Corresponding Author: Gabi U. Dachs
Mackenzie Cancer Research Group, Department of Pathology and Biomedical Science, University of Otago,
Christchurch, 2 Riccarton Ave, Christchurch 8140, New Zealand
Tel. +64-3-3640544 , E-Mail email@example.com
Limited Association Between Ascorbate Concentrations and Vitamin C Transporters in Renal Cell Carcinoma Cells and Clinical Samples
Margreet C.M. Vissersc
Eleanor R. Burgessa
Bridget A. Robinsona,d Gabi U. Dachsa
aMackenzie Cancer Research Group, Department of Pathology and Biomedical Science, University of Otago, Christchurch, New Zealand, bFianostics GmbH, Technologie- und Forschungszentrum, Wiener Neustadt, Austria, cCentre for Free Radical Research, Department of Pathology and Biomedical Science, University of Otago, Christchurch, New Zealand, dCanterbury Regional Cancer and Hematology Service, Canterbury District Health Board, Christchurch, New Zealand
Maintenance of whole-body ascorbate levels and distribution to different compartments is mediated primarily via sodium-dependent vitamin C transporters (SVCTs) . SVCTs are members of the solute carrier gene family 23 (SLC23) , with three isoforms identified thus far; SVCT1 and SVCT2 that transport ascorbate, and the orphan transporter SVCT3 with still unknown function . SVCT1 and SVCT2 are each comprised of 12 transmembrane domains and exert the cotransport of sodium and ascorbate in a ratio of 2:1 down an electrochemical sodium gradient which is maintained by K/Na+ exchange mechanisms . SVCT2 additionally relies on Ca2+ and Mg2+ for its activity . Expression of the different SVCT transport proteins is tissue and cell type-specific and is controlled by transcriptional regulation of SLC23 genes [3, 5] and post-translational regulation . The exact control mechanisms are still not fully understood.
SVCT1 (encoded by SLC23A1) is expressed in the epithelial tissue of kidney, intestine, liver, lung and skin. SVCT1 is described as a low affinity, high capacity transporter with a Km in the range of 65 –237 μM and Vmax around 15 pmol/min/cell, which makes it capable of efficient uptake of ascorbate from the diet . In comparison, SVCT2, encoded by SLC23A2, is expressed in almost every tissue and cell in the body and mediates whole body tissue uptake . SVCT2 is characterised as a low capacity, high affinity transporter with a Vmax ~1 pmol/min/cell and Km of 8–69 µM, that is suited to the maintenance of tissue homeostasis [4, 8].
The kidney plays a major role in the maintenance of whole-body ascorbate levels, with kidney epithelial cells expressing both SVCT isoforms . In the renal cortex, SVCT1 is situated in the brush-border membrane of the proximal tubule where it mediates re-uptake of ascorbate from the glomerular filtrate . Expression increases towards the distal regions of the tubules, and is hypothesised to be regulated by a decreasing ascorbate gradient along the tubular system . SVCT2 is expressed in all cells of the kidney, including the proximal tubular epithelial cells, although at lower levels than SVCT1, and is located intracellularly . Reports of SVCTs in cancer are sparse; only two studies have previously measured ascorbate transporter levels in human tumour tissue [11, 12], and SVCTs have never been investigated in renal cell carcinoma (RCC) tumours.
Worldwide, each year over 270,000 individuals are diagnosed with RCC, which is curable at an early stage but has limited treatment options when diagnosed at advanced stage, resulting in a 5-year survival of <10% [13, 14]. Several histological types for RCC are defined, including clear cell RCC (ccRCC), which is the most aggressive and most frequent (~70%) type, and papillary RCC (pRCC) which is less common (~15%) .
Recent evidence suggests that accumulation of ascorbate may differ in tumour compared to normal tissue. We have shown that both ccRCCs and pRCCs contained higher ascorbate levels than normal cortex tissue , unlike other cancers that showed the reverse [12, 17, 18]. In the analysis of tissue from patients with colorectal, endometrial or breast cancer, no association between ascorbate levels measured in tumour and matched normal tissue was apparent [12, 16-18]. Protein levels of SVCT1 was measured in human colon adenocarcinoma samples with similar levels to normal colon mucosa . SVCT1 and SVCT2 together with ascorbate were measured in clinical breast tumour tissue, showing no clear association between transporter levels and ascorbate content . No other human studies on SVCTs together with ascorbate in tumour tissue have been reported.
In this study we aimed to investigate the role of the two SVCTs in ascorbate uptake in RCC. SVCT protein levels and cellular location in response to ascorbate supplementation and withdrawal were determined in human ccRCC cell lines. SVCT patterns of staining and protein levels were also analysed in clinical samples of renal cancer and associated normal renal cortex, and compared to measured tissue ascorbate levels.
Materials and Methods
All chemicals were obtained from Sigma-Aldrich (St Louis, USA), unless otherwise specified. Cell lines were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). Bovine serum and antibiotics were from Life Technologies (Carlsbad, CA, USA). The following primary antibodies were used: anti-human SVCT1 (polyclonal rabbit, Aviva Systems, San Diego, CA, USA, OAAB09000), anti-human SVCT2 (polyclonal rabbit, Atlas Antibodies, Stockholm, Sweden, HPA052825) and anti-β-actin (monoclonal mouse, Sigma-Aldrich, A5316).
Renal cell lines
The human ccRCC cell lines Caki-1 (HTB-46), Caki-2 (HTB-47) and 786-0 were used at early passages in ATCC-recommended growth media (modified McCoy’s 5A for Caki-1 and Caki-2, Dulbecco›s Modified Eagle›s Medium (DMEM) for 786-0 cells) with 10% foetal bovine serum and 1% antibiotic-antimycotic solution (Sigma-Aldrich). All cells were regularly tested for mycoplasma by PCR .
Ascorbate uptake and measurement
Cells were grown to near confluence in multi-well plates. As growth media contains little or no ascorbate (McCoy’s contains 5.4 μM, DMEM contains 0 μM), freshly prepared sodium ascorbate was added to a final concentration of 50 or 500 μM. For measurements of intracellular ascorbate, cells were pelleted at a range of time points and processed for high-performance liquid chromatography with electrochemical detection (HPLC-ECD) analysis, as previously described . Briefly, 0.54 M perchloric acid containing diethylenetriamine penta-acetic acid was added to the cell extract, followed by ascorbate measurements using HPLC-ECD (Thermo Fisher Scientific, Waltham, MA, USA). Ascorbate concentration was assessed relative to standards (freshly prepared ascorbate, 1.25 to 40 μM).
Patient samples and ethics
Tissue samples, gifted to the Cancer Society Tissue Bank Christchurch (CSTB), were used with ethical approval from the University of Otago Human Ethics committee (reference code H14/020). This cohort of 73 ccRCC and 41 pRCC samples with matched renal cortex has previously been described . In addition, a separate cohort of formalin-fixed, paraffin embedded sections and microarrays (15 pRCC and 63 ccRCC) were received from the CSTB and analysed by immunohistochemistry.
Frozen tissue samples were ground to a fine powder in liquid nitrogen, homogenized with RIPA buffer (50 mM Tris pH 8.0, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxylate, 0.1% SDS, with complete proteinase inhibitor cocktail, Roche, Basel, Switzerland), and DNA content was measured as an indication of the cellular content, as previously described .
The blocking peptide for SVCT1 (sc-9924 P) resembles amino acids 1 - 30 and the peptide for SVCT2 (sc-31991 P) corresponds to amino acids 183 - 212 of the protein sequence (Santa Cruz, Dallas, TX, USA). For competition assays, antibodies were incubated with 5 times excess of blocking peptides by weight for 1 hour at room temperature before incubation on the membrane.
For cell lines, lysates equivalent to 20 μg protein, and for tissue, homogenates equivalent to 0.5 μg DNA, were loaded per well. Proteins were separated on 4 – 12% Bis-Tris Plus SDS gels (Life Technologies, Carlsbad, CA, USA) and transferred to membranes, as described before . Membranes were incubated with the following primary antibodies: anti-SVCT1 (1/1000), SVCT2 (1/500) and β-actin (1/10000), with horseradish peroxidase labelled secondary goat anti-rabbit/anti-mouse antibodies (Dako, Glostrup, Denmark, P0448 and P0449). Protein bands were detected using the ECL Prime Western Blotting Detection Reagent (GE Healthcare, Chicago, USA), captured using the Alliance 4.7 imaging system and quantified using ImageJ.
Cells seeded into 8-well chamber slides (Thermo Fisher Scientific, Waltham, MA, USA) were washed and fixed with 4% paraformaldehyde, permeabilised with 0.1% Triton X-100 and blocked with 1% bovine serum albumin. Transporters were detected using anti-SVCT2 at 1/500 and secondary fluorescent antibody (Donkey anti-rabbit IgG Alexa Fluor 598, 1/1000, Abcam, ab 150076). Cells were co-stained with CytoPainter Phalloiden-iFluor 488 (1/1000, Abcam, ab176753) and 4’,6-diamidino-2-phenylindole (DAPI, Thermo Fisher Scientific). Slides were covered with Vectashield Antifade Mounting Medium (Vector Laboratories, Burlingame, CA, USA) and fluorescence assessed with an Axio Imager 2 using the ApoTome (Zeiss, Oberkochen, Germany).
Sections cut at 3 μm were baked at 60°C, deparaffinised and pressure heated for antigen retrieval in Tris-EDTA buffer with 0.05% Tween 20. Cell and Tissue Staining kits (R&D Systems, Minneapolis, MN, USA) were used following manufacturer’s recommendations to stain for SVCT1 (1/200) and SVCT2 (1/200); negative controls lacked primary antibodies.
Data were analysed using GraphPad Prism 5, using the Shapiro-Wilk test for normality. Differences between treatment conditions in cell culture were tested by One-way ANOVA with Dunnett’s Multiple Comparison or Bonferroni post-test. Statistical significance between renal cortex and tumour data was tested with the non-parametric Wilcoxon matched-pairs signed rank test. Values of p < 0.05 were considered significant.
Ascorbate transporters in renal cell carcinoma cell lines
Specificity of the antibodies against SVCT1 and SVCT2 was determined by pre-absorbing antibodies with blocking peptides that prevent binding to the target epitope . For SVCT1, with a predicted molecular weight of 65 kDa, antibody blocking confirmed that the second immunoreactive band at 80 kDa was non-specific (Fig. 1A). SVCT2 was detected at 72 kDa, its predicted molecular weight, but also at ~100 kDa; both bands disappeared in the blocking assay and were therefore considered as specific for SVCT2 (Fig. 1A).
SVCT1 and SVCT2 proteins were confirmed in all three ccRCC cell lines (Fig. 1B). Protein levels of the two transporters varied between cell lines and between individual samples. 786-O cells appeared to have the highest levels of SVCT1 and lowest levels of both immunoreactive forms of SVCT2 compared to the other two cell lines. Of the three cell lines, Caki-2 cells showed strongest immunoreactivity for SVCT2 (Fig. 1C).
Ascorbate accumulation and loss over time was measured in the ccRCC cells by HPLC-ECD. Our previous data had shown that all three cell lines reached intracellular ascorbate saturation when incubated with 500 μM ascorbate . Therefore, cells were exposed for 16 h to doses of ascorbate that achieve suboptimal (50 μM) or optimal intracellular levels (500 μM), followed by removal of ascorbate from the culture medium with sampling for up to 24 h. Measurements were compared to cells that did not receive ascorbate (0 μM), which had low/undetectable intracellular ascorbate concentrations, as expected (Fig. 2). Intracellular ascorbate levels in all three cell lines increased significantly over time with exposure to both 50 μM and 500 μM ascorbate, and dropped noticeably once ascorbate was withdrawn (dotted lines in Fig. 2). Supplementation with 50 μM ascorbate resulted in a peak of 0.46 nmol/106 cells at 8 h in 786-0 cells, 1.85 nmol/106 cells at 16 h in Caki-1, and 3.73 nmol/106 cells at 8 h in Caki-2 cells. Higher supplementation (500 μM ascorbate) resulted in 2-11-fold higher intracellular concentrations at the same time points (5.43, 7.20 and 8.92 nmol/106 cells in 786-0, Caki-1 and Caki-2 cells, respectively).
Changes in transporter levels in response to varying ascorbate supply over time was monitored by Western blot (Fig. 2). Levels of SVCT1 and both immunoreactive forms of SVCT2 were variable over time in culture in the presence and absence of ascorbate. Neither transporter was significantly affected by the addition or removal of ascorbate (Fig. 2), with SVCT1 remaining particularly stable. However, in 786-O cells, SVCT2 (100 kDa) levels tended to increase during exposure to 500 μM ascorbate and to reduce during ascorbate withdrawal (p = 0.081; Fig. 2C).
When comparing SVCT protein levels from Fig. 1C and maximal ascorbate uptake in Fig. 2, it is noteworthy that the cell line (Caki-2) with the highest levels of SVCT2 proteins (72 and 100 kDa) also showed the highest maximal ascorbate accumulation following supplementation with 50 μM and 500 μM ascorbate. The cell line with the lowest levels SVCT2 proteins (786-0) also showed the lowest ascorbate accumulation, with Caki-1 showing intermediate protein and uptake characteristics. No such association was seen for SVCT1.
As there were no clear changes in overall protein levels of ascorbate transporters, possible differences in intracellular SVCT2 distribution were investigated. Immunofluorescence staining of SVCT2 was carried out in the three cell lines over a time period of either 5-60 min (short-term, Fig. 3) or 2-8 h (longer-term, Fig. 4) exposure to 500 μM ascorbate. Immunofluorescence showed immunoreactivity of SVCT2 that appeared to be concentrated in cytoplasmic ‘spots’ (Fig. 3, Fig. 4). During early time-points (up to 1 h) there appeared to be translocation to the nucleus or nuclear membrane in some cells (eg Caki-1 at 60 min, Fig. 3, Fig. 4). However, there was no clear translocation to or from the plasma membrane at any time over 8h, despite the clear increase in intracellular ascorbate accumulation over this time period (Fig. 2 C, F, I). Co-staining of actin filaments with fluorescence labelled Phalloidin was used to evaluate cell shape. There was no apparent co-localisation of SVCT2 with Phalloidin, indicating a relative lack of SVCT2 at the plasma membrane.
In summary, our data indicate that SVCT isoforms and protein modifications may differ between tumour and normal renal tissue. The SVCTs appear to be predominantly located at intracellular sites, and expression levels do not change appreciably in the presence or absence of ascorbate. Hence there is not a simple relationship between tissue ascorbate content and SVCT levels, and our data indicate that SVCT protein levels do not predict intracellular ascorbate accumulation in RCC. Also, ascorbate supply may not modify SVCT protein levels and there may be complex dynamic changes in sub-cellular localisation of the transporter, but any functional impact of such changes in renal cancer cells is unknown.
We thank the Cancer Society Tissue Bank (CSTB) in Christchurch for providing patient samples. CSTB is supported by the Canterbury West Coast Division of the Cancer Society of New Zealand.
GD conceived the study, CW, EB and MN collected the data, MV and EP helped analyse the data, and CW composed the draft manuscript. MV, BR and GD edited and refined the manuscript. All authors finalized the manuscript. GD, MV and BR obtained funding for the study.
This study was supported by the Cancer Research Trust NZ (GOT/1644/RPG), the Mackenzie Charitable Foundation (GD and EP), the Vitamin C for Cancer Trust (EB), the University of Otago (PhD Scholarship for CW) and the Centre for Translational Cancer Research, University of Otago (Summer studentship for MN).
Statement of Ethics
Human tissue samples were collected by the Cancer Society Tissue Bank (CSTB) Christchurch and used with ethical approval from the University of Otago Human Ethics committee (reference code H14/020). Use of samples for this study was approved by the CSTB board. All CSTB donors gave informed written consent for the use of their samples for research.
The authors have no conflicts of interest to declare.
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