Cadmium chloride (CdCl2), Cisplatin (DDP), and 2,7-dichlorofluorescin diacetate were obtained from Sigma-Aldrich/Merck KGaA. Dulbecco's modified Eagle's medium (DMEM) with high glucose, penicillin, streptomycin and fetal bovine serum (FBS) were obtained from Thermo Fisher Scientific, Inc. The MTT Cell Proliferation and Cytotoxicity Assay Kit was purchased from Beyotime Institute of Biotechnology. The following antibodies were used: Cleaved caspase-3 antibody [dilution, 1:1,000 for Western blot analysis (WB); cat. #9664; Cell Signaling Technology, Inc. USA (CST)], Bcl-2 (dilution 1:1,000 for WB; cat. #15071; CST), BAX (dilution 1:1,000 for WB; cat. #5023; CST), MMP-2 (dilution 1:1,000 for WB; cat. #4022; CST), MMP-9 (dilution 1:1,000 for WB; cat. #3852; CST), E-cadherin (dilution 1:2,000 for WB; cat. #3195; CST), FOXM1 (dilution 1:80 for IHC, 1:1,000 for WB; cat. no. ab232649; Abcam) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (dilution 1:1,000 for WB).

The human embryo immortalized osteoblast cell line Hfob1.19 and OS cell lines MG63, U2OS, 143B and SaoS2 were purchased from Yong Jin Biotech and cultured in DMEM containing 10% FBS, penicillin 100 U/ml and streptomycin 100 pg/ml, in 5% CO₂ at 37°C. Cells were assessed when in the logarithmic growth phase.

Cells in the logarithmic growth phase were plated into 6-well plates at a density of 5×10⁵ cells/well. After incubation for 12–24 h, the cells were treated with 20 µM cadmium chloride (CdCl₂) for 24 h and fixed in 2.5% glutaraldehyde solution overnight at 4°C. The cells were washed in PBS, fixed in 1% citric acid for 1–2 h, and dehydrated with ethanol. Cells were mounted using embedding agent, and the ultrastructural changes of the cells were observed under an electron microscope (magnification, ×1,000 and ×5,000).

Cells (1×10⁵ cells/ml) were seeded in 96-well plates at 200 µl per well. After the cells had grown to a confluent state, the culture medium was discarded and 200 µl of serum-free medium containing different final concentrations of CdCl₂ (0, 10, 20, 30, 40, 50 µM) or DDP (0, 5, 10, 15, 20, 25 µM) was added to each well. Three replicates were plated for each group. After 24 h of incubation at room temperature (RT), the culture medium was discarded. Then, 200 µl thiazole blue (0.5 mg/ml) was added to each well. After incubation for 4 h at RT, the waste solution was discarded and dimethyl sulfoxide (150 µl/well) was added and mixed thoroughly for 10 min; the absorbance A (wavelength: 570 nm) of each well was detected with a microplate reader. The cell inhibition rate and half maximal inhibitory concentration (IC₅₀) were calculated.

Cells were seeded into 96-well plates at 1×10⁵ cells per well, and cultured for 24 h at RT. Different concentrations of CdCl₂ were then added to the culture medium for different times. Control groups were treated with an equal volume of dimethyl sulfoxide (DMSO). MTT reagent (20 µl) was added to each well, and supernatants were discarded after 4 h. DMSO (150 µl) was added to each well to dissolve the MTT reagent and absorbances were measured at 490 nm. Inhibition rate formula: Inhibition rate (%) = (Control group value-Treatment group value)/Control group value ×100%.

A total of 1×10⁶ cells in serum-free medium were seeded into the upper chamber, while the lower chamber was maintained in 10% FBS medium. After incubation for 24 h at RT, migratory cells at the bottom of the upper chamber were fixed with 4% paraformaldehyde for 30 min, stained with 0.5% crystal violet for 15 min and then counted under an inverted microscope (magnification, ×400). Data were analyzed using ImageJ V1.8.0 software (National Institutes of Health).

Human FOXM1-specific silenced and overexpressing lentivirus and corresponding negative control lentivirus were purchased from Genechem. The sh-FOXM1 group was transfected with the specific silenced sequence, the FOXM1 group was transfected with the specific overexpressing sequence. The sh-NC and the vector group were transfected with corresponding negative control lentivirus. MG63 cells were seeded into 6-well plates and transfected with the indicated lentiviruses, and the multiplicity of infection (MOI) was 20. At ~30% confluency, the cells were infected for 10 h and the culture media was replaced. Fluorescence intensities were measured at 72 h post infection to confirm lentivirus transduction, which reached an efficiency of ~70-80%. Forty-eight hours after observing fluorescence, cells were collected and used for follow-up studies.

Adherent MG63 cells were fixed in 4% paraformaldehyde at 4°C for 15 min. Cells were treated with 0.1% Triton-X for 10 min and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) was performed for 1 h at 37°C to allow diaminobenzidine (DAB) development. Imaged sections showing brown granules in the nucleus were deemed TUNEL-positive cells. Five fields (×400) were selected for each slice and the nucleus and TUNEL-positive cells were then observed under a fluorescence microscope (Nikon), and the apoptosis rate (TUNEL-positive cells/nucleus) of five random fields were calculated.

For immunohistochemical analysis, mouse cancer tissues were fixed in 10% formalin at RT for 24 h and paraffin-embedded. Sections (5-µm thick) were dewaxed and dehydrated in a gradient ethanol series. Endogenous peroxidase activity was quenched by incubating sections in methanol containing 3% H₂O₂ for 10 min and heating sections in decanoic acid at 65°C for 10 h for antigen retrieval. Paraffin sections were added with anti-human FOXM1, followed by incubation overnight at 4°C. Then the sections were reacted with goat anti-rabbit lgG-HRP polymer (Beyotime Institute of Biotechnology) at 37°C for 20 min, followed by color exposure using DAB.

Total proteins were extracted from the tissues or cells. Protein concentrations were measured via bicinchoninic acid assays (Nanjing Keygen Biotechnology Co., Ltd.). Protein samples (50 µg) were separated on 10% polyacrylamide gels and transferred to Hybond^® polyvinylidene difluoride membranes (Millipore). Membranes were blocked in 5% fat-free milk for 2 h at room temperature and probed with primary antibodies (cleaved caspase-3, Bcl-2, Bax, MMP-2, MMP-9, E-cadherin, FOXM1 and GAPDH) overnight at 4°C. Membranes were labeled with the appropriate secondary antibody for 2 h, and protein bands were visualized using electrochemiluminescence hypersensitive luminescent solution. Data were analyzed using ImageJ software (V1.8.0; National Institutes of Health).

Cells or tissues were lysed in TRIzol reagent (Sigma-Aldrich/Merck KGaA) and air dried following the addition of chloroform, isopropanol, and ethanol. FOXM1 primers were: 5′-GGAGGAAATGCCACACTTAGCG-3′ and 5′-TAGGACTTCTTGGGTCTTGGGGTG-3. Samples were reverse transcribed into cDNA and RT-qPCR was performed using reverse transcription reagent on a LightCycler 96 (Roche Life Sciences) using Real-Time PCR Mix (Vazyme Biotech Co. Ltd.). The reaction conditions were: 95°C pre-heated for 3 min, 95°C denaturation for 30 sec, 60°C annealing for 30 sec and involving 40 cycles. Gene expression was assessed using the 2^−ΔΔCq method relative to GAPDH expression (13). Two independent experiments were performed in triplicate for each RT-qPCR.

Total RNA was isolated from MG63 cells using TRIzol (Invitrogen/Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. RNA purity was assessed using the ND-1000 (Nanodrop; Thermo Fisher Scientific, Inc.). Each RNA sample had an A260:A280 ratio above 1.8 and an A260:A230 ratio above 2.0. RNA integrity was evaluated using the Agilent 2200 TapeStation (Agilent Technologies). Briefly, rRNAs were removed from total RNA using the EpicenterRibo-Zero rRNA Removal Kit (Illumina, Inc.) and fragmented to approximately 200 bp. Subsequently, the purified RNAs were subjected to first-strand and second-strand cDNA synthesis followed by adaptor ligation and enrichment with a low-cycle according to the instructions of the NEBNext^® Ultra™ RNA Library Prep Kit for Illumina [New England BioLabs, Inc. (NEB)]. The purified library products were evaluated using the Agilent 2200 TapeStation and Qubit^® 2.0 (Thermo Fisher Scientific, Inc.) and then diluted to 10 pM for cluster generation in situ on the pair-end flow cell followed by sequencing (2×150 bp) HiSeq 3000 system (Illumina, Inc.). Clean reads were obtained after removal of reads containing adapter, poly-N and at low quality from raw data. Differential expression was assessed by DESeq using read counts as input. The Benjamini-Hochberg multiple test correction method was enabled. Differentially expressed genes were chosen according to the criteria of fold change >2 and adjusted P-value <0.05. All of the differentially expressed genes were used for heat map analysis.

Sixteen BALB/c nude mice (6 weeks, ~18 g, random sex) were obtained from the Experimental Animal Center of Southern China Medical University and used for tumor formation experiments. After MG63 cells were digested, they were washed twice with PBS to remove the residual medium. A total of 1×10⁶ cells were injected into the right forelegs of the nude mice subcutaneously. The status of nude mice and tumor growth were observed every week. When the transplanted tumor reached 60–80 mm³, the nude mice were randomly divided into 4 groups: i) negative controls [intraperitoneal (i.p.) injection of normal saline, once every day); ii) DDP group (peritoneal injection of cisplatin 5 mg/kg, once every other day); iii) cadmium chloride (CdCl₂) (i.p. injection of cadmium chloride 1 mg/kg), once every other day); and iv) DDP+ CdCl₂ (cisplatin 5 mg/kg + cadmium chloride 1 mg/kg, once every other day).

Drug administration was performed according to the above grouping. Every 2–3 nude mice were kept in a cage. The feeding conditions were as follows: room temperature was maintained at 26–28°C, relative humidity was maintained at 40–60%, and 10 h of light and 14 h of dark were kept daily. The drinking water, defecation and mental state of the nude mice were observed every day, and a vernier caliper was used to measure the longest and shortest diameters of the transplanted tumor every two days. The volume (V) of the subcutaneous transplanted tumor was calculated (V = L × W² × 0.5), and the tumor growth curve was drawn. Forty-eight hours after the last dose (after 2 weeks, about 23 g), mice were anesthetized by using carbon dioxide. The specific method is as follows: The nude mouse was placed in a closed chamber (50×40×40 cm), and the air in the chamber was gradually replaced by a mixture of carbon dioxide and oxygen. The flow rate of gas was 16,000 cm³/min and the ratio of carbon dioxide and oxygen was 6:4. After 20 min when the nude mice lost consciousness, the CO₂ concentration was then raised to 100% for 20 min until the nude mice died. The criteria for judging the death of the nude mice were: Stopped heartbeat, dilated pupils, and halted breathing. Tumor tissue was then removed, weighed and imaged. At the same time, blood was collected from the posterior orbital venous plexus of the nude mice, and blood routine and liver and kidney functions were measured. Automatic blood cell analyzer (ProCyte Dx; IDEXX, Inc.) was used to detect the following indicators: white blood cells (WBCs), red blood cells (RBCs), platelets (PLTs) and lymphocytes (LYMs). An automatic biochemical analyzer (Catalyst One; IDEXX, Inc.) was used to detect the following indicators: Aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), uric acid (UA), urea nitrogen (BUN), and creatinine (CR). The animal experiment was performed in strict accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The animal study complied with the ARRIVE guidelines and the AVMA euthanasia guidelines from 2013 (14,15). All protocols described in this study were reviewed and approved by the Ethics Committee of the Affiliated Yuebei People's Hospital of Shantou University Medical College (Shaoguan, Guangdong, China).

All data were analyzed using SPSS 17.0 software (SPSS, Inc.). Qualitative variables are expressed as counts (%), and quantitative variables are expressed as the mean ± standard deviation. Measurement data that conformed to the normal distribution were analyzed using independent sample t-test and the Mann-Whitney rank sum tests were used to analyze those that did not conform to a normal distribution, while for multiple specimens, statistical analysis was performed using one-way ANOVA and a post hoc test was performed after pairwise comparison test. On the premise of homogeneity of variance, the post hoc test was performed by Scheffe and Tukey method. Differences were statistically significant at the P<0.05 level.

To evaluate the cytotoxicity of both cadmium chloride (CdCl₂) and DDP in OS and normal cells, we first investigated the effects of CdCl₂ and DDP on MG63 human OS cells and hFOB1.19 human osteoblasts. MTT assays revealed that the cell death increased in a concentration-dependent manner (Fig. 1A-D). Compared to CdCl₂, DDP was more cytotoxic. Normal cells were more sensitive to the drugs than tumor cells. The half maximal inhibitory concentration (IC₅₀) values of CdCl₂ and DDP on MG63 cells were calculated to be 39.95±1.99 and 12.79±0.07 µM, respectively. To compare the effects of different concentrations of CdCl₂ on cell proliferation, MTT assays were performed. The results demonstrated that CdCl₂ at a concentration of 10 µM slightly inhibited cell proliferation, and when the concentration was greater than 20 µM, cell proliferation was significantly inhibited at day 3 and 4 compared with the untreated cells (Fig. 1E and F). The concentration of 20 µM of CdCl₂ was selected for subsequent experiments.

OS MG63 cells were treated with 20 µM CdCl₂ for 24 h, and apoptosis was assessed by electron microscopy. After treatment with CdCl₂, the characteristic morphological changes of apoptosis were observed which included: Shrinkage of cell membranes, concentrated chromatin and organelles, folding of endoplasmic reticulum and appearance of apoptotic bodies outside the membrane (Fig. 2A). MTT assay results showed that compared with the control (DMSO) group, cell proliferation was significantly decreased after treatment with CdCl₂ for 7 days (Fig. 2B). Meanwhile, cell apoptosis was significantly increased after treatment with DDP or CdCl₂ compared with the control group, and the combined use of DDP and CdCl₂ further promoted cell apoptosis (Fig. 2C-E). Western blot analysis confirmed these effects, as DDP+CdCl₂ treatment led to a loss of Bcl-2 expression and increased Bax and cleaved caspase-3 levels (Fig. 2F). Then, Transwell assays were performed to investigate the cell migration level. The results revealed that DDP+CdCl₂ treatment significantly inhibited MG63 cell migration ability compared with the use of DDP or CdCl₂ only, or the control group (Fig. 2G and H). Western blot results confirmed that the expression of MMP2 and MMP9 was significantly inhibited while E-cadherin was increased in the DDP+CdCl₂ treatment group compared with the use of DDP or CdCl₂ only, or the control group (Fig. 2I). This implies that DDP and CdCl₂ may downregulate the migration of MG63 through the Wnt pathway.

To investigate the mechanism(s) by which CdCl₂ inhibits OS, we performed gene chip screening of CdCl₂-treated MG63 cells and the untreated cells. Heatmaps were constructed based on the differentially expressed genes between the treatment groups (Fig. 3A, Table I), of which FOXM1 was identified as being significantly downregulated in response to treatment with CdCl₂. To investigate the effects of FOXM1 on OS cells, Western blotting was adopted to explore the expression of FOXM1 in OS cell lines. The results demonstrated that the expression of FOXM1 in MG63, U2OS, 143B and SaoS2 OS cells was higher than that in the hFOB.19 cells (Fig. 3B). Then, FOXM1-overexpressing and -silenced lentiviruses were constructed. Results of the Western blot analysis and qRT-PCR showed that the expression of FOXM1 in the sh-FOXM1 and the FOXM1 group were significantly lower and higher, respectively, than that in the control (shNC and Vector) groups (Fig. 3C and D). In the FOXM1-silenced (sh-FOXM1) cells, low concentrations of DDP significantly reduced OS cell viability (Fig. 3E). Conversely, high DPP concentrations were required to reduce the viability of FOXM1-overexpressing (FOXM1) cells when compared to the Vector group (Fig. 3F), thus confirming a role for FOXM1 in DPP-mediated cell death. DPP treatment of FOXM1-silenced MG63 cells induced cell apoptosis, while DPP treatment of FOXM1-overexpressing MG63 cells inhibited cell apoptosis (Fig. 3G and H). These results were verified by Western blot analysis. Compared with the control group, silencing of FOXM1 upregulated the expression of cleaved caspase-3 and Bax levels, and downregulated the expression of Bcl-2 levels. Addiitonally, overexpression of FOXM1 led to the opposite trend in regards to the expression of cleaved caspase-3, Bax and Bcl-2 levels (Fig. 3I).

To verify the regulatory relationship between CdCl₂ and FOXM1 in OS cells, MG63 cells were treated with various concentrations of CdCl₂, and the expression of FOXM1 was verified by qPCR and Western blot analysis. FOXM1 levels were decreased with increasing CdCl₂ concentrations at both the mRNA and protein levels (Fig. 4A and B). We next investigated the effects of FOXM1 expression on the response of OS cells to DDP+CdCl₂ treatment. qPCR and Western blotting results demonstrated that after MG63 cells were treated with DPP, FOXM1 expression was upregulated, and the combined use of DDP+CdCl₂ significantly suppressed the expression of FOXM1 in MG63 cells compared to the control cells (Fig. 4C and D). In FOXM1-silenced MG63 cells, MTT results showed that DDP led to an increase in the MG63 cell death rate compared to the shNC cells. DDP+CdCl₂ treatment of the sh-FOXM1 cells yielded no significant change in cell death. Meanwhile, DDP+CdCl₂ treatment of the shNC cells resulted in higher apoptosis compared with the DDP alone treated cells (Fig. 4E).

To confirm these findings in vivo, nude mice were implanted with OS MG63 cells (Fig. 5A), and the effects of DDP+CdCl₂ combination therapy were assessed after 14 days of treatment in the i) control (Con.), ii) DDP, iii CdCl₂ and iv) DDP+CdCl₂ groups. We first tested the safety of the DDP+CdCl₂ combination. DDP+CdCl₂ caused changes in blood routine and liver and kidney function in nude mice (Tables II and III). The results showed that DDP+CdCl₂ increased WBCs, PLTs, LYMs, ALT, ALP, UA, BUN, and CR levels compared with control group in venous blood. There was no significant effect on red RBCs or AST levels. Then, we measured the tumor volumes and weight (Fig. 5B and C). Compared with the control group and the use of DDP or CdCl₂ group, treatment with DDP+CdCl₂ reduced tumor volume and weight, and DDP+CdCl₂ had the most significant effect compared with the use of DDP or CdCl₂ alone. Tumor tissues were isolated, and FOXM1 expression was assessed by immunohistochemical (IHC) staining (Fig. 5D), Western blot analysis (Fig. 5E) and qPCR (Fig. 5F). Similar to the results of the in vitro experiments, DDP upregulated FOXM1 expression while CdCl₂ inhibited FOXM1 expression.