Abstract

AIM:

This study aims to investigate the effect of ATP, EGF and combination of those two to the Natrium Iodide Symporter (NIS) expression in MCF7, SKBR3 and HaCaT cell lines.

METHODS:

MCF7, SKBR3 and HaCaT cell lines were treated with ATP, EGF and combination of those two for 6, 12 and 24 hours. The expression of NIS mRNA was measured through quantitative-reverse transcription-polymerase chain reaction (qRT-PCR). The NIS protein expression was confirmed by immunocytofluorescence.

RESULTS:

NIS mRNA was expressed in SKBR3 and HaCaT cell lines but not in MCF7. The levels of NIS mRNA expression, after treatment by epidermal growth factor (EGF), adenosine Tri-Phosphate (ATP) or the combination of both for 6 and 12 hours were not significantly different from those of untreated cells. However, the treatment by a combination of ATP and EGF for 24 hours increases the level of NIS mRNA expression by 1.6 fold higher than that of the untreated cells (1.6241 ± 0.3, p < 0.05) and protein NIS expression increase significantly by the treatment than untreated cells (P < 0.05).

CONCLUSION:

The level of NIS expression varies among the different subtypes of breast cancer cell lines. MCF7 cell line is representing the luminal A subtype of breast cancer does not express NIS. Only SKBR3 cell line express NIS and this subtype might be suitable to receive radioiodine therapy as those cells expressing NIS. A combination treatment of EGF and ATP increases the expression of NIS mRNA and protein at the membrane in SKBR3 cells.

Keywords: Sodium iodide symporter, Breast cancer cell lines, Epidermal growth factor, Adenosine triphosphate

Introduction

Natrium Iodide Symporter is a plasma membrane glycoprotein. It transports two ions of sodium (Na+) and one of iodide (I-) and maintained by Na+/K+ ATPase [1], [2], [3]. In thyroid cells, NIS plays a pivotal role to accumulate iodine. Additionally, NIS is also expressed in breast cancer tissue and has been considered as a potential target for radioiodine therapy. However, NIS expression in breast cancer tissues had been reported to be varied between studies.

Furthermore, the molecular mechanism of NIS expression in breast cancer remains unclear [4]. Interestingly, it has been reported that NIS is highly expressed in invasive breast cancer tissues. A study by Tazebay et al. reported that indeed, there is a correlation between NIS expression and malignant transformation of human breast tissue [5], [6].

Other than the variation of NIS expression level between molecular subtypes of breast cancer, the location of NIS expression is another factor that may influence the uptake of radioiodine by breast cancer cells. Natrium Iodide Symporter supposed to be localised at membrane cell instead of in the cytoplasm to be fully functional as iodine co-transporter [7], [8]. Several agents were used to increasing NIS expression in breast cancer cells, and the effect of those agents had been proven could increase radioiodine uptake and cells susceptibility toward the treatment. A study reported, EGF increased NIS expression at membrane cell in the T47D cell line [9]. Another study reported that a combination of ATP with all-trans-Retinoid Acid (tRA) and hydrocortisone had increased NIS protein expression and cell membrane targeting in MCF7 cells [10].

In this study, we examined the effect of EGF and ATP for NIS mRNA and protein expressions in SKBR3 and MCF7 breast cancer cell lines and HaCaT a normal cell line. MCF7 and SKBR3 cell lines are representing the luminal A and HER2 type of breast cancer, respectively [11]. HaCaT cell line consists of normal human keratinocyte cells. It is used as a control cell line.

Material and Methods

Cell lines and culture conditions

SKBR3, MCF7 and HaCaT cell lines were used in this study. SKBR3 was obtained from the American Type Culture Collection (ATCC). MCF7 and HaCaT cell lines from faculty of Medicine, Universitas Padjadjaran, Bandung-Indonesia. MCF7 and HaCaT cells were cultured in RPMI 1640 medium (Sigma-Aldrich). SKBR3 was cultured in McCoy’s 5A medium (Sigma-Aldrich). Both cell culture mediums were supplemented with 10% fetal bovine serum (Sigma-Aldrich), 1% Penicillin, 1% Streptomycin and 1% amphotericin B. The cells were incubated at 37° C and supplied with 5% of carbon dioxide (CO2) until 80% confluence. The cells were incubated in serum-free medium overnight and followed by treatment of EGF 50 ng/ml (Abcam #ab9697), ATP 100μM (Sigma-Aldrich # 1388), and a combination of EGF and ATP, for 6, 12 and 24 hours. Each type of cell lines divided into four groups based on the type of treatments, such as ATP, EGF, combination ATP and EGF groups and without any treatment as a control group.

Quantitative real-time reverse transcriptase –PCR (qRT-PCR)

The cells were harvested at the appropriate time points and then followed by centrifuging at 1000 rpm for 4 minutes. The total RNA was isolated by using the RNeasy mini kit (Qiagen #74106) following the manufacturer’s instructions. RNA was quantified using Nanodrop 2000, and 5 ng of RNA was reverse transcribed and analysed by one-step real-time quantitative PCR using Rotor-Gene Quantitect probe RT-PCR (Qiagen # 204443) using NIS and beta-actin primers. The mRNA NIS expression fold change of treated cells to those untreated was analysed using a method described by Livak et al., (2-∆∆CT) [12]. Three independent qPCR assays were conducted in triplicate.

Immunocytofluoresence

The cells were seeded on coverslips in a well of a 24-wells culture plate and fixated by 4% paraformaldehyde for 15 minutes. This was followed by incubation with protein blocking agent fluorescein-isothiocyanate (FITC) for 15 minutes. The cells were rinsed twice with ice-cold phosphate-buffered saline (PBS) and followed by overnight incubation with 2µg/ml rabbit polyclonal antibody anti-NIS (Abcam, #ab83816) at 4°C. The cells were rinsed three times with PBS and incubated at room temperature with Goat anti-Rabbit IgG secondary polyclonal antibody, with dilution 1:1500 (Abcam, #ab6716) for one hour. The cells were rinsed three times with PBS. The coverslips were placed over slides and mounted with fluoroshield containing 4’, 6-diamidino-2-phenylindole (DAPI). The slides were inspected under immunofluorescence microscope (Olympus BX51) with 200 x magnification. Cells that only incubated with secondary antibody were used as a negative control.

Statistical analysis

Collected results are expressed as mean ± SD. Responses of treatments to NIS mRNA expression were calculated in numeric data. They were analysed by ANOVA method to compare means between groups and followed by the Turkey test. P < 0.05 was considered statistically significant when compared to controls.

NIS protein expression was analysed using the Her2/neu category. The results were divided based on the level of staining from 0 to 3. Level 0 was no staining, 1 was non-complete staining at the membrane and pale of > 10% tumour cells, 2 was complete staining around the membrane of ≤ 10% tumour cells, and 3 was complete staining at membrane of > 10% tumour cells. Level 0 and 1 were considered as negative, and level 2 to 3 were considered as positive [8], [13]. Mann-Whitney test was used to compare differences between treatment and control groups, P < 0.05 was considered statistically significant.

Results

Effect of EGF and ATP treatment on NIS mRNA expression by qRT-PCR

We treat all cell lines with ATP, EGF and the combination of both to induce the level of NIS mRNA expression. The data of NIS mRNA expression was calculated from the cycle threshold (CT) target toward CT of reference (beta-actin) and CT of control genes. The NIS mRNA expression fold change of treated cells to untreated was analysed.

The expression NIS mRNA is found in SKBR3 and HaCaT cells but not in treated or untreated of MCF7 cells. The levels of NIS mRNA expression in SKBR3 cells after treatment by EGF, ATP or the combination of both for 6 and 12 hours were not significantly different from those of untreated cells. However, the treatment by a combination of ATP and EGF for 24 hours increases the level of NIS mRNA expression by 1.6fold higher than that of the untreated cells (1.6241 ± 0.3, p < 0.05) (Figure 1). Furthermore, the treatments in the HaCaT cell line do not change the expression of NIS mRNA in treated and untreated cells.

Introduction

Natrium Iodide Symporter is a plasma membrane glycoprotein. It transports two ions of sodium (Na+) and one of iodide (I-) and maintained by Na+/K+ ATPase [1], [2], [3]. In thyroid cells, NIS plays a pivotal role to accumulate iodine. Additionally, NIS is also expressed in breast cancer tissue and has been considered as a potential target for radioiodine therapy. However, NIS expression in breast cancer tissues had been reported to be varied between studies.

Furthermore, the molecular mechanism of NIS expression in breast cancer remains unclear [4]. Interestingly, it has been reported that NIS is highly expressed in invasive breast cancer tissues. A study by Tazebay et al. reported that indeed, there is a correlation between NIS expression and malignant transformation of human breast tissue [5], [6].

Other than the variation of NIS expression level between molecular subtypes of breast cancer, the location of NIS expression is another factor that may influence the uptake of radioiodine by breast cancer cells. Natrium Iodide Symporter supposed to be localised at membrane cell instead of in the cytoplasm to be fully functional as iodine co-transporter [7], [8]. Several agents were used to increasing NIS expression in breast cancer cells, and the effect of those agents had been proven could increase radioiodine uptake and cells susceptibility toward the treatment. A study reported, EGF increased NIS expression at membrane cell in the T47D cell line [9]. Another study reported that a combination of ATP with all-trans-Retinoid Acid (tRA) and hydrocortisone had increased NIS protein expression and cell membrane targeting in MCF7 cells [10].

In this study, we examined the effect of EGF and ATP for NIS mRNA and protein expressions in SKBR3 and MCF7 breast cancer cell lines and HaCaT a normal cell line. MCF7 and SKBR3 cell lines are representing the luminal A and HER2 type of breast cancer, respectively [11]. HaCaT cell line consists of normal human keratinocyte cells. It is used as a control cell line.

Material and Methods

 

Identitas Publikasi Ilmiah

Tanggal Publikasi 15 Juni 2019
Edisi issue13
Volume 7
Halaman 2088
Tahun 2019
Status publikasi
Relasi Project Penelitian
Staf Pendukung Publikasi Ilmiah
Nama Sebagai

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