Artemisinins induce endoplasmic reticulum stress in acute leukaemia cells in vitro and in vivo

Abstract

Loss of endoplasmic reticulum (ER) homeostasis leads to ER stress, thus prolonged activation can lead to apoptosis. Herein, artesunate (ART) induced ER stress in leukaemia cells, resulting in eIF2α phosphorylation, activation of transcription factor 4, subsequent CHOP upregulation and XBP1 splicing. Furthermore, in vitro cyclin/CDKs reduction induced G1-phase arrest. An in vivo xenograft model showed a consistent pattern of ART in reducing tumour burden, supporting roles in the UPR pathway, which we speculate could lead to apoptosis by NOXA activation. Moreover, ART were capable of increasing the survival of mice. Taken together, our data indicate that ART may represent an interesting weapon to fight leukaemia.

Keywords: acute leukaemia; artemisinin; artesunate; endoplasmic reticulum stress.

FIGURE 1

ART derivatives induce apoptosis mediated by NOXA and activation of the ER stress pathway in leukemic cells. (A) U937 leukemic cells were exposed to increasing concentrations of ART, and cell viability was assessed by MTT following 24 and 48 h of treatment. (B) U937 cells were exposed to increasing concentrations of ART. Cell apoptosis was assessed by Annexin V and flow cytometry. (C) ART changes in NOXA protein after 24 h and (D) the expression of p‐eIF2α, ATF4 and CHOP were analysed in U937 cells treated with ART at different time points. GAPDH was used as loading controls. Relative luminescence units (RLU), compared to untreated cells, are shown (mean ± SEM, n = 3 [cell lines]). (E) XBP1 mRNA was analysed by standard RT‐PCR after ART treatment of U937 cells at different time points. The upper band represents 210 bp (unspliced) and the lower band represents 184 bp (spliced). (F) U937 cells were exposed to IC₅₀ of ART. FACS was used for determination of O₂ ^– production after 18 h. (G) U937 cells were exposed to increasing concentrations of ART for 24 h. FACS was used for determination of cell cycle distribution. (G–M) ART changes the expression of cell cycle‐related proteins after 24 h. Control cells were exposed to DMSO. Data were analysed by ANOVA, followed by post hoc comparisons (Tukey–Kramer test). *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001, significantly different from control cells

FIGURE 2

Effects of ART upon the activation of the PERK branch of the ER stress pathway of U937 xenograft tumours and upon the survival of the PML–RARa model. (A) Schematic diagram showing transplantation of 1 × 10⁷ U937 cells into NOD/SCID mice (Ethics Committee Number 4957‐1/2018) (day 0), followed by control or ART (200 mg/kg) i.p. injections on days 10, 12, 14, 16 and 18. (B) Representative images of tumours after ART treatment for 18 days (compared to controls), showing tumour size reduction. (C) The representative column diagrams show tumour volume weight and (D) tumour mass. The changes in mice weight (E) over the course of time of the experiment in the U937 xenograft model are shown. Western blot analyses of (F) CHOP, (G) CDK2, (H) NOXA, (I) p‐eIF2α, (J) p21, (K) p27, (L) cyclin A and (M) CDK4. (N) ART treatment significantly prolonged survival of the PML–RARa model, showing that ART has antileukemic activity in vivo. (O) Illustration of artesunate hypothetic mechanism in leukaemia cells. The bar graphs show means ± SEM of relative luminescence units (RLU), compared to vehicle mice (n = 5). GAPDH was used as the loading control. **p < 0.01, ***p < 0.001 and ***p < 0.0001, significantly different from control groups