Human Saos-2 and MG-63 cell lines were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). Berberine was purchased from Sigma-Aldrich (St. Louis, MO, USA). Cells were cultured in Dulbeccos modified Eagles medium (DMEM; HyClone Laboratories, Inc., Logan, UT, USA) supplemented with 10% (v/v) fetal bovine serum (FBS; Gibco, Carlsbad, CA, USA) in an atmosphere of 95% humidified air and 5% CO₂ at 37°C. Cells were investigated within 8 h of harvest. To detect the effects of berberine on osteosarcoma, cells were treated without or with berberine (Sigma-Aldrich), respectively. To detect the effects of caspase-1 on osteosarcoma, cells were treated without or with selective caspase-1 inhibitor N-Ac-Tyr-Val-Ala-Asp-CMK (Ac-YVAD-CMK) (Cayman Chemical, Ann Arbor, MI, USA), respectively.

Cell viability was determined by MTT assay according to the manufacturer's instructions. Briefly, cells (2×10⁴ cells/well) were seeded in a 96-well plate and treated differently based on the experimental purpose. Cells were washed with phosphate-buffered saline (PBS), and then 20 µl of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide solution (5 mg/ml) was added to each well. The plate was covered and shaken at room temperature, after which the medium was discarded. Next, dimethyl sulfoxide (DMSO) was added to each well (200 µl), and the solution was vigorously mixed to dissolve the purple tetrazolium crystals. The amount of produced purple formazan is proportional to the percentage of cell viability. The absorbance of each well was measured by automated microplate reader at a test wavelength of 490 nm. All experiments were repeated at least three times.

Western blotting was used to detect the expression levels of the proteins of interest. Drugs were diluted and added to cells for 24 h at 37°C before analysis by western blot. The cells were washed using ice-cold phosphate-buffered saline, and total protein was harvested with radioimmunoprecipitation assay buffer (RIPA) containing 1% protease inhibitor (Sigma-Aldrich). Protein (100 µg) per sample was separated using 12% SDS-PAGE, and then transferred into nitrocellulose membranes. The membrane was blocked with 5% non-fat milk (BD Biosciences, San Jose, CA, USA) and 0.1% Tween-20 in Tris-buffered saline and immunoblotted overnight using appropriate primary antibodies at 4°C with gentle shaking. After that, fluorochrome labelled secondary antibody (Alexa Fluor 800; LI-COR Biosciences, Lincoln, NE, USA) was used to identify the appropriate primary antibody. Immunoreactivity was detected with the Odyssey fluorescent scanning system (LI-COR Biosciences) and analyzed by Image Studio software. β-actin was used as a loading control.

Real-time PCR was used to measure caspase-1 and IL-1β mRNA levels. Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) from tissues and cells. First-strand cDNA was synthesized using a reverse transcriptase kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's instructions. Real-time PCRs were carried out with a SYBR-Green PCR Master Mix kit (Applied Biosystems) and performed on 7500 FAST Real-Time PCR system (Applied Biosystems, Carlsbad, CA, USA). GAPDH was used as an internal control. The following primers were used in the study. Caspase-1: forward, 5-ACACGTCTTGCCCTCATTATCT-3 and reverse, 5-ATAACCTTGGGCTTGTCTTTCA-3; IL-1β: forward, 5-CCCTGCAGCTGGAGAGTGTGG-3 and reverse, 5-TGTGCTCTGCTTGAGAGGTGCT-3; GAPDH: forward, 5-ATCACTGCCACCCAGAAGAC3 and reverse, 5-TTTCTAGACGGCAGGTCAGG-3.

Cells were seeded on coverslips in 6-well culture plates and grown overnight for adherence, then treated with different drugs, respectively. Apoptosis was measured by an In Situ Cell Death Detection kit according to the manufacturer's instructions (Roche Applied Science). In brief, cells were fixed with freshly prepared 4% paraformaldehyde for 60 min. The slides were rinsed with PBS and incubated in 0.1% Triton X-100 permeabilization solution for 2 min. Then slides were incubated with TUNEL reaction mixture for 60 min at 37°C in a humidified chamber. the rinsed slides were counterstained with DAPI. Cells were counted under a fluorescence microscopy and the green fluorescence staining cells were calculated as positive-staining cells. All experiments were repeated at least three times.

The experimental protocols were approved by the Ethic Committee of Harbin Medical University (Harbin, China). The use of animals followed the National Institutes of Health guide for the care and use of laboratory animals published by the US National Institutes of Health (NIH Publication no. 85–23, revised 1996). BALB/c-nu/nu mice, male, 5–6-week old weighing 18–20 g were used. The mice were housed with a regular 12-h light/12-h dark cycle and ad libitum access to standard rodent chow diet and were kept in a pathogen-free environment. For in vivo tracking, the Saos-2 and MG-63 cells were stably transfected with firefly luciferase. Saos-2 and MG-63 cells (1×10⁷ cells were suspended in 100 µl serum-free DMEM) were injected subcutaneously into the back of mice. Eight days post-implantation, the mice were randomly divided into three groups (n=6 for each group) and fed by oral gavage with saline, berberine (20 mg/kg/day), or berberine and intraperitoneal caspase-1 inhibitor Ac-YVAD-CHO (0.1 mg/kg/day). Tumor growth was monitored by luciferase activity in Saos-2 and MG-63 cells, and the emitted photons from the target site penetrated through the mammalian tissue and could be externally detected and quantified using a sensitive light imaging system. Mice were euthanized 21 days after treatment and the tumors were isolated for further detection.

Data are expressed as mean ± standard error of mean (mean ± SEM) and analyzed with SPSS 13.0 software. Statistical comparisons between the two groups were performed using the Students t-test. Statistical comparisons among multiple groups were performed using analysis of variance (ANOVA). A two-tailed P<0.05 was taken to indicate a statistically significant difference.

Based on previous studies on the relationship between osteosarcoma and inflammation, and the central role of caspase-1 in the process of inflammation (18–20), we first compared the expression level of caspase-1 in osteosarcoma tissues from six pairs of clinical osteosarcoma cases with peritumoral tissues. Then we found that the expression of caspase-1 was obviously elevated both in the mRNA and the protein level, which is consistent to our expectation (Fig. 1).

Next, we evaluated the effects of berberine on Saos-2 and MG-63 cells by MTT assay. MTT assay results demonstrated that berberine significantly inhibits the growth of Saos-2 and MG-63 cells in a time- and dose-dependent manner. As shown in Fig. 2A and B, the concentration of berberine at 80 µM could inhibit the cell viability to the greatest extent; and the viable cells at 48 h decreased more obviously than 24 h after treatment with 80 µM berberine. Thus, the following administration of berberine were all at 48 h with 80 µM. Fig. 2C and D shows that berberine significantly reduced osteosarcoma cell viability and caspase-1 inhibitor exerts similar effects, which suggest that caspase-1 may be involved in the inhibition of osteosarcoma cell growth caused by berberine.

To study the effect of berberine administration on osteosarcoma cell apoptosis, TUNEL assay was performed. Cells in the images with green nuclei were considered apoptotic. In Fig. 3, we found few cells with nuclei staining green in the control group. After being exposed to 80 µM berberine for 48 h, ~13.12% of cells showed apoptotic hallmarks. Moreover, caspase-1 inhibitor exert similar effect to berberine. In addition, co-incubation berberine with caspase-1 inhibitor exerted inhibitory effects to the greatest extent. Thus, these results indicated that berberine could induce apoptosis of osteosarcoma cells possibly by caspase-1 involved process.

Currently, the role of inflammation in cancers has caused widespread concern. It is believed that inflammation could promote the occurrence and development of cancer (21,22). Real-time PCR and western blot assay was performed to explore the molecular mechanism of berberine on the anti-osteosarcoma property. As we can see from the bar graphs (Figs. 4 and 5), caspase-1 mRNA and protein expression level were both downregulated in Saos-2 and MG-63 osteosarcoma cells after treated with berberine; caspase-1 inhibition extert similar effect to berberine on caspase-1 expression level. At the same time, to gain further insights into the mechanism of anti-osteosarcoma of berberine, we analyzed the expression level of caspase-1 downstream target IL-1β, which plays a decisive role in the formation of tumor inflammatory microenvironment. The results show that the expression of IL-1β was consistent with caspase-1. Furthmore, immunofluorescence staining analysis was used to further confirm the anti-osteosarcoma property of berberine. Accordingly, immunofluorescence results showed that the expression of caspase-1 and IL-1β were both downregulated compared with normal groups. Caspase-1 inhibition exterts similar effect to berberine on osteosarcoma cells (Fig. 6).

After eight days of post-implantation of the osteosarcoma cells, the mice were treated differently. Fig. 7 shows that the size of the osteosarcoma shrinks obviously after administration of berberine by oral gavage compared with the control group. Then, the tumor tissues were isolated for further mRNA and protein detection. The results from the tumor tissues were in accordance with the results from Saos-2 and MG-63 osteosarcoma cells (Fig. 8), which illustrate that berberine attenuates the activation of caspase-1/IL-1β signal pathway. In conclusion, these observations demonstrate that berberine could probably relieve the inflammation in tumor microenvironment and then results in apoptosis of osteosarcoma cells.