Hypoxia increases osteopontin expression in human osteosarcoma cells.

<p>MG63 osteosarcoma cells were treated with the chemical hypoxic agent CoCl₂ (100 µM). Osteopontin (OPN) mRNA (6 h) (A) and protein (24 h) (B) levels were increased by CoCl₂ treatment. Data are presented as the mean ± S.E.M. (n = 3), *p≤0.05, as compared with the control (con).</p

Osteopontin Upregulates the Expression of Glucose Transporters in Osteosarcoma Cells

<div><p>Osteosarcoma is the most common primary malignancy of bone. Even after the traditional standard surgical therapy, metastasis still occurs in a high percentage of patients. Glucose is an important source of metabolic energy for tumor proliferation and survival. Tumors usually overexpress glucose transporters, especially hypoxia-responsive glucose transporter 1 and glucose transporter 3. Osteopontin, hypoxia-responsive glucose transporter 1, and glucose transporter 3 are overexpressed in many types of tumors and have been linked to tumorigenesis and metastasis. In this study, we investigated the regulation of glucose transporters by osteopontin in osteosarcoma. We observed that both glucose transporters and osteopontin were upregulated in hypoxic human osteosarcoma cells. Endogenously released osteopontin regulated the expression of glucose transporter 1 and glucose transporter 3 in osteosarcoma and enhanced glucose uptake into cells via the αvβ3 integrin. Knockdown of osteopontin induced cell death in 20% of osteosarcoma cells. Phloretin, a glucose transporter inhibitor, also caused cell death by treatment alone. The phloretin-induced cell death was significantly enhanced in osteopontin knockdown osteosarcoma cells. Combination of a low dose of phloretin and chemotherapeutic drugs, such as daunomycin, 5-Fu, etoposide, and methotrexate, exhibited synergistic cytotoxic effects in three osteosarcoma cell lines. Inhibition of glucose transporters markedly potentiated the apoptotic sensitivity of chemotherapeutic drugs in osteosarcoma. These results indicate that the combination of a low dose of a glucose transporter inhibitor with cytotoxic drugs may be beneficial for treating osteosarcoma patients.</p></div

Knockdown of osteopontin decreases glucose transporters expression in a hypoxic osteosarcoma cell line.

<p>(A) Two OPN-shRNA plasmids (shOPN1 and shOPN2) and one empty vector (ev) plasmid were transiently transfected (24 h) in MG63 cells. OPN protein expression was downregulated by both shOPN1 and shOPN2. After treatment with the chemical hypoxia agent CoCl₂ (100 µM, 6 h), GLUT1 (B) and GLUT3 (C) mRNA expression was markedly upregulated in the empty vector (ev) group. This effect was significantly antagonized by OPN knockdown (shOPN1 and shOPN2) in MG63 cells. Data are presented as the mean ± S.E.M. (n = 4), *p≤0.05, compared with the empty vector group (ev) in the control group, #p≤0.05, compared with the empty vector group (ev) in the CoCl₂ treatment group.</p

Osteopontin regulates GLUT1 and GLUT3 expression via the αvβ3 integrin and MAPK pathways in osteosarcoma cells.

<p>OPN (10 ng/ml) increased GLUT1 and GLUT3 protein expression in MG63 cells. This effect was significantly antagonized by pretreatment with an anti-αvβ3 mAb (2 µg/ml) and PF573228 (5 µM, FAK inhibitor) (A). (B) MG63 cells were pretreated with PD98059 (20 µM), LY294002 (20 µM), SP600125 (20 µM), and SB203580 (20 µM) for 30 min and then stimulated with OPN (10 ng/ml, 24 h). OPN-induced increase of GLUT1 and GLUT3 protein expression was significantly antagonized by LY294002, SP600125, and SB203580. (C) OPN (10 ng/ml) increased the phosphorylation of AKT, JNK, and p38 in a time-dependent manner, and pretreatment with an anti-αvβ3 mAb (2 µg/ml) inhibited OPN-induced AKT, JNK, and p38 phosphorylation. Data are presented as the mean ± S.E.M. (n = 3). *p≤0.05, compared with the control group (con), #p≤0.05, compared with OPN treatment alone.</p

Osteopontin increases GLUT1 and GLUT3 expression in osteosarcoma cell lines.

<p>OPN (24 h) increased GLUT1 (A) and GLUT3 (B) protein levels in a concentration-dependent manner in MG63 osteosarcoma cells. OPN (10 ng/ml, 24 h) also increased GLUT1 and GLUT3 protein expression in U-2OS (C) and 143B (D) osteosarcoma cells. Data are presented as the mean ± S.E.M. (n = 4), *p≤0.05, as compared with the control group (con).</p

Osteopontin increases glucose uptake in MG63 osteosarcoma cells.

<p>2-NBDG, a fluorescent d-glucose analog, was used as an indicator of glucose uptake. Note that treatment with OPN (100 ng/ml) for 24 h enhanced 2-NBDG uptake into MG63 cells, as shown by confocal microscopy (A) and flow cytometric analysis (B).</p

The cytotoxic effect of chemotherapeutic drugs is enhanced by combination with a glucose transporter inhibitor.

<p>(A) Treatment of MG63 cells with phloretin (100 µM), daunomycin (1 µM), 5-Fu (10 µM), etoposide 10 µM, or methotrexate (10 µM) alone for 24 h induced a low level of cell death. However, the combination of phloretin with chemotherapeutic drugs (daunomycin, 5-Fu, etoposide, and methotrexate) markedly increased cell death in three osteosarcoma cell lines: MG63, U-2OS, and 143B. Representative photographs are shown in panel B. Data are presented as the mean ± S.E.M. (n = 4). *p≤0.05, compared with the control (con), #p≤0.05, compared with the respective treatment of the chemotherapeutic drug alone.</p

Hypoxia increases the expression of glucose transporters in human osteosarcoma cells.

<p>(A) The mRNA levels of glucose transporter (GLUT) 1, 2, 3, 4, 6, 8, 10, and 12 were evaluated using quantitative PCR. After treatment with CoCl₂ (100 µM, 6 h), GLUT 1, 2, and 3 mRNA levels were increased. CoCl₂ (100 µM, 24 h) also increased GLUT1 (B) and GLUT3 (C) protein levels in MG63 cells. Data are presented as the mean ± S.E.M. (n = 3), *p≤0.05, compared with the control group (con).</p

Local Immunosuppressive Microenvironment Enhances Migration of Melanoma Cells to Lungs in DJ-1 Knockout Mice

<div><p>DJ-1 is an oncoprotein that promotes survival of cancer cells through anti-apoptosis. However, DJ-1 also plays a role in regulating IL-1β expression, and whether inflammatory microenvironment built by dysregulated DJ-1 affects cancer progression is still unclear. This study thus aimed to compare the metastatic abilities of melanoma cells in wild-type (WT) and DJ-1 knockout (KO) mice, and to check whether inflammatory microenvironment built in DJ-1 KO mice plays a role in migration of cancer cells to lungs. First, B16F10 melanoma cells (at 6×10⁴) were injected into the femoral vein of mice, and formation of lung nodules, levels of lung IL-1β and serum cytokines, and accumulation of myeloid-derived suppressor cells (MDSCs) were compared between WT and DJ-1 KO mice. Second, the cancer-bearing mice were treated with an interleukin-1 beta (IL-1β) neutralizing antibody to see whether IL-1β is involved in the cancer migration. Finally, cultured RAW 264.7 macrophage and B16F10 melanoma cells were respectively treated with DJ-1 shRNA and recombinant IL-1β to explore underlying molecular mechanisms. Our results showed that IL-1β enhanced survival and colony formation of cultured melanoma cells, and that IL-1β levels were elevated both in DJ-1 KO mice and in cultured macrophage cells with DJ-1 knockdown. The elevated IL-1β correlated with higher accumulation of immunosuppressive MDSCs and formation of melanoma module in the lung of DJ-1 KO mice, and both can be decreased by treating mice with IL-1β neutralizing antibodies. Taken together, these results indicate that immunosuppressive tissue microenvironment built in DJ-1 KO mice can enhance lung migration of cancer, and IL-1β plays an important role in promoting the cancer migration.</p></div

IFN-γ and I-TAC reduce the survival of dopaminergic neurons in primary midbrain neuron-glia mixed cultures but not in neuron-enriched cultures.

<p>(A) Midbrain neuron-glia mixed cultures were derived from E14 rat embryos and treated with recombinant IFN-γ protein (30 and 300 ng/ml) or I-TAC protein (1 and 10 ng/ml) on Day-7 cultures for 48 hours. LPS (500 ng/ml) was used as positive control. Note that IFN-γ and I-TAC reduced the survival of TH-positive neurons in neuron-glia mixed cultures (B) The neuron-enriched cultures were obtained from E14 rat embryos and treated with IFN-γ and I-TAC on Day-7 cultures for 48 hours. Note that IFN-γ and I-TAC did not affect survival of TH-positive neurons in neuron-enriched cultures. The dopaminergic neurons (TH-positive) were counted and normalized as percentage of TH-positive neurons in control cells. Data were presented as mean ± S.E.M. (n = 4–5 for each group) * p<0.05 as compared with control.</p