SCREEN-WELL (FDA-approved drug library V2) was purchased from Enzo Life Sciences (Enzo, NY, USA). Dulbecco’s modified Eagle’s medium (DMEM) and Roswell Park Memorial Institute (RPMI) 1640 medium were purchased from Gibco BRL (Grand Island, NY). Fetal bovine serum (FBS) was purchased from GenDEPOT (Barker, TX). Primary antibodies for cleaved caspase 3 and cleaved PARP were purchased from Cell Signaling Technology (Danvers, MA). Antibodies against β-actin were purchased from Santa Cruz Biotechnology (Dallas, TX). Bicinchoninic acid (BCA) protein assay reagent was purchased from Pierce Biotechnology (Rockford, IL).

Human osteosarcoma Saos-2 and U2OS cell lines, obtained from the Korean Cell Line Bank (Seoul, Korea), were maintained at 37°C in a humid atmosphere of 5% CO₂ and 95% air in RPMI and DMEM, respectively. Each medium contained 10% FBS and 1% antibiotics.

Saos-2 and U2OS cells were plated in six-well plates at densities of 500 and 300 cells per well, respectively. After 24 h, they were treated with different concentrations of colchicine for 48 h. The medium was then changed, and the cells were incubated for an additional 12 days in drug-free medium. The colonies that formed were fixed with 3.7% formaldehyde at room temperature for 10 min and then stained with 0.05% crystal violet. Colonies with >10 cells were counted under a light microscope. The experiments were carried out in triplicate.

Intracellular ROS levels were measured by staining the cells with 2-7-dichlorofluorescein diacetate (DCF-DA; Molecular Probes). Cells were treated with DMSO or colchicine for 48 h and treated with 5 μM DCF-DA 37°C for 1 h. The fluorescence intensity of the cells was monitored via flow cytometry using a FACSCalibur apparatus (BD Biosciences, San Jose, CA).

Saos-2 and U2OS cells were collected, washed with phosphate-buffered saline (PBS), and dissociated with Accutase solution (Sigma-Aldrich, St. Louis, MO). The cells were then counted and washed with PBS containing 2% FBS and 0.1% Tween-20. Apoptosis in cultured cells was assessed by flow cytometry after Annexin V-FITC/propidium iodide (PI) double staining using the Annexin-V FITC Apoptosis Detection Kit I (BD Biosciences, San Jose, CA) according to the manufacturer’s instructions.

The cells were washed with cold PBS, and the pellet was collected. Cells were then suspended in 1× cell lysis buffer (Cell Signaling Technology, Danvers, MA) enriched with 0.1 mM PMSF and protease inhibitor (Roche, Basel, Switzerland) and incubated on ice for 30 min. After centrifugation at 13,000 g for 15 min, the supernatant was collected as a whole cell lysate. Lysates containing equal amounts of protein were prepared and analyzed using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). The blots were then blocked with 5% fat-free dry milk–TBST (Tris-based saline buffer containing 0.1% Tween-20) for 1 h at room temperature and probed overnight using the indicated primary antibody (1/5,000 dilution for β-actin and 1/1,000 dilution for the rest). The blots were then washed three times with TBST buffer for 10 min each. The washed blots were then incubated with 1:5,000 diluted horseradish peroxidase-conjugated secondary antibodies (Thermo Fisher Scientific, MA, USA). The blots were washed three times with TBST buffer for 10 min each, and the transferred proteins were detected after incubation with the ECL substrate detection reagent.

Transwell migration assays were used to determine the migratory and invasive abilities of osteosarcoma cells after colchicine treatment. Saos-2 cells (1 × 10⁴ cells) and U2OS cells (5 × 10³ cells) were seeded into the apical side of the transwell chamber with a non-coated membrane using an 8-μm pore (Corning, NY, USA). For invasion assays, Saos-2 and U2OS cells were plated at the same density as mentioned above with a Matrigel-coated membrane (BD Biosciences, San Jose, CA, USA). To create a chemotactic gradient, serum-free media were added to the upper chamber, while 10% FBS was added to the lower chamber media. After 48 h, the cells were fixed in methanol for 5 min, stained with 0.05% crystal violet, washed with PBS, and allowed to dry. Images of the cells that migrated through the pores were taken with an optical microscope, and the cells were counted. Migration and invasion assays were carried out in sextuple and triplicate, respectively.

Library preparation and sequencing of control or colchicine-treated total RNAs and library construction were performed using the QuantSeq 3’ mRNA-Seq Library Prep Kit (Lexogen, Inc., Austria) according to the manufacturer’s instructions. In brief, 500 ng of total RNA was prepared for each sample, an oligo-dT primer containing an Illumina-compatible sequence at its 5’ end was hybridized to the RNA, and reverse transcription was performed. After degradation of the RNA template, second-strand synthesis was initiated by a random primer containing an Illumina-compatible linker sequence at its 5’ end. The double-stranded library was purified using magnetic beads to remove all reaction components. The library was amplified to add the complete adapter sequences required for cluster generation. The final library was purified from the PCR components. High-throughput sequencing was performed as single-end 75 sequencing using a NextSeq 500 (Illumina, Inc., USA). Data analysis of QuantSeq 3’ mRNA-Seq reads was aligned using Bowtie2 (15). Bowtie2 indices were either generated from the genome assembly sequence or the representative transcript sequences for alignment to the genome and transcriptome. The alignment file was used for assembling transcripts, estimating their abundance, and detecting the differential expression of genes. Differentially expressed genes were determined based on the counts from unique and multiple alignments using coverage in Bedtools (16). The read count (RC) data were processed based on the quantile normalization method using EdgeR within R (R Development Core Team, 2016) using Bioconductor (17). Gene classification was based on searches of the DAVID and Medline databases. Data mining and graphic visualization were performed using ExDEGA (Ebiogen Inc., Korea).

Cell viability was detected using a CCK-8 solution (Dojindo, Kumamoto, Japan) according to the manufacturer’s protocol. Briefly, the Saos-2 and U2OS cells were counted and seeded at a density of 5 × 10⁴ cells/well in 96-well plates. One day after seeding, cells were incubated with different concentrations (0.01, 0.05, 0.1, 0.5, 1, and 5 μM) of colchicine. After 48 h of treatment, 10 μl of CCK-8 was added, and the plates were incubated at 37°C for 2 h. The absorbance per well was measured at a wavelength of 450 nm. Each experiment was sextupled independently.

All animal experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals. Five-week-old BALB/c nude mice were acclimatized for 1 week under standard temperature, humidity, and timed lighting conditions at the animal care facility. Five mice were used for each group. Saos-2 cells were injected subcutaneously into the flank at a density of 5 × 10⁶ cells/ml in PBS/Matrigel (7:3, v/v) in a total volume of 100 µl. The mice were divided into control and treatment groups once the tumor volume reached 150 mm³. Vehicle (PBS) or colchicine (10 μM) was injected (i.t.) at 100 µl. Tumor volumes were measured every 3–4 days from the day of colchicine injection for 15 days. Tumor volumes were calculated using the following formula: length × width × 0.52. The mice were sacrificed 15 days after colchicine treatment.

Data were analyzed using the GraphPad Prism 6 software and are expressed as mean ± standard deviation. A Mann–Whitney U test was used to compare the means of two groups and a Kruskal–Wallis test with Tukey’s post-hoc test was used when the means of two or more variables (i.e., induction and concentration) were compared. Results were considered significant when p < 0.05.

We assessed 772 compounds in an FDA-approved library for their ability to reduce the viability of osteosarcoma cells. Two human osteosarcoma cell lines (U2OS and Saos-2) were used to select the lead compound ( Figure 1A ). Initially, osteosarcoma cell lines were treated with each compound at a concentration of 10 μM for 48 h and screened using the CCK-8 assay. Compounds that were originally developed as chemotherapeutics were excluded. Thirty compounds that demonstrated the most effective inhibitory effects were selected from both Saos-2 and U2OS cell lines for a second screening. Subsequently, five compounds were identified that inhibited the growth of both Saos-2 and U2OS cells to below 50% compared to that of the control ( Figure 1B ), and treated with different concentrations (0.01, 0.05, 0.1, 0.5, 1, 5, and 10 μM) to confirm the dose-dependent cytotoxic effect ( Figure 1C ). Based on previous research and clinical use, colchicine was selected as the lead compound ( Figure 1D ). Colchicine has been actively investigated beyond the scope of its original use, including in cancer (18–21). In line with these previous studies, colchicine was of prime interest among all the candidates tested. We examined the effects of colchicine on the viability of Saos-2 and U2OS cells. Treatment of the cells with colchicine for 48 h resulted in cytotoxic effects in both osteosarcoma cell lines ( Figure 1E ).

Colony-forming assays characterize the ability of cancer cells to grow into colonies through independent growth and clonal expansion. Thus, we evaluated the effect of colchicine on the clonogenic survival of osteosarcoma cells. As shown in Figure 2A , different concentrations (20 and 30 nM) and 30 nM of colchicine significantly reduced the colony formation of both Saos-2 and U2OS cells in a dose-dependent manner, respectively. ROS has been suggested to induce cancer cell cycle arrest and apoptosis (22). The effect of colchicine on ROS production in colon cancer (HT-29) cells has already been discussed (19). Therefore, we also hypothesized that we would see a surge in ROS levels in osteosarcoma cells after colchicine treatment. In the 2’,7’-dichlorofluorescin diacetate (DCF-DA) ROS assay, colchicine treatment (30 nM) induced a change in intracellular ROS levels compared to the control ( Figure 2B ) in both Saos-2 and U2OS cells. To examine whether apoptosis impairs cell viability via colchicine treatment, flow cytometry analysis with annexin V-FITC/PI was used to assess the percentage of apoptotic cells. Similar to the cell viability assay findings, colchicine significantly reduced the percentage of live cells and increased total apoptosis in both Saos-2 and U2OS cells at 10 and 30 nM ( Figure 2C ). Similarly, treatment with colchicine increased the expression of cleaved caspase 3 and cleaved PARP in Saos-2 and U2OS cells ( Figure 2D ). These results indicated that colchicine exerts anticancer effects by inhibiting proliferation and inducing apoptosis in osteosarcoma cells.

Enhanced cell mobility and invasiveness are hallmarks of cancer (23). Cell migration and invasion are propelling factors that provide a wide array of cellular mechanisms under the metastatic cascade (24). Therefore, to investigate the role of colchicine in cell migration and invasion, we performed Transwell cell migration and Matrigel invasion assays. Colchicine treatment (10 or 30 nM) significantly inhibited cell migration, as determined by transwell migration of both Saos-2 and U2OS cells ( Figure 3A ). We also found that colchicine treatment (10 or 30 nM) dramatically inhibited the invasion of Saos-2 and U2OS cells ( Figure 3B ). Overall, these results indicate that colchicine inhibits the migration and invasion of osteosarcoma cells in vitro.

To explore relevant biological functions, GO biological process (GOBP) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed on the upregulated and downregulated genes after colchicine treatment and analyzed using the DAVID bioinformatics tools. As shown in Figure 4A , there was a significant involvement of gene related to the cell cycle, including microtubule-based process and G2/M transition of the mitotic cell cycle in Saos-2 cells. This finding provides a plausible explanation for the anticancer effects of colchicine through the regulation of microtubule-based processes in osteosarcoma cells. KEGG analysis revealed that the pathways involved in the colchicine-treated cells were related to gap junctions and apoptosis. These data further support the possibility that colchicine induces apoptosis and inhibits the migration and invasion of osteosarcoma cells, as previously described. The GOBP and KEGG analyses in U2OS cells found that colchicine treatment significantly affected cancer-related pathways compared to that of the control ( Figure 4B ).

To verify the inhibitory effect of colchicine on osteosarcoma cells in vivo, BALB/c nude mice were injected subcutaneously with Saos-2 cells into the flank. Intraperitoneal administration of colchicine (10 μM) significantly reduced tumor volume compared to that in the vehicle-treated group ( Figures 5A, B ). The inhibitory effect of colchicine on the growth of Saos-2 cells was accompanied by a marked decrease in proliferating cell nuclear antigen (PCNA) expression ( Figure 5C ).