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

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 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

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 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

The Use of Alendronate Is Associated with a Decreased Incidence of Type 2 Diabetes Mellitus—A Population-Based Cohort Study in Taiwan

<div><p>Purpose</p><p>Bone remodeling has been linked to glucose metabolism in animal studies, but the results of human trials were inconclusive. Bisphosphonates may play a role in glucose metabolism through their impacts on bone remodeling enzymes. In this study, we aimed to examine the influence of alendronate usage on the incidence of type 2 diabetes mellitus (DM) among osteoporotic patients.</p><p>Methods</p><p>A retrospective cohort study was designed to include osteoporotic patients without DM from a population-based cohort containing 1,000,000 subjects. Patients treated with alendronate (exposed group, N=1,011) were compared with those who received no treatment (age and gender matched non-exposed group, N=3,033). Newly diagnosed DM was identified from medical records by International Classification of Diseases, Ninth Revision, Clinical Modification (ICD-9CM) code. The incidence of DM in both groups was calculated for comparison.</p><p>Results</p><p>The non-exposed group had a significantly higher incidence of DM (Odds ratio 1.21, 95% confidence interval 1.03~1.41) when compared with the exposed group. In subgroup analysis, the DM risk reduction in exposed group was only significant among those younger than 65 years and those without hypertension or dyslipidemia. Patients who were prescribed alendronate more than or equal to 3 times had demonstrated a significant reduction in DM risk.</p><p>Conclusions</p><p>Our study showed alendronate might yield a protective effect for incident DM. This effect became insignificant in patients with older age, dyslipidemia or hypertension. The underlying mechanism needs further exploration with prospective data for confirmation of the observed findings.</p></div

Flowchart of our study.

<p>Flowchart of our study.</p