Radioiodide uptake and sodium iodide symporter expression in breast carcinoma

Breast cancer is a common malignancy in women all over the world and novel therapeutic approaches are required for the treatment of patients who become refractory to conventional therapies. Thyroid cancer is being treated successfully with radioiodine since many years. The iodide is transported inside the thyroid epithelial cell via sodium iodide symporter (NIS) which is a trans-membrane protein. The present study was aimed to explore the uptake of radioiodide (RAI) and the expression of NIS in breast tissues of invasive ductal carcinoma patients. Breast tissues from tumor region (Tu-Br) as well as corresponding normal region (N-Br) were collected from patients of invasive ductal carcinoma. In vitro RAI uptake, its efflux and NIS expression were studied. The uptake of RAI (1.98+/-1.75 x 10(5) cpm/g) in Tu-Br was significantly higher as compared to that observed in N-Br (0.31+/-0.27 x 10(5) cpm/g) and fast efflux was observed in the tissue samples. NIS gene expression was positive in 41.66% (10/24) samples of Tu-Br. None of the N-Br samples expressed NIS gene. In 14 samples of Tu-Br, RAI uptake as well as NIS expression was studied. In 50% of these Tu-Br samples RAI uptake as well as of NIS gene expression was positive. The results indicate that RAI uptake is significantly higher in breast tumor tissues as compared to their normal counterpart and in future radioiodine may be an important agent for treatment of breast cancer.     

Induction of sodium/iodide symporter (NIS) expression and radioiodine uptake in non-thyroid cancer cells

Background

This study was designed to explore the therapeutic potential of suppressing MAP kinase and PI3K/Akt pathways and histone deacetylase (HDAC) to induce the expression of sodium/iodide symporter (NIS) and radioiodine uptake in non-thyroid cancer cells.

Methods

We tested the effects of the MEK inhibitor RDEA119, the Akt inhibitor perifosine, and the HDAC inhibitor SAHA on NIS expression in thirteen human cancer cell lines derived from melanoma, hepatic carcinoma, gastric carcinoma, colon carcinoma, breast carcinoma, and brain cancers. We also examined radioiodine uptake and histone acetylation at the NIS promoter in selected cells.

Results

Overall, the three inhibitors could induce NIS expression, to various extents, in melanoma and all the epithelial carcinoma-derived cells but not in brain cancer-derived cells. SAHA was most effective and its effect could be significantly enhanced by RDEA119 and perifosine. The expression of NIS, at both mRNA and protein levels, was most robust in the melanoma cell M14, hepatic carcinoma cell HepG2, and the gastric carcinoma cell MKN-7 cell. Radioiodine uptake was correspondingly induced, accompanied by robust increase in histone acetylation at the NIS promoter, in these cells when treated with the three inhibitors.

Conclusions

This is the first demonstration that simultaneously suppressing the MAP kinase and PI3K/Akt pathways and HDAC could induce robust NIS expression and radioiodine uptake in certain non-thyroid human cancer cells, providing novel therapeutic implications for adjunct radioiodine treatment of these cancers.     

Effect of prolactin on sodium iodide symporter expression in mouse mammary gland explants

Iodide accumulates in milk at a concentration that is more than an order of magnitude higher than the iodide concentration in maternal plasma. In earlier studies from our laboratory, we have shown that prolactin (PRL) enhances iodide accumulation by two- to threefold in cultured mammary tissues taken from pregnant mice. In the present studies, we demonstrate via Western blotting techniques that prolactin elevates the quantity of the sodium iodide symporter (NIS) in cultured mouse mammary tissues. In time-course studies, the onset of the PRL effect of NIS accumulation was found to be between 4 and 16 h after addition of PRL to the explants. The lowest PRL concentration that elicited a significant response was 1 ng/ml, and a maximum effect was elicited with PRL concentrations >100 ng/ml. Actinomycin D, cycloheximide, and thiocyanate abolished the PRL effect on NIS accumulation, whereas perchlorate was without effect. These studies suggest that the PRL stimulation of iodide accumulation in milk is mediated, at least in part, by the PRL stimulation of NIS accumulation in mammary gland tissues. These studies further demonstrate that the PRL effect on NIS accumulation occurs via an RNA protein synthesis-dependent mechanism.     

人钠/碘同向转运体基因cDNA的克隆与序列测定

为研究人钠／碘同向转运体(hNIS)的生物学性能和用于肿瘤放射性碘治疗的可能性,运用反转录聚合酶链反应(RT—PCR)从人甲状腺组织总RNA中扩增出hNIS基因cDNA序列,将其克隆至pUCm-T载体中。序列分析证实克隆片段与献报道的hNIS基因cDNA序列完全一致,说明已成功克隆到hNIS基因cDNA。     

Effect of ATP depletion and temperature on the transferrin-mediated uptake and release of iron by BeWo choriocarcinoma cells.

We have recently described the transferrin-mediated uptake and release of iron by BeWo cells [van der Ende, du Maine, Simmons, Schwartz & Strous (1987) J. Biol. Chem. 262, 8910-8916]. We now extend our studies of the mechanisms responsible for uptake and release of iron by these cells. Following preloading, 59Fe release was maximal (about 12%) after about 4 h. Replacement of the extracellular medium with an equal volume of fresh medium either prior to or following the time at which equilibrium was reached further stimulated 59Fe release. Both the rate and maximum amount of iron release decreased if longer loading times were used. Preincubation of BeWo cells for 15 min with 10 mM-sodium cyanide and 50 mM-2-deoxyglucose prior to the determination of 59Fe release did not alter the amount released into medium (which did not contain a high-affinity iron chelator). However, under these conditions, the uptake of 59Fe was dramatically inhibited as a result of prolongation of the transferrin-transferrin-receptor complex recycling time. These results demonstrate that the release of iron from BeWo cells is independent of cellular ATP levels, whereas iron uptake is ATP-dependent. Rates of both 59Fe release and 59Fe uptake were temperature-dependent. Analysis of these data via an Arrhenius plot suggests a single rate-limiting step for the release and uptake processes between 0 and 37 degrees C. The apparent energies of activation of these processes are very similar (approx. 59.0 kJ/mol for iron release and 50.6 kJ/mol for iron uptake), which raises the possibility that the release and uptake of iron share a common thermodynamically rate-limiting step. Possible mechanisms involved in iron release out of the cell and out of the endosome are discussed.     

Density-dependent inhibition of expression of syncytiotrophoblastic markers by cultured human choriocarcinoma (BeWo) cells

In the presence of methotrexate, cultured human choriocarcinoma (BeWo) cells undergo a differentiative response that resembles normal trophoblastic development. In the current study, the effects of cell number and population density on drug-induced conversion of BeWo cells from the cytotrophoblastlike to the syncytiotrophoblastlike phenotype were investigated using as markers of differentiation formation of "giant" cells, a process shown to require exogenous purines, and expression of placental (heat-stable) alkaline phosphatase. Giant cell formation, assessed by determination of cell volumes, was reduced in crowded cultures, and addition of hypoxanthine to growth media partially restored methotrexate-induced cell enlargement. Cellular uptake of methotrexate, assessed by following the loss of methotrexate from cell culture fluids during drug exposures, was two-threefold greater in sparsely populated than in densely populated cultures. Although the concentration of methotrexate in culture fluids of crowded cultures declined during exposures of 48 hr, the amount of extracellular drug remaining at 48 hr was well above the threshold for induction of the differentiative response. When culture population was held constant and population density was manipulated by varying the substratum available to cells, methotrexate-induced cell enlargement was inversely related to population density. Expression of placental alkaline phosphatase, salvage of exogenous hypoxanthine, and synthesis of RNA were also reduced at high population densities. These results indicate that expression of markers of methotrexate-induced differentiation of BeWo cells was inhibited in a density-dependent manner that may have been related to reduced cellular uptake of the inducing agent and of exogenous nutrients (purines) from culture fluids.     

Real-time imaging of myoblast transplantation using the human sodium iodide symporter

The quantification of the graft success is a key element to evaluate the efficiency of cellular therapies for several pathologies such as Duchenne muscular dystrophy. This study describes an approach to evaluate the success of myoblast transplantation (i.e., survival of the transplanted cells and the muscle fibers formed) by real-time imaging. C2C12 myoblasts were first transfected with a plasmid containing the human sodium iodide symporter (hNIS) gene. Specific uptake of the radioactive sodium pertechnetate (Na99mTcO4) by the hNIS-positive myoblasts was demonstrated in vitro, while only background level of Na99mTcO4 was observed within the control cells. The cells were then transplanted into the tibialis anterior (TA) muscle of mdx (X-linked dystrophic) mice. Following intraperitoneal administration of Na99mTcO4, scintigraphies were performed to detect hNIS-dependent Na99mTcO4 uptake within the TA. This approach permitted to evaluate the progression of the transplantation and the graft success without having to biopsy the animals during the follow-up period.     

Insulin and leptin do not affect fatty acid uptake and metabolism in human placental choriocarcinoma (BeWo) cells

Placental transport of long chain polyunsaturated fatty acids is important for fetal growth and development. In order to examine the effects of leptin and insulin on fatty acid uptake by the placenta, placental choriocarcinoma (BeWo) cells were used. BeWo cells were incubated for 5h at 37 degrees C in the absence or presence of different concentrations of insulin (0.6, 60, and 100 ng) or leptin (10 ng) with 200 microM of various radiolabeled fatty acids (docosahexaenoic acid, arachidonic acid, eicosapentaenoic acid, and oleic acid, mixed with 1:1 bovine serum albumin (fat free). After incubation, the uptake and distribution of these fatty acids into different cellular lipid fractions were determined. The uptakes of oleic, eicosapentaenoic, arachidonic, and docosahexaenoic acids were 15.36+/-4.1, 19.95+/-3.6, 28.56+/-8.1, and 62.25+/-9.5 nmol/mg of protein, respectively, in BeWo cells. Incubation of these cells with insulin (0.6 or 60 ng/ml) or leptin (10 ng/ml) did not significantly alter uptake of any of these fatty acids (P>0.5). Insulin or leptin also did not affect beta oxidation of fatty acids in these cells. In contrast, leptin (10 ng/ml) and insulin (0.60 ng/ml)) stimulated the uptake of oleic acid (7.4+/-2.3 nmol/mg protein) in human adipose cells, SGBS cells by 1.28- and 2.48-fold (P<0.05), respectively. The distribution of fatty acids in different cellular lipid fractions was also not affected by these hormones. Our data indicate that unlike adipose tissue, fatty acid uptake and metabolism in placental trophoblasts is not regulated by insulin or leptin.     

Mammary radioiodine accumulation due to functional sodium iodide symporter expression in a benign fibroadenoma

The sodium iodide symporter (NIS) has been characterized to mediate the active transport of iodide not only in the thyroid gland but also in various non-thyroidal tissues, including lactating mammary gland and the majority of breast cancers, thereby offering the possibility of diagnostic and therapeutic radioiodine application in breast cancer. In this report, we present a 57-year-old patient with multifocal papillary thyroid carcinoma, who showed focal radioiodine accumulation in a lesion in the right breast on a posttherapy (131)I scan following radioiodine therapy. CT and MR-mammography showed a focal solid lesion in the right breast suggestive of a fibroadenoma, which was confirmed by histological examination. Immunostaining of paraffin-embedded tumor tissue sections using a human NIS antibody demonstrated NIS-specific immunoreactivity confined to epithelial cells of mammary ducts. In conclusion, in a thyroid cancer patient we identified a benign fibroadenoma of the breast expressing high levels of functionally active NIS protein as underlying cause of focal mammary radioiodine accumulation on a posttherapy (131)I scan. These data show for the first time that functional NIS expression is not restricted to lactating mammary gland and malignant breast tissue, but can also be detected in benign breast lesions, such as fibroadenomata of the breast.     

RhoE is regulated by cyclic AMP and promotes fusion of human BeWo choriocarcinoma cells

Fusion of placental villous cytotrophoblasts with the overlying syncytiotrophoblast is essential for the maintenance of successful pregnancy, and disturbances in this process have been implicated in pathological conditions such as pre-eclampsia and intra-uterine growth retardation. In this study we examined the role of the Rho GTPase family member RhoE in trophoblast differentiation and fusion using the BeWo choriocarcinoma cell line, a model of villous cytotrophoblast fusion. Treatment of BeWo cells with the cell permeable cyclic AMP analogue dibutyryl cyclic AMP (dbcAMP) resulted in a strong upregulation of RhoE at 24 h, coinciding with the onset of fusion. Using the protein kinase A (PKA)-specific cAMP analogue N(6)-phenyl-cAMP, and a specific inhibitor of PKA (14-22 amide, PKI), we found that upregulation of RhoE by cAMP was mediated through activation of PKA signalling. Silencing of RhoE expression by RNA interference resulted in a significant decrease in dbcAMP-induced fusion. However, expression of differentiation markers human chorionic gonadotrophin and placental alkaline phosphatase was unaffected by RhoE silencing. Finally, we found that RhoE upregulation by dbcAMP was significantly reduced under hypoxic conditions in which cell fusion is impaired. These results show that induction of RhoE by cAMP is mediated through PKA and promotes BeWo cell fusion but has no effect on functional differentiation, supporting evidence that these two processes may be controlled by separate or diverging pathways.     

Hydrocortisone and purinergic signaling stimulate sodium/iodide symporter (NIS)-mediated iodide transport in breast cancer cells

The sodium/iodide symporter (NIS) mediates a remarkably effective targeted radioiodide therapy in thyroid cancer; this approach is an emerging candidate for treating other cancers that express NIS, whether endogenously or by exogenous gene transfer. Thus far, the only extrathyroidal malignancy known to express functional NIS endogenously is breast cancer. Therapeutic efficacy in thyroid cancer requires that radioiodide uptake be maximized in tumor cells by manipulating well-known regulatory factors of NIS expression in thyroid cells, such as TSH, which stimulates NIS expression via cAMP. Similarly, therapeutic efficacy in breast cancer will likely depend on manipulating NIS regulation in mammary cells, which differs from that in the thyroid. Human breast adenocarcinoma MCF-7 cells modestly express endogenous NIS when treated with all-trans-retinoic acid (tRa). We report here that hydrocortisone and ATP each markedly stimulates tRa-induced NIS protein expression and plasma membrane targeting in MCF-7 cells, leading to at least a 100% increase in iodide uptake. Surprisingly, the adenyl cyclase activator forskolin, which promotes NIS expression in thyroid cells, markedly decreases tRa-induced NIS protein expression in MCF-7 cells. Isobutylmethylxanthine increases tRa-induced NIS expression in MCF-7 cells, probably through a purinergic signaling system independent of isobutylmethylxanthine's action as a phosphodiesterase inhibitor. We also observed that neither iodide, which at high concentrations down-regulates NIS in the thyroid, nor cAMP has a significant effect on NIS expression in MCF-7 cells. Our findings may open new strategies for breast-selective pharmacological modulation of functional NIS expression, thus improving the feasibility of using radioiodide to effectively treat breast cancer.     

The effects of hypoxanthine on methotrexate-induced differentiation of cultured human choriocarcinoma (BeWo) cells

When cultured human choriocarcinoma (BeWo) cells are exposed to methotrexate, proliferation ceases and cells undergo a complex differentiative response that resembles development of normal trophoblast. Although thymidylate starvation has been shown to be causative in methotrexate-induced expression of syncytiotrophoblastic markers by BeWo cells, the role of purine deprivation is uncertain since previous studies utilized growth media containing exogenous purines. This work investigated the effects of hypoxanthine on methotrexate-induced cell enlargement, expression of placental alkaline phosphatase, and morphological differentiation to the syncytiotrophoblast-like phenotype. When methotrexate exposures (1 microM, 48 h) were conducted in a purine-free basal medium supplemented with dialyzed fetal bovine serum, RNA synthesis was greatly reduced and cell enlargement did not occur. Specific methods for removing purines (charcoal extraction and xanthine oxidase treatment) decreased the ability of serum to support cell enlargement during methotrexate exposures, whereas addition of hypoxanthine to culture fluids restored its ability to support maximal increases in cell mass, confirming that purines were the factors lost during dialysis. In contrast, morphologically differentiation to the syncytiotrophoblast-like phenotype and increased expression of placental alkaline phosphatase were unaffected by the availability of purines during exposure to methotrexate.     

Regulation of ADRP expression by long-chain polyunsaturated fatty acids in BeWo cells, a human placental choriocarcinoma cell line

Transplacental transfer of maternal fatty acids is critical for fetal growth and development. In the placenta, a preferential uptake of fatty acids toward long-chain polyunsaturated fatty acids (LCPUFAs) has been demonstrated. Adipose differentiation-related protein (ADRP) is a lipid droplet-associated protein that has been ascribed a role in cellular fatty acid uptake and storage. However, its role in placenta is not known. We demonstrate that ADRP mRNA and protein are regulated by fatty acids in a human placental choriocarcinoma cell line (BeWo) and in primary human trophoblasts. LCPUFAs of the n-3 and n-6 series [arachidonic acid (20:4n-6), docosahexaenoic acid (22:6n-3), and eicosapentaenoic acid (20:5n-3)] were more efficient than shorter fatty acids at stimulating ADRP mRNA expression. The fatty acid-mediated increase in ADRP mRNA expression was not related to the differentiation state of the cells. Synthetic peroxisome proliferator-activated receptor and retinoic X receptor agonists increased ADRP mRNA level but had no effect on ADRP protein level in undifferentiated BeWo cells. Furthermore, we show that incubation of BeWo cells with LCPUFAs, but not synthetic agonists, increased the cellular content of radiolabeled oleic acid, coinciding with the increase in ADRP mRNA and protein level. These studies provide new information on the regulation of ADRP in placental trophoblasts and suggest that LCPUFA-dependent regulation of ADRP could be involved in the metabolism of lipids in the placenta.     

Arachidonic acid stimulates internalisation of leptin by human placental choriocarcinoma (BeWo) cells

Arachidonic acid at 100 nM stimulated internalisation of 125I-leptin in human placental choriocarcinoma (BeWo) cells by 3-fold compared with controls. In contrast, eicosapentaenoic acid at similar concentration decreased internalisation of leptin by 2-fold. Use of ibuprofen and indomethacin (inhibitors of prostaglandin synthesis) inhibited the stimulatory effect of arachidonic acid. Prostaglandin E(2), a cyclooxygenase metabolite of arachidonic acid, stimulated internalisation of leptin by these cells. All these data demonstrate that stimulation of leptin internalisation by arachidonic acid in placental trophoblasts may be mediated via prostaglandin E(2).     

Concanavalin-A stimulates human chorionic gonadotropin (hCG) and hCG-alpha secretion by human choriocarcinoma cells

The plant lectin Concanavalin-A stimulates the secretion of human chorionic gonadotropin and free alpha subunit by cultured human choriocarcinoma cells in a dose dependent and time dependent manner. This stimulation is prevented by alpha-methyl-D-mannopyranoside, a Concanavalin-A specific hapten sugar. This is the first report of a lectin stimulating the secretion of a glycoprotein hormone. Since the stimulation likely occurs subsequent to interactions at the membrane level, Concanavalin-A may represent a probe for studying membrane-related events involved in the control of human chorionic gonadotropin secretion.     

Insulin stimulates placental leucine aminopeptidase/oxytocinase/insulin-regulated membrane aminopeptidase expression in BeWo choriocarcinoma cells

Placental leucine aminopeptidase (P-LAP), a cystine aminopeptidase that is identical to insulin-regulated membrane aminopeptidase, hydrolyzes oxytocin, which results in the loss of oxytocin activity. We previously isolated genomic clones containing the human P-LAP promoter region, which included two sites homologous to the 10-bp-insulin responsive element (IRE) that was identified on the phosphoenolpyruvate carboxinase gene. We therefore postulated that insulin regulates P-LAP expression via these IREs and investigated this notion using BeWo choriocarcinoma trophoblastic cells cultured in the presence of insulin. Insulin increased P-LAP activity in a time- and dose-dependent manner. Physiological concentrations of insulin at 10(-7) M exhibited the most potent effect on P-LAP activity. Western blotting demonstrated that 10(-7) M insulin increased P-LAP protein levels. Semi-quantitative RT-PCR and Southern blotting showed that insulin also increased P-LAP mRNA, which was abrogated by prior exposure to cycloheximide. Luciferase assay did not reveal any regulatory regions within 1.1 kb upstream of the P-LAP gene that could explain the insulin-induced P-LAP mRNA accumulation. These findings indicate that insulin induces P-LAP expression in trophoblasts, and that it acts via de novo synthesis of other proteins, which partially contradicts our initial hypothesis.     

Acyl-CoA thioesterase activity in human placental choriocarcinoma (BeWo), cells: effects of fatty acids

The effects of fatty acids on acyl-CoA thioesterase activity and peroxisome proliferator-activated receptor gamma (PPARgamma), a regulator of lipid metabolism, were investigated in placental choriocarcinoma (BeWo) cells. Substrate preference for acyl-CoA thioesterase was in the following order; gamma-linolenoyol-CoA>/=arachidonoyol-CoAz.Gt;palmitoyl-CoA>/=linoleyol-CoA. However, when these cells were incubated with fatty acids, acyl-CoA thioesterase activity was increased by both conjugated linoleic and gamma linolenic acids, but not by docosahexaenoic and eicosapentaenoic acids. In addition, these fatty acids also increased expression of PPARgamma in these cells, suggesting a putative relationship between free fatty acid generated by acyl-CoA thioesterase and expression of PPARgamma. Since expression of PPARgamma is critical for feto-placental growth, these fatty acids may be important during pregnancy.     

Expression of the thyroid sodium/iodide symporter in Xenopus laevis oocytes

Poly(A+) RNA isolated from FRTL-5 cells (a continuous line of cultured and fully functional rat thyroid cells (Ambesi-Impiombato, F. S., Parks, L. A. M., and Coons, H. G. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 3455-3459] was injected into Xenopus laevis oocytes, and the expression of the Na+/I- symporter in the plasma membrane was assayed by measuring the Na+-dependent ClO4--sensitive uptake of 125I. Expression of the Na+/I- symporter was detected as a 7-fold average increase in transport over background, 5-6 days after injection. Poly(A+) RNA was subsequently fractionated by sucrose gradient centrifugation, and fractions were assayed for their ability to induce I- transport activity. The poly(A+) RNA encoding the Na+/I- symporter was found in a fraction containing messages of 2.8-4.0 kilobases in length.