Original Article Withaferin a Activates Stress Signalling Proteins in High Risk Acute Lymphoblastic Leukemia

Int J Clin Exp Pathol 2015;8(12):15652-15660 www.ijcep.com /ISSN:1936-2625/IJCEP0016078 Original Article Withaferin A activates stress signalling proteins in high risk acute lymphoblastic leukemia Li-Huan Shi1, Xi-Jun Wu2, Jun-Shan Liu1, Yin-Bo Gao3 1Key Laboratory of Paediatric Blood Diseases, Children’s Hospital of Zhengzhou, Zhengzhou 450053, China; 2Guizhou Province Key Laboratory of Regenerative Medicine, Guizhou Medical University, Guiyang 550004, China; 3Department of Science and Technology, Children’s Hospital of Zhengzhou, Zhengzhou 450053, China Received September 13, 2015; Accepted October 23, 2015; Epub December 1, 2015; Published December 15, 2015 Abstract: Withaferin A, the principal bio-active component isolated from the Withaniasomnifera, has shown promis- ing anti-leukemic activity in addition to anti-invasive and anti-metastatic activity. The present study demonstrates the effect of withaferin A on the cell cycle status and the phosphorylation/activation of proteins involved in signal transduction in t(4;11) and non-t(4;11) acute lymphoblastic leukemia (ALL) cell lines after treatment with withaferin A. The cells after treatment with the vehicle or 25 μM withaferin A for 1, 2, 4 and 8 h were examined using flow cyto- metric analysis. The results revealed that withaferin A treatment induced cell growth arrest at the S to G2/M phase transition of the cell cycle. Withaferin A treatment also induced the phosphorylation of stress signalling proteins, including the p38 mitogen-activated protein kinase, the c-Jun N-terminal kinase, c-Jun, the heat shock protein 27 and protein kinase B within 0 to 16 h. These results were observed using multiplex technology and Western blotting analysis. Thus withaferin A induces stress response leading to cell death. Therefore, withaferin A can be a potent therapeutic agent for the treatment of high risk ALL with chromosomal translocation t(4;11). Keywords: Withaniasomnifera, antileukemic activity, signal transduction, cell death, translocation Introduction phorylation of vimentin ser56. Not only the withaferin but its analogs also show vimentin- Withaniasomnifera root extract contains 14 targeting and anti-invasive activity. Importantly, withanolides, among which the most abundant WFA has potent anti-metastatic efficacy that is Withaferin A (Figure 1) [1, 2]. The extract has results in vimentin ser56 phosphorylation, with a long traditional medicinal importance in East minimal toxicity to normal tissues [14]. Indian medicine [3]. It is reported that withaferin A induces apoptosis in vimentin express- Despite advancement in molecular biology the ing tumour cells and inhibits soft tissue sarco- chromosomal abnormality is associated with ma growth and local recurrence in xenografts poor prognosis [15, 16]. About 60-85% of [4]. It inhibits vimentin-dependent proteasome infants, 2% of children and 3-6% of adults are activity at micromolar concentration [3, 5]. In diagnosed with acute lymphoblastic leukemia breast and prostate cancers withaferin A has (ALL). ALL is resistant to conventional chemo- shown potent pro-apoptotic and anti-tumour therapeutics; hence the need for a novel effec- activity [6-8]. It exhibits anti-tumour activity tive therapeutic strategy is unmet. Previously, through a variety of targets including NF-Kb, parthenolide has been reported to induced BCL-2, FOXO3A, Hsp90, phosphorylated STAT3 apoptotic cell death in SEM and RS4; 11 cell and annexin II27 [9-13]. Withaferin A is known lines established from patients with ALL carry- to possess anti-invasive and anti-metastatic ing the t(4;11)(q21;q23) chromosomal translo- activity. It inhibits cancer cell motility and inva- cation [17]. Parthenolide induces apoptosis in sion with negligible effects on proliferation in t(4;11) ALL cells at a rapidly compared to the breast cancer at nanomolar concentrations. It REH leukemia cells which have no transloca- leads to vimentin disassembly and hyperphos- tion. Increased generation of ROS including Withaferin A activates stress signalling cycle analysis. FACSCanto fluorescence-activated cell sorter (FACS) with FACSDiva software (Becton-Dickinson, San Jose, CA) was used for analysis of the resulting nuclei. For each sam- ple stained with PI twenty thousand events were collected. ModFit LT software (Verity Software House, Inc., Topsham, ME) was used for the generation of cell cycle profiles. Multiplex measurements of phosphoproteins We used the Bio-PlexPhosphoprotein Detection kit (Bio-Rad, Hercules, CA) to examine the phosphorylation of various proteins. The cells after Figure 1. Structure of withaferin A. treatment with 25 μM withaferin A or DMSO (control) were lysed at 1, 2, 4, 8 and 16 h. Cell nitric oxide, superoxide anion and hypochlorite lysates were prepared with lysate buffer sup- anion was reported in these cells [17]. The plied with the kit as per the manufacturer’s pro- present study was designed to investigate the tocol. The concentration of proteins was mea- effects of withaferin A on intracellular protein sured using the Bio-Rad DC Protein Assay and signalling events leading to apoptosis in leuke- Bovine Á-globulin was used as the standard. mia cells. We observed an enhancement of The measurements were performed in tripli- activation (phosphorylation) of several proteins cates using Bio-Plex multiplex flow cytometry involved in the stress response, including the instrument (Bio-Rad). All the results were pre- heat shock protein 27 (HSP27), protein kinase sented as the mean of the median bead inten- B (Akt), c-Jun N-terminal kinase (JNK), c-Jun and sities normalized to the protein concentration the p38 mitogen-activated protein kinases in each well. (p38 MAPK) on withaferin A treatment. Thus Western blot analysis of total and phosphopro- withaferin A is a potent therapeutic agent for teins ALL by activating stress response proteins. The cells after treatment with DMSO or 25 μM Materials and methods withaferin A were lysed, and using dye-binding Cell lines and reagents method (Bio-Rad) protein concentration was determined. The proteins were resolved on The established SEM and RS4;11 cell lines are 15% SDS-PAGE and transferred to nitrocellu- from patients with high-risk pre-B cell ALL with lose membranes. Digitonin-based subcellular chromosomal translocation t(4;11)(q21;q23) fractionation technique was used for cytosolic [14, 15]. The REH cell line was obtained from and mitochondrial fractions. Onto DS-PAGE the American Type Culture Collection (Ma- equal volumes of cytosolic and mitochondrial nassas, VA). The cell lines were maintained in fractions were resolved and transferred to RPMI-1640 (Invitrogen, Carlsbad, CA) supple- nitrocellulose membranes. The membranes mented with 10% fetal bovine serum (Sigma, after incubation with primary antibody were St. Louis, MO). Withaferin A and other common washed, and then incubated with horseradish chemicals were purchased from Sigma (Sigma, peroxidase anti-mouse or horseradish peroxi- St. Louis, MO). dase anti-rabbit antibodies. Rabbit polyclonal antibodies against phosphorylated and total Cell cycle analysis Akt, p38 MAPK, JNK, c-Jun, phosphorylated HSP27 and mouse antibody against total Cells at a density of 2 × 105/ml were treated HSP27 (Cell Signalling Technology, Danvers, with DMSO or 25 μM withaferin A for 1, 2, 4 and MA) were used. Enhanced chemiluminescence 8 h. The cells were then collected, lysed in a system was used for visualization of immunore- hypotonic solution containing 1 mg/ml sodium active bands. The primary antibodies used citrate, 0.1% Triton X-100, and 50 μg/mL prop- were: thymidylate synthase (TS), acetyl-H3, idium iodide (PI, Sigma) and examined for cell poly-(ADP-ribose)-polymerase (PARP), g-tubulin, 15653 Int J Clin Exp Pathol 2015;8(12):15652-15660 Withaferin A activates stress signalling 15654 Int J Clin Exp Pathol 2015;8(12):15652-15660 Withaferin A activates stress signalling Figure 2. Effect of withaferin A (WFA) on cell cycle arrest. SEM, REH andRS4 leukemia cell lines were treated with DMSO (control) or 25 μM withaferin A for 1 (A), 2 (B), 4 (C) and 8 h (D), and lysed in hypotonic buffer containing PI. The nuclei were collected on a flow cytometer and the percentage of cells in the G1, S and G2/M phases were calculated using ModFit LT software. The data presented are from 3 separate experiments. Figure 3. Effect of withaferin A (WFA) on cell cycle in SEM cells after 8 h of treatment. The cells treated with DMSO (control) or 25 μM withaferin A for 8 h were lysed in hypotonic buffer containing PI. Cell cycle histogram profiles were generated with ModFit LT software. platelet-derived endothelial growth factor (TP) In SEM cells treatment with withaferin A lead to and GAPDH, cleaved caspase 3 and BAX- a significant increase in the population of cells antibodies from Cell Signalling Technology in subG1 phase after 8 h (Figure3). (Boston, MA, USA). Withaferin A treatment activates stress re- Statistical analysis sponse proteins All the results are expressed as the mean of To determine early signalling events involved in three independent experiments. The statistical withaferin A induced leukemic cell apoptosis multiplex technology was used. The phosphory- significance of differences was determined by lation status of each protein involved in signal two-sided Student’s t-test and one-way ANOVA. transduction at 0, 1, 2, 4, 8 and 16 h after with- The differences were considered statistically aferin A treatment was determined. The results significant at P > 0.05. Sigma Stat software showed phosphorylation of several proteins (Systat Software Inc., San Jose, California, USA) involved in the cellular stress responses lead- was used for all statistical evaluations. ing to the apoptosis. In withaferin A treated cells the levels of phosphorylated p38 MAPK Results was significantly higher compared to the control cells (Figure 4A). In SEM and RS4;11 cells Withaferin A induces cell cycle arrest the level of phosphorylated JNK markedly higher than REH cells which in turn expressed high- The preliminary results revealed that withaferin er level compared to untreated cells (Figure A induces apoptosis in t(4;11) ALL cell lines at a 4B).

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