Elsevier

Experimental Eye Research

Volume 139, October 2015, Pages 108-114
Experimental Eye Research

Upregulation of sodium iodide symporter (NIS) protein expression by an innate immunity component: Promising potential for targeting radiosensitive retinoblastoma

https://doi.org/10.1016/j.exer.2015.07.011Get rights and content

Highlights

  • Y79 and Weri-Rb-1 retinoblastoma cell lines differentially expressed lactoferrin receptor mRNA.

  • Y79 and Weri-Rb-1 efficiently internalized iron free and iron saturated bovine lactoferrin forms.

  • Bovine lactoferrin (bLf) upregulated sodium iodide symporter expression in retinoblastoma cells.

  • bLf induced NIS activity can be applied in diagnostic imaging.

  • bLf's activity may lead towards the development of more safer treatments for retinoblastoma.

Abstract

Retinoblastoma (RB), a malignant tumour of the eye arising from developing retina, is the most frequent primary intraocular malignancy of childhood. Its primary management with chemotherapy involves combination regimen of etoposide, vincristine and carboplatin and intra vitreal chemotherapy using melphalan when vitreous seeds develop. Radiotherapy is another effective mode in treating RB. We recently explored the notion if radiotherapy in RB can be mediated via Sodium Iodide Symporter (NIS), an intrinsic membrane glycoprotein which is a key regulator of iodide access to thyroid gland. Its expression has been exploited successfully for diagnostic imaging and molecular radionuclide-based therapy of thyroid cancer. We determined that NIS is expressed endogenously in RB tumour tissues, and in retinoblastoma cell lines Y79 and Weri-Rb-1, and therefore made an attempt to enhance the endogenously low expression of NIS protein in both Y79 and Weri-Rb-1 cells. Here we report about the potential of bovine lactoferrin (bLf) which is a known chemo preventive and emerging safe anti-cancer bio drug, as well as a natural transcriptional activator of genes, to enhance the endogenous expression of NIS in Y79 and Weri-Rb-1 cells. Real time PCR revealed that both cell lines express mRNA of lactoferrin receptors while flow cytometry and confocal microscopy showed the cells efficiently internalize bLf which upregulates NIS expression. These findings highlight an important step that could be taken towards the development of less harmful approaches for the treatment of RB by employing natural supplement bLf (with its clinically proven safe profile), and warrants further studies in future, focussing on enhancing NIS expression in RB cells and NIS functional assays in these cells.

Introduction

Therapy for cancer has eluded medical and scientific community for centuries. Many newer and complicated therapies have been introduced but only few established. One of the reasons for many failed attempts at achieving cure for cancer is lack of targeted treatments and also unwarranted side effects. NIS is an intrinsic plasma membrane glycoprotein which plays a pivotal role in thyroid hormonogenesis by mediating iodide transport into thyroid cells (Dai et al., 1996, Smanik et al., 1996). For many years, radiotherapy has been the primary and most successful treatment for follicular cell derived thyroid carcinoma (Dohán et al., 2003). I131 is used for ablation of cancer tissue after thyroidectomy, if NIS expressed in the metastatic tissues is functional (Arturi et al., 2000). NIS-mediated iodide accumulation within the thyroid gland has played an important role for diagnostic nuclear imaging as well as for highly efficient molecular radionuclide-based targeted therapy of malignant thyroid diseases (L 1996).

RB, is the commonest paediatric intraocular malignancy, and many of the RB patients in developing/under resource countries are often diagnosed late which mostly end up in enucleation (removal of eye) leading to full or complete loss vision of a patient in childhood. RB, though a rare form of pediatric cancer is highly fatal if left untreated as a result of metastasis to bone, brain and lymphnodes. RB is a radiosensitive tumour and a mild form of radiotherapy (brachytherapy) is used clinically to treat small RB tumours. We recently explored the notion that in order to mediate radiotherapy via NIS in RB, the possibility of NIS presence in RB tumours could prove useful in clinics, similar to the successful diagnostics and therapy of thyroid tumours. In line with this, NIS expression at protein level was explored in RB tumours. We found that NIS protein is expressed at a higher percentage in 100% of tumours analysed (n = 41), and the expression is associated to invasiveness of the tumours and at endogenous levels (Data not shown, Manuscript under review).

In the past two decades, studies on NIS expression in many cancers (thyroidal and extrathyroidal) reveal that there are multiple regulatory levels of NIS functional expression. These include transcriptional (gene), translational, posttranslational, distribution to intracellular organelles and targeting to the plasma membrane (Kogai and Brent, 2012). Currently, two strategies are used to induce NIS expression in cancer cells. One is transfer of NIS gene using viral and non-viral vectors. Second strategy is to stimulate the functional activity of endogenous NIS using stimulating agents thought to be having an up-regulatory effect on NIS expression along with enhancing the iodide uptake ability (Alotaibi et al., 2010). Use of several compounds with known tumour-inhibitory effects has been shown to partially succeed in inducing endogenous NIS expression in thyroid and breast cancers. With relevance to breast cancer, though NIS expression was higher, only a small fraction of cells possess the ability to take up radioiodide suggesting that further augmentation of the functional activity is required (Wapnir et al., 2003). For enhancement of NIS function, various ligands and hormones were identified and one among them was a group of ligands called retinoids (Tanosaki et al., 2003). The most influential ligand of the retinoid family is all-trans-retinoic acid (tRA) which increased the NIS expression at transcriptional level and I transport up to 10 fold. MCF-7 cell xenograft study revealed that on systemic tRA treatment at 160 mg/kg/day for 5 days, a 15 fold increase in NIS expression and I uptake was observed (Kogai et al., 2004). Stimulation of NIS expression at transcriptional level was shown not only in breast cancer cells but also in human follicular thyroid carcinoma cell lines like FTC-133 and FTC-238 (Schmutzler et al., 2002).

Similar to tRA's transcriptional activity, lactoferrin (Lf) a natural bio macromolecule functions as a transcriptional trans-activator (He and Furmanski, 1995). Lf is a glycoprotein which belongs to the transferrin protein family. Being a protein involved in innate immune responses, it is found in high concentrations in human milk and other mammalian exocrine secretions (Kanwar et al., 2008, Kanwar and Kanwar, 2013, Stelwagen et al., 2009, Tomita et al., 2009). Bovine milk derived Lf (bLf) is a multifunctional therapeutic protein with various properties like immunomodulatory, anti-cancer, anti-inflammatory, anti-microbial and anti-oxidant (Burrow et al., 2011, Kanwar and Kanwar, 2013, Tomita et al., 2009). It can act also as a cell growth/cell differentiating agent as well as a transcriptional agent. Due to its iron binding properties, Native bLf is approximately 15–20% saturated, and it can be modified to forms with different levels of iron: Apo-bLf (<5% iron saturation) and Holo-bLf (100% iron saturated). With the successful clinical trials, multifunctional glycoprotein bLf is gaining attention as a safe nutraceutical and biologic drug targeting cancer, chronic-inflammatory, viral and microbial diseases (Ebrahim et al., 2014, EFSA Panel on Dietetic Products and NaAN, 2012, Rulis, 2001, Tsuda et al., 2010).

Lf is also thought to act as a transcriptional activator once it binds to the nucleus (He and Furmanski, 1995). Another transcriptional activity of Lf was shown in increasing gene expression of IL-1β (Son et al., 2002) and TGF-β1 (Liao et al., 2012). More recently, we have reported that bLf in its iron saturated and native form downregulates the gene expression of survivin (the key cancer target), in colon cancer cells (Kanwar et al., 2015, Kanwar and Kanwar, 2013). Studies from our laboratory employing osteoarthritis in vitro and in vivo models, have recently shown that internalization of bLf through receptor mediated endocytosis, into the cartilage cell and nucleus can regulate gene transcription in favour of cartilage cell growth and repair and alleviate inflammation (Samarasinghe et al., 2014). Though, many studies including the one from our laboratory have shown that Lf activates genes at transcriptional level, none has shown the effect on NIS gene expression. Here we report the potential of bLf (in its native, iron saturated and iron free forms) to upregulate NIS expression. We explore this by analysing the internalisation efficiency of bLf forms in RB ATCC derived cell lines Y79 and Weri-Rb-1 after investigating the presence of Lf receptors on Y79 and Weri-Rb-1. Finally, the NIS expression was measured in these cells by Western blotting after treatment with bLf forms for 48 h.

Section snippets

Maintenance of Y79 and Weri-Rb-1 cell lines

Human Retinoblastoma cell lines, Y79 (HTB-18) and Weri-Rb-1 (HTB 169) were purchased from American Type Culture Collection (ATCC, U.S.A). The cell lines were propagated in Roswell Park Memorial Institute 1640 (RPMI-1640) medium (Invitrogen/Life Technologies) supplemented with 10% fetal bovine serum (Invitrogen/Life Technologies) and 1% Penicillin-Streptomycin mixture (Invitrogen/Life Technologies) in a fully humidified incubator containing 5% CO2 at 37 °C.

RNA isolation from Y79 and Weri-Rb-1 cells

Total RNA was isolated from 5 × 105 Y79

qRT-PCR revealed the differential expression of Lf receptors in Y79 and Weri-Rb-1 cells

In this study, we hypothesized that bLf could upregulate the NIS protein expression in Y79 and Weri-Rb-1 cells, both are established RB cell lines from ATCC. Initially, the status of Lf receptors was analysed in Y79 and Weri-Rb-1 cells at mRNA level in order to determine the possibility of receptor mediated cellular uptake of bLf. Based on qRT-PCR delta CT value, LRP2 mRNA level was found to be higher followed by LRP1 and TfR in Y79 cells (Fig. 1A). On the other hand, the mRNA expression of LRP

Discussion

We report here the unprecedented potential of bLf in upregulating the NIS protein expression. The multifunctional effects of Lf molecule have been suggested to depend on its target cells and on the presence of specific receptors (Ebrahim et al., 2014, Jiang et al., 2011, Kanwar and Kanwar, 2013). This was indeed observed in the current study, where the absence of TfR mRNA might be responsible for the comparatively lower uptake in Weri-Rb-1 cells than Y79 cells. We also found that both untreated

Conflicts of interest

The authors report no conflict of interests.

Acknowledgements

We thank fully acknowledge the funding from Australia–India Strategic Research Grants (BF 030016 & 060042), Deakin India Research Initiative (DIRI) and Board of Research in Nuclear Sciences, India Project No- 2009/35/17/BRNS to carry out this study. We acknowledge Deakin University for providing scholarship to Ms. Jaisy Samuel and Mr. Narinderbir Singh under PhD student program (Code H960; Student ID- 211042535 and Code H960; Student ID- 210018516 respectively).

References (29)

  • M. Demeule et al.

    Involvement of the low-density lipoprotein receptor-related protein in the transcytosis of the brain delivery vector Angiopep-2

    J. Neurochem.

    (2008)
  • O. Dohán et al.

    The sodium/iodide symporter (NIS): characterization, regulation, and medical significance

    Endocr. Rev.

    (2003)
  • F. Ebrahim et al.

    Identification of unprecedented anticancer properties of high molecular weight biomacromolecular complex containing bovine lactoferrin (HMW-bLf)

    PloS One

    (2014)
  • EFSA Panel on Dietetic Products et al.

    Scientific opinion on bovine lactoferrin

    EFSA J.

    (2012)
  • 1

    Jaisy Samuel and Narinderbir Singh share first authorship.

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