SaOS2 Osteosarcoma Cells as an In Vitro Model for Studying the Transition of Human Osteoblasts to Osteocytes

The central importance of osteocytes in regulating bone homeostasis is becoming increasingly apparent. However, the study of these cells has been restricted by the relative paucity of cell line models, especially those of human origin. Therefore, we investigated the extent to which SaOS2 human osteosarcoma cells can differentiate into osteocyte-like cells. During culture under the appropriate mineralising conditions, SaOS2 cells reproducibly synthesised a bone-like mineralised matrix and temporally expressed the mature osteocyte marker genes SOST, DMP1, PHEX and MEPE and down-regulated expression of RUNX2 and COL1A1. SaOS2 cells cultured in 3D collagen gels acquired a dendritic morphology, characteristic of osteocytes, with multiple interconnecting cell processes. These findings suggest that SaOS2 cells have the capacity to differentiate into mature osteocyte-like cells under mineralising conditions. PTH treatment of SaOS2 cells resulted in strong down-regulation of SOST mRNA expression at all time points tested. Interestingly, PTH treatment resulted in the up-regulation of RANKL mRNA expression only at earlier stages of differentiation. These findings suggest that the response to PTH is dependent on the differentiation stage of the osteoblast/osteocyte. Together, our results demonstrate that SaOS2 cells can be used as a human model to investigate responses to osteotropic stimuli throughout differentiation to a mature osteocyte-like stage.     

Induction of cell death of human osteogenic sarcoma cells by zoledronic acid resembles anoikis

The aim of this study was to investigate the cytotoxic activity of the third-generation nitrogen-containing bisphosphonate zoledronic acid (ZOL) as a single agent, and in combination with clinically relevant anticancer drugs, in a panel of human osteogenic sarcoma cell lines (HOS, BTK-143, MG-63, SJSA-1, G-292, and SAOS2). We found that ZOL, when used alone, reduced cell number in a dose- and time-dependent manner, due either to cell cycle arrest in S-phase or to the induction of apoptosis. In the sensitive HOS, BTK-143, and G-292 cell lines, genomic DNA fragmentation and morphological changes characteristic of apoptosis were evident, and cells became nonadherent. Induction of apoptosis in osteosarcoma cells by ZOL was associated with caspase activation. However, coaddition of the broad-spectrum caspase inhibitors, z-VAD-fmk, Boc-D-fmk, or the caspase-3-specific inhibitor z-DEVD fmk, failed to protect these cells from ZOL-induced apoptosis. Our data support a ZOL-specific induction of cell apoptosis that involves cell detachment (anoikis), and in which caspase activation occurs secondarily to, and is redundant as a mediator of cell death. The addition of geranylgeraniol, an intermediate of the mevalonate pathway, suppressed the ZOL-induced apoptosis, suggesting that the cytotoxic effects of ZOL in osteosarcoma cells were mediated by the mevalonate pathway. While treatment of osteosarcoma cells with the chemotherapeutic agents doxorubicin or etoposide decreased cell viability, combination of these agents with ZOL did not significantly augment apoptosis in any of the cell lines tested. These observations suggest that ZOL has direct effects on the proliferation and survival of osteosarcoma cells in vitro, which has implications for future therapy of osteosarcoma.     

Targeted Disruption of the CXCL12/CXCR4 Axis Inhibits Osteolysis in a Murine Model of Myeloma‐Associated Bone Loss

The plasma cell (PC) malignancy, multiple myeloma (MM), is unique among hematological malignancies in its capacity to cause osteoclast (OC)‐mediated skeletal destruction. We have previously shown that elevated plasma levels of PC‐derived CXCL12 are associated with presence of X‐ray detectable osteolytic lesions in MM patients. To further investigate this relationship, plasma levels of CXCL12 and βCrossLaps, a marker of bone loss, were measured. A strong correlation between levels of CXCL12 and OC‐mediated bone resorption was identified. To confirm the OC‐activating potential of MM PC‐derived CXCL12 in vivo, we established a model of MM‐mediated focal osteolysis, wherein MM PC lines, such as RPMI‐8226, were injected into the tibias of nude mice. Implanting RPMI‐8226 gave rise to osteolytic lesions proximal to the tumor, resulting in a 5% decrease in bone volume (BV) compared with vehicle control. Importantly, bone loss was significantly inhibited with systemic administration of the CXCL12/CXCR4 antagonist T140. Furthermore, implanting CXCL12‐overexpressing RPMI‐8226 cells resulted in a 13% decrease in BV and was associated with increased OC recruitment proximal to the tumor, increased serum matrix metalloproteinase activity, and increased levels of collagen I degradation products. These findings confirm our hypothesis that MM PC‐derived CXCL12 stimulates the recruitment and activity of OC, thereby contributing to the formation of MM osteolytic lesions.     

Apo2l/Tumor necrosis factor-related apoptosis-inducing ligand prevents breast cancer-induced bone destruction in a mouse model

Breast cancer is the most common carcinoma that metastasizes to bone. To examine the efficacy of recombinant soluble Apo2 ligand (Apo2L)/tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) against breast cancer growth in bone, we established a mouse model in which MDA-MB-231 human breast cancer cells were transplanted directly into the marrow cavity of the tibiae of athymic nude mice producing osteolytic lesions in the area of injection. All vehicle-treated control animals developed large lesions that established in the marrow cavity, eroded the cortical bone, and invaded the surrounding soft tissue, as assessed by radiography, micro-computed tomography, and histology. In contrast, animals treated with recombinant soluble Apo2L/TRAIL showed significant conservation of the tibiae, with 85% reduction in osteolysis, 90% reduction in tumor burden, and no detectable soft tissue invasion. Tumor cells explanted from Apo2L/TRAIL-treated animals were significantly more resistant to the effects of Apo2L/TRAIL when compared with the cells explanted from the vehicle-treated control animals, suggesting that prolonged treatment with Apo2/TRAIL in vivo selects for a resistant phenotype. However, such resistance was readily reversed when Apo2L/TRAIL was used in combination with clinically relevant chemotherapeutic drugs, including taxol, etoposide, doxorubicin, cisplatin, or the histone deacetylase inhibitor suberoylanilide hydroxamic acid. These studies show for the first time that Apo2L/TRAIL can prevent breast cancer-induced bone destruction and highlight the potential of this ligand for the treatment of metastatic breast cancer in bone.     

Calcitonin decreases the adherence and survival of HEK-293 cells by a caspase-independent mechanism

We recently reported that calcitonin (CT) can profoundly inhibit the growth of HEK-293 cells transfected with the human calcitonin receptor (hCTR). We also obtained preliminary evidence that suggested a role for CT in cell survival, and in the present study we have investigated the pro-apoptotic action of CT, which we observe in conditions of low serum concentration. Under these conditions, we have found that CT treatment of HEK-293 cells stably transfected with the insert-negative form of the human CTR (HR12 cells) caused a time-dependent decrease in cell number associated with loss of cellular attachment. Loss of cellular adherence in CT-treated cultures caused programmed cell death, as shown by Annexin V staining of cells, failure of cells to exclude Trypan Blue dye, condensation and cleavage of nuclear DNA, and appearance of hypodiploid cells in fluorescence-activated cell sorting (FACS) analysis. The accumulation of non-adherent cells and cell death was concomitant with increased intracellular activity of caspase-3. However, inhibition of caspase activation in HR12 cells did not prevent CT-mediated loss of attachment and did not maintain the viability of non-adherent cells, indicating that caspase activation accompanied, but was probably not the cause of, the loss of cell viability. Neither the effects of CT on cell survival nor the activation of caspase-3 were observed in serum-replete conditions, suggesting that serum-derived factors provide protection of cells from CT-induced apoptosis. The inhibitory effects of CT on cell growth were found previously to be related to activation of Erk1/2 MAP kinase. In the present experiments, it was found that the Erk1/2 inhibitor, PD 98059, inhibited the CT-induced loss of cellular adherence and the consequent reduction in cell numbers. These results demonstrate that CT can negatively affect cell survival and they identify roles for cell adherence and MAP kinase activation in this process.     

Involvement of p21(Waf1/Cip1) and its cleavage by DEVD-caspase during apoptosis of colorectal cancer cells induced by butyrate

Butyrate, a short chain fatty acid produced in the colon, inducesapoptosis in cancer cell lines by a sequential process involvinginhibition of histone deacetylase, de novo protein synthesisand activation of DEVD-caspase, a major effector of apoptoticDNA fragmentation and membrane blebbing. We now show, in LIM1215 colorectal cancer cells, that butyrate, in addition toactivating DEVD-caspase and inducing apoptosis, also increasesexpression and cleavage of the universal cyclin-dependent kinaseinhibitor p21^Waf1/Cip1 and leads to hypo-phosphorylation ofretinoblastoma protein. Accompanying these molecular changeswas a progressive loss of G₀/G₁ and S phase cells. Expressionof p21 had similar kinetics to that of the essential proteinrequired for DEVD-caspase activation, indicating parallel effectsof butyrate on anti-apoptotic and pro-apoptotic mechanisms.LIM 1215 cells, which were resistant to butyrate-induced apoptosis,were selected by three cycles of exposure to butyrate and removalof floating apoptotic cells. These cells showed markedly enhancedp21 expression and were in cell cycle arrest as determined byflow cytometry. On the other hand, subsequent culture of thesecells for 2–3 days in the absence of butyrate resultedin down-regulation of p21 and restoration of sensitivity toapoptosis by butyrate. Western blots of butyrate-treated cellsundergoing apoptosis consistently demonstrated a 15 kDa band(p15) that was not present in control cultures. This band becameapparent immediately after the onset of DEVD-caspase activation,was enriched in the floating apoptotic cell population whencompared with the adherent, non-apoptotic cells and was absentin butyrate-resistant cells lacking DEVD-caspase activity. Peptidecaspase inhibitors partially blocked appearance of p15. Herewe show, for the first time, that p21 is a target of effectorcaspases in colorectal cancer cells and that the resistanceto butyrate-induced apoptosis is characterized by failure ofp21 cleavage. Abbreviations: CDK, cyclin-dependent kinase; CRC, colorectal cancer; FCS, fetal calf serum; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HBSS, Hank's balanced salt solution; PBS, phosphate-buffered saline; Rb, retinoblastoma protein; zDEVD-AFC, z-Asp-Glu-Val-Asp-7-amino-4-trifluoromethyl coumarin; zDEVD-fmk, z-Asp-Glu-Val-Asp-fluoromethyl ketone; zVAD-fmk, z-Val-Ala-Asp-fluoromethyl ketone. ³ To whom correspondence should be addressed Email: pzalewski{at}medicine.adelaide.edu.au     

The histone deacetylase inhibitor, suberoylanilide hydroxamic acid, overcomes resistance of human breast cancer cells to Apo2L/TRAIL

While the apoptosis-inducing ligand Apo2L/TRAIL is a promising new agent for the treatment of cancer, the sensitivity of cancer cells for induction of apoptosis by Apo2L/TRAIL varies considerably. Identification of agents that can be used in combination with Apo2L/TRAIL to enhance apoptosis in breast cancer cells would increase the potential utility of this agent as a breast cancer therapeutic. Here, we show that the histone deacetylase inhibitor, suberoylanilide hydroxamic acid (SAHA), can sensitize Apo2L/TRAIL-resistant breast cancer cells to Apo2L/TRAIL-induced apoptosis. Importantly, neither Apo2L/TRAIL alone, nor in combination with SAHA, affected the viability of normal human cells in culture. Apo2L/TRAIL-resistant MDA-MB-231 breast cancer cells, generated by long-term culture in the continuous presence of Apo2L/TRAIL, were resensitized to Apo2L/TRAIL-induced apoptosis by SAHA. The sensitization of these cells by SAHA was accompanied by activation of caspase 8, caspase 9 and caspase 3 and was concomitant with Bid and PARP cleavage. The expression of the proapoptotic protein, Bax, increased significantly with SAHA treatment and high levels of Bax were maintained in the combined treatment with Apo2L/TRAIL. Treatment with SAHA increased cell surface expression of DR5 but not DR4. Interestingly, SAHA treatment also resulted in a significant increase in cell surface expression of DcR1. Taken together, our findings indicate that the use of these 2 agents in combination may be effective for the treatment of breast cancer.