCH and studying the underlying mechanism.

Materials and methods: Anti-proliferative activity of BSO and HCH alone or in combination against a number of leukemic (K562, KCL22, KU812, U937, Molt4), non-leukemic (A549, MIA-PaCa2, PC-3, HepG2) cancer cell lines and normal cell lines (NIH3T3, Vero) was measured by MTT assay. Apoptotic activity in CML cell line K562 was detected by flow cytometry (FCM) after staining with annexin V-FITC/propidium iodide (PI), detection of reduced mitochondrial membrane potential after staining with JC-1, cleavage of caspase- 3 and poly (ADP)-ribose polymerase proteins by western blot analysis and translocation of apoptosis inducing factor (AIF) by confocal microscopy. Intracellular reduced glutathione (GSH) was measured by colorimetric assay using GSH assay kit. 2',7'-dichlorodihydrofluorescein diacetate (DCF-DA) and 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM) were used as probes to measure intracellular increase in ROS and nitric oxide (NO) levels respectively. Multiple techniques like siRNA transfection and pharmacological inhibition were used to understand the mechanisms of action.

Results: Non-apoptotic concentrations of BSO significantly potentiated HCH-induced apoptosis in K562 cells. BSO potentiated apoptosis-inducing activity of HCH in CML cells by caspase-dependent as well as caspase-independent but apoptosis inducing factor (AIF)-dependent manner. Enhanced depletion of intracellular GSH induced by combined treatment correlated with induction of ROS. Activation of ROS- dependent JNK played a crucial role in ERK1/2 activation which subsequently induced the expression of inducible nitric oxide synthase (iNOS). iNOS- mediated production of NO was identified as an effector molecule causing apoptosis of CML cells.

Conclusion/significance: BSO synergizes with HCH in inducing apoptosis of CML cells through the GSH-ROS-JNK-ERK-iNOS pathway.

Figure 1. BSO potentiates HCH induced cytotoxicity in leukemic cells.

(A) K562 cells were incubated with varying concentrations of HCH (left panel) and BSO (right panel) for 48 h and viability was determined by (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Structures of HCH and BSO are shown within the respective graphs. * p<0.05 compared to treatment with vehicle control; ** p<0.01 compared to vehicle control. (B) K562 cells were simultaneously treated with varying concentration of BSO and HCH for 48 h. * p<0.05 compared to HCH treatment alone; ** p<0.01 compared to treatment with HCH alone; *** p<0.001 compared to HCH treatment alone; # p<0.01 compared to treatment with vehicle control or BSO. (C) Leukemic cell lines were incubated with BSO and HCH either alone or in combination for 48 h. *** p<0.001 compared to treatment with vehicle control or either of the compounds alone. (D) Same as (C) except that non-leukemic cancer cell lines were used instead of leukemic cell lines. (E) Normal cell lines or normal human peripheral blood mononuclear cells (hPBMC) were incubated with BSO and HCH either alone or in combination for 48 h. (F) PBMC from one normal donor and 3 CML patients were incubated with BSO and HCH either alone or in combination for indicated time periods and viability was determined by trypan blue dye exclusion assay. ** p<0.01 and *** p<0.001 compared to treatment with vehicle control or either of the compounds alone respectively. (A)-(F) Data represent mean ± SD of quadruplicate wells. (G) Isobologram analysis for the determination of synergy using Calcusyn software.

Figure 2. Sequential administration of BSO potentiates HCH- induced apoptosis in caspase dependent manner.

(A) K562 cells were treated with 100 µM BSO and 10 µM HCH either alone or in combination for indicated times and apoptosis was measured by annexin V/PI binding assay. Dot plots are representative of two similar experiments. (B) hPBMC were treated as indicated for 36 h and apoptosis was measured by annexin V/PI binding assay. Representative of two similar experiments. (C) BSO (100 µM) was administered 24 h prior addition of HCH (10 µM) and vice versa in K562 cells and incubated for 24 h for measurement of apoptosis by annexin V/PI binding assay. Representative of two similar experiments. (D) K562 cells were incubated with 100 µM BSO and 10 µM HCH or their combination for 24 h. Whole cell lysates were subjected to western blot analysis using indicated antibodies. Antibody to actin was used as loading control. (E) K562 cells were pre-treated with pan-caspase inhibitor Z-VAD-FMK (50 µM), caspase 8 inhibitor Z-IETD-FMK (50 µM) and caspase-9 inhibitor LEHD-CHO (25 µM) before treatment with BSO (100 µM) and HCH (10 µM). After 36 hours, cell viability was assessed using MTT assay. Every column represents mean ± SD of three experiments. *** p<0.001 compared to treatment with vehicle control; * p<0.05 compared to treatment with no caspase inhibitors.

Figure 3. Combination of BSO and HCH induces AIF translocation in caspase-independent manner.

K562 cells were treated as indicated and processed for confocal microscopy as stated in Materials and Methods. Scale bar, 10 µm.

Figure 4. BSO potentiates HCH- induced apoptosis by depleting intracellular GSH.

(A) K562 cells were treated as indicated for measurement of intracellular GSH as described in Materials and Methods. Data represent mean ± SD of three experiments. ** p<0.01 and *** p<0.001compared to treatment with BSO alone. (B) K562 cells and hPBMC were treated as indicated for 24 h and intracellular GSH was measured. Data represent mean ± SD of three experiments. # p<0.01 compared to vehicle control; ** p<0.01 compared to BSO alone. (C) K562 cells were treated as indicated for different time points and intracellular H₂O₂ were measured by flow cytometry after DCF-DA staining. Data represent mean ± SD of three experiments. ** p<0.01 compared to treatment with BSO or HCH alone. (D) K562 cells were preincubated with varying concentrations of glutathione monoethyle ester (GME) for 1 h and further incubated with BSO (100 µM) and HCH (10 µM) as indicated for 36 h. Percent apoptotic cells were calculated by flow cytometry after annexinV-PI staining. Data represent mean of three experiments. ** p<0.01 compared to treatment in absence of GME. *** p<0.001 compared to treatment in absence of GME. (E) K562 cells were pre-incubated with 2 mM GME for 1 h and further incubated with BSO (100 µM) and HCH (10 µM) in combination for 36 h. Mitochondrial membrane potential was assessed in a flow cytometer after staining with JC-1 dye. Dot plots and histograms are representative of two similar experiments.

Figure 5. Combination of BSO and HCH induces NO production in CML cells that causes apoptosis.

(A) K562 cells were treated as indicated for different time periods and then intracellular nitric oxide (NO) was measured by flow cytometry after staining with DAF-FM. Data represent mean ± SD of three experiments. ** p<0.01 compared to treatment with either BSO or HCH alone. *** p<0.001 compared to treatment with either BSO or HCH alone. (B) Representative histograms of DAF-FM staining in K562 cells and hPBMC after treatment as indicated for 24 h. (C) K562 cells were pretreated with 100 µM cPTIO for 2 h followed by incubation with combination of 100 µM BSO and 10 µM HCH for 24 h. Cells were then subjected to annexin V/PI binding assay by flow cytometry (left panel). Histograms show measurement of NO in the presence or absence of cPTIO after indicated treatment (right panel). Representative of two similar experiments. (D) K562 cells were treated as indicated for 24 hours after pretreatment with 100 µM cPTIO for 2 h. The whole cell lysates were then immunoblotted with indicated antibodies. (E) K562 cells were pre-incubated with indicated concentrations of GME followed by treatment with BSO and HCH as indicated for 24 h. Analysis of intracellular NO was done by flow cytometry after staining with DAF-FM. Histograms are representative of two similar experiments.

Figure 6. Apoptosis induced by combination of BSO and HCH is mediated by JNK dependent ERK1/2 activation.

(A) K562 cells were treated as indicated for different time periods and cell lysates were subjected to western blot analysis with indicated antibodies. (B) K562 cells were pretreated with JNK inhibitor SP600125 (20 µM), p38 inhibitor SB203580 (20 µM), ERK inhibitor PD98059 (40 µM) for 1 h before treatment with BSO (100 µM) and HCH (10 µM) in combination. After 36 h of incubation, cells were subjected to Annexin V/PI binding assay by flow cytometry. Data represent mean ± SD of three experiments. *** p<0.001 compared to treatment in absence of MAPK inhibitors. (C) Knockdown of JNK by specific JNK1 siRNA attenuates apoptosis. JNK1 protein level was shown after knockdown (upper panel). Annexin V/PI binding assay by flow cytometry in K562 cells after transfection with indicated siRNAs (lower panel). Dot plots are representative of two similar experiments. (D) Knockdown of ERK by specific ERK2 siRNA attenuates apoptosis. ERK2 protein level was shown after knockdown (upper panel). Annexin V/PI binding assay by flow cytometry after knocking down ERK2 (lower panel). Dot plots are representative of two similar experiments (E) K562 cells were transfected with indicated siRNAs for 48 h and then treated with BSO plus HCH for 18 h. Protein expression and phosphorylation of JNK and ERK1/2 were analysed by western blot on whole cell lysates. (F) K562 cells were pre-incubated with or without 2 mM GME for 1 h and further incubated with BSO (100 µM) and HCH (10 µM) in combination for 18 h. The level of each protein and phosphorylation status was analyzed as indicated by western blot. (G) K562 cells were transfected with indicated siRNAs for 48 h and then treated with BSO plus HCH for 24 h. Analysis of intracellular NO was done by flow cytometry after staining with DAF-FM. Histograms are representative of two similar experiments.

Figure 7. Nitric oxide is produced by inducible nitric oxide synthase (iNOS); iNOS expression is dependent on ERK 1/2 and JNK activation.

(A) K562 cells were treated as indicated for varying time periods and the whole cell lysates were subjected to western blot analysis with indicated antibodies. (B) After transfection of K562 cells with indicated siRNAs followed by indicated treatments for 24 h, cells were stained with DAF-FM for measurement of NO. Representative of two similar histograms. (C) K562 cells were transfected with control siRNA or indicated NOS siRNAs for 48 h and then treated with combination of BSO (100 µM) and HCH (10 µM) for 24 h. Whole cell lysates were subjected to western blot for the expression of indicated proteins to confirm knockdown of representative NOS isoforms. (D) K562 cells were subjected to annexin V/PI binding assay by flow cytometry after 36 h. Representative of two similar histograms. (E) K562 cells were transfected with JNK1 or ERK2 siRNA. Cells were then treated with combination of BSO (100 µM) and HCH (10 µM) for 18 h. Cell lysates were then immunoblotted with anti-iNOS antibody. (F) iNOS siRNA transfected K562 cells were treated with combination of BSO (100 µM) and HCH (10µM) for 18 h and subjected to immunoblot analysis with indicated antibodies. (G) K562 cells were pre-incubated with GME for 1 h before treatment with combination of BSO and HCH for 18 h and cell lysates were then immunoblotted with anti-iNOS antibody. (H) GSH measurement was done in iNOS transfected K562 cells after combined treatment with BSO and HCH for 18 h. Data represent mean ± SD of three experiments. *** p<0.001 compared to vehicle control.