Xiaoping Duan

Osterix, a transcription factor for osteoblast differentiation, mediates antitumor activity in murine osteosarcoma

Osterix is a novel zinc finger-containing transcription factor that is essential for osteoblast differentiation and bone formation. We hypothesized that osterix might have a role in osteosarcoma tumor growth and metastasis. Northern blot analysis showed that the mRNA level of osterix was decreased in two mouse osteosarcoma cell lines compared with its level in normal mouse osteoblasts. Osterix expression was also decreased in three human osteosarcoma cell lines. Transfection of the osx gene into the mouse osteosarcoma cells inhibited tumor cell growth in vitro and in vivo and significantly reduced tumor incidence, tumor volume, and lung metastasis following intratibial injection. Osterix expression was also associated with decreased osteolysis. Using an in vitro migration assay, osterix suppressed the migration of tumor cells to lung extracts. These results suggest that osterix expression may play a role in osteosarcoma tumor growth and metastasis.

Increased Fas Expression Reduces the Metastatic Potential of Human Osteosarcoma Cells

Purpose: The process of metastasis requires the single tumor cell that seeds the metastatic clone to complete a complex series of steps. Identifying factors responsible for these steps is essential in developing and improving targeted therapy for metastasis. Resistance to receptor-mediated cell death, such as the Fas/Fas ligand pathway, is one mechanism commonly exploited by metastatic cell populations. Experimental Design and Results: LM7, a subline of the SAOS human osteosarcoma cell line with low Fas expression, was selected for its high metastatic potential in an experimental nude mouse model. When transfected with the full-length Fas gene (LM7-Fas), these cells expressed higher levels of Fas than the parental LM7 cells or LM7-neo control-transfected cells. These cells were also more sensitive to Fas-induced cell death than controls. When injected intravenously into nude mice, the LM7-Fas cell line produced a significantly lower incidence of tumor nodules than control cell lines. Lung weight and tumor nodule size were also decreased in those mice injected with LM7-Fas. Levels of Fas were quantified in osteosarcoma lung nodules from 17 patients. Eight samples were Fas negative, whereas the remaining 9 were only weakly positive compared with normal human liver (positive control). Conclusions: Our results demonstrate that altering Fas expression can impact the metastatic potential of osteosarcoma cells. We conclude that the increase of Fas on the surface of the LM7 osteosarcoma cells increased their sensitivity to Fas-induced cell death in the microenvironment of the lung, where Fas ligand is constitutively expressed. Thus, loss of Fas expression is one mechanism by which osteosarcoma cells may evade host resistance mechanisms in the lung, increasing metastatic potential. Fas may therefore be a new therapeutic target for osteosarcoma.

Murine bone marrow-derived mesenchymal stem cells as vehicles for interleukin-12 gene delivery into ewing sarcoma tumors

This study evaluated the therapeutic efficacy of interleukin 12 (IL‐12) gene therapy in Ewing sarcoma and whether murine mesenchymal stem cells (MSCs) could serve as vehicles for IL‐12 gene delivery.

Intranasal interleukin-12 gene therapy enhanced the activity of ifosfamide against osteosarcoma lung metastases

Cyclophosphamide (CTX) and ifosfamide (IFX) are alkylating agents used to treat osteosarcoma (OS). It was previously demonstrated that the sensitivity of OS cells to 4‐hydroperoxycyclophosphamide (4‐HC, the active metabolite of CTX) is augmented by interleukin (IL)‐12 in vitro through a mechanism involving the Fas/FasL pathway. The purpose of these studies was to determine whether this synergistic effect is operational in vivo.

Association of αvβ3 integrin expression with the metastatic potential and migratory and chemotactic ability of human osteosarcoma cells

Introduction. Expression of adhesion molecules such as αvβ3 integrin has been associated with the metastatic potential of tumor cells. The purpose of this study was to determine whether αvβ3 expression correlated with the metastatic potential of human osteosarcoma cells. Materials and methods. We developed a series of sublines (LM2–LM7) from human osteosarcoma SAOS parental cells, with progressively increasing potential to form lung metastases in nude mice after intravenous injection. SAOS parental and LM2 cells were poorly metastatic, but LM7 cells resulted in visible metastatic lung nodules by 6–8 weeks. We quantified αvβ3 integrin expression using flow cytometry. Results. αvβ3 expression correlated with the metastatic potential of the cells, with LM7 cells showing the highest expression. LM7 cell adhesion to vitronectin decreased after treatment with echistatin, a RGD-containing peptide antagonist of αvβ3. LM7 cells demonstrated higher chemotactic activity than SAOS cells to a homogenate made from lung tissue. This chemotactic activity was also inhibited by echistatin. These data indicated that αvβ3 was critical for the migration of LM7 cells to the lung homogenate. Chemotaxis to a liver homogenate was the same for LM7 and SAOS cells. Migration of LM7 cells through lung endothelial cells was higher than that through liver endothelial cells, and echistatin again inhibited this migration. Conclusions. αvβ3 integrin expression may play a role in the metastatic potential of osteosarcoma cells by enhancing the ability of the cells to migrate specifically to the lung. αvβ3 integrin may therefore be a potential new target for osteosarcoma.

Suppression of Ewing's Sarcoma Tumor Growth, Tumor Vessel Formation, and Vasculogenesis Following Anti–Vascular Endothelial Growth Factor Receptor-2 Therapy

Purpose: We previously showed that bone marrow cells participate in new tumor vessel formation in Ewings sarcoma, and that vascular endothelial growth factor 165 (VEGF165) is critical to this process. The purpose of this study was to determine whether blocking VEGF receptor 2 (VEGFR-2) with DC101 antibody suppresses tumor growth, reduces tumor vessel formation, and inhibits the migration of bone marrow cells into the tumor. Experimental Design: An H-2 MHC-mismatched bone marrow transplant Ewings sarcoma mouse model was used. Bone marrow cells from CB6F1 (MHC H-2b/d) mice were injected into irradiated BALB/cAnN mice (MHC H-2d). TC71 Ewings sarcoma cells were s.c. injected 4 weeks after the bone marrow transplantation. Mice were then treated i.p. with DC101 antibody or immunoglobulin G (control) twice a week for 3 weeks starting 3 days after tumor cell injection. Results: DC101 antibody therapy significantly reduced tumor growth and tumor mean vessel density (P < 0.05) and increased tumor cell apoptosis. Decreased bone marrow cell migration into the tumor was also shown after DC101 therapy as assessed by the colocalization of H-2Kb and CD31 using immunohistochemistry. DC101 inhibited the migration of both human and mouse vessel endothelial cells in vitro. Conclusion: These results indicated that blocking VEGFR-2 with DC101 antibodies may be a useful therapeutic approach for treating patients with Ewings sarcoma.

VEGF165 promotes the osteolytic bone destruction of Ewing's sarcoma tumors by upregulating RANKL

The purpose of this study was to determine whether vascular endothelial growth factor-165 (VEGF165) contributed to the osteolytic process in Ewings sarcoma. VEGF165 induced osteoclast formation from murine bone marrow cells. Tartrate-resistant acid phosphatase (TRAP) staining demonstrated significantly fewer osteoclasts in VEGF-inhibited TC/siVEGF7-1 tumors compared to TC71 parental or TC/si-control tumors. Receptor activator NF-kappaB (RANKL), a critical osteoclastogenic factor, was decreased in TC/siVEGF7-1 cells. Incubation of these cells with recombinant VEGF165 upregulated RANKL in a dose- and time-dependent manner. The induction of (RANKL) by VEGF165 was also demonstrated in MC3T3-E1 mouse osteoblast cells and bone marrow stromal cells. This upregulation was transcriptionally mediated by an effect on the RANKL promoter. Both VEGF and EWS/FLI-1 increased RANKL promoter activity. Taken together, these data suggest that modulation of RANKL by VEGF165 may be one of the mechanisms responsible for the osteolytic process induced by Ewings sarcoma cells. VEGF165 may, therefore, play an important role in modulating RANKL gene expression in the bone marrow microenvironment during the metastatic process, thereby contribution to tumor induced bone lysis.

Interleukin-12 Enhances the Sensitivity of Human Osteosarcoma Cells to 4-Hydroperoxycyclophosphamide by a Mechanism Involving the Fas/Fas-Ligand Pathway

Cyclophosphamide (CY) and its derivative ifosfamide are alkylating agents used to treat osteosarcoma (OS). The purpose of these studies was to determine whether alkylating agents affect the expression of Fas ligand (FasL) and whether interleukin 12 enhances the sensitivity of human OS cells to alkylating agents. 4-Hydroperoxycyclophosphamide (4-HC), the preactivated CY compound, and 4-hydroperoxydidechlorocloclophosphamide (4-HDC), its nonalkylating analogue, human OS LM6 cells, and a clone of cells derived by transfection with the interleukin 12 gene (LM6-#6) were used for these studies. Incubation of LM6 and LM6-#6 with 10 μm 4-HC increased the expression of FasL mRNA (2.5- and 3.0-fold, respectively). By contrast, 4-HDC, Adriamycin (ADR), cisplatin (CDP), and methotrexate (MTX) had no effect on FasL mRNA expression. Increased FasL expression after treatment with 4-HC was also demonstrated by immunohistochemistry and flow cytometry. Drug-induced FasL was functional and mediated cell death. We examined the effect of FasL up-regulation by 4-HC on LM6 and LM6-#6 cells. Flow cytometry showed that LM6-#6 cells expressed 2.2-fold more Fas than LM6 cells. Cytotoxicity of 4-HC, 4-HDC, ADR, CDP, and MTX on LM6, LM6-neo, and LM6-#6 were quantified. Colony-forming assay revealed an IC50 of 2.10 μm for 4-HC in LM6-neo cells compared with 0.41 μm in LM6-#6 cells. The IC50 for 4-HDC, ADR, CDP, and MTX were not significantly different between the two cell lines. We concluded that the increased expression of Fas enhanced LM6-#6 sensitivity to 4-HC. These data indicate that Fas/FasL may be involved in the cytotoxic pathway of CY. Combining biological agents with chemotherapeutic agents that have complementary Fas/FasL pathway actions may offer new therapeutic alternatives.

Intratumor murine interleukin-12 gene therapy suppressed the growth of local and distant Ewing's sarcoma.

We evaluated the effect of interleukin-12 (IL-12) gene therapy using an Ewings sarcoma animal model in T-cell-deficient nude mice. Subcutaneous injection of TC71 cells resulted in tumor development by day 5. Mice were treated with a single intratumor injection of adenovirus β-galactosidase (Ad.β-gal) or adenovirus murine IL-12 (Ad.mIL-12) (2 × 109 PFU) and killed 1–7 days later. Reverse transcriptase-polymerase chain reaction analysis of tumor tissue demonstrated peak expression of IL-12 p35 and p40 at 48 h, which persisted up to 7 days. For in vivo therapy, mice received intratumor Ad.β-gal or Ad.mIL-12 twice weekly for 2.5 weeks starting on day 6. Ad.mIL-12-treated tumors were significantly smaller (median volume, 19.7 mm3; range, 3.41–159.5 mm3) than Ad.β-gal-treated tumors (median volume, 3214.9 mm3; range 1679.9–5909.8 mm3, P<0.003) on day 31. The weight of Ad.mIL-12-treated tumors was also lighter than the Ad.β-gal-treated tumors (median, 2 mg; range, 1–5 mg versus median, 1960 mg; range 1640–5230 mg, P<0.01). Ad.mIL-12 therapy significantly prolonged the survival time and also inhibited the growth of an untreated tumor on the contralateral side. Immunohistochemistry analysis of the IL-12-treated tumors demonstrated IL-12 expression with increased Fas, Fas ligand and tumor cell apoptosis. CD31 and vascular endothelial growth factor expression were decreased. These data suggest that IL-12 gene therapy may be useful in the treatment of Ewings sarcoma.