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

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

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 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

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

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

High Protein Diet and Huntington's Disease

<div><p>Huntington’s disease (HD) is a neurodegenerative disorder caused by the <i>huntingtin</i> (<i>HTT</i>) gene with expanded CAG repeats. In addition to the apparent brain abnormalities, impairments also occur in peripheral tissues. We previously reported that mutant Huntingtin (mHTT) exists in the liver and causes urea cycle deficiency. A low protein diet (17%) restores urea cycle activity and ameliorates symptoms in HD model mice. It remains unknown whether the dietary protein content should be monitored closely in HD patients because the normal protein consumption is lower in humans (~15% of total calories) than in mice (~22%). We assessed whether dietary protein content affects the urea cycle in HD patients. Thirty HD patients were hospitalized and received a standard protein diet (13.7% protein) for 5 days, followed by a high protein diet (HPD, 26.3% protein) for another 5 days. Urea cycle deficiency was monitored by the blood levels of citrulline and ammonia. HD progression was determined by the Unified Huntington’s Disease Rating Scale (UHDRS). The HPD increased blood citrulline concentration from 15.19 μmol/l to 16.30 μmol/l (<i>p</i> = 0.0378) in HD patients but did not change blood ammonia concentration. A 2-year pilot study of 14 HD patients found no significant correlation between blood citrulline concentration and HD progression. Our results indicated a short period of the HPD did not markedly compromise urea cycle function. Blood citrulline concentration is not a reliable biomarker of HD progression.</p></div

Influence of dietary protein content on the urea cycle in Huntington’s disease (HD) patients.

<p>Blood citrulline (A) and ammonia (B) levels in HD patients given the standard protein diet (13.7%) and high protein diet (26.3%). The data for the 23 non-HD subjects (Con) were taken from a previous report [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127654#pone.0127654.ref013" target="_blank">13</a>]. All HD patient data were plotted. Data are presented as mean ± standard deviation (SD) and were analyzed by paired <i>t</i> test. *<i>p</i> < 0.05.</p