Luteolin (purity, ≥98%) was purchased from Sigma-Aldrich (cat. no. L9283). DMSO (cat. no. D8371; Beijing Solarbio Science & Technology Co., Ltd.) was used to dissolve with luteolin as the mother solution. Oxaliplatin was purchased from Tai-Tianqing Pharmaceutical Co., Ltd. (cat. no. H20143263). Oxaliplatin was dissolved with PBS (P1020; Beijing Solarbio Science & Technology Co., Ltd.). Luteolin and/or oxaliplatin were diluted to the required concentration (luteolin: 10, 20, 30, 40, 50, 60, 70 and 80 µM; and oxaliplatin: 5, 10, 20, 30, 40, 50, 60, 70 and 80 µM) with RPMI-1640 complete medium. RPMI-1640 complete medium (cat. no. 31800; Beijing Solarbio Science & Technology Co., Ltd.) containing 0.1% DMSO was used as a control.

The MFC cell line was purchased from the National Infrastructure of Cell Line Resource (cat. no. 1101MOU-PUMC000143). The cells were cultured with RPMI-1640, containing 10% fetal bovine serum (cat. no. REF10091-48; Gibco; Thermo Fisher Scientific, Inc.), and incubated in an incubator (HF90/HF240; Heal Force) at 5% CO₂ at 37°C. The cells were treated with different concentrations of luteolin and/or oxaliplatin for 24 h in the subsequent experiments.

The effect of luteolin and/or oxaliplatin on MFC cell viability was determined by CCK-8 assay (24). Briefly, MFC cell suspension (100 µl/well) was seeded at a density of 6,000-7,000 cells/well in a 96-well plate. After incubation for 24 h, the supernatant was discarded, and the cells were treated with luteolin (10, 20, 30, 40, 50, 60, 70, and 80 µM) and oxaliplatin (5, 10, 20, 30, 40, 50, 60, 70, and 80 µM) (total volume 200 µl/well) for 24 h. Based on the CCK-8 cell proliferation and cytotoxicity assay kit (cat. no. CA1210; Beijing Solarbio Science & Technology Co., Ltd.) protocol, 100 µl testing solution (CCK-8: Complete RPMI-1640, 1:10) was added to each well. After incubation at 37°C in 5% CO₂ in the dark for 1 h, the absorbance at a wavelength of 450 nm was detected via a Thermo 3001 multi-function microplate reader (Infinite 200 PRO; Tecan Austria GmbH). IC₅₀ indicated the drug concentration resulted in 50% reduction in cell survival. The Chou-Talalay CI method and Compusyn 2.0 software were used to calculate the CI and the dose reduction index (DRI). The CI was calculated as (D)₁/(Dx)₁ + (D)₂/(Dx)₂, where (Dx)₁ and (Dx)₂ were the doses of drug 1 and 2 alone that inhibit x%, while (D)₁ and (D)₂ were the portions of drug 1 and 2 in combination to achieve x%. This equation was used to quantitatively depict the synergism (CI <1), additive effect (CI=1), and antagonism (CI >1) (25).

DNA contents analysis was conducted by PI staining and flow cytometry (26). MFC cells were seeded in a 6-well plate at 2.5×10⁵ cells/well and incubated at 37°C in a 5% CO₂ incubator for 24 h. After treatment with luteolin (20 µM) and/or oxaliplatin (5 µM) (total volume 3 ml/well) for one day, the cells were collected and fixed in 75% ethanol at 4°C for 4–5 h. In accordance with the manufacturer's instructions of the DNA content quantitation assay kit (cat. no. CA1510; Beijing Solarbio Science & Technology Co., Ltd.), the fixed cells were stained by PI staining solution (50 ng/ml) and RNase A (0.1 mg/ml) at 37°C in the dark for 30 min. FACSCanto II flow cytometer (BD Biosciences) recorded the DNA content in MFC cells. The data were analyzed by FACSDiva software (version 6.1.3; BD Biosciences).

MFC cells were seeded in a 6-well plate at 2.0×10⁵ cells/well. The cells were treated with luteolin (20 µM) and/or oxaliplatin (5 µM) (total volume 3 ml/well) for 24 h. Based on Hoechst-33258 stain solution (cat. no. C0021; Beijing Solarbio Science & Technology Co., Ltd.) instructions (14), the cells were incubated with 500 µl staining working solution at room temperature in the dark for 5 min and soaked in PBS three times for 5 min. Inverted fluorescence microscopy (DMI3000; Leica Microsystems GmbH) was used to detect blue nuclei.

Apoptosis induced by luteolin was examined by fluorescein isothiocyanate (Annexin V-FITC) and propidium iodide (PI) double-staining assay (27). MFC cells were seeded in a 6-well plate at 2.5×10⁵ cells/well and incubated at 37°C in 5% CO₂ for 24 h. The cells were treated with luteolin (20 µM) and/or oxaliplatin (5 µM) (total volume 3 ml/well) for 24 h. After the treatment, the supernatant was discarded. In line with the instructions of the Annexin V-FITC/PI apoptosis detection kit (cat. no. CA1020; Beijing Solarbio Science & Technology Co., Ltd.), the cells were pre-processed and stained using Annexin V-FITC/PI in the dark for 30 min. The fluorescence intensity was measured using FACSCanto II flow cytometer (BD Biosciences), and the apoptotic rates were analyzed using FACSDiva software (version 6.1.3, BD Biosciences).

Subsequent to the indicated drug treatment for 24 h, the supernatant was discarded. The cells were rinsed with PBS, and stained with 10 µM DCFH-DA solution in the dark for 30 min in accordance with the reactive oxygen species assay kit (cat. no. CA1410; Beijing Solarbio Science & Technology Co., Ltd.) protocol (28). Inverted fluorescence microscopy (DMI3000, Leica) was used to record the morphological changes. The stained cells were analyzed by FACSCanto II flow cytometer (BD Biosciences). The fluorescence mean value in P2 from the flow cytometer was collected using FACSDiva software (version 6.1.3, BD Biosciences), which was used for quantitative analysis. The data were presented as fold increase normalized to the control group.

MMP was measured using a mitochondrial membrane potential assay kit with JC-1 (cat. no. M8650; Beijing Solarbio Science & Technology Co., Ltd.) (29). The treated MFC cells were stained with JC-1 fluorescence working solution in accordance with the protocol. MMP of stained cells was measured by FACSCanto II flow cytometer (BD Biosciences) and analyzed by FACSDiva software (version 6.1.3, BD Biosciences). The relative ratio of P2 (red fluorescence) to P3 (green fluorescence) was used for quantitative analysis of MMP change in MFC cells.

A total of 3×10⁶ MFC cells were inoculated into 100-mm culture dishes. After overnight culture, the cells were incubated with luteolin (20 µM) and/or oxaliplatin (5 µM) (total volume 7 ml/well) for 24 h. Then, the cells were rinsed with PBS and lysed on ice for 30 min with RIPA buffer (cat. no. R0010; Beijing Solarbio Science & Technology Co., Ltd.) containing 0.1 M PMSF (cat. no. P0100; Beijing Solarbio Science & Technology Co., Ltd.) and protease phosphatase inhibitor (cat. no. P1261; Beijing Solarbio Science & Technology Co., Ltd.). The cell lysate was centrifuged at 12,000 × g (Thermo Scientific™ Sorvall™ Legend™ Micro 21R Microcentrifuge; Thermo Fisher Scientific, Inc.) for 15 min at 4°C, and the supernatant was collected for the subsequent tests. The protein quantification was performed using the BCA protein assay kit (cat. no. PC0020; Beijing Solarbio Science & Technology Co., Ltd.) (30). The data were obtained at the absorbance of 562 nm by a microplate reader (Infinite 200 PRO, Tecan, Tecan Austria GmbH). The amount of protein was calculated in accordance with the prescribed computational formula of the kit's protocol.

In total, 50 µg cell lysates from each group were loaded per lane and separated by SDS-PAGE (6% spacer gel and 10% separating gel). Next, cell lysates in the gel were transferred to PVDF membranes (cat. no. ISEQ00010; MilliporeSigma). The transferred membranes were blocked with Tris-buffered saline (10 mM Tris-Cl; pH 7.4), containing 0.5% Tween-20 and 5% skimmed dry milk, at room temperature for 2 h, and then incubated with primary antibodies at 4°C overnight (31), respectively. The primary antibodies used were: β-actin mouse monoclonal antibody (cat. no. TA-09; 1:2,000; OriGene Technologies, Inc.), Bcl-2 rabbit polyclonal antibody (cat. no. ab196495, 1:1,000; Abcam), BCL-2-associated X protein (Bax) rabbit monoclonal antibody (cat. no. ab182734; 1:1,000; Abcam), cyclin A2 rabbit monoclonal antibody (cat. no. ab181591; 1:2,000; Abcam), cyclin B1 rabbit monoclonal antibody (cat. no. ab32053; 1:1,000; Abcam), cyclin-dependent kinase-1 (CDK1) rabbit monoclonal antibody (cat. no. ab133327; 1:20,000; Abcam), tumor necrosis factor receptor-associated protein 1 (TRAP1) rabbit polyclonal antibody (cat. no. 10325-1-AP; 1:2,000; ProteinTech Group, Inc.), cell division cycle 25 homolog C (CDC25C) mouse monoclonal antibody (cat. no. 66912-1-lg; 1:2,000; ProteinTech Group, Inc.), extracellular-regulated protein kinases1/2 (ERK1/2) rabbit polyclonal antibody (cat. no. 9102s; 1:1,000; Cell Signaling Technology, Inc.) and p-ERK1/2 mouse monoclonal antibody (cat. no. 9106s; 1:1,000; Cell Signaling Technology, Inc.). The membranes were rinsed with TBST and then probed with appropriate secondary antibodies (peroxidase-conjugated goat anti-mouse IgG (H+L); cat. no. ZB-2305; 1:50,000; OriGene Technologies, Inc.; and goat anti-rabbit IgG H&L; ab6721; cat. no. 1:20,000; Abcam). The immunoreactive protein bands were visualized with the gel imaging analysis system (BioSpectrum 510 Imaging System Motorized Platform). Scanning gray analysis was analyzed by Photoshop CC 2019 software (Adobe Systems Inc.). The grayscale value of each band was used to plot histograms.

The experiments were performed at least 3 times. Data were analyzed using the SPSS 21.0 software package (version 21.0, SPSS Inc.), and presented as mean ± standard deviation (SD). Scanning gray analysis was calculated by Photoshop CC software (Adobe Systems Inc.) Statistical differences were calculated using ANOVA followed by Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

To investigate the inhibitory effects of luteolin and/or oxaliplatin on mouse forestomach carcinoma MFC cells, CCK-8 assay was performed on MFC cells exposed to a series of luteolin (10, 20, 30, 40, 50, 60, 70, and 80 µM) and oxaliplatin concentrations (5, 10, 20, 30, 40, 50, 60, 70, and 80 µM) for 24 h. Luteolin and oxaliplatin effectively exerted inhibitory effects on MFC cells proliferation in a dose-dependent manner (Fig. 1A and B). Based on the cell viability curve, 50 and 20 µM were calculated as the IC₅₀ values of luteolin and oxaliplatin to MFC cells, respectively. The CI was used to quantitatively depict the synergism (CI <1), additive effect (CI=1), and antagonism (CI >1) in the combined treatment (25). DRI is a measure of how many folds the dose of each drug in a synergistic combination may be reduced at a given effect level when compared with the doses of each drug alone. A greater DRI value indicates a greater dose reduction of the single drug to create the same effect (14). According to the Compusyn analysis in Table I, the CI of luteolin (20 µM) and oxaliplatin (5 µM) was 0.801, suggesting that luteolin and oxaliplatin exerted synergic effects on inhibiting MFC cell proliferation. DRIs for luteolin (20 µM) and oxaliplatin (5 µM) was >1, indicating that the dose reduction led to toxicity reduction in the therapeutic applications (25). The data from CCK-8 assays showed that the combined treatment with luteolin (20 µM) and oxaliplatin (5 µM) exerted a much stronger anticancer effect than the monotherapies (Fig. 1C). Oxaliplatin at 10 µM did not exhibit much stronger inhibitory effects on cell proliferation compared with 5 µM oxaliplatin (Fig. 1D). The morphological change was observed under a light microscope, showing that the cell number was more markedly reduced after the combined treatment than after the monotherapy (Fig. 1E).

Cell cycle arrest is one of the main causes of the inhibition of cell proliferation (1,32). We examined whether cell cycle arrest had an impact on the combined treatment. MFC cells were treated with luteolin (20 µM) and/or oxaliplatin (5 µM) for 24 h and stained with PI prior to flow cytometry analysis. As shown in Fig. 2A, the combined treatment significantly induced G₂/M phase arrest and exhibited much stronger effects on G₂/M phase arrest than any other single drug did (Fig. 2B). Next, western blotting was used to test the expression levels of the key proteins related to G₂/M phase arrest in the treated MFC cells. The combined therapy of MFC cells markedly reduced the expression levels of cyclin B1, and CDK1 compared with controls and any other monotherapy (Fig. 2C and D). These data indicated that luteolin and oxaliplatin effectively induced MFC cell cycle arrest by downregulating the expression levels of certain key proteins, such as cyclin B1 and CDK1. The combined treatment had significant effects on triggering G₂/M phase arrest (P<0.01). Thus, the combined treatment with luteolin and oxaliplatin possibly suppressed cell proliferation via inducing G₂/M phase arrest.

TRAP1, ERK1/2, and CDC25C are closely related to G₂/M cell cycle arrest and apoptosis (33,34). Thus, the changes in expression levels of TRAP1, P-ERK1/2/ERK1/2, and CDC25C proteins in MFC cells treated with luteolin and/or oxaliplatin as mentioned before were examined by western blot analysis. As shown in Fig. 3, the combined treatment reduced the expression levels of TRAP 1 (Fig. 3A), P-ERK1/2 (Fig. 3B) and CDC25C (Fig. 3C) protein more effectively than the monotherapies. Compared with the control and oxaliplatin group, the combined treatment significantly reduced the TRAP1 and CDC25C proteins expressions (*P<0.05). The combined treatment also significantly downregulated ERK1/2 phosphorylation, thereby suppressing ERK1/2 activation. Thus, the combined treatment with luteolin and oxaliplatin induced G₂/M cell cycle arrest through suppression of TRAP 1/P-ERK1/2/CDC25C in MFC cells.

The artificial regulation of apoptosis remains a considerable focus of attention in cancer treatment (35). Thus, whether apoptosis was involved in anticancer activities of luteolin and/or oxaliplatin was examined. MFC cells were exposed to luteolin (20 µM) and/or oxaliplatin (5 µM), and stained by Hoechst-33258 staining. Images captured from a fluorescence inversion microscope system showed that the combined treatment group manifested a more apparent apoptotic morphology, such as karyopyknosis and chromosome condensation, than other groups (Fig. 4A). By contrast, in the control group, the cells were still in the process of mitosis. The quantitative analysis was used to measure the apoptotic rate by Annexin-V FITC/PI double staining and flow cytometry. The percentage of apoptotic cells in the combined therapy group was much higher than that in the control group (Fig. 4B and C), but for 20 µM luteolin and 5 µM oxaliplatin as monotherapies, the result was not significant compared with the control. Usually, the balance between proapoptotic and antiapoptotic protein regulators is a critical point to determine whether a cell undergoes apoptosis (36). Thus, the protein expression levels of Bcl-2 and Bax were assessed by western blot analysis (Fig. 4D). In accordance with the results of flow cytometry, the ratio of Bcl-2/Bax was markedly lower in the combined group (Fig. 4E). Thus, the combined treatment exerted stronger effects on inducing apoptosis in MFC cells.

ROS are the primary secondary messengers, and are involved in many biological processes, including apoptosis and cell cycle (37). Therefore, the role of ROS in these processes was examined. DCFH-DA staining and flow cytometry were performed to examine ROS accumulation in all of the groups. Based on the fluorescence inversion microscope system, stronger green fluorescence was evident in the combined group than in the other groups (Fig. 5A). The results from the flow cytometry showed that P2 peaks of the drug groups moved to the right compared with the control group, and quantitative analysis verified that the combined treatment was able to more effectively induce ROS accumulation in MFC cells than any monotherapy (Fig. 5B and C). Thus, the combined treatment exerted stronger effects on inducing ROS accumulation in MFC cells. Mitochondria are the main cell organelles where ROS are generated. MMP becomes abnormal when ROS generation exceeds the threshold value, and mitochondrial function is damaged by ROS over-capacity (37). JC-1 fluorescence staining and flow cytometry were applied to examine the MMP change of MFC cells exposed to drug treatments. The results showed that the combined treatment significantly induced MMP reduction, compared with any monotherapy (Fig. 5D and E). Thus, it was suggested that luteolin and oxaliplatin jointly exerted destructive effects on MMP and destroyed mitochondrial function in MFC cells.