Curcumin (cat. no. S1848; Selleck Chemicals, Houston, TX, USA) was dissolved in dimethyl sulfoxide (DMSO) and stored at 4°C as a stock solution. Cisplatin (cat. no. S1166; Selleck Chemicals) was dissolved in normal saline and stored at room temperature. RPMI-1640 medium, fetal bovine serum (FBS), penicillin, streptomycin and all other cell culture reagents were obtained from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). Primary antibodies against cleaved-poly (ADP-ribose) polymerase (PARP) (cat. no. 9548), caspase-3 (cat. no. 9665), cleaved caspase-3 (cat. no. 9664), caspase-8 (cat. no. 4927), cleaved caspase-8 (cat. no. 8592), caspase-9 (cat. no. 9504), PI3K (cat. no. 4257), AKT serine/threonine kinase (Akt) (cat. no. 4691), phosphorylated (p)-Akt (Ser473) (cat. no. 9271), mammalian target of rapamycin (mTOR) (cat. no. 2972), p-mTOR (Ser2448) (cat. no. 5536), p70 ribosomal protein S6 kinase (p70S6K) (cat. no. 2708), p-p70S6K (Thr389) (cat. no. 9234) and Histone H3 (cat. no. 4499) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Primary antibodies against β-actin (cat. no. YT0099) and GAPDH (cat. no. YT5052) were purchased from ImmunoWay Biotechnology Company (Plano, TX, USA). Primary antibodies against apoptosis-inducing factor (AIF) (cat. no. ab1998), B-cell lymphoma (Bcl)-extra large (xL) (cat. no. ab32370), Bcl-2-associated X protein (Bax) (cat. no. ab32503), cluster of differentiation (CD)31 (cat. no. ab28364) and Ki67 (cat. no. ab15580) were purchased from Abcam (Cambridge, MA, USA). Horseradish peroxidase (HRP)-conjugated secondary antibodies [Anti-rabbit immunoglobulin G (IgG), HRP-linked Antibody #7074 or Anti-mouse IgG, HRP-linked Antibody #7076] were purchased from Cell Signaling Technology, Inc. Polyvinylidene difluoride (PVDF) membranes and western blot luminescence reagents were purchased from EMD Millipore (Billerica, MA, USA). Cell cycle assay kit and cell apoptosis kit were purchased from Nanjing KeyGen Biotech Co., Ltd. (Nanjing, China). Alexa Fluor-555-conjugated goat anti-rabbit IgG antibody (cat. no. A27039), and the nuclear and cytoplasmic extraction kit were purchased from Thermo Fisher Scientific, Inc.

The RN5 murine malignant mesothelioma cell line (13), which was kindly gifted by Dr Marc de Perrot's Laboratory (Toronto General Hospital, Toronto, ON, Canada), was maintained in RPMI-1640 medium supplemented with 10% FBS and 100 µg/ml penicillin-streptomycin. The cells were maintained at 37°C in the presence of 5% CO₂.

The inhibitory effects of curcumin were determined using the sulforhodamine B (SRB) assay. Briefly, RN5 cells were seeded at 4×10³ cells/well in 96-well flat-bottom plates for 24 h at 37°C. The cells were then treated with curcumin or cisplatin at various concentrations for 72 h at 37°C in the presence of 5% CO₂. The optical density (OD) was measured at 510 nm using a microplate reader. The percentage of cells that were killed was calculated using the following formula: 100% - ODsample/ODcontrol. Half maximal effective concentration (EC₅₀) values were calculated using Sigmaplot 12.0 software (Systat Software Inc., San Jose, CA, USA).

Cells were seeded in a 10-cm dish (10⁶ cells/dish) and were incubated at 37°C for 24 h. Cells were then treated with vehicle control (DMSO 0.1%), various doses of curcumin (15, 20 and 25 µM) or cisplatin (30 µM) for 24 h, after which, cells were lysed with extraction buffer (cat. no. FNN0011; Thermo Fisher Scientific, Inc.) supplemented with protease inhibitor (cat. no. P8340; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), phosphatase inhibitor (cat. no. 4906845001; Roche Diagnostics, Basel, Switzerland) and 1 mM phenylmethylsulfonyl fluoride. Protein concentration was determined using the Pierce™ bicinchoninic acid protein assay kit (Thermo Fisher Scientific, Inc.). Equal volumes of protein (15-40 µg) were separated by 10% SDS-PAGE and were electrophoretically transferred onto PVDF membranes. The membranes were blocked with 5% non-fat milk in Tris-buffered saline (TBS) with 0.1% Tween-20 and were agitated for 1 h at room temperature. Subsequently, the membranes were incubated overnight at 4°C with specific primary antibodies (1:1,000, in 5% non-fat dry milk or bovine serum albumin (cat. no. B2064; Sigma-Aldrich; Merck KGaA) in TBS with 0.1% Tween-20). The membranes were washed three times with TBS containing 0.1% Tween-20 solution and were then incubated with the secondary antibodies (1:5,000, in 5% non-fat milk in TBS with 0.1% Tween-20). After secondary antibody incubation, the membranes were washed three times with TBS with 0.1% Tween-20 and the proteins were detected using the Luminata Forte Western HRP substrate (EMD Millipore), according to the manufacturer's protocol. Band intensity was analyzed using ImageJ 1.51 software (National Institutes of Health, Bethesda, MD, USA), according to previous studies (14,15).

RN5 cells were seeded in a 6-well plate at a cell density of 2×10⁵ cells/well. The cells were treated with various concentrations of curcumin for 24 h, whereas cisplatin (30 µM) was used to treat cells as a positive control. After treatment, cells were harvested, washed twice with PBS and fixed in 70% ethanol at −20°C overnight. After washing, the cells were suspended in PBS containing 50 µg/ml propidium iodide (PI) and were incubated at 4°C for at least 4 h. Cell cycle analysis was performed using flow cytometry. Fluorescence was measured using the BD FACSCanto II flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) and cell cycle progression was estimated using Modfit LT 4.1 Software (Verity Software House, Inc., Topsham, ME, USA).

RN5 cells were seeded in a 6-well plate at a cell density of 2×10⁵ cells/well. The cells were treated with various concentrations of curcumin for 24 h, whereas cisplatin (30 µM) was used to treat cells as a positive control. The cells were then collected and washed twice with cold PBS. Annexin V-PI (AV-PI) double staining was performed according to the manufacturer's protocol. Apoptosis was analyzed using the BD FACSCanto II flow cytometer (BD Biosciences) and FlowJo version 10.0.7 (FlowJo LLC, Ashland, OR, USA).

To evaluate the in vivo effects of curcumin, the effects of curcumin on tumor growth were investigated in tumor-bearing mice. The animal study was approved by the Committee on the Ethics on Animal Experimentation of The Second Hospital of Shandong University [no. KYLL-2017(LW) 011, approved February 3, 2017; Jinan, China]. A total of 20 male C57BL/6J mice (weight, 20-25 g; age, 6 weeks) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China) and were housed under pathogen-free conditions. The mice were maintained in a temperature-controlled room (22-24°C) with a relative humidity between 55 and 65% under a 12-h light/dark cycle. Food and water were freely available. RN5 cells (5×10⁶ cells in 100 µl PBS) were injected subcutaneously into the dorsal flank of each mouse. When the tumors reached volumes of 60 mm³, the animals were randomly assigned into four groups. The mice were treated five times every 5 days with intraperitoneal administration of i) 200 µl solvent (PBS/cremophor/DMSO/ethanol, 15:3:1:1); ii) curcumin at a low dose (200 mg/kg); or iii) curcumin at a high dose (500 mg/kg). As a positive control, another group of mice was treated with cisplatin at a dose of 5 mg/kg. Cisplatin was dissolved in normal saline at a final concentration of 1 mg/ml. Body weight was measured every day in order to monitor the toxicity of the treatments. Tumor volumes were determined using the following formula: a × b²/2, where 'a' is the longest diameter and 'b' is the shortest diameter. The mice were euthanized by CO₂ inhalation when the tumor in the solvent-treated group grew to 15 mm in diameter. The tumor samples were then collected and were fixed in 10% formalin neutral buffer solution for at least 24 h at room temperature. Sections were then paraffin-embedded.

RN5 cells were seeded into a 10-cm Petri dish (10⁶ cells/dish) and were incubated at 37°C for 24 h. Cells were then treated with vehicle control (DMSO 0.1%), three doses of curcumin (15, 20 and 25 µM) for 24 h. After treatment, cells were trypsinized, and nuclear and cytoplasmic fractions were isolated according to the manufacturer's protocol, using NE-PER^® Nuclear and Cytoplasmic Extraction reagents (Thermo Fisher Scientific, Inc.). After isolation of nuclear and cytoplasmic fractions, western blotting was performed using antibodies specific for the nuclear fraction of AIF and Histone H3.

Paraffin- embedded sections (5 µm) of tumor tissues were mounted on gelatin-coated histological slides, deparaffinized with xylene, and rehydrated in a descending series of alcohol (100-70%) and distilled water at room temperature for 5 min. Antigen retrieval was performed by microwaving the slides in 10 mM citrate buffer (pH 6.0) for 20 min. Endogenous peroxidase activity was quenched by incubation with 3% H₂O₂ for 20 min, after which, the sections were blocked with 1.5% goat serum (Beyotime Institute of Biotechnology, Shanghai, China) for 60 min at room temperature to prevent non-specific binding. The sections were then incubated overnight at 4°C with primary antibodies against CD31 and Ki67 (1:100). The slides were washed three times with PBS containing 0.1% Tween-20 (5 min/wash) and were incubated with a secondary antibody (1:10,000) [Goat anti-Rabbit IgG H&L (HRP), cat. no. ab205718; Abcam] for 1 h at room temperature. Immunohistochemical staining for CD31 and Ki67 was further performed using the avidin-biotin peroxidase complex method, with diaminobenzidine (DAB) as a chromogenic substrate. Immunostained sections were counterstained with hematoxylin staining solution (Beyotime Institute of Biotechnology) for 40 sec at room temperature, and were then examined by light microscopy.

To semi-quantitatively analyze tumor cell proliferation and apoptosis in tumor sections, the H-score was used to evaluate the expression of Ki67-positive cells, as previously reported (16,17). The H-score was calculated using the following formula: (0 × percentage of negative staining) + (1 × percentage of weak staining) + (2 × percentage of moderate staining) + (3 × percentage of strong staining). Staining intensity was classified as follows: Negative staining (no staining), weak staining (light yellow), moderate staining (yellowish brown) and strong staining (tan). The values ranged between 0 and 300. CD31-positive area was used to analyze angiogenesis.

For TUNEL staining of tumor tissue samples, proteolytic digestion was conducted by incubating the sections for 10 min at room temperature with proteinase K (10 µg/ml), after which, they were fixed in 4% paraformaldehyde solution for 5 min at room temperature. After rinsing with PBS, the samples were permeabilized in equilibration buffer for 5 min. Samples were then incubated with recombinant terminal deoxynucleotidyl transferase reaction mix for 60 min at 37°C, washed in saline sodium citrate solution for 15 min, and then quenched with 0.3% H₂O₂ in PBS for 5 min. After washing with PBS, the samples were incubated with HRP-labeled streptavidin for 30 min at room temperature and DAB was applied as a chromogenic substrate. The sections were then dehydrated through a graded series of alcohol, immersed in xylene and mounted with coverslips. The sections were examined under an Olympus BX43 light microscope (Olympus Corporation, Tokyo, Japan). The H-score was used to evaluate the expression of TUNEL-positive cells, as aforementioned.

RN5 cells were seeded onto glass coverslips at 2×10⁵ cells in a 6-well plate overnight at 37°C in the presence of 5% CO₂. Subsequently, cells were treated with 15, 20 or 25 µM curcumin at 37°C for 24 h, after which, cells were rinsed with cold PBS and fixed with 4% paraformaldehyde for 30 min at room temperature. Fixed cells were washed with PBS and were permeabilized with 1% Triton X-100 in PBS for 10 min. Cells were then blocked with goat serum for 30 min at room temperature and were incubated with AIF primary antibodies (1:500) overnight at 4°C. After washing with PBS three times (5 min/wash), fixed cells were incubated with Alexa Fluor-555-conjugated goat anti-rabbit IgG antibody at room temperature for 1 h. After washing three times with 0.1% Tween-20 in PBS, the coverslips were mounted with mounting medium containing DAPI (Abcam). Cells were examined under an Olympus BX43 fluorescence microscope (Olympus Corporation).

The data are presented as the means ± standard deviation. All of the statistical analyses were performed using Sigmaplot 12.0. Statistical comparisons between the control and treatment groups, as well as differences within the experimental groups at different doses, were conducted using one-way analysis of variance followed by least-significant difference or Tukey's tests. P<0.05 was considered to indicate a statistically significant difference.

To investigate the inhibitory effects of curcumin on RN5 cell viability, RN5 cells were treated with increasing concentrations of curcumin. Cisplatin was used as a positive control. Cell viability was determined following treatment with increasing concentrations of curcumin for 72 h (Fig. 1A). The results demonstrated that curcumin induced a significant inhibition in RN5 cell viability in a dose-dependent manner, as measured by the SRB assay at 72 h, relative to control cells (Fig. 1A). The EC₅₀ value of curcumin at 72 h was 19.1±1.3 µM, whereas the EC₅₀ value of cisplatin was 7.09±1.14 µM (Fig. 1B). These findings indicated that curcumin may inhibit MPM cell viability in a dose-dependent manner. In addition, 5 µM cisplatin killed ~50% of RN5 cells after 72 h of treatment, whereas 20 µM curcumin provoked similar effects, thus suggesting that cisplatin is more cytotoxic to RN5 cells than curcumin.

Previous studies have reported that curcumin induces G₁/S or G₂/M cell cycle arrest in numerous types of human cancer cells (18,19). In order to investigate whether curcumin induced a cell cycle arrest in RN5 cells, cell cycle distribution was examined following treatment with curcumin for 24 h. As shown in Fig. 1C, <10% of RN5 cells were in G₂/M phase in the DMSO groups. Conversely, 18, 35 and 41% of cells were in G₂/M phase in the 15, 20 and 25 µM curcumin groups after 24 h, respectively. These findings indicated that 25 µM curcumin resulted in an obvious G₂/M cell phase arrest. Curcumin may inhibit cell viability in a dose-dependent manner and may induce G₂/M cell phase arrest.

It is well accepted that cell cycle arrest at G₂/M phase leads to apoptotic cell death. The present study assessed curcumin-induced RN5 cell apoptosis using flow cytometry. AV-PI staining indicated that treatment with 15 µM curcumin markedly increased early and late apoptosis. Treatment of RN5 cells with 15, 20 and 25 µM curcumin for 24 h induced an increase in the percentage of AV- and PI-positive cells (Fig. 2A).

Apoptosis is induced via two main routes, involving either the caspase-dependent or caspase-independent pathway. It has previously been demonstrated that mitochondria or activation of the death receptor pathway converge to induce the activation of caspases, which are the final executioners of cell death (20). However, treatment of RN5 cells with increasing doses of curcumin did not affect the expression levels of activated caspase-3, -8 or -9 after 24 h (Fig. 2B).

Increased levels of the proapoptotic protein Bax and decreased levels of the anti-apoptotic protein Bcl-xL were observed in response to curcumin (Fig. 2C). Furthermore, cleaved-PARP expression was also increased (Fig. 2C). These results demonstrated that curcumin-induced apoptosis may be mediated by the mitochondrial pathway.

Notably, caspase-independent forms of apoptosis are mediated by AIF. To explore whether AIF nuclear translocation was involved in curcumin-induced apoptosis, subcellular AIF protein expression was measured by western blotting and AIF nuclear translocation was detected by immunofluorescence (Fig. 3A and B). Nuclear AIF expression was significantly increased after 24 h curcumin treatment in a dose-dependent manner. In addition, AIF-positive immunofluorescence was increased in the nucleus in a dose-dependent manner. These results indicated that the caspase-independent AIF nuclear translocation-mediated apoptotic pathway may be involved in curcumin-induced cell death.

The PI3K-Akt-mTOR signaling pathway has a critical role in various cellular processes (21), including survival, proliferation, apoptosis and angiogenesis. Previous studies on cancer have revealed that curcumin inhibits the PI3K-Akt-mTOR signaling pathway (22,23). Targeting the PI3K-Akt-mTOR signaling pathway, which is downstream of several activated receptor tyrosine kinases (RTKs) in mesothelioma, seems to be an emerging therapeutic strategy (24). As shown in Fig. 4A and B, treatment with increasing doses of curcumin resulted in significant downregulation of PI3K, p-Akt, p-mTOR and p-p70S6K; however, no significant differences were detected with regards to the total expression levels of Akt, mTOR and p70S6K. These results suggested that the PI3K-Akt-mTOR signaling pathway may be involved in curcumin-induced apoptosis.

Tumor volume was significantly reduced following three injections of cisplatin and 500 mg/kg curcumin. The mean volumes of subcutaneous tumors in the cisplatin- and high dose curcumin-treated mice were decreased compared with in the vehicle control group (Fig. 5A and B). The experiment was terminated and the mice were sacrificed on day 25 following tumor cell injection. The mean tumor volumes in mice treated with cisplatin and a high dose of curcumin were 58 and 72 mm³, respectively, whereas the tumor volumes in mice treated with the solvent and a low dose of curcumin were 161 and 120 mm³ on day 25. The body weight of the mice was also monitored, in order to evaluate the potential side effects of curcumin. As shown in Fig. 5C, there was no obvious body weight loss in mice treated with curcumin throughout the experiment, thus indicating that curcumin did not exert evident systemic toxicity at the doses used in this investigation. Body weight loss in mice treated with cisplatin reflected the toxicity of the drug towards normal tissue (Fig. 5C). These results suggested that curcumin may inhibit tumor growth in vivo with no obvious toxicity.

As tumor cell proliferation and inhibition of apoptosis are attributable to tumor growth, Ki-67 immunohistochemistry and TUNEL analysis were performed on tumor sections. As shown in Fig. 6A and B, treatment with cisplatin or a high dose of curcumin affected the number of proliferating Ki-67-positive cells. In addition, using TUNEL cell death detection, it was revealed that tumors from the cisplatin and high-dose curcumin groups contained an increased number of TUNEL-positive apoptotic cells. CD31 immunostaining was also conducted to evaluate tumor angiogenesis following curcumin treatment in mice. The positive area of CD31 was significantly lower in the high dose curcumin-treated group (Fig. 6C). These results indicated that curcumin may inhibit tumor growth through inducing apoptosis, and inhibiting cell proliferation and angiogenesis.