Curcumol (racemate, purity ≥96.7%) was donated by Haimen Desihang Co., Ltd. (Zhejiang, China). Curcumol was dissolved in absolute ethyl alcohol and diluted in Dulbecco's modified Eagle's medium (DMEM) culture medium with 1% alcohol to treat cells. AG-120 (ivosidenib) was purchased from Selleck Chemicals (Houston, TX, USA). Dimethyl sulfoxide (DMSO) was purchased from Aladdin Reagent Co. (Los Angeles, CA, USA). Fetal bovine serum (FBS), DMEM and Trypsin-EDTA were all purchased from Gibco (New York, NY, USA). Methylthiazolyldiphenyl-tetrazolium bromide (MTT) was purchased from Gen-View Scientific Inc. (Calimesa, CA, USA).

Annexin V-FITC Apoptosis Detection kit, Cell Cycle and Apoptosis Analysis kit, MMP assay kit with JC-1 and Reactive Oxygen Species Assay kit were purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China). A Hoechst 33258 kit was purchased from Kaiji Biotechnology Co., Ltd. (Jiangsu, China). The antibodies against IDH1, IDH2 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were purchased from Proteintech Group, Inc. (Chicago, IL, USA). PrimeScript™ RT reagent kit with gDNA Eraser (Perfect Real-Time), SYBR^® Premix Ex Taq™ II (Tli RNaseH Plus), RNAiso Plus and diethyl pyrocarbonate (DEPC)-treated water were purchased from Takara (Shiga, Japan). The primers of IDH1, IDH2 and GAPDH were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China).

The human GC cell line MGC-803 and the human embryonic lung fibroblast cell line MRC-5 were purchased from the Institute of Biochemistry and Cell Biology, SIBS, of the Chinese Academy of Sciences (CAS, Shanghai, China). Both cell lines were cultured in DMEM supplemented with 10% FBS at 37°C in a humidified atmosphere with 5% CO₂.

Cell viability was detected by MTT assay. The MGC-803 and MRC-5 cells were cultured in 96-well plates at a density of 3×10³ cells/100 µl/well. After incubation for 24 h, the cells were treated with: DMEM culture medium, 1% alcohol (DMEM culture medium with 1% alcohol) and curcumol at concentrations of 20, 40, 60, 80, 100, and 120 µM or AG-120 at concentrations of 40, 60, 80 and 100 µM for different time-points, including 24, 48 and 72 h. NAC was used to detect whether curcumol inhibited cell proliferation by increasing ROS. The cells were treated with the control (DMEM culture medium), 1% alcohol (DMEM culture medium with 1% alcohol), curcumol (80 µM), NAC (5 mM), curcumol (80 µM) + NAC (5 mM) for 24, 48 and 72 h. Then 10 µl of 0.5 mg/ml MTT were added to each well and the mixture was cultured at 37°C for an additional 4 h. Subsequently the culture medium was replaced with 100 µl DMSO to dissolve the formazan crystals. After shaking for 10 min, the absorbance of each well was determined at 570 nm by a plate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Five replicate wells were designed for each sample. This experiment was repeated three times and the cell viability was calculated as follows: cell viability rate (%) = (experimental OD value - zero set OD value)/(control OD value - zero set OD value) × 100. The 50% inhibitory concentration (IC₅₀) was calculated by GraphPad Prism 6 (GraphPad Software Inc., La Jolla, CA, USA).

The MGC-803 cells in logarithmic growth phase were cultured in a 6-well plate and treated with curcumol (0, 20, 40, 60, 80 and 100 µM) for 24 h. Then the cells in each well were stained using Hoechst 33258 and the changes in the nuclei of cells were observed and photographed with a fluorescence microscope (Leica Microsystems, Wetzlar, Germany).

MGC-803 cells were seeded in 6-well plates at a density of 1×10⁵ cells/well. After being cultured with various concentrations (0, 20, 40, 60 and 80 µM) of curcumol for 24 h, the cells were collected and stained using an Annexin V-FITC Apoptosis Detection kit (including PI) according to the manufacturer's instructions. Then the cells were analyzed using a flow cytometer (Beckhman Coulter Inc, Brea, CA, USA).

Similar to the apoptosis assays by Annexin V/PI staining, the cells treated with curcumol for 24 h were collected. Then the cells were stained with PI according to the manufacturer's instructions. The cell cycle distribution was detected a the flow cytometer and was analyzed using FlowJo software (FlowJo, LLC, Ashland, OR, USA).

The MMP assay was performed to evaluate the level of the polarization/depolarization of the mitochondrial membrane using the JC-1 dye. MGC-803 cells were treated with curcumol (0, 20, 40, 60 and 80 µM) at a density of 1×10⁵ cells/well in a 6-well plate. After 24 h, the cells were collected and stained using JC-1 according to the manufacturer's instructions. Finally, the cells were analyzed on a flow cytometer and the results were analyzed using FlowJo software.

The ROS assay was performed to detect the intracellular ROS production from the curcumol-treated MGC-803 cells. Briefly, the cells were seeded at a density of 1×10⁵ cells/well in a 6-well plate and incubated for 24 h. The cells were then treated with curcumol (0, 20, 40, 60 and 80 µM, NAC, NAC + 80 µM curcumol) for another 24 h. Then the cells were loaded with DFCH-DA and harvested for the ROS assay according to the manufacturer's instructions. Finally, the assay results were analyzed on a flow cytometer and the data were analyzed using FlowJo software.

MGC-803 cells were cultured in a 6-well plate at a density of 2×10⁵ cells/well with different curcumol concentrations, including 0, 20, 40, 60 and 80 µM. AG-120 (80 µM) treatment was used as a positive control. After 48 h, the cells were collected in lysis buffer and incubated on ice for 20 min. The lysates were centrifuged at 4°C for 20 min and then the supernatant was transferred to a new 1.5-ml test tube. To ensure the equal loading of protein (40 µg) for each group, protein quantification (Thermo Fisher Scientific, Waltham, MA, USA) was performed to detect the total protein. Then the samples were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and were transferred to polyvinylidene fluoride (PVDF) membranes. Next the membranes were blocked with 5% skim milk for 1 h at room temperature. Subsequently, the membranes were washed in TBST and incubated with antibodies of GAPDH (1:1,000 dilution), IDH1 (1:5,000 dilution) and IDH2 (1:2,500 dilution) overnight at 4°C. On the second day, the membranes were washed in TBST and then incubated at room temperature for 1 h with the appropriate secondary antibody (goat anti-mouse IgG for GAPDH and goat anti-rabbit IgG for IDH1 and IDH2). The bands were visualized using an enhanced chemiluminescence (ECL) system (Thermo Fisher Scientific) and Kodak XBT-1 film. Gray value analysis was performed using ImageJ software (National Institute of Mental Health, Bethesda, MD, USA). The variation in the gray value was expressed as fold changes compared to the control in the blot.

Total RNA was isolated from the MGC-803 cells treated with curcumol (0, 20, 40, 60 and 80 µM) using the RNAiso Plus. The AG-120-treated cells (80 µM) were used as a positive control.

After incubation at 45°C for 2 min, genomic DNA was removed in the reaction with a total volume of 10 µl consisting of 2 µl gDNA eraser buffer, 1 µl gDNA eraser, an appropriate amount of RNA (up to 1 µg) and nuclease-free water. cDNA was synthesized in the reaction with a total volume of 20 µl consisting of 10 µl of the reaction liquid of the last step, 4 µl PrimeScript buffer, 1 µl reverse transcriptase, 1 µl RT Primer Mix and 4 µl nuclease-free water. The reaction conditions included 37°C for 15 min, followed by 85°C at 50 sec and ending at 4°C. Real-time RT-PCR was performed using the Thermal Cycler Real-Time system (Thermo Fisher Scientific). The reaction was performed at a final volume of 10 µl containing 5 µl SYBR Premix Ex Taq II, 1 µl of appropriate primer (10 µM), 1 µl cDNA (50 ng/µl) and nuclease-free water. The cycling conditions included one cycle at 95°C for 30 sec, followed by 40 cycles of 5 sec at 95°C and 20 sec at 60°C. The sequences of the primers were as follows: GAPDH forward, 5′-CAGGAGGCATTGCTGATGAT-3′ and reverse, 5′-CAGGAGGCATTGCTGATGAT-3′; IDH1 forward, 5′-ACTTGCACATGACTGGAACG-3′ and reverse, 5′-TCCTGCGGCCTAAACAGTAT-3′; IDH2 forward, 5′-AGAGTGGAGCCATGACCAAG-3′ and reverse, 5′-TGTCCAGGTTGCTCTTGATG-3′.

All of the data are presented as the mean ± standard deviation (SD). The rate of survival and the IC₅₀ values were analyzed by GraphPad Prism 6.0 (GraphPad Software Inc.). A two-tailed independent sample t-test was used to assess the level of significance between the means. A P-value of <0.05 was considered to indicate a statistically significant difference.

Human embryonic lung fibroblast MRC-5 cells treated with curcumol as a control exhibited light toxicity. AG-120 which is an inhibitor of IDH1, was used as a positive control in MGC-803 cells. Curcumol inhibited the viability of MGC-803 cells with an IC₅₀ value of 114.60±3.55 µM at 24 h, 66.36±1.26 µM at 48 h and 29.00±1.14 µM at 72 h. The viability of MGC-803 cells was suppressed by curcumol in a time- and concentration-dependent manner (Fig. 1A). Fig. 1B revealed that curcumol was slightly effective on MRC-5 cells, thus signifying that curcumol has light toxicity on non-tumor cells. AG-120 inhibited the viability of MGC-803 cells with an IC₅₀ value of 58.19±1.14 µM at 24 h, 75.40±1.04 µM at 48 h and 67.24±1.14 µM at 72 h. As time progressed, the antitumor effect of curcumol improved compared to the AG-120 treatment. This group demonstrated that the MGC-803 cells were also suppressed by AG-120, however the potency was lower than that of curcumol at low concentrations (Fig. 1C). In addition, NAC was capable of reversing the inhibitory effect of curcumol on cell proliferation (Fig. 1D).

To assess the level of apoptosis in the MGC-803 cells treated with curcumol, we performed Hoechst 33258 fluorescence staining following curcumol treatment. The apoptotic morphological changes of MGC-803 cells treated with curcumol for 24 h were observed and compared to those of the control cells treated with DMEM and 1% ethyl alcohol. Curcumol at 40, 60, 80 and 100 µM induced significant morphological changes in the MGC-803 cells, including a decrease in cell volume, intercellular junction disappearance and formation of apoptotic bodies (Fig. 2). Annexin V-FITC/PI double staining followed by FACS analysis confirmed the dose-dependent apoptosis-inducing effect of curcumol. The results revealed a dose-dependent increase in the apoptosis rate for cells treated with 40, 60 and 80 µM of curcumol compared to the control cells (Fig. 3).

After curcumol was confirmed to inhibit MGC-803 cell growth, we next examined whether the growth inhibitory effect of curcumol was related to cell cycle arrest. PI staining followed by FACS analysis demonstrated the effect of curcumol on cell cycle progression in MGC-803 cells. MGC-803 cells treated with curcumol at 60 and 80 µM resulted in a small accumulation of cells in the G2/M phase and a decrease in the S-phase cell population. This result revealed a G2/M phase cell cycle arrest of gastric cells upon exposure to curcumol (Fig. 4). However the cycle arrest of curcumol was not statistically significant.

An MMP decrease is an early manifestation of apoptosis. As an additional verification of the effects of curcumol, we evaluated the MMP of MGC-803 cells after 24 h of curcumol treatment. Most of the negative control cells were stained red by JC-1, indicating an intact mitochondrial membrane. In contrast, cells treated with curcumol exhibited increasing amounts of green fluorescence. The effect was more obvious with 80-µM treatment. Analysis of the red/green fluorescence light density ratio revealed that the decrease was dose-dependent and significant for the 40, 60 and 80 µM concentrations of curcumol. CCCP-treated cells were used as a positive control (Fig. 5).

It has been confirmed that excess ROS promotes cell death and curcumol upregulates ROS to induce apoptosis in tumors (16). Thus, we further examined the effects of curcumol on ROS production in MGC-803 cells. The level of ROS in the MGC-803 cells was assessed using the DCFH-DA probe and flow cytometry in the FL1-H channel (Fig. 6A). The result revealed that the level of ROS increased significantly after treatment with curcumol for 24 h at concentrations of 60 or 80 µM (p<0.05). NAC was capable of decreasing the increase in ROS caused by curcumol. Rosup treated cells were used as a positive control (Fig. 6B).

Our previous research revealed that curcumol increased the levels of ROS to promote cell death. However a study demonstrated that IDH1-dependent reductive carboxylation mitigated the intracellular ROS level and protected tumor cells from ROS damage. In order to explore the molecular mechanism through which curcumol inhibits cell proliferation, the expression levels of IDH1 and IDH2 were determined by western blotting. The expression levels of IDH1 were downregulated after cells were treated with 60 or 80 µM of curcumol, but the expression levels of IDH2 were not altered by curcumol. AG-120 (80 µM) treatment was used as a positive control (Fig. 7). The result of RT-qPCR confirmed the same tendency in the gene expression of IDH1 and IDH2. In contrast to the negative control group, the expression level of IDH1 was significantly decreased after 48 h of treatment with curcumol at 40, 60 and 80 µM (Fig. 8). The results may reveal a new molecular mechanism in gastric cell apoptosis induced by curcumol.