Parthenolide Induces Apoptosis in Committed Progenitor AML Cell line U937 via Reduction in Osteopontin

Abstract

Background: Interfering with cell proliferation and survival is a critical role for antineoplastic drugs leading to cell death through induction of apoptosis. Alternative treatments with herbal extracts offer insights into acute myeloid leukemia (AML) therapy. Parthenolide (PTL), an extract from feverfew, induces apoptosis in primary human leukemia stem cells (LSCs) and bulk leukemic cell populations. Osteopontin (OPN) preserves cell viability in response to anticancer agents and its receptors could be utilized for therapeutic targeting of cancer cells.

Methods: U937 cells were cultured in RPMI 1640 with concentrations of 2, 4, 6, 8, and 10 µM PTL for 20-24 hours for MTT assays. Apoptosis assays were performed with Annexin V-Alexa Fluor-488/PI as Annexin V+/PI- and Annexin V+/PI+ to measure early and late apoptosis, respectively. Quantitative real-time PCR was used to measure OPN gene expression using the 2(-ΔΔCt) method. The PTL-treated cells were stained with FITC-CD38 antibody for flow cytometry analyses. Data were compared using one-way analysis of variance (ANOVA) by SPSS 19.

Results: Parthenolide inhibited growth of U937 cells with IC25 and IC50 values of 4 and 5.8 µM, respectively. Death induction with PTL was apoptotic. Flow cytometry showed a significant decrease in the percentage of CD38+ U937 cells in response to PTL. Osteopontin gene expression decreased in response to PTL.

Conclusion: PTL induced apoptosis and reduced OPN gene expression in U937 cells.

Keywords: AML cell line U937; Osteopontin; Parthenolide.

Fig. 1

Parthenolide induced apoptosis in U937 cells (a) Dose-response curves with different concentrations of PTL using MTT assay/24 h were generated. (b) The performance of the Annexin-V/PI staining on treated cells with PTL. (c) Representative dot-plot diagrams of AV/PI flow cytometry. The graphs represent three independent experiments (mean±SD). *P<0.05, **p<0.01, ***p<0.001 (compared with control or comparisons depicted).

Fig. 2

Parthenolide reduced the CD38+ population of U937 cells. Flow cytometric analysis of PTL-treated/24 h U937 cells stained with monoclonal antibody FITC-CD38.

Fig. 3

Parthenolide decreased OPN expression in U937 cells. Evaluation of mRNA expression of OPN relative to HPRT, using real time RT-PCR after treatment of U937 cells with PTL for 24h. Three independent experiments were performed (mean ± SD). *P < 0.05, **p < 0.01, ***p < 0.001 (compared with control).