For the present study, 10 (6-week-old) male NOD-SCID (BALB/cSIc (nu/nu), nude mice were procured from Japan SLC, Inc. They were housed in similar environmental and nutritional conditions. Mice were sacrificed at 11 weeks of age, according to the standard protocols of Jeju National University, Jeju-si, Jeju-do, Korea. The research proposal and the relevant experimental procedures were approved by the institutional review board of the Department of Animal Biotechnology, Jeju National University, Jeju-si, Jeju-do, Korea. All animal studies were conducted to induce tumors. In order to design tumorigenesis induction experiments, the mice were divided into three groups. Two mice were grouped as a negative control (NC), the rest were divided in two treatment groups, the Cal72 group (n=4) and the SaOS-2 group (n=4). Furthermore, 4×10⁶/ml of SLCICs were suspended in Matrigel and injected subcutaneously into the right lower flank of the nude mouse in both the treatment groups.

Human osteosarcoma Cal72 and SaOS-2 cell lines were purchased from Korean Cell Bank (Seoul, Korea). These cell lines were routinely cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Hyclone, South Logan, UT, USA), 1% antibiotic-antimycotic (Gibco, Invitrogen, Carlsbad, CA, USA) at humidified 37°C and 5% CO₂ atmosphere. The cells were sub-cultured after attaining confluency of 80%. For the present study, the cells in passages 9 and 12 were used. Human bone marrow cells (hBMCs) were also used as control.

The population of CD133 expressed in Cal72 and SaOS-2 cells was isolated by anti-human CD133 MicroBead kit (Miltenyi Biotec Corp., Auburn, CA, USA) using Midi MACS separator (Miltenyi Biotec Corp.) by following the user instruction manual. The hBMCs were isolated from the adult human bone marrow (Jeju National University Hospital, Jeju, Korea) and were separated by Ficoll-paque density gradient centrifugation (GE Healthcare Life Science, Piscataway, NJ, USA). The potential hBMCs were cultured in DMEM supplied with 10% FBS, 1% antibiotic-antimycotic and 10 nM epithelial growth factor (Sigma-Aldrich Corp., St. Louis, MO, USA). The hBMCs from 3rd and 4th passage were confirmed as normal control cells against the efficacy of BRM270.

A pellet of 1×10⁷ cells was cultured to 80% confluency, then cells were treated with BRM270. Later, BRM270-treated or non-treated vs lipopolysaccharide (LPS) + BRM270 treated samples of Cal72 and SaOS-2 SLCICs were lysed with 400 μl of 10 mM KCl, 0.2 mM EDTA, 1.5 mM MgCl₂, 0.5 mM DTT and 0.2 mM PMSF at 4°C for 10 min. The lysate was centrifuged for 5 min at 12,000 × g and supernatants were stored as cytosolic extract at −80°C. The resulting pellet was re-suspended in 100 μl of ice-cold 20 mM HEPES (pH 7.9), 420 mM NaCl, 1.5 mM MgCl₂, 20% (v/v) glycerol, 0.5 mM DTT, 0.2 mM EDTA and 0.2 mM PMSF. After incubation at 4°C for 20 min, the lysate was centrifuged for 10 min at 12,000 × g and the supernatant was stored as a nuclear extract at −80°C. The concentration of cytosolic and nuclear extract was determined using a BCA kit (Bio-Rad, Richmond, CA, USA).

Suspended cells (100 μl) at density of 5,000 cells/well were seeded in 96-well plate (Nunc™, Wiesbaden, Germany). After 24 h of recovery the cells were incubated in humid atmosphere at 37°C and 5% CO₂ and treated with BRM270. The BRM270 was procured from the Biological Response Modifier International Heath Town Corp., Korea. The cells were treated with varying concentrations of BRM270 such as 15.6–125 μg/ml and doxorubicin (Dox) 5 μM/ml in MTT assay while 125 μg/ml in rest of the experiments were incubated for 24 h. The enhanced cell viability assay was conducted with EZ-CyTox kit (Daeil Lab Service Co., Seoul, Korea) to measure the viable cells. EZ-CyTox solution (10 μl) was added to each well and then cells were incubated for 4 h at 37°C and 5% CO₂. The light absorbance was determined at 450 nm by the Model 680 microplate-reader (Bio-Rad, Berkeley, CA, USA).

CD133 expressing Cal72 and SAOS2 cells (1×10⁶ cells) were seeded in 6-well microtitre plate (Nunc Nunclon™ Delta, USA). Then the cells were treated with the 125 μg/ml concentrations of BRM270 for 24 h. For analysis of genomic DNA, attached and floating cells in the supernatant were harvested and collected. Genomic DNA was extracted by AccuPrep^® Genomic DNA Extraction kit (Bioneer). DNA (5 μg) was separated on a 1.2% agarose gel. DNA in the gel was stained with ethidium bromide and was visualized under UV light.

Apoptotic morphological changes in the nuclear chromatin of the cancer stem cells were stained by Hoechst 33258. The cells were seeded in sterile 6-well plates (Nunc Nunclon Delta). After overnight growth, cells were treated with BRM270. Later, the cells were washed with 1X phosphate-buffered saline (10X PBS Gibco, Life Technologies™, USA) and were fixed with 4% paraformaldehyde for 10 min followed by incubation with 50 μM Hoechst 33258 staining solution for 5 min. After three washes with cold PBS, the cells were viewed under a fluorescence microscope (Olympus, IX-70, and Japan).

Cells (1×10⁵) suspended in 2 ml fresh media were seeded in each well of a 6-well flat-bottomed microtiter plate and incubated overnight. Then BRM270 with 125 μg/ml was added. After 24 h, cells were harvested and washed twice with pre-cold PBS followed by re-suspension in 1X binding buffer at a concentration of 1×10⁶ cells/ml. One hundred microliters of such solution (1×10⁵ cells) was mixed with 5 μl of Annexin V-FITC and 5 μl of propidium iodide (PI, BD Biosciences, San Jose, CA, USA) according to the manufacturer’s instructions. The mixed solution was incubated at room temperature (25°C) in the dark for 15 min. Then 400 μl of 1X dilution buffer was added to each tube. Analysis was performed by Beckman Coulter FC500 Flow Cytometry System with CXP Software (Beckman Coulter, Fullerton, CA, USA) within 1 h of the treatment of cells with dilution buffer.

The analysis of cell cycle was detected by PI staining and analysis was performed by flow cytometry using a fluorescence-activated cell sorting (FACS) caliber (Becton-Dickinson). Subsequent to the treatment with 125 μg/ml and 5 μM/ml concentrations of BRM270 and Dox for 24 h, the hBMCs, CD133 expressing Cal72 and SaOS-2 SLCICs were harvested at concentration of 1×10⁶ cells/ml. The cells were fixed in 70% ethanol and incubated at 4°C overnight. The fixed cells were washed twice with cold PBS and then incubated for 30 min with Ribonuclease A (#R-5125, Sigma, 8 μg/ml) and PI (10 μg/ml). Then the cell samples were transferred to meshed blue capped tubes (BD Falcon™ Tubes #352235). Later, the fluorescent signals were detected through the FL2 channel and the proportion of DNA that was present in the various phases was analyzed using ModfitLT Version 3.0 (Verity Software House, Topsham, ME, USA).

The cells were detached by 0.5% trypsin-EDTA (Gibco) and total RNA was extracted by easyBlue (Intron Biotech, Seongnam-si, Gyeonggi-do, Korea). The purified RNA was quantified by using photometer (Bio-Rad Hercules, CA, USA). Purified RNA (1 μg) was subjected to first strand cDNA synthesis using Superscript III first-strand cDNA synthesis kit and Oligo dT primer (Invitrogen). The cDNA was subjected to qPCR for the quantification of the relative transcript levels of NF-κB (p65) and its inhibitor (IκBα), structural maintenance of chromosome protein 2 (SMC-2), inflammatory cytokine IL-6 as well as pro-apoptotic gene/anti-apoptotic gene caspase-8 (Casp-8) using the specific primers (Table I).

CD133 expressing Cal72 and SaOS-2 SLCICs were grown in 4-well chambered slide with glass coverslips (SPL Life Sciences Co., Ltd., Korea, 30104 PS/Glass/PP). The cells were cultured with BRM270 (125 μg/ml) and as non-treated (negative control) in DMEM high glucose medium for 24 h. The next day cells were washed with cold 1X PBS and were fixed with 4% paraformaldehyde for 15 min. Following this step the cells were permeabilized with 0.2% Triton X-100 for 10 min. After washing with the PBS, cells were blocked with 1% bovine serum albumin (BSA) for 30 min. Further, the cells were incubated with primary antibodies for anti-p-NF-κB/p65 Ser536, anti-rabbit-CD133 (Prominin 1), SMC2 and Cdk6 (rabbit IgG, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). All these antibodies were used at 1:200 dilutions. Subsequently, the cells were incubated with Alexa Fluor 488 (donkey anti-rabbit) secondary antibodies (both from Santa Cruz Biotechnology) for 1 h at room temperature in the dark. Cells were then washed with cold PBS and were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, Invitrogen). After incubation with DAPI for 5 min again cells were washed with cold 1X PBS. After addition of 1 ml mounting media, cells were observed under a fluorescence microscope (Olympus, Milan, Italy) with adaptable filter consistent with Alexa Flour 488, FICT or PE.

Total proteins were isolated by ice-cold RIPA buffer PMSF (Sigma-Aldrich Corp., USA) and ready to use protein inhibitor cocktails (Fermentas, Thermo Scientific, Rockford, IL, USA). Protein concentrations were calculated by using Pierce^® BCA Protein Assay kit (Thermo Scientific) following the manufacturer’s instructions. Sixteen micrograms of protein were then loaded per well. Proteins were separated on 12% polyacrylamide gel and transferred to polyvinyline difluoride membranes (PVDF, Sigma-Aldrich Corp.) in the Bio-Rad western blotting system (Bio-Rad, Berkeley, CA, USA). The membrane was incubated with the primary antibodies β-actin (1:1,000), 1:200 diluted anti-p-NF-κB antibody, anti-human-IL-6 antibody (rabbit IgG), Pro-apoptotic protein Casp-8 was detected using 1:200 anti-casp-8 antibody (rabbit IgG), Cdk6 (C-21) (rabbit IgG) and structural maintenance chromosomes protein-2 (SMC-2) (sc-10709 rabbit polyclonal IgG). All the antibodies used were procured from Santa Cruz Biotechnology. The proteins of interest were detected by an enhanced chemiluminescence detection kit following the manufacturer’s instructions using LAS4000 machine (GE Healthcare).

After 6 days of injection tumors were visible. Each tumor was measured with Vernier calipers and the volume of the tumor (mm³) was calculated by the following formula:

The size and the extent of metastasis of tumor under in vivo conditions was detected using LI-COR Pearl animal image analyzer (LI-COR Biosciences, USA). All the mice were sacrificed for the collection of the tumors.

The relative quantitative expressions of the genes by real-time qPCR and western blotting were analyzed by the analysis of variance (ANOVA). The significant differences between the mean expressions of different genes at P<0.05 were analyzed by Tukey’s b-test. The values are expressed as mean ± SEM.

In an attempt to identify SLCICs, CD133 expressing Cal72 cells were analyzed. The cells were characterized on the basis of expression of CD133⁺ on their cell surface, as found in number of malignancies. To verify the CD133⁺ as an SLCIC marker, cells were stained with monoclonal antibody human anti-CD133-FITC with iso-control and showed overexpression in Cal72 cells (Fig. 1A). Similarly, Oct4 with higher invasiveness and proliferative rate was overexpressed in immunocytochemistry of SLCICs in tumor sphere assay in vitro. Formation of tumor spheroids of high grade was also confirmed by the Oct4 expression in SLCICs in vitro (Fig. 1B) and in vivo (Fig. 1C). Moreover, the CD133⁺ expressed on the cell surface of cancer cells (Fig. 1A) can proliferate and metastasize in in vivo. Palpable tumors were visible after 7 days of injection and to endorse the tumorigenesis capability of CD133⁺ expressing SLCICs, the tumors were visualized by LI-COR Biosciences Pearl impulse IR-fluorophore, IRDye^® 800CW 2-deoxyglucose optical imaging system. In this approach the size, grade as well as the metastasis of the tumor were distinctly observed (Fig. 1C). In contrast, the SLCICs are often characterized by a high metabolic rate exemplified by a dramatically elevated glucose uptake. The IRDye 800CW 2DG was used for its ability to target several different tumor types for example solid tumors, colon tumors and soft tumors. This biological activity has been exploited by non-invasive imaging system using infrared pseudo-colored fluorescent signals. Further, pseudo-colored fluorescent signals were superimposed on a white light image using glucose analogues such as ¹⁸F-2-deoxy-D-glucose (due to high glucose metabolism in tumor sites) to generate a tumor-localized signal (Fig. 1C). Higher intensity signals are in red, while lower signal intensity is in blue (Fig. 1C). For more valid confirmation, cells from solid subcutaneous tumor and CD133 expressing SLCICs were stained with anti-human CD133-FITC conjugate after one day of their isolation and were further assessed by FACS. The CD133⁺ rich SLCICs were selected according to forward scatter (FSC) and side scatter (SSC) property using the gated region R4 to remove the cell debris, residual platelets and red cell contamination. FACS analysis showed the presence of a variable fraction of CD133⁺ cell populations in tumor-bearing nude mouse samples, 18.31 and 48.22%, respectively. However, 2.05 and 16.12% CD133⁺ were observed in Cal72 SLCICs. (Fig. 1D). Moreover, these SLCICs are CD133⁺ rich tumor initiating populations which can be characterized by flow cytometry. A significant (P<0.05) quantitative transcript level of CD133 in both Cal72 (0.87%) and SaOS-2 (0.79%) SLCICs, respectively, was analyzed by qPCR (Fig. 1E).

Cell cycle progression is highly regulated at several checkpoints to ensure proper DNA synthesis and chromosome segregation. It has become increasingly clear that the deregulation of CDK pathways causes unscheduled proliferation that contributes to oncogenic transformation. In the present study, we showed that efficacy of BRM270 against Dox-resistant SLCICs in a dose- and time-dependent manner induced morphological cell shrinkage of DNA and apoptosis (Fig. 2A and B). BRM270 selectively inhibits cell proliferation by inducing programmed cell death (PCD) and mitotic cell death (MCD) due to unrepaired DNA damage during premature apoptosis (Fig. 2B and F). Initially the morphology of chromatin condensed in the cultured cells undergoing apoptosis was compared with that of in vitro triggered DNA fragmentation in nuclei isolated from the corresponding non-apoptotic cells (Fig. 2B and F). Therefore after 24 h of exposure, cells treated with BRM270 and Dox in a dose-dependent manner, were observed for DNA fragmentation and chromatin condensation by confocal fluorescence microscope (Fig. 2A and B). Further, the genomic DNA was extracted and was separated on 1.2% agarose gel. To confirm the extent of genomic DNA fragmentation by BRM270, the DNA was run in the gel stained with ethidium bromide (EtBr) and was visualized under UV light (Fig. 2F). It showed that hallmark of apoptotic cell death is the cleavage of chromosomal DNA at inter-nucleosomal sites into fragments of multiples sizes (Fig. 2B and F). For confirmation of DNA damage-induced mitotic cell death, CD133⁺ expressing SLCICs treated with BRM270 and Dox, were stained with Hoechst 33258. It was observed that BRM270 interrupts the microtubule cytoskeletal formation in Dox resistant SLCICs, formed chromosomal condensation (pyknosis), nuclear fragmentation (karyorrhexis) (Fig. 2B, 5 and 6, red arrows) and cytoplasmic shrinkage (Fig. 2B, 2 and 5, yellow arrows). BRM270 displayed potent cytotoxicity in Dox resistant SLCICs by changed morphology of cells (Fig. 2A, 2 and 5). In addition, BRM270 shows selective inhibition of the neoplasm. Cytotoxicity was analyzed by clorometric assay 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT assay) (Fig. 2C) where it showed its ability to kill the CD133⁺ expressing SLCICs. MTT assay analysis showed that at the concentration of 125 μg/ml, after 24 h exposure with BRM270, 4.51% (CD133⁺ expressing Cal 72 SLCICs) and 7.253% (SaOS-2 SLCICs) viability was recorded. Further, to evaluate the cytotoxic effect of BRM270 on a normal human cell line, hBMCs were used. At the similar conditions as those of cancer cell lines, hBMCs showed 93.41% viability with respect to control (Fig. 2A, 7–9 and C). Similarly, in Dox treated group 17.95, 13.56 and 79.99% viability was observed in Cal72, SaOS-2 and hBMCs, respectively (Fig. 2D). It showed that Dox is more toxic in normal cells compare with BRM270 (Fig. 2C and D). Furthermore, CD133⁺ and Oct3/4 in Cal72 SLCICs have indicated stemness while after treatment with BRM270 a significant (P<0.05) decrease in their levels was found (Fig. 2E). The cell cycle progression in both parental and Dox-resistant SLCICs after 24 h exposure of BRM270 was observed (Fig. 2A, 2 and 5). Flow cytometry analysis showed a significant (P<0.05) decrease in cell population from G0–G1 phases (Fig. 3). A significant decrease (17.09%) was recorded in the Cal72-SLCICs population after their treatment with BRM270 as compared to 6.77% decrease in the cell population after their exposure to Dox. The 15.49% (BRM270 treated) and 5.88% (Dox treated) concomitant accumulation or transit of cells in the G2/M checkpoint (BRM270–15.49%, Dox-5.88%) was recorded in CD133⁺ expressing Cal72 SLCICs. Again, SaOS-2 SLCIC cell samples were collected and the cells were stained with propidium iodide to analyze cell cycle arrest which divulged transition from G0–G1 (loss 4.82%) into G2/M phase (transit 15.09%) while in normal hBMCs, checkpoints for transition of G2/M are G0–G1 (loss 0.2%) into G2/M phase (transit 4.31%) while in Dox treated G0–G1 (loss 6.63%) to G2/M (6.47%), respectively (Fig. 3). This finding suggests that the anticancer drug Dox arrests cells at G0. Further it was also observed that transit to G2/M checkpoint by Dox is less than the NF-κB novel inhibitor BRM270 in both SaOS-2 and Cal72 SLCICs while Dox showed more cytotoxicity to normal hBMCs compared to BRM270.

BRM270 induced IL-6 mediated apoptosis and the expression of pro-apoptotic proteins was observed in SLCICs. Apoptotic cell death was measured with fluorescein isothiocyanate-conjugated Annexin V and PI staining at 12 and 24 h after treated with BRM270. Apoptosis induced by BRM270 was studied in Cal72 SLCICs and SaOS-2 SLCICs. The lower right quadrant in Fig. 4A indicates early apoptotic cells. In addition, the number of apoptotic cells (Annexin V-positive cells) increased in both CD133⁺ expressing Cal72 and SaOS-2 SLCICs after 24-h exposure to BRM270. The Annexin V-FITC assay showed that ~16.75 and 12.14% in 12 h, while 33.39 and 29.32% in 24 h underwent apoptosis with the exposure to BRM270 in both Cal72 and SaOS-2 SLCICs, respectively. No significant (P<0.05) difference in the necrotic cells (PI-positive and Annexin V-negative cells) was observed between the groups (Fig. 4A). On the basis of time of exposure to BRM270 in both cell lines, significant differences were observed. The number of early apoptotic cells (Annexin V-positive and PI-negative cells) was ~2-fold greater in both cell lines at 24 h exposure than the 12 h of exposure (Fig. 4A). Furthermore, protein lysates were analyzed by western blotting using IL-6-mediated regulation of pro-apoptosis protein Casp-8 and SMC2 in Cal72 SLCICs (Fig. 4B–E). Similarly, qPCR revealed the relative quantitatively downregulated levels of IL-6 and SMC2 whereas increased transcript levels of Casp-8 were observed in both Cal72 SLCICs and SaOS-2 SLCICs (Fig. 5C, E and F). These results suggest that down-regulation of IL-6 and SMC2 increased Casp-8 activity and enhanced BRM270-induced apoptosis in CD133 expressing Cal72 and SaOS-2 SLCICs. To demonstrate the direct effects of BRM270 to regulate NF-κB signal trafficking, before exposure to BRM270 the cells were given treatment with LPS. Later, the blot expressions of Casp-8 and SMC2 was studied in the cells treated with and without LPS after 24 h exposure to BRM270. However, there is significant difference (P<0.05) in the blot expression of both BRM270 with LPS and only BRM270 treated groups (Fig. 4B). Moreover, the relative transcriptomic levels of apoptotic gene Casp-8 increased more significantly (P<0.05) in Cal72 SLCICs than LPS treated group after treatment with BRK270. At the same time, a significant (P<0.05) decrease in the transcriptomic levels of IL-6 were recorded in both groups. Moreover, a significant decrease in the transcript levels of SMC2 was recorded in the group without treatment with LPS as compare to cell lines treated with LPS after exposure to BRM270 (Fig. 4B, C and E).

The relationships between the level of IL-6 with the expression of Cdk6, NF-κB and Casp-8 in CD133 expressing Cal72 SLCICs were investigated (Figs. 5 and 6) to understand the role of IL-6 in crosstalk between NF-κB signaling cascades in SLCICs mediated cancer progression and MDR. On comparison of the transcriptional levels of candidate genes after BRM270 treatment with respect to NC, the Cdk6 showed significantly higher transcript levels in Cal72 SLCICs than the BRM270 treated group (Fig. 5B). Similarly, NF-κB was also overexpressed in non-BRM270 treated SLCICs while after treatment it was suppressed simultaneously with Cdk6 (Figs. 5A, B and 6F). Further, to investigate the effect of BRM270 we targeted the IL-6 induced NF-κB activation pathway. IL-6 is reported to enhance the stemness of the oncogenic cells when demonstrating the MDR to Dox. An attempt was made to find the efficacy of BRM270 on SLCICs in a dose-dependent manner (Figs. 1E and 2D). Although, it recruits the Cdk6 to catalyze the cyclin-D which upregulates the cell cycle in G0/G1 phase (Fig. 3) in cancer stem cells. It has been observed that CD133⁺ SLCICs induces tumorigenesis via NF-κB signaling where Cdk6 is co-localized with p65 (Fig. 6F). Moreover, qPCR analysis has demonstrated the efficacy of BRM270 which suppressed the expression of p65 and Cdk6 significantly (P<0.05) in vitro (Fig. 5A and B). It further arrests the cell cycle at G1 phase and transit to G2/M phase (Fig. 3). In the LPS treated group IL-6 overex-pressed the stemness property of CD133⁺ SLCICs (Figs. 2E and 6E). It also exerted MDR under in vitro conditions (Fig. 2A–C). On the contrary, non-LPS treated group demonstrated higher cytotoxicity, which in turn led to decrease in the proliferation rate and viability of the SLCICs in Dox and BRM270 treated groups (Fig. 2A–C). Significantly higher blot expression of IκBα, p65 and IL-6 was observed towards the enhanced tumorigenicity under in vivo and in vitro conditions (Figs. 2A–C and 6A). On treatment with BRM270 in the SLCICs and in the nude mouse model, in vitro and in vivo conditions, a dramatically downregulated expression of NF-κB was observed. Findings from qPCR and western blotting also supported the concept of co-suppression of Cdk6 and p65 (Figs. 2A–C, 5A–C and 6A and F).