VLX1570 induces apoptosis through the generation of ROS and induction of ER stress on leukemia cell lines

Abstract

A novel proteasome deubiquitinase inhibitor, VLX1570, has been highlighted as a promising therapeutic agent mainly for lymphoid neoplasms and solid tumors. We examined in vitro effects of VLX1570 on eight myeloid and three lymphoid leukemia cell lines. From cell culture studies, 10 out of 11 cell lines except K562 were found to be susceptible to VLX1570 treatment and it inhibited cell growth mainly by apoptosis. Next, to identify the signaling pathways associated with apoptosis, we performed gene expression profiling using HL-60 with or without 50 nmol/L of VLX1570 for 3 hours and demonstrated that VLX1570 induced the genetic pathway involved in "heat shock transcription factor 1 (HSF1) activation", "HSF1 dependent transactivation", and "Regulation of HSF1 mediated heat shock response". VLX1570 increased the amount of high molecular weight polyubiquitinated proteins and the expression of HSP70 as the result of the suppression of ubiquitin proteasome system, the expression of heme oxygenase-1, and the amount of phosphorylation in JNK and p38 associated with the generation of reactive oxygen species (ROS) induced apoptosis and the amount of phosphorylation in eIF2α, inducing the expression of ATF4 and endoplasmic reticulum (ER) stress dependent apoptosis protein, CHOP, and the amount of phosphorylation slightly in IRE1α, leading to increased expression of XBP-1s in leukemia cell lines. In the present study, we demonstrate that VLX1570 induces apoptosis and exerts a potential anti-leukemic effect through the generation of ROS and induction of ER stress in leukemia cell lines.

Keywords: VLX1570; acute myeloid leukemia; endoplasmic reticulum stress; proteasome deubiquitinase; reactive oxygen species.

FIGURE 1

VLX1570 inhibits the proliferation of leukemia cells and induces apoptosis in most cell lines. (A) Eleven cell lines, including eight myeloid leukemia cell lines (HL‐60, MDS‐L, F‐36P, THP‐1, MOLM13, OCI‐AML3, MV4‐11, K562) and three lymphoid cell lines (MOLT‐4, Jurkat, U937), were cultured with VLX1570 (0‐100 nmol/L) for the indicated times. The cell count was measured by MTT assay. The value without VLX1570 was adjusted to 100%. The data represent the mean values with SD from five independent experiments. Solid line, 24 h; dotted line, 48 h. (B) These cell lines were cultured with different concentrations of VLX1570 (0‐100 nmol/L) for the indicated times and apoptosis was assessed by flow cytometry using annexin V‐FITC/PI staining. The single‐positive fraction for annexin V‐FITC (white bar) implies early apoptosis and the double‐positive fraction for annexin V‐FITC/PI (black bar) implies late apoptosis. The data represent the mean values with SD from three independent experiments. (C) Cell lysates of each cell line treated with or without 100 nmol/L of VLX1570 for 12 h were analyzed by immunoblotting analysis for detection of cleaved PARP (cPARP). The amount of β‐actin is shown as a loading control

FIGURE 2

The antiproliferative effect is related closely to the inhibition of proteasome ubiquitin processing by VLX1570. The cells were treated with or without 50 or 100 nmol/L of VLX1570 for 6 h and cell lysates were analyzed by immunoblotting analysis for detection of high molecular weight polyubiquitinated proteins (A) and HSP70 (B). The amount of β‐actin is shown as a loading control

FIGURE 3

GSEA revealed the activation of the gene sets associated with HSF activation in VLX1570‐treated HL‐60 cells. (A) VLX1570 (50 nmol/L)‐treated or untreated HL‐60 cells were harvested at 3 h. Gene expression profiling was examined in triplicate experiments and obtained data were used for GSEA. The gene set “HSF1 activation”, “HSF1‐dependent transactivation”, and “Regulation of HSF1‐mediated heat shock response” were strongly upregulated by VLX1570 treatment and several statistical values are also presented. (B) Heat map presentation of affected genes included in the gene set is shown in the triplicate experiments (VLX1570 50 nmol/L: 50 nmol/L of VLX1570‐treated; control: untreated)

FIGURE 4

VLX1570 induces ROS generation. (A) HL‐60, MDS‐L, MOLT‐4, and Jurkat cells were treated with indicated doses of VLX1570 for 6 h and the cell lysates were analyzed by immunoblotting analysis for detection of HO‐1. (B) HL‐60 cells were treated with or without 100 nmol/L of VLX1570 or 5 mmol/L of NAC for 3 h and stained with 0.1 μmol/L of CM‐H2DCFDA for 30 min at 37℃. ROS levels were determined by flow cytometry. (C) HL‐60 and MV4‐11 cells were treated with VLX1570 in the absence or presence of NAC for 24 h. The treated cells were stained by Annexin V‐FITC/PI and analyzed by flow cytometry. (D) HL‐60 and MOLT‐4 cells were treated with or without 100 nmol/L of VLX1570 for 3 h and stained with 10 μmol/L of HPF for 30 min at 37℃. ROS levels were determined by flow cytometry. (E) HL‐60 and MOLT‐4 cells were treated with or without indicated doses of VLX1570 for 24 h and stained with 0.2 μmol/L of HYDROP^TM for 30 min at 37℃. ROS levels were determined by flow cytometry. (F) HL‐60 and MOLT‐4 cells were treated with or without indicated doses of VLX1570 for the indicated times and stained with 0.2 μmol/L of HYDROP^TM for 30 min at 37℃. ROS levels were determined by flow cytometry. (G) HL‐60, MV4‐11, and OCI‐AML3 cells were exposed to 100 nmol/L of VLX1570 for 24 h. Mitochondrial membrane potential was detected by rhodamine 123 staining and flow cytometry. Solid line, negative control; dotted line, cells treated with rhodamine 123. (H) HL‐60, MDS‐L, MOLT‐4, and Jurkat cells were treated with indicated doses of VLX1570 for 3 h and the cell lysates were analyzed by immunoblotting analysis for detection of JNK, phospho‐JNK, p38, and phospho‐p38. β‐actin was used as a loading control. (I) HL‐60 and OCI‐AML3 cells were treated with indicated doses of VLX1570 for 12 h and the cell lysates were analyzed by immunoblotting analysis for detection of cleaved caspase‐3, ‐6, and ‐9. β‐actin was used as a loading control. In B, D, E, F, statistical differences were evaluated and presented by asterisks if P values were less than .05

FIGURE 5

VLX1570 exerted an antiproliferative effect on leukemia cells by apoptosis involved in ER stress induction. (A), (B) HL‐60, MDS‐L, MOLT‐4 and Jurkat cells were treated with 0‐500 nmol/L of VLX1570 for 3 h and the cell lysates were analyzed by immunoblotting analysis for detection of XBP‐1s, CHOP, ATF4, BiP, eIF2α, phospho‐eIF2α, and phospho‐IRE1α. For detection of the expression of XBP‐1s, ATF4, and CHOP, the nuclear fraction was extracted as described in the Materials and methods section. β‐actin was used as a loading control. The number of protein bands was measured by densitometry and the ratio was adjusted as 1.0 in the untreated sample and the changes of the ratio of treated samples relative to untreated sample were indicated

FIGURE 6

Proteasome inhibition by VLX1570 induces ROS generation and the activation of unfolded protein response under ER stress followed by apoptosis. VLX1570 induces apoptosis mainly through ROS stress signaling and the unfolded protein response pathway under ER stress. Regarding the PERK signaling pathway, VLX1570 increases the amount of phospho‐eIF2α and the expression of the downstream molecules, ATF4 and CHOP, induction of ER stress‐dependent apoptosis. As another pathway, excess accumulation of polyubiquitinated proteins on the mitochondria induces ROS generation, followed by phosphorylation of JNK and p38, leading to caspase‐3‐ and 9‐dependent apoptosis