4T1.2 mouse mammary tumor cells (obtained from Dr R. Anderson at Peter MacCallum Cancer Institute, Melbourne, Australia), MDA-MB-231 human breast cancer cells [American Type Culture Collection (ATCC), Manassas, VA, USA (25)], and MCF-7 human breast cancer cells (26) were cultured in Dulbecco's modified Eagle's medium (Corning Incorporated, Corning, NY, USA). RAW264.7 pre-osteoclast cells (ATCC), MC3T3 osteoblast-like cells (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), and MLO-A5 osteocyte-like cells (obtained from Dr L. Bonewald at Indiana University, Indianapolis, IN, USA) were grown in α minimum essential medium (MEM; Gibco; Thermo Fisher Scientific. Inc., Waltham, MA, USA). Bone marrow-derived cells, obtained by flushing the tibia and femur from one female 10-week old BALB/c mouse and separating with low-density gradient centrifugation (27), were cultured in αMEM with 10 ng/ml macrophage colony-stimulating factor (PeproTech, Inc., Rocky Hill, NJ, USA) for 2 days, and the surface-attached cells were used as osteoclast precursors. The experimental procedures using animals were approved by the Indiana University Animal Care and Use Committee and were in compliance with the Guiding Principles in the Care and Use of Animals endorsed by the American Physiological Society (28). The culture media contained 10% fetal bovine serum (FBS; Atlanta Biologicals, Flowery Branch, GA, USA) and antibiotics (50 U/ml penicillin and 50 µg/ml streptomycin; Life Technologies; Thermo Fisher Scientific, Inc.). MLO-A5 cells were grown with 5% FBS and 5% bovine calf serum. Cells were maintained at 37°C and 5% CO₂ in a humidified incubator, and treated with AZD7762 (0.1–10 µM), PD407824 (0.1–20 µM), or thapsigargin (1 µM; Tocris Bioscience, Bristol, UK).

Cellular proliferation was evaluated using an MTT assay (Sigma-Aldrich; Merck KGaA), according to the manufacturer's protocol. The relative cell proliferation was determined as a ratio of the absorbance at 570 nm of each sample and control, as measured using a plate reader. To quantify 3D spheroid cell viability, the CellTiter-Glo 3D Cell Viability Assay (Promega Corporation, Madison, WI, USA) was used, according to the manufacturer's protocol, measuring luminescence with a plate reader.

To induce spheroid formation, ~5,000 cells were cultured in a U-bottom low-adhesion 96-well plate (S-Bio, Hudson, NH, USA). MLO-A5 osteocyte spheroids were bio-printed onto a needle array using a Regenova 3D Bioprinter (Cyfuse Biomedical K.K., Tokyo, Japan) (22) to generate bone constructs mineralized in osteogenic medium (70 µM ascorbic acid and 5 mM β-glycerophosphate) (27). Each construct contained 18 osteocyte spheroids (3×3×2 configuration).

To evaluate motility in a monolayer culture, a wound healing scratch motility assay was performed as described previously (29). In brief, cells were plated in 12-well plates and, the following day, scratching was performed using a plastic tip. The areas newly occupied with cells in the scratched zone were determined 24 h post-scratching using images obtained using a microscope (magnification, ×40; Nikon Eclipse TS100, Nikon Corporation, Tokyo, Japan), which were scanned using Adobe Photoshop (CS2; Adobe Systems, Inc., San Jose, CA, USA) and quantified with Image J v1.51p (National Institutes of Health, Bethesda, MD, USA).

Using RAW264.7 pre-osteoclast cells, an osteoclast differentiation assay was conducted in 24-well plates with 30 ng/ml receptor activator of nuclear factor-κB ligand (RANKL) in the presence and absence of 0.1–2 µM AZD7762 and PD407824. For bone marrow-derived cells, cells were cultured in 10 ng/ml macrophage colony stimulating factor (M-CSF; Peprotech, Inc., Rocky Hill, NJ, USA) for 2 days prior to the addition of RANKL (27). During the 5-day experiments, the culture medium was replaced once on day 3. Adherent cells were fixed with a citrate, acetone and formaldehyde solution for 30 sec at room temperature and stained for 20 min at 37°C with a tartrate resistant acid phosphate (TRAP)-staining kit, according to the manufacturer's protocol (Sigma-Aldrich; Merck KGaA). TRAP-positive multinucleated cells (>3 nuclei) were identified as mature osteoclasts (30).

A pit formation assay was conducted similarly using 24-well culture plates coated with hydroxyapatite (31). After 6 days, cells were removed with 5% sodium hypochlorite for 5 min at room temperature, and images of the wells were captured with a phase-contrast inverted microscope at ×40 magnification. Using ImageJ, the areas of the wells no longer coated with hydroxyapatite were measured, and the pit area fraction was calculated by normalizing to the total image area.

MC3T3 cells were seeded on 24-well tissue-culture plates at 6.5×10⁴ cells/well. Upon becoming fully confluent, osteogenic medium (70 µM ascorbic acid and 5 mM β-glycerophosphate) was added, with 0–0.5 µM AZD7762, and the cells were cultured for 5 weeks, changing the media every other day. The effect of AZD7762 on mineralization was evaluated by staining at room temperature for 3 min with 1% Alizarin Red S solution (Sigma-Aldrich; Merck KGaA) and imaged at ×40 magnification with an inverted microscope. Alizarin red staining was quantified by measuring the image grey-scale values using ImageJ.

Total RNA was extracted using an RNeasy Plus mini kit (Qiagen, Inc., Valencia, CA, USA) and RT was conducted (37°C for 2 h; 85°C for 5 min) with a high-capacity cDNA reverse transcription kit (Applied Biosystems; Thermo Fisher Scientific, Inc.). RT-qPCR was performed using Power SYBR green PCR master mix kits (Applied Biosystems; Thermo Fisher Scientific, Inc.) with the PCR primers listed in Table I. RNA interference was conducted using small interfering (si)RNA specific to PERK (cat. no. 100334), Chk1 (cat. no. 160184) and Chk2 (cat. no. 78549; Life Technologies; Thermo Fisher Scientific, Inc.). A negative siRNA (Silencer Select #1; Life Technologies; Thermo Fisher Scientific, Inc.) was used as a nonspecific negative control. Cells were transiently transfected with siRNA (130 pmol per 6-cm dish) using Lipofectamine^® RNAiMAX (Life Technologies; Thermo Fisher Scientific, Inc.) in Opti-MEM I medium. The medium was replaced with regular culture medium after 24 h, and the silencing efficiency was measured by immunoblotting 48 h post-transfection.

Cells were lysed in a radioimmuno-precipitation assay buffer with protease inhibitors (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and phosphatase inhibitors (Calbiochem; Merck KGaA). Following measuring of the protein concentration using a Bicinchoninic Acid Protein Assay kit (Thermo Fisher Scientific, Inc.), ~10 µg isolated proteins/lane were fractionated using 10–15% SDS gels and electrotransferred to polyvinylidene difluo-ride membranes (EMD Millipore, Billerica, MA, USA). Membranes were blocked in 2% blotting-grade blocker (Bio-Rad Laboratories, Inc., Hercules, CA, USA) for 1 h at room temperature or overnight at 4°C. Antibodies against alkaline phosphate (cat. no. sc-28904; Santa Cruz Biotechnology, Inc.), cathepsin K (cat. no. sc-48353; Santa Cruz Biotechnology, Inc.), PERK (cat. no. sc-13073; Santa Cruz Biotechnology, Inc.), nuclear factor of activated T cells cytoplasmic 1 (NFATc1; cat. no. sc-7294; Santa Cruz Biotechnology, Inc.), Chk1 (cat. no. 2360; Cell Signaling Technology, Inc., Danvers, MA, USA), phosphorylated (p)-Chk1 (cat. no. 90178; Cell Signaling Technology, Inc.), Chk2 (cat. no. 2662; Cell Signaling Technology, Inc.), eIF2α (cat. no. 9722; Cell Signaling Technology, Inc.), p-eIF2α (cat. no. 9721; Cell Signaling Technology, Inc.), microtubule-associated proteins 1A/1B light chain (LC)3A/B II (cat. no. 4108; Cell Signaling Technology, Inc.), caspase 3 (cat. no. 9662; Cell Signaling Technology, Inc.), cleaved caspase 3 (cat. no. 9661; Cell Signaling Technology, Inc.), p38 (cat. no. 9212; Cell Signaling Technology, Inc.), p-p38 (cat. no. 9211; Cell Signaling Technology, Inc.), p-PERK (cat. no. 3179; Cell Signaling Technology, Inc.), p53 (cat. no. MA5-12557; Invitrogen; Thermo Fisher Scientific, Inc.), p-Chk2 (cat. no. SAB4504366; Invitrogen; Thermo Fisher Scientific, Inc.), and β-actin (cat. no. A5441; Sigma-Aldrich; Merck KGaA) were used. Primary antibodies were incubated for 1 h at room temperature or overnight at 4°C at a 1:1,000 dilution, except Chk2, p-Chk2, p-PERK (all 1:500), p53 (1:200) and β-actin (1:10,000), while anti-rabbit (cat. no. 7074; Cell Signaling Technology, Inc.) and anti-mouse (cat. no. 7076; Cell Signaling Technology, Inc.) secondary antibodies were incubated for 45 min at room temperature at a 1:2,000 dilution. Protein expression levels were assayed using a SuperSignal west femto maximum sensitivity substrate (Thermo Fisher Scientific, Inc.).

To evaluate the potential role of GTPases in cellular motility, the activity of RhoA GTPase was determined by FRET imaging. In response to 2 µM AZD7762 and PD407824, energy transfer was measured in a RhoA-specific cyan fluorescent protein (CFP)-yellow fluorescent protein (YFP) biosensor (32). The filter sets (Semrock; IDEX Health & Science, LLC, Rochester, NY, USA) were chosen for CFP excitation at 438±24 nm (center wavelength ± bandwidth), CFP emission at 483±32 nm and YFP emission at 542±27 nm. Using a fluorescence microscope (Nikon Corporation, Tokyo, Japan) time-lapse images were acquired at an interval of 5 min. The emission ratio of YFP/CFP for individual cells was computed to determining the activity levels using NIS-Elements software v3.2 (Nikon Corporation).

Micro-CT was performed using Skyscan 1172 (Bruker-MicroCT, Kontich, Belgium) (33). The printed bone constructs were wrapped in Parafilm to maintain hydration and placed in a plastic tube. Scans were performed at pixel size 6 µm. Using manufacturer-provided software, the images were reconstructed (nRecon v1.6.9.18), rotated (DataViewer v1.5.0), and the bone mineral density of the mineralized bone construct was determined by calculating the mean attenuation coefficient and calibrated to two hydroxyapatite phantoms of known density (CTAn v1.11.4.2).

The data are expressed as the mean ± standard deviation of three to four replicates. One-way analysis of variance was employed to examine the statistical significance among groups, and Fisher's protected least significant difference was conducted as a post hoc test to evaluate the pairwise comparisons using R 3.4.3 (R Foundation for Statistical Computing, Vienna, Austria). P<0.05 was considered to indicate a statistically significant difference.

The present study first examined the effects of AZD7762, an inhibitor of Chk1 and Chk2, on the proliferation and migration of 4T1.2 mammary tumor cells. As the representative images of 4T1.2 cells illustrate, in response to 1 µM AZD7762, the cells underwent apoptosis (blebbing and cell shrinkage) and autophagy (increase in vacuole-like structures in the cytoplasm and membrane expansion), and the relative proliferation of the tumor cells was significantly reduced in a dose-dependent manner (Fig. 1A and B). Western blot analysis revealed that p-eIF2α was elevated in addition to cleaved caspase 3 (apoptosis marker) and LC3A/B II (autophagy marker) (Fig. 1C and D), while the expression levels of p-Chk1 and p-Chk2 were reduced. The scratch assay for examining cellular motility demonstrated that the healing of the wound area was decreased by 0.5 and 1 µM AZD7762 (Fig. 1E and F), indicating that AZD7762 reduces the motility of tumor cells. Notably, the wound area was also enlarged as a result of AZD7762-driven cell death.

Since tumor growth may differ according to the culture conditions, the present study subsequently examined the effects of AZD7762 on 3D spheroids solely composed of 4T1.2 cells (Fig. 1G). Initially, the spheroids were irregularly shaped with a rough surface. They gradually compacted, forming a necrotic core at the center and a quiescent shell around the core with a proliferating surface. In response to 0.5 to 10 µM AZD7762, the spheroids significantly altered in appearance. While the spheroids exhibited extended quiescent zones at the lower concentrations (0.5 and 1 µM AZD7762), it increased the necrotic core at the higher concentrations (5 and 10 µM AZD7762). AZD7762 reduced the cross-sectional area of the tumor spheroids and decreased 3D cell viability (Fig. 1H and I).

To evaluate its effect on other types of breast cancer, the present study also examined the responses of two human breast cancer cell lines, MDA-MB-231 and MCF-7, to AZD7762. MDA-MB-231 cells are estrogen receptor-negative, while MCF-7 cells are estrogen receptor-positive. The results revealed that AZD7762 inhibited the proliferation of the two cell lines, as measured by MTT assay, in a monolayer cell culture, and decreased the sizes of the 3D tumor spheroids, although 4T1.2 and MDA-MB-231 cells were more sensitive to AZD7762 compared with MCF-7 cells (Fig. 2).

Osteoclasts serve a pivotal role in bone homeostasis. Using RAW264.7 pre-osteoclast cells and osteoclast precursors from bone marrow-derived cells, the present study evaluated the effect of AZD7762 on the proliferation and differentiation of osteoclasts. Treatment with AZD7762 significantly reduced the proliferation of RAW264.7 cells and primary osteoclast precursors (Fig. 3A and B). The half-maximal inhibitory concentration for AZD7762 in bone marrow-derived osteoclasts was estimated as 0.54 µM by fitting an exponential curve to the MTT results (data not shown). In RAW cells, AZD7762 also downregulated NFATc1, a master transcription factor for osteoclastogenesis, in addition to cathepsin K, a marker of bone resorption (Fig. 3C). It was additionally observed that the expression level of p-eIF2α was elevated, together with cleaved caspase 3, in RAW cells (Fig. 3C and D). In primary osteoclast precursors, AZD7762 suppressed the RANKL-driven upregulation of NFATc1 and elevated cleaved caspase 3 expression (Fig. 3E). As demonstrated by TRAP staining for identifying multi-nucleated osteoclasts, AZD7762 significantly reduced the number of multi-nucleated TRAP-positive osteoclast cells in a dose-dependent manner (Fig. 3F and G). Furthermore, AZD7762 decreased the 3D cell viability of RAW264.7 spheroids in a dose-dependent manner (Fig. 3H). Consistent with the inhibitory effect of AZD7762 on RAW264.7 cultured cells, AZD7762 at 0.5, 1, 5 and 10 µM decreased the cross-sectional area of RAW264.7 spheroids at 24–72 h (Fig. 3I). Finally, osteoclast activity measured by a pit formation assay was reduced by 0.5 and 1 µM AZD7762 (Fig. 3J).

The present study additionally examined the effect of AZD7762 on osteoblast activity. AZD7762 reduced the relative proliferation of MC3T3 osteoblast-like cells in response to 0.1, 0.5, 1 and 2 µM, although the degree of reduction was smaller compared with RAW264.7 pre-osteoclast cells (Fig. 4A). AZD7762 increased the mRNA levels of osteogenesis-associated genes, including osterix, osteocalcin (OCN), and alkaline phosphatase (ALP) at 24 and 48 h (Fig. 4B and C); it also elevated the protein expression levels of p-eIF2α, p-p38 and ALP (Fig. 4D and E).

In order to evaluate any differential effects of Chk1 and Chk2 inhibition, PD407824, a selective inhibitor of Chk1, was used. The responses to PD407824 were largely similar to those to AZD7762. PD407824 at 0.1, 0.5 and 2 µM reduced the relative proliferation of 4T1.2 cells (Fig. 5A), and the expression level of p-eIF2α was elevated in a dose-dependent manner (Fig. 5B). Furthermore, the scratch assay with 4T1.2 mammary tumor cells revealed that PD407824 suppressed cell motility by inhibiting the healing of the wounded area (Fig. 5C). To evaluate the potential mechanism of p-eIF2α upregulation, the expression of p-PERK, a kinase induced by stress to the (ER), was evaluated. First, thapsigargin, an ER stress inducer, elevated p-eIF2α and p-PERK expression after 3 h of incubation (Fig. 5D). Second, partial silencing of PERK by RNA interference downregulated p-eIF2α (Fig. 5E), and PD407824 and AZD7762 elevated the expression level of p-PERK (Fig. 5F). Collectively, these data indicated that the elevation of p-eIF2α may be induced by p-PERK, an eIF2α selective kinase.

In addition to its effect on tumor cells, PD407824 affects RAW264.7 pre-osteoclasts similarly to AZD7762. PD407824 at 0.1, 0.5 and 2 µM reduced the relative proliferation of RAW264.7 cells (Fig. 5G). In MC3T3 cells, PD407824 at 0.1 and 0.5 µM did not significantly alter the relative cell proliferation, although a higher dosage of 2 µM decreased the number of MC3T3 cells (Fig. 5H). The RANKL-driven differentiation of RAW264.7 cells was also suppressed by 0.1, 0.5 and 2 µM PD407824 in a dose-dependent manner (Fig. 6A and B).

A major difference between AZD7762 and PD407824 was their effects on genes involved in bone formation. While AZD7762 elevated the mRNA expression levels of osterix and ALP, PD407824 at 2 µM reduced their expression levels (Fig. 6C). Furthermore, the effect of PD407824 on the protein expression levels of p53, p-p38, and ALP was not notable (Fig. 6D). To evaluate the potential involvement of RhoA GTPase in the regulation of cellular motility with Chk1 inhibitors, its activity level was evaluated using a RhoA GTPase FRET biosensor. In response to 2 µM AZD7762 and PD407824, significant reductions in RhoA activity were observed after 60 min (Fig. 6E).

The present study demonstrated that AZD7762 serves an inhibitory role in bone-resorbing osteoclasts. To examine its role in bone-forming osteoblasts, Alizarin red staining was performed using MC3T3 cells. The results demonstrated that in response to 0.05, 0.1 and 0.5 µM AZD7762 for 5 weeks, staining intensity was elevated in a dose-dependent manner (Fig. 6F and G). It was also observed that AZD7762 decreased the expression of p53 (Fig. 6H). Furthermore, 3D bone constructs with MLO-A5 osteocyte-like cell spheroids were bio-printed, and X-ray imaging was conducted after a 2-week incubation in osteogenic medium. The results revealed that bone mineral density was elevated in response to 0.05 and 0.1 µM AZD7762 (Fig. 7A–C). To evaluate the mediators of the differential effects of AZD7762 and PD407824 on p53 expression, partial silencing of Chk1 and Chk2 was conducted via siRNA in MC3T3 cells. Following silencing of Chk2, the AZD7762-mediated upregulation of ALP and downregulation of p53 were muted. However, the same alterations were not observed in Chk1-silenced cells (Fig. 7D). A proposed regulatory mechanism for AZD7762 is illustrated in Fig. 8, in which Chk1 and Chk2 are inhibited by AZD7762, as opposed to only Chk1 inhibition by PD407824.