Curcuminoid B63 induces ROS-mediated paraptosis-like cell death by targeting TrxR1 in gastric cells.

Gastric cancer is one of the leading causes of cancer-related deaths. Chemotherapy has improved long-term survival of patients with gastric cancer. Unfortunately, cancer readily develops resistance to apoptosis-inducing agents. New mechanisms, inducing caspase-independent paraptosis-like cell death in cancer cells is presently emerging as a potential direction. We previously developed a curcumin analog B63 as an anti-cancer agent in pre-clinical evaluation. In the present study, we evaluated the effect and mechanism of B63 on gastric cancer cells. Our studies show that B63 targets TrxR1 protein and increases cellular reactive oxygen species (ROS) level, which results in halting gastric cancer cells and inducing caspase-independent paraptotic modes of death. The paraptosis induced by B63 was mediated by ROS-mediated ER stress and MAPK activation. Either overexpression of TrxR1 or suppression of ROS normalized B63-induced paraptosis in gastric cancer cells. Furthermore, B63 caused paraptosis in 5-fluorouracil-resistant gastric cancer cells, and B63 treatment reduced the growth of gastric cancer xenografts, which was associated with increased ROS and paraptosis. Collectively, our findings provide a novel strategy for the treatment of gastric cancer by utilizing TrxR1-mediated oxidative stress generation and subsequent cell paraptosis.

Introduction

Gastric cancer is the second leading cause of cancer-related deaths worldwide [1]. Most patients are asymptomatic in the early stages of the disease and most cases are diagnosed with distant metastasis. At this advanced stage, gastric cancer is largely incurable [2], [3]. Targeted chemotherapy has increased long-term survival of patients with gastric cancer [4]. However, severe adverse effects and complications arising from chemotherapy pose yet another clinical challenge [5]. Therefore, new drugs and/or new therapeutic combinations are needed for the treatment of patients with gastric cancer. We have previously developed a novel analog of curcumin, 1,5-bis(2-methoxyphenyl) penta-1,4-dien-3-one (B63, Fig. 1A), which inhibited human non-small cell lung cancer (NSCLC) cells while lacking any observable toxicity in normal cells [6]. We also found that B63 afforded this inhibitory activity against lung cancer cells through induction of ER stress-mediated apoptotic pathway [6]. Although this compound is being in pre-clinical evaluation as an anti-cancer agent, the precise mechanism of its anti-cancer actions is still unclear.

Fig. 1

B63 reduces gastric cancer cell viability and generates ROS. (A) Chemical structure of compound B63. (B) Effect of B63 on the viability of human gastric cancer cells. Cells were treated with increasing concentrations of B63 for 24 h and cell viability was measured by MTT assay. IC50 values in three different cell lines are shown. (C) Intracellular ROS generation in SGC-7901 cells exposed to B63. Cells were challenged with 5, 10, or 20 μM B63 for 2 h and then stained with ROS probe DCFH-DA. NAC pretreatment was carried out at 5 mM for 1 h. Representative histogram is shown. (D) Quantification of ROS levels in SGC-7901 cells as determined by DCFH-DA probe [n = 3; *P < 0.05, **P < 0.01 compared to DMSO; #P < 0.05 compared to B63-20]. (E) Representative images of cells stained with DCFH-DA. Cells were treated as indicated in panel C [scale bar = 20 µm]. (F) Quantification of DCFH-DA staining intensity [n = 3; *P < 0.05, ***P < 0.001 compared to DMSO; ##P < 0.01 compared to B63-20].

Since cancer cells may develop different adaptive mechanisms to escape apoptotic cell death, candidates with new anti-cancer strategies or mechanisms need be developed in the fight against cancer. Among new mechanisms, inducing caspase-independent paraptosis-like cell death in cancer cells is presently emerging as a potential direction [7]. Paraptosis, a new form of non-apoptotic cell death, is characterized by a process of cytoplasmic vacuolization that begins with progressive swelling of endoplasmic reticulum (ER) and mitochondria [8], [9], [10], [11]. This form of cell death typically does not respond to caspase inhibitors nor does it involve formation of the apoptotic characteristics such as pyknosis, DNA fragmentation, or caspase activation [8], [12]. Paraptosis is known to require new protein synthesis, and recent studies have confirmed the key protein AIP-1/Alix as an inhibitor of paraptosis [12]. Paraptosis has recently been proposed as an emerging therapeutic strategy to overcome apoptosis-based resistance and to effectively inhibit drug-resistant tumor growth [7], [13], [14]. It has been reported that exposure of glioblastoma cell lines to curcumin caused morphological change characteristic of paraptosis cell-death [15]. In addition, curcumin causes breast cancer cell death primarily through paraptosis, which is in turn mediated through the generation of reactive oxygen species (ROS) [16], [17]. Induction of caspase-independent cell death in cancer cells by compounds may be an effective strategy to develop new anti-cancer agents and overcome resistance.

The purpose of this present study was to examine the effect of B63 on gastric cancer cells. Our previous study already showed that B63 inhibits NSCLC. Here we hypothesized that B63 will be effective in preventing gastric cancer growth. To test our hypothesis, we utilized gastric cancer cells in culture and in a xenograft model. Our study shows that B63 inhibits the growth of gastric cancer cells and induces primarily a paraptosis-like mode of cell death. We also found that pro-paraptotic activity of B63 in gastric cancer cells was mediated through directly targeting thioredoxin reductase-1 (TrxR1) protein and increasing cellular ROS level. Interestingly, B63 was also effective against chemotherapy-resistant gastric cancer lines. Together, our results suggest that B63 may provide an effective therapeutic strategy for gastric cancer.

Materials and methods

Cell culture and reagents

Human gastric cancer cell lines SGC-7901, BGC-823, and SNU-216, human non-small cell lung cancer (NSCLC) H1975 and H460 cells, human pancreatic cancer Patu8988T cells, and human triple negative breast cancer (TNBC) MDA-MB-231, were purchased from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). 5-flurouracil-resistant lines were prepared from BGC-823 as described by us previously [18]. MDA-MB-231 was cultured in L-15 Medium (Gibco, Eggenstein, Germany); other cell lines were cultured in RPMI 1640 media (Gibco, Eggenstein, Germany) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Hyclone, Logan, UT), 100 units/mL penicillin, and 100 µg/mL streptomycin.

Antioxidant N-acetyl cysteine (NAC), L-Glutathione reduced (GSH), Catalase, Trolox, Catechin hydrate (CTH), Lipoic acid (LAD), Vitamin E (Vita-E), Butylated hydroxyanisole (BHA), 2,6-Di-tert-butyl-4-methylphenol (BHT), protein synthesis inhibitor cycloheximide (CHX), caspase inhibitor Z-VAD-FMK, and 5-fluoruracil (5-FU) were all purchased from Sigma-Aldrich (St. Louis, MO). Mitogen-activated protein kinase kinase (MAPKK, MEK) inhibitor U0126, pan-p38 inhibitor doramapimod (BIRB 796), and broad-spectrum c-Jun N-terminal kinase (JNK) inhibitor SP600125 were purchase from Selleck Chemicals (Houston, TX). Reactive oxygen species (ROS) inducer auranofin (AF) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Fluorescein isothiocyanate (FITC) Annexin V Apoptosis Detection Kit I and propidium iodide (PI) were purchased from BD Pharmingen (Franklin Lakes, NJ). ROS probes 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA), 3-Amino,4-aminomethyl-2′,7′-difluorescein diacetate (DAF-FM DA), and dihydroethidium (DHE) were obtained from Beyotime Biotech (Nantong, China). Malondialdehyde (MDA) determination assay was also obtained from Beyotime Biotech. Antibodies against cell division cycle protein 2 (Cdc2), Bcl-2, Bax, cyclin B1, thioredoxin reductase 1 (TrxR1), cleaved poly ADP ribose polymerase (PARP), murine double minute (MDM-2), Ki67, and GAPDH were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against activating transcription factor-4 (ATF-4), eukaryotic initiation factor 2 (EIF2α, and phospho/p-EIF2α), Alix, p-P38, P38, extracellular signal-regulated kinase (ERK and p-ERK), CCAAT/enhancer-binding protein homologous protein (CHOP), c-Jun N terminal kinase (JNK and p-JNK), and cleaved caspase3 were purchased from Cell Signaling Technology (Danvers, MA). Horseradish peroxidase (HRP)-conjugated secondary antibodies were obtained from Santa Cruz Biotechnology.

Cell viability assay

To measure viability of cells, we plated 8 × 103 cells per well of a 96-well plate. Cells were allowed to attach overnight in complete growth media and were then treated with B63 or 5-FU (dissolved in DMSO; diluted in RPMI medium) for 24 or 48 h before performing the MTT assay.

Determination of intracellular reactive oxygen species (ROS)

Intracellular ROS contents were measured by flow cytometry utilizing DCFH-DA, DAF-FM-DA, and DHE, respectively. Briefly, 5 × 105 cells were plated on 60-mm dishes, allowed to attach overnight, and then treated with B63 (5, 10 or 20 μM) for 3 h. Antioxidants (NAC, GSH, etc) pretreatment, where indicated, was carried out for 1 h. Cells were stained with 10 μM DCFH-DA, 5 μM DAF-FM-DA, or 1 μM DHE at 37 °C for 30 min in the dark, respectively. DCF, DAF, or DHE fluorescence (produced in the presence of ROS) was analyzed using flow cytometry. Images were also captured using Nikon epifluorescence microscope equipped with a digital camera (Nikon, Japan).

Cell apoptosis analysis

For cell death analysis, cells were treated with B63 (5, 10 or 20 μM) for 24 h. Cells were then harvested, and apoptotic cell death was evaluated by double staining cells with FITC conjugated Annexin V and PI for 10 min.

Western blot analysis

Lysates from cells and tumor tissues were prepared and protein levels determined using the Bradford assay (Bio-Rad, Hercules, CA). Proteins were separated by 10% SDS-PAGE and transferred to poly-vinylidene difluoride transfer membranes. The blots were blocked for 2 h at room temperature with freshly prepared 5% nonfat milk in TBST and then incubated with specific primary antibodies overnight at 4 °C. HRP-conjugated secondary antibodies and ECL substrate (Bio-Rad) were used for detection.

Cell transfections for gene silencing or overexpression

To knockdown ATF4 expression, SGC-7901 and BGC-823 cells were plated in 6-well plates at a density of 5 × 104 and cultured for 24 h. siRNA against ATF4 or non-targeting control were transfected at a final concentration of 50 pmol mL−1 using lipofectamine 3000 reagent (Invitrogen, CA). Culture medium was replaced with fresh medium after 6–8 h and cells were incubated for an additional 36 h. Then, cells were treated with 20 μM B63 for 3 h and used for subsequent experiments. siRNA oligonucleotides were synthesized by GenePharma(Shanghai, China). A pool of two siRNA against ATF4 were used. Sequence 1: (sense: 5′-GCCUAGGUCUCUUAGAUGATT-3′, antisense: 5′-UCAUCUAAGAGACCUAGGCTT-3′); Sequence 2: (sense: 5′-GCGUAGUUCGCUAAGGUGATT-3′, antisense: 5′-UCACCUUAGCGAACUACGCTT-3′).

To express TrxR1, the recombinant plasmid vector coding TrxR1 protein was obtained from Addgene (Plasmid #38863, Addgene, Cambridge, MA). The TXNRD1 plasmid was transfected into SGC-7901 and BGC-823 using Lipofectamine 3000 reagent (Invitrogen). After 24 h post transfection, TXNRD1 expression in SGC-7901 and BGC-823 cells was confirmed by Western blotting analysis.

Electron microscopy

SGC-7901 cells were treated with vehicle control (DMSO) or 20 µM B63. NAC pretreatment where applicable was carried out for 1 h. Following treatment, cells were fixed with 2.5% glutaraldehyde overnight at 4 °C. The cells were then post-fixed in 1% OsO4 at room temperature for 60 min, stained with 1% uranyl acetate, dehydrated through graded acetone solutions, and embedded in epon. Areas containing cells were block-mounted and cut into 70 nm sections and examined with an electron microscope (H-7500, Hitachi, Ibaraki, Japan).

Thioredoxin reductase-1 activity assays

TrxR1 activity was determined at room temperature using SpectraMax M5 microplate reader (Molecular Devices, USA). NADPH-reduced TrxR1 (160 nM) was incubated with various concentrations of B63 for 2 h at room temperature in 96-well plates. A mixture of TE buffer (50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 50 mL) containing 5,50-dithiobis(2-nitrobenzoate) (DTNB) and NADPH was added to achieve final concentrations of 2 mM and 200 mM, respectively. The linear increase in absorbance at 412 nm during the initial 3 min was recorded. The same amounts of DMSO (1%, v/v) were added to the control experiments and the activity was expressed as the percentage of the control.

TrxR1 activity in cells was measured by end-point insulin reduction assay as described previously [19], [20]. Briefly, 100 μg total proteins were incubated in a final reaction volume of 50 μL, containing 100 mM Tris-HCl (pH 7.6), 0.3 mM insulin, 660 μM NADPH, 3 mM EDTA, and 15 μM E. coli-derived Trx (Sigma) for 1 h at 37 °C. The reaction was terminated by adding 200 μL of 1 mM 2,4-dinitrochlorobenzene (DTNB). A blank sample, containing everything except Trx, was treated in the same manner. The absorbance at 412 nm was measured, and the blank value was subtracted from the corresponding absorbance value of the sample. The activity was expressed as the percentage of the control.

Molecular docking of B63 to the TrxR1 structural model

CovalentDock, which was written based on Autodock, was implemented to predict the interaction of B63 binding to TrxR1 [21]. The crystal structure of human TrxR1 used for this docking was obtained from the Protein Data Bank (PDB ID 2ZZ0, chain A) and prepared by using PyMOL, including removing water molecules and adding hydrogens. Then, minimization was performed to avoid local collision. A grid box of 60 × 60 × 60 points centering on the coordinate of −29.11, −1.26, and −6.55 was implemented, which enclose the whole redox motif. Other parameters were set as default during the docking.

Gastric cancer xenografts

Animal studies conducted are in compliance with the ARRIVE guidelines [22], [23]. All experimental procedures were approved by the Institutional and Local Committee on the Care and Use of Animals of Wenzhou Medical College. All animals received humane care according to the National Institutes of Health (USA) guidelines.

Five-week-old athymic BALB/c nu/nu female mice (18–22 g; N = 18) were purchased from Vital River Laboratories (Beijing, China). Animals were housed at a constant room temperature with a 12/12-hr light/dark cycle and fed a standard rodent diet. The mice were randomly divided into four experimental groups. All investigators performing analyses were blinded to the experimental group allotment. SGC-7901 cells were subcutaneously injected into the right flank of each mouse at 1 × 106 cells in 0.1 mL PBS. When tumors reached a volume of 100–200 mm3, mice were treated by intraperitoneal (i.p.) injection of 20 mg/kg B63 or 50 mg/kg curcumin (in a solution of PBS with 6% castor oil) once every two days. Control mice received vehicle alone. All mice were treated for 14 days. The tumor volumes were determined by measuring length (l) and width (w) and calculating volume (V = 0.5 × l × w2) at the indicated time points. At the end of study, mice were sacrificed after being anesthetized by intraperitoneal injection with pentobarbital (50 mg/kg). Tumor specimens were harvested and weighed. Samples were then processed for histology and protein assays.

Tissue staining

Harvested tumor tissues were fixed in 10% formalin and embedded in paraffin. Specimens were sectioned at 5 µm thickness. Tumor sections were stained using routine immunohistochemical techniques with primarily antibodies against p-EIF2α (1:100), cleaved caspase 3 (1:100), Alix (1:100) or ki-67 (1:200). HRP-conjugated secondary antibodies and diaminobenzidine (DAB) were used for detection. Tumor specimens as well as heart, liver and kidney tissues were also stained with hematoxylin and eosin (H&E).

Frozen tissue sections were used for immunofluorescence detection of ROS. Sections were incubated with DCFH-DA. Slides were counterstained with DAPI and images were captured.

Malondialdehyde (MDA) assay

Tumors tissue lysates were prepared, and protein concentrations were determined by the Bradford assay. Malondialdehyde (MDA) levels were measured by using Lipid Peroxidation MDA assay kit (Beyotime Institute of Biotechnology).

Statistical analysis

Statistical analyses were performed only when a minimum of n = 3 independent samples were acquired. All data are reported as mean±SEM. Statistical analysis was performed with GraphPad Prism 6.0 software (GraphPad, San Diego, CA, USA). We used one-way ANOVA followed by Dunnett's post hoc test when comparing more than two groups of data, and one-way ANOVA, non-parametric Kruskal–Wallis test followed by Dunn's post hoc test when comparing multiple independent groups. When comparing two groups, the unpaired Student's t-test was used. A p value < 0.05 was considered statistically significant.

Results

B63 reduces gastric cancer cell viability and induces reactive oxygen species

We first wanted to establish the effect of B63 on gastric cancer cells. Three gastric cancer lines, SGC-7901, BGC-823, and SNU-2016 were utilized. We exposed the cells to increasing concentrations of B63 for 24 h and measured cell viability. In all three cell lines, B63 reduced viability to half at approximately 9–12 µM (Fig. 1B). As a comparison, curcumin, the parent compound of B63, reduced viability by half but only with concentrations greater than 30 µM (data not shown). Exposure of gastric cancer cells to B63 for 48 h yielded IC50 values in 6–7 µM range (Supplementary Fig. S1).

Our recent studies have shown that several curcumin analogs increased reactive oxygen species (ROS) level to induce cell death in gastric and colon cancer cells [24], [25], [26]. Curcumin also induces cell death through an increase in oxidative stress in gastric cancer cells [27], [28]. Therefore, we determined whether reduced viability in gastric cancer cells following exposure to B63 was associated with increased generation of ROS. We measured ROS generation in cells through 2′,7′–dichlorofluorescin diacetate (DCFH-DA) staining. DCFH-DA undergoes hydrolysis and then ROS-mediated oxidation to a fluorescent state, providing a measure of ROS levels in cells. Our results show that exposure of SGC-7901 or BGC-823 gastric cancer cells to B63 does indeed increase ROS levels (Fig. 1C, D, Supplementary Fig. S2A, S2B). This increased ROS generation can also be qualitatively appreciated in cells with B63 challenge using fluorescence intensity assay detected by a fluorescence microscope (Fig. 1E, F, and Supplementary Fig. S2C, S2D). However, pre-treated with the N-acetyl cysteine (NAC), a specific ROS inhibitor, for 1 h significantly reversed the B63-induced increase in ROS levels in both cell lines (Fig. 1C-F and Supplementary Fig. S2A-D). We also measured ROS generation using other ROS probes, DAF-FM-DA and DHE. Interestingly, neither DAF-FM-DA nor DHE probes detected the B63-induced ROS in SGC-7901 cells (Supplementary Fig. S3). To identify the ROS species induced by B63, we used different ROS scavengers, including NAC, BHT, Trolox, GSH, Catalase, BHA, CTH, Vita-E, and LAD to block B63-induced ROS. Interestingly, only thiol-containing antioxidants NAC and GSH could reverse ROS generation induced by B63 in SGC-7901 cells, while other ROS scavengers could not achieve this effect (Supplementary Fig. S4). These data suggested that B63 induced ROS generation probably via targeting thioredoxin redox system.

Thioredoxin/thioredoxin reductase is a potential target of B63

Recent studies have shown that thioredoxin/thioredoxin reductase (TrxR) system contributes to tumor cell resistance to oxidative stress-induced cell death [29]. TrxR1 is overexpressed in human cancers and plays a role in regulating intracellular redox balance [30]. Our previous studies also showed that curcumin analogs B19 and EF24 can directly target TrxR1 to increase ROS level in gastric cancer cells [25], [26]. This prompted us to determine whether the structurally similar B63 target TrxR system to generate ROS. To gain insight into this potential mechanism, we determined TrxR1 enzymatic activity using human recombinant TrxR1 protein in a cell-free system, as well as in SGC-7901 cell lysates prepared following exposure of cells to increasing concentrations of B63. Our results show that B63 suppresses TrxR1 activity in a dose-dependent manner (Fig. 2A, B). These results indicate that TrxR1 may be one of the targets of B63. Hence, we carried out molecular simulation docking of B63-TrxR1 complex using AutoDock. Our result in Fig. 2C shows that B63 can insert into the C-terminal active site of TrxR1 and forms a strong covalent bond with Cys-497 of TrxR1 protein, resulting in an occupation in TrxR1 redox active center to block the natural enzymatic recognition. These findings are certainly suggestive of TrxR1 as a target of B63. If true, then overexpression of TrxR1 would be expected to dampen the effect of B63. To test this, we over-expressed TrxR1 in gastric cancer cells (Fig. 2D) and measured ROS level. Our results show that TrxR1 overexpression reduces B63-induced ROS generation (Fig. 2E, F). Importantly, this overexpression also significantly increased the IC50 values obtained in our viability test following B63 challenge (Fig. 2G). These findings show that B63 may target TrxR1 to generate ROS and lead to reduced cell viability.

Fig. 2

B63 targets and inactivates TrxR1 in human gastric cancer cells. (A, B) TrxR1 enzyme activity was measured using recombinant human TrxR1 protein in cell-free system and in lysates prepared from cells, respectively. Cell-free TrxR1 inhibition by B63 was determined by the DTNB assay utilizing recombinant human TrxR1 (A), and in SGC-7901 lysates (B) by end-point insulin reduction assay [n = 3; *P < 0.05, **P < 0.01, ***P < 0.001 compared to DMSO]. (C) Molecular interaction between B63 and TrxR1 was simulated by docking software. (D) Western blotting analysis of TrxR1 overexpression in SGC-7901 and BGC-823 cells [Con = no transfection, veh = control vector, TrxR1 = TXNRD1 plasmid transfection]. Lower panels show densitometric quantification [n = 3; **P < 0.01, ***P < 0.001 compared to DMSO]. (E) Effect of overexpressing TrxR1 on B63-mediated ROS production in gastric cancer cells. ROS levels were detected by DCFH-DA staining. Representative flow cytometry histograms are shown (F) Quantification of ROS levels as determined by DCFH-DA staining [n = 3; *P < 0.05, **P < 0.01 compared to indicated comparator]. (G) Effect of expressing TrxR1 in SGC-7901 and BGC-823 cells on viability altered by B63.

Decreased viability in gastric cancer cells by B63, is for the most part, through induction of paraptosis-like cell death

We further examined cell death induction by B63. We examined cell death by staining cells with Annexin V/PI. Our results show that B63 induces Annexin V/PI double-positivity in gastric cancer cell in a dose-dependent manner (Fig. 3A-B and Supplementary Fig. S5A-B). Induction of cell death was not seen in cells pretreated with NAC, indicating a key role of ROS generation in gastric cell death. Analysis of protein mediators of apoptosis showed increased levels of cleaved Poly (ADP-ribose) polymerase (PARP) in cells exposed to B63 (Fig. 3C and Supplementary Fig. S5C). PARP is a target of active caspase-3 and these results indicate caspase-3 activation. In support of this notion, levels of Bcl2 protein were also suppressed by B63 (Fig. 3C and Supplementary Fig. S5C). Importantly, NAC pretreatment largely prevented these changes.

Fig. 3

Apoptosis is not critically involved in the B63-induced gastric cancer cell death. (A) Effect of B63 on SGC-7901 gastric cancer cell death as assessed by Annexin V/PI staining. Cells were exposed to B63 for 24 h. NAC pretreatment was carried out at 5 mM for 1 h. (B) Quantification of cell death following Annexin V/PI staining. The percentage of cells shown are double positive for Annexin V and PI [n = 3; *P < 0.05, **P < 0.01 compared to DMSO; #P < 0.05 compared to B63-20]. (C) Western blot analysis of apoptosis-associated proteins in cells challenged with B63 for 15 h. GAPDH was used as loading control. (D) SGC-7901 cells were pretreated with pan-caspase inhibitor z-VAD for 30 min before exposure to 20 µM B63 or 4 µM ROS inducer AF for 24 h. Cell death was assessed by Annexin V/PI staining. (E) Quantification of cell death as determined by Annexin V/PI staining. Cells were treated as indicated in panel D [n = 3; **P < 0.01, ***P < 0.0001 compared to indicated comparator]. (F) SGC-7901 cells were pretreated with a pan-caspase inhibitor z-VAD at 20 μM for 30 min. Cells were then exposed to 20 μM B63 for 12 h. Phase contrast images were captured. Arrows indicate cytoplasmic vacuolation [scale bar = 20 µm].

We initially hypothesized that B63 induced a ROS-mediated caspase-dependent apoptosis in gastric cancer cells. To achieve this goal, we inhibited caspases by a caspase inhibitor, Z-VAD fluoromethyl ketone [31]. We also used auranofin (AF) as a comparison. Previous studies have shown that AF acts as an inhibitor of TrxR1, like B63, and causes production of ROS and caspase-dependent cell death [32]. Interestingly, our studies show that caspase inhibition reduces the level of cell death induced by B63, but this prevention is not robust (Fig. 3D, E). This rescue by caspase inhibition was less than 20%. In comparison, caspase inhibition rescued more than 50% of cell death induced by AF. These results indicated that there must be another contributing mode of cell death following B63 exposure of gastric cancer cells. Microscope analysis of cells revealed that, rather than the typical morphological changes characteristic of apoptosis, B63 induced the formation of massive vacuoles inside of the cells (Fig. 3F and Supplementary Fig. S6)

Our results showing insufficient rescue of cell death by caspase inhibition and cytoplasmic vacuolation pointed to paraptosis-like cell death as the primary mode of programmed cell death in B63-treated gastric cancer cells. To probe this possibility, we exposed gastric cancer cells to B63 in the presence of cycloheximide (CHX). Studies have shown that CHX inhibits paraptosis [10] as protein synthesis is required for this process [8]. Phase contrast images show cytoplasmic vacuolation in cells exposed to B63 but not when cells were pre-treated with CHX (Fig. 4A, Supplementary Fig. S7A). This vacuolation was also inhibited in cells by NAC indicating an upstream role of ROS. Although various forms of programmed cell death are difficult to decipher conclusively and exclusively, a few surrogate markers may help in the identification. One such marker of paraptosis is Alg-2 interacting protein X (Alix) [33], [34]. Alix has been shown to inhibit paraptosis but not apoptosis [12]. Based on this negative feedback relationship, we anticipated reduced levels of Alix in cells upon B63 challenge. This finding would support the induction of paraptosis-like cell death. As expected, our results show dose-dependent decrease in Alix protein levels by B63 (Fig. 4B). Western blot analysis also showed that the decrease in Alix levels by B63 is normalized when cells are pretreated with NAC or CHX (Fig. 4C and Supplementary Fig. S7B). Collectively, these findings show that B63 induces ROS-mediated cytoplasmic vacuolation and suppression of Alix. We also examined the effects of other ROS scavengers (BHT, Trolox, GSH, and Catalase) on B63-induced parapotosis in SGC-7901 cells. As shown in Supplementary Fig. S8A-F, only GSH reversed B63-induced cell vacuolation and Alix reduction in SGC-7901 cells, while BHT, Trolox, and Catalase failed. These data are consistent with the fact that only NAC and GSH blocked B63-induced ROS (Supplementary Fig. S4), indicating that only Thiol-containing antioxidants can suppress B63-induced paraptosis-like cell death in gastric cancer cells.

Fig. 4

B63 induces ROS-mediated paraptotic cell death. (A) Representative phase contrast images of SGC-7901 cells exhibiting characteristics of paraptosis-like cell death. Cells were pretreated with 5 mM NAC or 20 μM CHX before being challenged with B63 for 12 h. Arrows showing cytoplasmic vacuolation [scale bar = 20 µm]. (B) Western blot analysis of Alix, a negative regulator of paraptosis. SGC-7901 cells were exposed to 20 μM B63 for the indicated times. Lysates were then probed for Alix levels. GAPDH was used as loading control. Lower panel showing densitometric quantification of the immunoblots [n = 3; *P < 0.05, **P < 0.01, ***P < 0.001 compared to 0 h]. (C) Effect of NAC and CHX on B63-mediated suppression of Alix protein levels in SGC-7901 cells. Cells were treated as indicated in panel A. GAPDH was used as loading control [n = 3; ***P < 0.001, ##P < 0.01 compared to B63]. (D) Representative phase images of cells overexpressing TrxR1. SGC-7901 transfected with TrxR1 expression vector were challenged with B63 for 12 h. Arrows showing cytoplasmic vacuoles [TrxR1 = TrxR1 vector, veh = control vehicle vector; scale bar = 20 µm]. (E) Western blotting analysis of Alix in cells transfected with Alix expression vector [Con = no transfection, veh = control vehicle vector, Alix = Alix plasmid transfection]. Lower panel showing densitometric quantification of immunoblots [n = 3; **P < 0.01 compared to Vector control]. (F) Representative phase images of cells overexpressing Alix exposed to B63. Cells were exposed to 20 μM B63 for 12 h. Arrows showing cytoplasmic vacuoles [scale bar = 20 µm].

As our studies revealed that TrxR1 is a potential target of B63 and is involved in ROS generation, we assayed for cytoplasmic vacuolation by B63 after TrxR1 overexpression. Our results show that TrxR1 overexpression reduces B63-induced cytoplasmic vacuoles, confirming an important role of TrxR1 in B63-induced ROS and paraptosis-like cell characteristics (Fig. 4D, Supplementary Fig. S9). Finally, we overexpressed Alix in gastric cancer cells (Fig. 4E) and show a reduction in characteristic features of paraptosis in gastric cancer cells (Fig. 4F).

We have previously reported the anti-cancer activity of B63 in NSCLCs [6]. According to this work, we would like to see if B63 will induce paraptotic phenotype in NSCLCs and other aggressive cancer models. We examined cytoplasmic vacuolation and Alix expression in B63-treated NSCLC H1975 and H460 cells, triple negative breast cancer MDA-MB-231 cells, and pancreatic cancer PATU-8988T cells. As shown in the Supplementary Fig. S10A-H, we observed similar results that B63 significantly induced paraptosis-like cell death and inhibited Alix expression in these cancer cell lines. These data indicate that paraptosis-like cell death may be a universal mechanism in B63's cancer treatment.

Endoplasmic stress response is initiated by B63 in gastric cancer cells and contributes to paraptosis-like cell death

Previous reports have indicated that paraptotic death is accompanied by formation of vacuoles that arise from endoplasmic reticulum (ER) [10], [35]. Increased ROS levels also increase the levels of unfolded proteins in the ER and induce ER stress response [36]. Unfolded protein response induces protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK)-mediated phosphorylation of eukaryotic initiation factor-2α (eIF2α). Phosphorylated-eIF2α then blocks protein translation but allows preferential translation of activating transcription factor 4 (ATF4). ATF4 is a key transcription factor in the ER stress pathway and mediates the induction of the pro-death transcriptional regulator CCAAT/-enhancer-binding protein homologous protein (CHOP). It is likely that ER stress played a role in ROS-mediated paraptotic cell death in gastric cancer cells. We measured the levels of key proteins involved in ER stress response in gastric cancer cells exposed to B63. Our results show increased levels of phosphorylated eIF2α, ATF4, as well as CHOP in response to B63 (Fig. 5A and Supplementary Fig. S11). Induction of these proteins was also prevented by NAC, linking oxidative stress and ER stress (Fig. 5B and Supplementary Fig. S12).

Fig. 5

B63 induces ER stress-dependent paraptosis in gastric cancer cells. (A) Western blot analysis of ER stress-related proteins in SGC-7901 cells. Cells were exposed to 20 µM B63 for indicated time points. Proteins levels of phosphorylated eIF2α, ATF4, and CHOP were determined. GAPDH and eIF2α were used as internal controls. (B) Effect of inhibiting ROS by NAC on B63-induced ER stress in SGC-7901 cells. Cells were pretreated with NAC for 1 h before exposure to increasing concentrations of B63 for 3 h and 8 h. (C) Electron microscopy images of SGC-7901 cells exposed to B63. Arrows pointing to ER [images shown in 10,000 or 20,000 magnifications]. (D) Western blot analysis of ATF4 and Alix protein following transfection of SGC-7901 cells with siRNA against ATF4 before 20 μM B63 treatment [Con siRNA = negative control siRNA]. (E) Densitometric quantification of Alix protein levels in panel D [n = 3; *p < 0.05 and **p < 0.01]. (F) Assessment of Annexin V/PI staining positive cells following knockdown of ATF4 and exposure to 20 μM B63 [n = 3; **P < 0.01]. (G) Representative phase contrast images of cells exposed to B63 for 12 h after ATF4 knockdown. Arrows pointing to cytoplasmic vacuoles [scale bar = 20 µm].

In addition to induction of proteins associated with the ER stress pathway, we noted perturbations in the morphology of ER through electron microscopy. Compared to control (DMSO-treated) cells, the ER in cells exposed to B63 exhibited swelling (Fig. 5C). These changes were not observed in cells pretreated with NAC. Both, morphological change and induction of ER stress proteins, suggest that ROS-mediated ER stress pathway causes cell death in gastric cancer cells. To confirm these findings, we knocked down the expression of ATF4 (Fig. 5D, E, Supplementary Fig. S13A) and then challenged the cells to B63. ATF4 silencing decreased the level of cell death following B63 treatment (Fig. 5F, Supplementary Fig. S13B-D). ATF4 knockdown also decreased cytoplasmic vacuolation, indicating that ER stress plays a role in B63-induced paraptosis-like cell death (Fig. 5G, Supplementary Fig. S14).

Paraptosis-like cells death in gastric cancer is mediated through mitogen activated-protein kinase pathway

Activation of mitogen-activated protein kinase (MAPK) has been implicated in paraptosis-like cell death and cytoplasmic vacuolation-mediated cell death in cancer cells [12]. We assessed MAPK activation by measuring phosphorylated forms of ERK, p38, and JNK. Exposure of gastric cancer cells to B63 induced a rapid increase in MAPK protein phosphorylation (Fig. 6A). Pretreatment of cells with NAC reduced B63-induced MAPK activation (Fig. 6B). Furthermore, inhibition of MAPK arms by pharmacological small-molecule inhibitors (Fig. 6C) prevented B63-induced cytoplasmic vacuolation in gastric cancer cells (Fig. 6D) in SGC-7901 cells. Similar results were also obtained in BGC-823 cells (Supplementary Fig. S15). These results confirmed the involvement of MAPKs pathway in paraptosis-like cell death in gastric cancer cells.

Fig. 6

B63 causes activation of MAPKs to induce paraptosis. (A) Activation of MAPK proteins as assessed by immunoblot of phosphorylated forms. SGC-7901 and BGC-823 cells were challenged with 20 µM B63 for the indicated times. p-JNK, p-P38 and p-ERK were determined by Western blot assay. JNK, P38 and ERK were used as internal control. (B) Effect of NAC on B63-induced activation of MAPKs. SGC-7901 cells were pretreated with NAC for 1 h before challenging the cells with B63 for 3 h at increasing concentrations. Western blot assay was used for determination. (C) Effect of pharmacological inhibitors of MAPKs on B63-induced MAPK activation. SGC-7901 cells were pre-treated with 20 μM ERK inhibitor U0126, pan-p38 inhibitor BIRB, and broad-spectrum JNK inhibitor SP600125 for 1 h, and then exposed to 20 µM B63 for 3 h. Western blot assay was used for determination. (D) Phase images of SGC-7901 cells pretreated with MAPK inhibitors 1 h prior to B63 challenge of 12 h. Arrows highlighting cytoplasmic vacuoles [scale bar = 40 µm].

B63 prevents gastric cancer growth in mice

Next, we assessed the in vivo effectives of B63 using a human gastric cancer xenograft mouse model. We implanted SGC-7901 cells in BALB/c mice and orally treated the mice with B63. For these studies, we also treated the mice with curcumin as a comparator. Treatment of mice with B63 reduced gastric cancer growth as evidenced by lower tumor volumes and weights (Fig. 7A, B). The effect appeared to be more pronounced than curcumin. Harvested tumor specimens aid in the appreciation of the effect of B63 on gastric cancer growth (Fig. 7C). Hematoxylin and Eosin (H&E) staining of resected tumor specimens showed features consistent with paraptosis-like cell death (Fig. 7D). These inhibitory effects were observed without any alterations to body weight measurements (Supplementary Fig. S16).

Fig. 7

B63 inhibits SGC-7901 xenograft growth in mice. (A) Measurement of tumor volumes at indicated time points following implantation of SGC-7901 cells in BALB/c mice. Mice treated with B63 or curcumin (comparator) showed reduction in tumor volumes. (B) Measurement of harvested tumor weights at the conclusion of the study [ns = not significant compared to control; **P < 0.01, ***P < 0.001]. (C) Photographs of resected tumor tissues from mice. (D) Representative hematoxylin and eosin staining of harvested tumor specimens from mice. B63 induced cytoplasmic vacuolation is indicated by arrows [scale bar = 20 µm]. (E) Levels of oxidative stress marker MDA in tumor tissue lysates showing increased lipid peroxidation products upon B63 treatment [ns = not significant compared to control; ***P < 0.001 compared to control]. (F) Staining of tumor tissue sections with ROS probe DCFH-DA (green). Tissues were counterstained with DAPI (Blue). Increased fluorescence intensity is indicative of increased ROS levels is seen in tumor specimens resected from B63-treated mice [scale bar = 50 µm]. (G) Immunohistochemical staining of tumor specimens for cell proliferation marker Ki-67 and paraptosis marker Alix. Immunoreactivity was detected by DAB chromogen (brown). Inserts showing higher magnification [scale bar = 100 µm].

The in vivo model we employed does not provide dynamic assessment of all the mechanisms we have identified in cultured cells. However, we are able to confirm the key findings. Our analyses show increased oxidative stress as determined by malondialdehyde (MDA) levels (Fig. 7E). We also measured ROS generation by staining the tissues with DCFH-DA, which showed increased level of ROS in tissues from mice treated with B63 (Fig. 7F, Supplementary Fig. S17). Furthermore, B63 treatment was associated with reduced proliferation (Ki67), induction of ER stress (eIF2α), and potential induction of paraptotic cell death (Alix) (Fig. 7G, Supplementary Fig. S18). These data suggest that B63 induces ROS, halts gastric cancer growth, induces ER stress, and leads to paraptosis-like cell death in vivo.

Inhibition of gastric cancer growth by B63 may be effective compared to traditional chemotherapeutic agents

Most chemotherapeutic formulations exert inhibitory activity in cancer by inducing apoptosis. However, cancer cells inherently have or acquire many mechanisms that aide in evasion from apoptosis. We wanted to determine whether B63, which induces paraptosis-like cell death based on our results, is able to target resistant gastric cancer cell lines. To test this, we generated 5-fluoruracil (5-FU)-resistant clones by a step-wise dose escalation study as reported by us previously [18]. Cell viability in the presence of 5-FU clearly showed that derived clones are resistant to 5-FU (Fig. 8A). Interestingly, exposure of these resistant cells to B63 showed effective reduction of viability (Fig. 8B). The IC50 values obtained did not differ between resistant and parental cells. Phase contrast images show that B63 induces cytoplasmic vacuolation in 5-FU resistant cells, indicating initiation of paraptosis-like features (Fig. 8C). Finally, 5-FU resistance leads to slight upregulation of Alix in SGC-7901 cells (Fig. 8D), while B63 treatment time-dependently reduces Alix in 5-FU-resistant gastric cancer cells (Fig. 8E). These findings suggest that B63 may effectively inhibit apoptosis-resistant gastric cancer via inducing paraptosis-like cell death.

Fig. 8

B63 overcomes chemotherapy resistance through induction of paraptosis. (A, B) Effects of 5-fluorouracil (5-FU) and B63 on gastric cancer cell viability. Cells were made resistant to 5-FU through a dose-escalation assay. Parental cells (WT) and 5-FU-resistant lines (5-FU-R) were then exposed to increasing concentrations of 5-FU or B63 for 48 and 24 h, respectively. (C) Phase images of 5-FU-R cells exposed to B63. Arrows indicating cytoplasmic vacuoles. Pretreatment of resistant lines with NAC and CHX prevented vacuolation [scale bar = 20 µm]. (D) Effect of B63 exposure on Alix protein levels in 5-FU-resistant lines. GAPDH was used as loading control. (E) Alix protein levels in parental (WT) and 5-FU resistant lines (5-FU-R) without exposure to B63. GAPDH was used as loading control.

Discussion

Cancer therapeutic drugs aim to halt the growth of cancer cells and to induce apoptosis. Unfortunately, cancer cells develop different adaptive mechanisms to escape apoptotic cell death. Recently, interest in inducing other forms of programmed cell death mechanisms in cancer cells has emerged. Some natural products including curcumin have been reported to induce caspase-independent, paraptotic programmed cell death in cancer cells. Since poor bioavailability and pharmacological kinetics of curcumin, our laboratory has previously developed novel analogs of curcumin which exhibit enhanced pharmacokinetics and bioavailability. Here we show that B63, directly targeting TrxR1, induces ROS-mediated paraptosis-like cell death in gastric cancer cells. Extensive cytoplasmic vacuolation associated with increased ROS production and ER stress is evident in cells exposed to B63. We also showed that treatment of mice implanted with gastric cancer cells with B63 reduces tumor growth. These findings suggest that B63, a pharmaco-enhanced curcumin analog, is a potential therapeutic candidate for gastric cancer via a paraptotic mechanism (Fig. 9).

Fig. 9

Schematic illustration of the underlying mechanism of B63's anti-cancer activity.

One of the central players behind the inhibitory activity of B63 was TrxR1-mediated ROS. Altering ROS levels has emerged as a strategy against cancer. Indeed, ROS levels are elevated in a number of human cancers [37], [38], [39]. Gastric cancer also exhibits increased mucosal expression of ROS compared with normal mucosa [40]. Interestingly, a lot of evidences also indicate that further increases in ROS may be detrimental to cancer cells. Conventional chemotherapy drugs including doxorubicin, etoposide, and cisplatin/paclitaxel can also induce apoptosis in cancer cells by interfering with the redox balance of the cell [41], [42]. In our studies, we report that B63 induces ROS in cultured gastric cancer cells and in gastric tumors in mice. Consistent with recent studies highlighting the utility of ROS to target cancer cells, we show that B63 essentially halts the growth of gastric cancer and induces cell death, primarily through ROS generation. In addition, perhaps one of the mechanisms which make ROS an attractive target in cancer therapeutics is the intricate relationship between ROS and ER stress. We know that misfolded proteins can elicit ER stress and the unfolded protein response, which eventually results in ROS accumulation [43]. The same applies to ROS eliciting ER stress. The role of ER stress in ROS-induced apoptosis has been demonstrated in a variety of cell types [44], [45]. In response to ROS caused by anti-cancer agents, accumulation of unfolded or misfolded proteins triggers a cellular adaptive ER stress to initiate cell death [25], [26], [45]. We previously showed that B63 is capable of at least partially inducing ER stress-dependent apoptosis in human lung cancer cells [6]. In the present study, we have demonstrated that ER stress in gastric cancer cells can be normalized by reducing ROS through NAC, illustrating the link between ROS and ER stress in targeting cancer.

More importantly, we found TrxR1 is one of targets of B63 for its pro-oxidative action. The thioredoxin system plays a crucial role in the redox regulation of multiple intracellular processes [46]. This system regulates DNA replication and repair, protein synthesis and folding, and intracellular redox balance by counteracting ROS, and eventually influences cell growth, differentiation, and death [46]. TrxR1 has been evidenced to be over-expressed and constitutively active in various kinds of cancer cells [47]. TrxR1 now becomes major target for anticancer drugs and increasing attention to developing novel inhibitors of TrxR1 as potential antitumor agents has been witnessed in the past decade [48]. Here, our cell-free measurement on enzyme activity identified TrxR1 as a target of B63 and revealed that B63 induces ROS and subsequent paraptosis by, at least, inhibiting this enzyme. Previously, we also found two structurally similar compounds, B19 [26] and EF24 [25], could directly target TrxR1 and induce ROS in cancer cells. These also strengthened our conclusion that B63 induces ROS via targeting TrxR1. We also found that overexpression of TrxR1 reversed B63-induced paraptosis. This is the first time a relationship between TrxR1 and paraptosis has been shown. In addition to showing that B63 may potentially provide therapeutic benefit against gastric cancer, our results advance our understanding of anti-cancer mechanisms of curcumin analogs. Both curcumin and its analogs are being used in preclinical studies and clinical trials and have been documented to target multiple proteins and signaling pathways. It is possible that B63 targets other proteins, in addition to TrxR1, in halting gastric cancer growth. These future studies are certainly warranted.

One of the important findings of our study is that B63-mediated cell death effectively targets conventional chemotherapy-resistant gastric cancer. We also found that B63 induced paraptotic phenotype in NSCLC cells, triple negative breast cancer, and pancreatic cancer cells. Pharmacological therapies targeting cancer have primarily focused on classical caspase-mediated apoptosis. Therefore, many chemotherapeutic drugs have been designed to activate intrinsic and extrinsic pathways of classical apoptosis [49], [50]. Identification of agents triggering other forms of programmed cell death may help in our attempts to combat gastric cancer, particularly resistant gastric cancer. Recently, a potent paraptosis-inducing nanomedicine is reported that causes quick nonapoptotic death of cancer cells, overcoming apoptosis-based resistance and effectively inhibiting drug-resistant tumor growth [14]. Based on our in vitro studies, B63 may have clinical utility against gastric cancer. We show that 5-FU resistant gastric cancer cells are effectively targeted by B63. The mechanism appears to be induction of paraptosis. Future researches should be noted to test the effects of B63 on 5-FU-resistant gastric cancer cells in vitro and to see its reversal against gastric cancer cells resistant to other apoptosis-inducing chemotherapy. In addition, a limitation of this study is that we did not measure the type of ROS induced by B63. We can see that the cell death induced by AF, which also activates ROS, was significantly inhibited by caspase-3 inhibitor Z-VAD (Fig. 2D-E), indicating AF-induced cell death is caspase-dependent. Thus, the ROS types induced by AF and B63 may be different, so that these two ROS inductors displayed different functions in causing apoptosis and paraptosis. We also tried to narrow which ROS species would be primarily involved in B63-dependent cell death. The results are interesting. Only thiol-containing antioxidants (NAC and GSH) could reverse ROS generation induced by B63 in SGC-7901 cells, while other seven ROS scavengers could not achieve this effect. Consistently, only NAC and GSH inhibited B63-induced paratotic phenotype in SGC-7901 cells. These data indicated that 1) the lipid ROS or lipid peroxide may not be involved in B63 treatment, and 2) ROS species in glutathione and thioredoxin systems may be responsible in B63 actions. Previous studies indicate an overlap of glutathione and thioredoxin systems in maintaining intracellular redox balance, and the glutathione system can act as a backup of the thioredoxin system [51]. These data further support that B63 is a TrxR1 inhibitor and suggest that ROS species in glutathione and thioredoxin systems may triggers paraptosis-like cell death, despite the precise mechanism of B63-induced specific ROS species needs further investigation.

In conclusion, this study has enhanced the applicability of B63 and uncovered a potential target (TrxR1) and a new mechanism (paraptosis) in gastric cancer treatment. Our study, for the first time to our knowledge, shows that B63, via targeting TrxR1 and increasing ROS, induces both caspase-independent paraptosis modes of death. The major contributor in gastric cancer cells was found to be caspase-independent programmed cell death. We show that suppression of ROS normalized B63-induced paraptosis in gastric cancer cells. Furthermore, B63 treatment reduced the growth of gastric cancer xenografts.

Supplementary information

Supplemental information includes 18 figures. All the other data are available from the authors on request.