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.

Herein, we report our in vitro and in vivo results regarding the use of two ARS derivatives in leukaemia, artesunate (ART) and artemether (ARM), which are water and oil soluble, respectively. The endoplasmic reticulum (ER) represents a complex membranous network that mediates the folding and trafficking of transmembrane and secretory proteins [6]. The unfolded protein response (UPR) pathway consists of three ER transmembrane proteins, including inositol-requiring protein-1 (IRE1), PKR-like ER kinase (PERK) and activating transcription factor 6 (ATF6). Loss of ER homeostasis leads to ER stress, which can be induced by various pathophysiological insults, including oxidative stress [7]. Severe or prolonged ER stress and uncontrollable UPR can activate ER stress-associated cell death signalling [8]. Therefore, activation of ER stress could represent a strategy to lead cancer cells to death and control cancer progression [9].
In our study, we detected decreased proliferation of the leukaemia cell lines, U937 (IC 50 of 3.59 μM) ( Figure 1A) and HL-60 (IC 50 of F I G U R E 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 IC 50 of ART. FACS was used for determination of O 2 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 S1F).
ART presented a higher potential to kill leukemic cells, compared to ARM ( Figure S1G,H). Our data showed a strong NOXA expression in U937 cells after 24 h of ART treatment ( Figure 1C). The increase of NOXA expression has been shown by other [10]. Therefore, our data corroborate that of other ARS derivatives.
Interestingly, our data showed an activation of the PERK and IRE1 branches in vitro, which increase proteins, such as p-eIF2α, ATF4, CHOP, and XBP1 splicing. An increase in phosphorylation of α-subunit of the eukaryotic translation initiation factor-2 (p-eIF2α), which is believed to sense accumulating misfolded protein and attenuation of global translation, was detected transiently after ART treatment, followed by an increase in the transcription factor 4 (ATF4), 6 h later. ATF4 has the ability to target CHOP and switch to a terminal outcome in cases where ER stress response is not resolved [11]. Studies indicated 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 NOXA as an important activator of apoptosis in response to ER stress [12]. Finally, an increase in CHOP protein expression was observed at 6 h after ART treatment, which was maintained until 15 h in U937 ( Figure 1D) and HL-60 cells ( Figure S1J). In addition, we observed an increase of XBP1 splicing in U937 ( Figure 1E) and HL-60 ( Figure S1I) cultures after ART treatment with a continuous increase until 9 h followed by a decrease. We further observed an enhanced in sXBP1 protein expression in the HL-60 cell line after 6 h followed by another increase after 12 and 15 h after ART treatment ( Figure S1J). Thus, our data corroborate the results recently published by Moses and colleagues [13] and suggest that the effects of ART on leukaemia cell lines are mediated, at least in part, by activation of ER stress, thereby reaffirming that CHOP levels may serve as a biomarker for artemisinin actions.
Multiple disturbances can cause accumulation of unfolded proteins in the ER, such as redox regulation induced by oxidants, leading to protein unfolding and misfolding [8]. We measured superoxide levels in the Additionally, in our xenograft model (Figure 2A), ART treatment (200 mg/kg/i.p.) reduced tumour growth ( Figure 2B). Our data demonstrated a reduction in tumour volume (from 1461 ± 178 to 702 ± 216 mm 3 , p = 0.0071) ( Figure 2C) and in tumour mass at the end point (from 1.37 ± 0.12 to 0.73 ± 0.15 g, p = 0.0044) in ARTtreated mice, compared with mice treated with 5% sodium bicarbonate in saline solution ( Figure 2D). No differences were observed in mice weight ( Figure 2E). Additionally, an increase in CHOP (p = 0.0012) ( Figure 2F) and a decrease in CDK2 (p = 0.039) ( Figure 2G Finally, a survival analysis of the acute promyelocytic leukaemia model [14,15] treated with ART was conducted. For the generation of this model, NOD/SCID mice were sub-lethally irradiated with 2 Gy, and 1 × 10 6 leukemic cells were intravenously injected into the tail vein [16]. Blood counts were monitored weekly and, after the confirmation of leukaemia (12th day), mice (n = 10) were submitted to daily intraperitoneally injections of ART (25 mg/kg, i.p.) until death.
An extended survival of ART-treated mice (p = 0.0027) was observed, compared to untreated mice ( Figure 2N).
In summary, our data demonstrate a consistent pattern of ART in reducing tumour burden in the xenograft model, supporting roles in UPR pathway, principally the PERK branch. We speculated that the CHOP-target gene NOXA regulated the cell fate. Moreover, ART was capable of increasing the survival of the PML-RARa model. ART reduced leukaemia cell growth, accompanied by increased apoptosis, G1-phase cell cycle arrest and the reduction of CDKs and cyclin A in vitro. Herein, we hypothesized an important direct antileukemic potential for ART-type drugs ( Figure 2O). Recently, we demonstrated an indirect antileukemic effect of ART through the modulation of monocytes to a tumoricidal phenotype [17]. Taken together, our data indicate that ART may represent an interesting weapon to fight leukaemia.