Effects of 3OTPCA on phosphatidylserine externalization and caspase-3 cleavage in U937 cells.

<p>(A) Typical histogram and (B) the percentage of apoptotic/necrotic cells in U937 cells treated with 40 μM 3OTPCA for the time indicated. Cells were stained with annexin V-FITC and PI followed by flow cytometry. (C) Caspase-3 cleavage in cells 24 h after 3OTPCA treatment at the concentration indicated. The data represent the mean ± SD (N = 3). **<i>p</i> < 0.01 vs. CT (Student`s t-test).</p

RNA-seq analysis and hierarchical clustering of 1402 transcripts differentially expressed by > 5.0 in U937 cells treated with 3OTPCA.

<p>Strand NGS software was used to perform hierarchical clustering (squared Euclidean distance and Ward’s linkage). Each transcript is represented by a single row of colored bars. Intensity in the green and red color range indicates down-regulated and up-regulated transcripts, respectively. The number in parentheses indicates the number of transcripts.</p

GO analysis of genes that were up-regulated in U937 cells treated with 3OTPCA.

<p>GO analysis of genes that were up-regulated in U937 cells treated with 3OTPCA.</p

Effects of 3OTPCA or thapsigargin on the elevation of intracellular calcium ion concentration in U937 cells.

<p>U937 cells were treated with 40 μM of 3OTPCA or 500 nM of thapsigargin, and then cells were loaded with the calcium-binding dye Fluo-3 AM, and fluorescence was measured by flow cytometry. The data represent the mean ± SD (N = 3). *<i>p</i> < 0.05 **<i>p</i> < 0.01 vs. CT (Student`s t-test).</p

Effects of 3OTPCA on cell viability and DNA fragmentation.

<p>(A) Percentage of viable cells in U937 cells treated with 0–80 μM 3OTPCA for 24 h. Cell viability was monitored using a CCK-8 assay. (B) Morphological change of nuclei was monitored by DAPI staining and fluorescence microscopy at 400× magnification. U937 cells were treated with various concentrations of 3OTPCA for 24 h. (C) DNA fragmentation in U937 cells treated with 3OTPCA for 24 h at the concentration indicated. (D) DNA fragmentation in U937 cells treated with 40 μM 3OTPCA for 3–24 h. (E-F). DNA fragmentation in Jurkat (E) and Molt-4 cells (F) treated with 3OTPCA for 24 h at the concentration indicated. The data represent the mean ± SD (N = 5). **<i>p</i> < 0.01 vs. CT (Student`s t-test).</p

Changes in the expression of UPR proteins, phospho-p38 MAPK, and other apoptotic proteins in U937 cells treated with 40 μM 3OTPCA (3OT) or 500 nM thapsigargin (Tg) for the time indicated.

<p>(A) Time-course analysis of UPR protein expression and p38 MAPK phosphorylation in response to 3OT or Tg. (B) Dose-dependent analysis of caspase-8 cleavage and Bcl family protein expression in cells 24 h after treatment with 3OTPCA. Changes in the expression of these proteins were determined by western blot analysis.</p

3-O-<i>trans-p</i>-coumaroyl-alphitolic acid, a triterpenoid from <i>Zizyphus jujuba</i>, leads to apoptotic cell death in human leukemia cells through reactive oxygen species production and activation of the unfolded protein response

<div><p>3-O-<i>trans-p</i>-coumaroyl-alphitolic acid (3OTPCA), a triterpenoid isolated from the plant <i>Zizyphus jujuba</i> (ZJ), is known to be cytotoxic to cancer cells; however, the molecular mechanism underlying 3OTPCA-induced cell death remains unknown. Here, we provide novel evidence that 3OTPCA induces apoptotic cell death in human leukemia cells. We found that 3OPTCA induces DNA fragmentation within 24 h after treatment in U937 cells, which was also observed in other leukemia cell lines, including Molt-4 and Jurkat cells. We then investigated other parameters involved in apoptosis, including phosphatidylserine externalization and caspase-3 cleavage in U937 cells treated with 3OTPCA. 3OTPCA caused significant DNA fragmentation, annexin-V binding, and caspase-3 cleavage, indicating that 3OTPCA exerts cytotoxicity through apoptosis induction. RNA-seq analysis revealed that the expression of transcripts associated with the unfolded protein response (UPR), such as spliced XBP-1 and CHOP, were up-regulated by 3OTPCA treatment. 3OTPCA-induced UPR activation may be due to endoplasmic reticulum (ER) stress because both 3OTPCA and thapsigargin, an endoplasmic Ca²⁺ transport ATPase inhibitor, increased intracellular calcium levels. 3OTPCA down-regulated the expression of Bcl-2, a target of CHOP, and led to the loss of the mitochondrial membrane, indicating that the intrinsic (mitochondrial) apoptotic pathway was triggered by 3OTPCA, likely through UPR activation. Furthermore, we found that 3OTPCA induced superoxide anion generation and, following p38 MAPK phosphorylation, caspase-8 cleavage without affecting Fas expression. It also induced subsequent Bid cleavage, which may enhance the apoptosis triggered by the intrinsic pathway. These findings reveal for the first time that 3OTPCA induces apoptotic cell death through the generation of reactive oxygen species and activation of UPR.</p></div

Schematic summery of the pathways involved in 3OTPCA-induced apoptosis.

<p>Schematic summery of the pathways involved in 3OTPCA-induced apoptosis.</p

Effects of SB203580 or MnTBAP on 3OTPCA-induced p38 MAPK phosphorylation and DNA fragmentation.

<p>(A, B) Effect of SB203580 on caspase-3/8 cleavage, p38 MAPK phosphorylation (A), and DNA fragmentation (B). U937 cells were pre-incubated with 200 μM SB203580 for 1 h and then co-incubated with 40 μM 3OTPCA followed by immunoblotting and DNA fragmentation assay. (C) Intracellular O₂⁻ and hydroxyl radicals in cells 1 h after 3OTPCA treatment. (D-F) Effect of MnTBAP on DNA fragmentation (D), intracellular O₂⁻ anion production (E), phospho-p38 MAPK, and CHOP expression (F). Cells were pre-incubated with 200 μM MnTBAP for 1 h and then co-incubated with 40 μM 3OTPCA for 24 h followed by the DNA fragmentation assay (D), 1 h followed by staining with HE for flow cytometry (E), or 3 h followed by immunoblotting of p-p38 and 6 h followed by immunoblotting of CHOP (F), *<i>p</i> < 0.05, **<i>p</i> < 0.01. Results are presented as the mean ± SD (n = 3).</p

The structure of 3-O-<i>trans-p</i>-coumaroyl-alphitolic acid (3OTPCA) isolated from <i>Zizyphus jujuba</i>.

<p>The structure of 3-O-<i>trans-p</i>-coumaroyl-alphitolic acid (3OTPCA) isolated from <i>Zizyphus jujuba</i>.</p