Limited Association Between Ascorbate Concentrations and Vitamin C Transporters in Renal Cell Carcinoma Cells and Clinical Samples.

BACKGROUND/AIMS: Maintenance of whole-body ascorbate levels and distribution is mediated via sodium-dependent vitamin C transporters (SVCTs). The kidney is one of a few organs that express both SVCT1 and SVCT2. Recent evidence suggests that accumulation of ascorbate may be different in tumour compared to normal tissue, but data on SVCT levels in tumours is sparse. METHODS: The role of the two SVCT isoforms in ascorbate uptake in renal cell carcinoma (RCC) was investigated in vitro and in clinical samples. In three human RCC cell lines, we investigated SVCT protein levels and cellular location in response to ascorbate supplementation and withdrawal. In clinical RCC samples (n=114), SVCT patterns of staining and protein levels were analysed and compared to ascorbate levels. RESULTS: In cell culture, transporter levels and cellular location were not modified by ascorbate availability at any time up to 8h, although basal SVCT2 levels governed maximal ascorbate accumulation. In clinical samples, SVCT1 protein levels in papillary RCC (pRCC) were similar to matched normal renal cortex, but were increased in clear-cell RCC (ccRCC). Native SVCT2 (72 kDa) was significantly decreased in both pRCC and ccRCC tissues compared to cortex (p<0.01), whereas a modified form of SVCT2 (100 kDa) was significantly increased (p<0.001). There was no association between the transporters (SVCT1, native or modified SVCT2) and ascorbate concentrations in either normal or tumour tissues. SVCT1 and SVCT2 displayed diffuse cytoplasmic staining in both pRCC and ccRCC tumour cells, with cortex showing distinct membrane staining for SVCT1. CONCLUSION: We observed a re-distribution of ascorbate transporters in tumour tissue compared to normal cortex and a shift from native to modified SVCT2 in cell culture and clinical samples. Data presented here show that SVCT protein levels do not appear to predict intracellular ascorbate accumulation in RCC.

Comparative analysis of sodium coupled vitamin C transporter 2 in human osteoarthritis grade 1 and grade 3 tissues.

BACKGROUND: Nutrient levels are known to influence the development of osteoarthritis (OA), presumably by modulating levels of matrix biosynthesis and degradation. These processes may be affected by ascorbic acid (AA), an antioxidant which acts as a cofactor for numerous biochemical reactions and is essential for post-translational modifications of collagen. In this study we examined the expression of SVCT2, the only known Sodium coupled vitamin C transporter isoform present in articular cartilage, in human articular cartilage explants derived from both normal and osteoarthritis articular cartilage. METHODS: OA1 and OA3 human articular cartilage was carefully dissected and macroscopically graded for degeneration via the Collins scale. The tissue samples were histologically examined by Hematoxylin and Eosin and Safranin O and Fast Green staining. SVCT2 expression analysis was performed at mRNA level by quantitative real time PCR and at a protein level by immunohistochemistry. RESULTS: Our quantitative real time PCR showed marked variation in the expression of SVCT2 in human osteoarthritic articular cartilage. SVCT2 expression was significantly down-regulated (p = 0.0001) in the Collins grade 3 (OA3) compared to Collins grade 1 (OA1) tissue. Furthermore, slides stained with fluorescent antibodies to SVCT2 demonstrated greatly reduced fluorescence for the SVCT2 transporter on the chondrocyte plasma membrane in the osteoarthritic tissue samples. CONCLUSIONS: These findings demonstrate that the expression of SVCT2 transporter is significantly altered in human osteoarthritic tissues (OA3). The modulation of this transporter could therefore potentially influence the prevention, management and treatment of osteoarthritis.

Dynamic expression of the sodium-vitamin C co-transporters, SVCT1 and SVCT2, during perinatal kidney development.

Isoform 1 of the sodium-vitamin C co-transporter (SVCT1) is expressed in the apical membrane of proximal tubule epithelial cells in adult human and mouse kidneys. This study is aimed at analyzing the expression and function of SVCTs during kidney development. RT-PCR and immunohistochemical analyses revealed that SVCT1 expression is increased progressively during postnatal kidney development. However, SVCT1 transcripts were barely detected, if not absent, in the embryonic kidney. Instead, the high-affinity transporter, isoform 2 (SVCT2), was strongly expressed in the developing kidney from E15; its expression decreased at postnatal stages. Immunohistochemical analyses showed a dynamic distribution of SVCT2 in epithelial cells during kidney development. In renal cortex tubular epithelial cells, intracellular distribution of SVCT2 was observed at E19 with distribution in the basolateral membrane at P1. In contrast, SVCT2 was localized to the apical and basolateral membranes between E17 and E19 in medullary kidney tubular cells but was distributed intracellularly at P1. In agreement with these findings, functional expression of SVCT2, but not SVCT1 was detected in human embryonic kidney-derived (HEK293) cells. In addition, kinetic analysis suggested that an ascorbate-dependent mechanism accounts for targeted SVCT2 expression in the developing kidney during medullary epithelial cell differentiation. However, during cortical tubular differentiation, SVCT1 was induced and localized to the apical membrane of tubular epithelial cells. SVCT2 showed a basolateral polarization only for the first days of postnatal life. These studies suggest that the uptake of vitamin C mediated by different SVCTs plays differential roles during the ontogeny of kidney tubular epithelial cells.

SVCT-2 in breast cancer acts as an indicator for L-ascorbate treatment.

L-ascorbate (L-ascorbic acid, vitamin C) clearly has an inhibitory effect on cancer cells. However, the mechanism underlying differential sensitivity of cancer cells from same tissue to L-ascorbate is yet to be clarified. Here, we demonstrate that L-ascorbate has a selective killing effect, which is influenced by sodium-dependent vitamin C transporter 2 (SVCT-2) in human breast cancer cells. Treatment of human breast cancer cells with L-ascorbate differentially induced cell death, dependent on the SVCT-2 protein level. Moreover, knockdown of endogenous SVCT-2 via RNA interference in breast cancer cells expressing high levels of the protein induced resistance to L-ascorbate treatment, whereas transfection with SVCT-2 expression plasmids led to enhanced L-ascorbate chemosensitivity. Surprisingly, tumor regression by L-ascorbate administration in mice bearing tumor cell xenograft also corresponded to the SVCT-2 protein level. Interestingly, SVCT-2 expression was absent or weak in normal tissues, but strongly detected in tumor samples obtained from breast cancer patients. In addition, enhanced chemosensitivity to L-ascorbate occurred as a result of caspase-independent autophagy, which was mediated by beclin-1 and LC3 II. In addition, treatment with N-acetyl-L-cysteine, a reactive oxygen species (ROS) scavenger, suppressed the induction of beclin-1 and LC3 II, implying that the differential SVCT-2 protein-dependent L-ascorbate uptake was attributable to intracellular ROS induced by L-ascorbate, subsequently leading to autophagy. These results suggest that functional SVCT-2 sensitizes breast cancer cells to autophagic damage by increasing the L-ascorbate concentration and intracellular ROS production and furthermore, SVCT-2 in breast cancer may act as an indicator for commencing L-ascorbate treatment.