Curcumin (batch number, SLBN7214V; molecular weight, 368.39 kDa) was purchased from Sigma-Aldrich; Merck KGaA. Fluorouracil (5-Fu; batch number, FA170415) was obtained from Shanghai Xudong Haipu Pharmaceutical Co., Ltd., and cisplatin (DDP; batch number, 8A0075B02) was purchased from Qilu Pharmaceutical Co., Ltd. The anti-CD11b-PE/Cy7 (cat. no. ab218786) and anti-Gr-1-FITC (cat. no. ab25024) antibodies were purchased from Abcam. PBS, DMEM and BSA were purchased from Sigma-Aldrich; Merck KGaA. HRP-conjugated goat anti-mouse IgG antibody (cat. no. SA00001-1), HRP-conjugated goat anti-rabbit IgG antibody (cat. no. SA00001-2) and HRP-conjugated donkey anti-goat IgG antibody (cat. no. SA00001-3) were purchased from ProteinTech Group, Inc. Protease inhibitors and protein phosphatase inhibitors were purchased from Roche Diagnostics.

The human liver cancer cell lines (HepG2, Huh-7 and MHCC-97H) were purchased from The Cell Bank of Type Culture Collection of The Chinese Academy of Sciences and stored at the Cell Center of Xiangya Medical College of Central South University (Changsha, China). The cells were cultured in DMEM supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) and 1X penicillin/streptomycin, and maintained in a humified atmosphere at 37°C with 5% CO₂. Curcumin was dissolved in DMSO to make a 9.6 mg/ml stock solution. HepG2, Huh-7 and MHCC-97H cells were co-incubated with curcumin (1.2, 2.4, 4.8 or 9.6 µg/ml) for 24 and 48 h at 37°C. In addition, HepG2 cells in the exponential growth phase were transplanted into mice to generate tumors as described subsequently.

Huh-7, MHCC-97H and HepG2 cells were seeded into 96-well plates at a density of 4,000 cells/well. Once attached, the cells were treated with 1.2, 2.4, 4.8 or 9.6 µg/ml curcumin for 24 or 48 h at 37°C. Cell viability was analyzed using CCK-8 reagent (cat. no. C0038; Beyotime Institute of Biotechnology) according to the manufacturer's protocol. Briefly, 10 µl CCK-8 reagent was added into each well, and cells were incubated for 1 h in the dark. Subsequently, the absorbance was measured at a wavelength of 450 nm using a microplate reader (BioTek Instruments, Inc.).

The cells were collected by trypsinization and adjusted to a density of 3×10⁵ cells/ml. The cell samples were washed twice with PBS via centrifugation for 10 min at 1,000 × g at 4°C, and resuspended in 1 µg anti-CD11b-PE/Cy7 (dilution, 1:100) and anti-Gr-1-FITC (dilution, 1:100) antibodies. Following 30 min of incubation at 37°C in the dark, the samples were performed on a Gallios flow cytometer (Beckman Coulter, Inc.) and analyzed using FlowJo V.10 software (Tree Star, Inc.). MDSCs were identified based on double positivity for CD11b and Gr-1.

BALB/c-nu nude mice [specific pathogen-free (SPF) grade; male; weight, 18–22 g; age, 21–28 days] were provided by Hunan SJA Laboratory Animal Co., Ltd. Mice were raised in an animal room (SPF grade) at Xiangya Medical College (Changsha, China). The animal experiment protocols were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (2011) (31) and approved by the Animal Research Ethics Committee of Central South University (approval no. 2018SYNKW0181; Changsha, China). A total of 28 mice were maintained at 25.0±2.0°C and 55.0±5.0% relative humidity with a 12-h light/dark cycle, and ad libitum access to food and water. Following acclimation for 1 week, 1×10⁷ HepG2 cells in 0.1 ml saline were injected into the right flank from the back of the mice to form subcutaneous xenograft tumors. Mice were divided into the following 4 groups (n=7/group) on the 5th day after injection when the tumor volume of HepG2 cells reached ~50 mm³: i) Model group (injected with cancer cells and administered intragastrically with the equivalent volume of 0.5% carboxymethylcellulose sodium, served as the negative control); ii) curcumin low dose group (Cur L; injected with cancer cells and administered intragastrically with 120 mg/kg curcumin daily); iii) curcumin high dose group (Cur H; injected with cancer cells and administered intragastrically with 240 mg/kg curcumin daily); and iv) chemotherapy group (injected with cancer cells and intraperitoneally injected with 50 mg/kg 5-Fu + 5 mg/kg DDP once a week; positive control). The regimens of 5-Fu+DDP and curcumin were based on their clinical application and a previous study, respectively (32,33). Furthermore, 240 mg/kg curcumin used in mice is equivalent to ~1.3 g/day in humans based on the body surface area (m²/kg) conversion between humans and mice, with a postulated adult body weight of 75 kg (human dosage=(240 mg/kg/14.16) ×75 kg), which was considered safe for humans according to a phase I clinical trial (34). The body weight and tumor volume of each mouse were measured every 3 days over a period of 15 days. The tumor volume was calculated using the following equation: Tumor volume=length × width × width/2. Animals were euthanized when >20% of weight loss was recorded, tumors were 1.0–1.5 cm in diameter or severe disease signs (e.g., difficulty breathing or paralysis) were observed. On day 15, a total of 28 mice (n=7/group) were sacrificed by intraperitoneal injection with an overdose of sodium pentobarbital (200 mg/kg), followed by cervical dislocation and rigor mortis as confirmation of death. The duration between injection and final tumor growth measurement was 20 days. The tumor tissues were removed, weighed and immediately stored at −80°C for further analysis. The maximum tumor diameter and volume observed in the present study was 15.83 mm and 516.32 mm³, respectively.

Total protein was extracted from tissues using 200 µl RIPA lysis buffer (Beyotime Institute of Biotechnology). Total protein was quantified by BCA and proteins (50 µg/lane) were separated via 10–12% SDS-PAGE and distinguished according to their molecular weights: TLR4, 75 kDa; TNF-α, 17 kDa; nuclear factor-κB kinase α (IKKα), 88 kDa; nuclear factor-κB kinase β (IKKβ), 85 kDa; phosphorylated (p-)IKKα, 88 kDa; p-IKKβ, 85 kDa; NF-κB, 65 kDa; MyD88, 33 kDa; IL-6, 24 kDa; IL-1β, 35 kDa; prostaglandin E2 (PGE2), 53 kDa; cyclooxygenase-2 (COX-2), 74 kDa; vascular endothelial growth factor (VEGF), 25 kDa; and β-actin, 42 kDa. The separated proteins were subsequently transferred onto nitrocellulose membranes, which were blocked with 5% BSA at room temperature for 2 h and incubated with the following primary antibodies at 4°C overnight: Anti-TLR4 (dilution, 1:700; cat. no. ab13556; Abcam), anti-TNF-α (dilution, 1:2,000; cat. no. 17590–1-AP; ProteinTech Group, Inc.), anti-IKKα (dilution, 1:10,000; cat. no. ab32041; Abcam), anti-IKKβ (dilution, 1:200; cat. no. 15649-1-AP; ProteinTech Group, Inc.), anti-p-IKKα (dilution, 1:500; cat. no. ab38515; Abcam), anti-p-IKKβ (dilution, 1:500; cat. no. ab59195; Abcam), anti-NF-κB (dilution, 1:500; cat. no. 10745-1-AP; ProteinTech Group, Inc.), anti-MyD88 (dilution, 1:500; cat. no. 23230-1-AP; ProteinTech Group, Inc.), anti-IL-6 (dilution, 1:500; cat. no. 21865-1-AP; ProteinTech Group, Inc.), anti-IL-1β (dilution, 1:200; cat. no. 16806-1-AP; ProteinTech Group, Inc.), anti-PGE2 (dilution, 1:1,000; cat. no. ab2318; Abcam), anti-COX-2 (dilution, 1:500; cat. no. 12375-1-AP; ProteinTech Group, Inc.), anti-VEGF (dilution, 1:500; cat. no. 19003-1-AP; ProteinTech Group, Inc.) and β-actin (dilution, 1:5,000; cat. no. 60008-1-Ig; ProteinTech Group, Inc.). Following the primary antibody incubation, the membranes were incubated with anti-rabbit secondary antibody (dilution, 1:6,000; cat. no. A0208; Beyotime Institute of Biotechnology) or HRP-conjugated goat anti-mouse IgG antibody (dilution, 1:500; cat. no. SA00001-1; ProteinTech Group, Inc.) for 1 h at room temperature. The protein bands were visualized using an ECL Western Blotting Substrate kit (cat. no. P0018FS; Beyotime Institute of Biotechnology). Bands were semi-quantified using ImageJ software (ImageJ 1.8.0; National Institutes of Health).

Mouse Granulocyte Colony Stimulating Factor and mouse Granulocyte Macrophage Colony Stimulating Factor ELISA kits were respectively used to determine G-CSF (cat. no. CSB-E04564m; Cusabio Biotech Co., Ltd.) and GM-CSF levels (cat. no. CSB-E04569m; Cusabio Biotech Co., Ltd.) in tumor tissues according to the manufacturer's protocols.

The tumor tissues were fixed in 10% neutral buffered formalin for 24 h at room temperature, and then embedded in paraffin. The paraffin-embedded tumor tissue sections (thickness, 5 µm) were deparaffinized in xylene, rehydrated using an ascending series of alcohol in the following sequence: 100% ethanol for 3 min, 90% ethanol for 3 min, 80% ethanol for 3 min and 70% ethanol for 3 min and subsequently flushed with distilled water, followed by washing thoroughly with 0.01 M PBS (pH 7.2–7.6). For antigen retrieval, trypsin antigen retrieval solution was directly pipetted onto the tissue on the slide for 10 min at 37°C. Subsequently, endogenous peroxidase activity was quenched by a 30-min incubation in methanolic hydrogen peroxide (2.5%). After being sealed with 5% BSA for 1 h at room temperature, the slides were incubated overnight at 4°C with the following primary antibodies: Anti-CD31 (dilution, 1:500; cat. no. 11265-1-AP; ProteinTech Group, Inc.), anti-α-smooth muscle actin (αSMC; dilution, 1:300; cat. no. ab21027; Abcam) and anti-VEGF (dilution, 1:500; cat. no. 19003-1-AP; ProteinTech Group, Inc.). Following the primary antibody incubation, the sections were incubated with the corresponding HRP-conjugated anti-rabbit secondary antibody (dilution, 1:500; cat. no. A0208; Beyotime Institute of Biotechnology) or HRP-conjugated donkey anti-goat IgG antibody (dilution, 1:500; cat. no. SA00001-3; ProteinTech Group, Inc.) at 37°C for 30 min. Subsequently, the slides were stained with 3,3′-diaminobenzidine at 25°C for 5 min. All sections were observed under a light or fluorescence microscope (magnification, ×400) and analyzed using HistoQuest 4.0 software (TissueGnostics GmbH) as previously described (35).

Statistical analysis was performed using SPSS version 21.0 software (IBM Corp.) and the results are presented as the mean ± SD, except cell viability assay data which are presented as the mean ± SEM. All data were from at least three independent triplicate experiments. Statistical differences among groups were analyzed using one-way ANOVA followed by Tukey's multiple comparisons test and the normality of data was determined using a Kolmogorov-Smirnov test. P<0.05 was considered to indicate a statistically significant difference.

To investigate the role of curcumin in regulating liver cancer cell proliferation, the effects of treatment with 1.2–9.6 µg/ml curcumin on the viability of Huh-7, MHCC-97H and HepG2 cells were determined. The results revealed a dose-dependent reduction in cell viability following 24 and 48 h of treatment (Fig. 1A and B). To verify the results in vivo, a HepG2 ×enograft nude mouse model was used. Similarly, 120 and 240 mg/kg/day curcumin progressively decreased the tumor volume compared with the model group, which was measured for 15 days (Fig. 1C). Treatment with curcumin was observed to cause a decrease in body weight on the 10th day; however, the body weight subsequently increased and was not affected by curcumin treatment by the 15th day (Fig. 1D). As expected, the curcumin-induced decline in tumor volume was also associated with a marked decrease in tumor weight, revealing comparable tumor growth inhibition efficacy between curcumin and chemotherapy treatments, which indicated the therapeutic potential of curcumin in liver cancer (Fig. 1F). The evaluation of tumor histology using hematoxylin and eosin staining indicated that the groups treated with curcumin (120 and 240 mg/kg/day) and the chemotherapy treatment group exhibited necrosis and lower cellularity compared with the model group (Fig. 1H). As shown in Fig. 1E and G, the in vivo and ex vivo images of the tumors further indicated the tumor-suppressive effects of curcumin.

MDSCs serve a crucial role in the immunology of the majority of tumors, and are responsible for inhibiting the immune function of T cells and promoting angiogenesis (36). It was hypothesized that curcumin may inhibit the expansion of MDSCs, which would subsequently enhance immune function. To test this hypothesis, flow cytometry was used to determine CD11b⁺Gr-1⁺ expression in mouse xenograft tumor tissues. As shown in Fig. 2A and B, compared with the model group, treatment with 120 or 240 mg/kg curcumin or chemotherapy significantly reduced the percentage of MDSCs (in 7 mice). Notably, the treatment with curcumin at both a low (120 mg/kg) and high (240 mg/kg) dose exhibited a comparable inhibitory effect on MDSCs, suggesting that there were no dose-dependent effects when comparing low and high doses of curcumin. These results suggested that curcumin may inhibit the expansion of MDSCs in mice xenograft tumor tissues, but not in a dose-dependent manner.

To investigate whether the inhibitory effect of curcumin on MDSCs could be attributed to M-CSF and GM-CSF secretion, ELISAs were used to determine the secretory levels of GM-CSF and G-CSF. As illustrated in Fig. 2C and D, treatment with Cur H and chemotherapy notably reduced G-CSF and GM-CSF levels compared with the model group, whereas Cur L only significantly decreased GM-CSF levels. These data indicated that curcumin may suppress M-CSF and GM-CSF secretion, which could lead to the inactivation of MDSCs, and thereby restrict cancer cell proliferation.

To investigate the latent molecular mechanism underlying the curcumin-modulated activity of MDSCs, the expression levels of proteins associated with the TLR4/NF-κB signaling pathway were analyzed using western blotting. The results revealed that both curcumin and chemotherapy treatment significantly decreased TLR4, MyD88, p-IKKα/IKKα, p-IKKβ/IKKβ and NF-κB expression compared with the model group. Notably, high doses of curcumin (Cur H) exhibited greater effects than the treatment with chemotherapy in terms of the inhibition of the TLR4/NF-κB signaling pathway, except the ratios of p-IKKα/IKKα and p-IKKβ/IKKβ (Fig. 3A-E). Considering that the TLR4/NF-κB signaling pathway is the major inflammatory signaling pathway that mediates the expression levels of numerous inflammatory cytokines in cancer (37), and that inflammation serves an important role in cancer progression and MDSC recruitment and activation (38), the expression levels of TNF-α, IL-6, IL-1β, PGE2 and COX-2 in xenograft tumor tissues were analyzed. As shown in Fig. 3, treatment with curcumin at two doses (Cur L and Cur H) and chemotherapy significantly blocked the increased expression of TNF-α, IL-6, IL-1β, PGE2 and COX-2 compared with the model group (Fig. 3F-I). Notably, Cur H was at least comparable or superior to the chemotherapy treatment in terms of its ability to downregulate the expression levels of the proteins. These data indicated that curcumin may suppress the TLR4/NF-κB signaling pathway and control the inflammatory response, which may be the underlying mechanism inhibiting MDSC activity and thus, inhibiting cancer progression.

The angiogenic programming of neoplastic tissue is a multidimensional process that is regulated by cancer cells in concert with a variety of tumor-associated stromal cells and their bioactive products. MDSCs promote tumor angiogenesis by producing pro-angiogenic growth factors, such as VEGF (39). The results of the western blot analysis demonstrated that 120 and 240 mg/kg curcumin, particularly Cur H, significantly downregulated VEGF expression in vivo compared with that in the model group (Fig. 4A). To further verify the anti-angiogenic effect of curcumin in vivo, the excised tumor tissues were analyzed using IHC for CD31 expression (a platelet endothelial cell adhesion molecule) and VEGF, and by immunofluorescence using anti-αSMC (an arterial smooth muscle cell marker) and anti-CD31 antibodies. The observed downregulated expression levels of VEGF, CD31 and αSMC in the tumor tissues indicated that angiogenesis may be suppressed by curcumin treatment (Fig. 4B). Overall, these findings indicated that curcumin may inhibit VEGF and CD31-induced angiogenesis in vivo.