Uptake and effect of radioiodine exposure on SKBR-3 cell lines

Radioiodine has been known as an adjuvant therapy for thyroid cancer. Beside for thyroid cell, radioiodine is reported to be taken up by breast cell. It may be proposed as an alternative for breast cancer therapy. Objective: The study aim was to analyze an uptake of radioiodine and cell proliferation rate of breast cancer cell after the exposure of radioiodine. Methods: This study used SKBR-3 cell line as representative of Human Epidermal Growth Factor Receptor (HER2+) subtype. The cells were planted in DMEM medium added 10% fetal bovine serum (FBS), panstrep 1% and amphotericin B. The cells were grown until 80% confluent and then stripped. Subsequently the cells were sub-cultured on plates 12 and 6 wells respectively for measured uptake of radioiodine and cell proliferation rate after the exposure. Radioiodine uptake was calculated by sum efflux of 125 Iodine (125I) and the amount of uptake after the addition of 95% ethanol. Cell proliferation rate was measured by calculated by using clonogenic assay after iodine-131 (131I) exposure. Results: The average of iodine uptake was 194±50 cpm/106 cell. The average cell proliferation after radioiodine exposure was 54%±5%; Conclusions: Radioiodine is taken by SKBR-3 cells and it reduced cell proliferation rate. This finding shows an opportunity for radioiodine as an alternative therapy for breast cancer. Another studies are needed to understand of cell death the mechanism.


INTRODUCTION
Treatment of breast cancer consists of surgery followed by adjuvant therapy such as hormone therapy, radiation, chemotherapy and immunotherapy. The cost of adjuvant therapy, especially chemotherapy and immunotherapy can reach hundreds of millions. 1 The financing of the treatment of breast cancer that continues to increase creates significant financial problems for patients and their families.
The type of adjuvant therapy is determined by the molecular types of breast cancer. Each molecular type of breast cancer has a different prognosis and response to chemotherapy. The HER2+ subtype is a group which is sensitive to chemotherapy but has limited response to the therapy. It has a worse prognosis than the luminal group. 2,3 Other adjuvant therapies are needed to increase the disease-free period and life expectancy for breast cancer patients, especially in the HER2+ subtype.
The ideal cancer adjuvant therapy, which is selective in target cells with minimal side effects and low prices, will make a very significant contribution to the management of breast cancer.
Radioiodine ( 131 I) has been used for more than 60 years as adjuvant therapy for thyroid cancer and has become an integrated part of its management. 4 The therapy has increased the disease-free period and life expectancy in thyroid cancer patients with recurrent or metastatic cases. The ability of the thyroid gland to capture and maintain iodide in cells is the basis for the use of 131 I for the diagnosis and treatment of thyroid cancer. 5 SKBR-3 cell line is a breast cancer HER2+ subtype. A study reported that, SKBR-3 cell expresses Natrium Iodide Symporter (NIS) which has important role for iodine transport cross the membrane. 6,7 The purpose of this study is to analyzed an uptake of 131 I and cell proliferation rate of SKBR-3cell after the exposure of 131 I.

METHODS
SKBR-3 cell line was used in this study. The study was conducted at the cell culture Laboratory of Radioisotope and Radiopharmaceutical Technology (PTRR) of National Nuclear Body of Indonesia. SKBR-3 cell line from ATCC (American Type Culture Collection). Radioiodine 125I was used measure the uptake and 131I was used to measure the toxicity.

Cell Culture
Cells were planted on 25 cm 3 flask in DMEM medium, added 10% fetal bovine serum (FBS), panstrep 1% and amphotericin B. Cells were grown until 80% confluent and then stripped. Subsequently the cells were sub-cultured on plates 12 and 6 well for measured uptake and toxicity of radioiodine.

Measurement of Radioactive Iodine Uptake
Measurements of radioiodine uptake were carried out by modification of methods by Knostnam. 8 Cells were planted on 12 culture plates, 200,000 cell/well for 24 hours. Each well added 2µC 125 I in (Hanks' balance salt solution) HBSS + 10mM HEPES (PH 7.3) and incubated at 37°C, CO2 level 5% for one hour. Then, the cells were trypsinazed and distributed in 1 mL (106 cell)/tube. Efflux was counted every 15 minutes until 1 hour and followed by incubation in 1 mL of 95% ethanol at room temperature for 20 minutes to release intracellular 125 I. The uptake was calculated by sum of efflux calculations and the amount of uptake after the addition of 95% ethanol. The studies were carried out three times repeated in two separate experiments.

Cell Proliferation Rate
The toxic effect was assessed by clonogenic assay. It refers to the procedure performed by Kogai et al. 9 Cells were planted on culture plates with 12 wells and wait until 80% confluent. Each well added 1µC ml of 131 I in HBSS + 10μM NaI + 10mM 3), then the cells were incubated at 37°C, with CO2 levels of 5% for 7 hours. After the incubation period was complete, the cells were quickly washed with cold HBSS and were thinned with 0.05% trypsin EDTA. The cells were replanted on a well 6 culture plate with a number of 1000 cells/well. After ten days, the cells were washed with PBS, fixed and stained with a mixture of 0.5% crystal violet in 50/50 methanol/water for 30 minutes. Then the cell is washed with water and dry in room temperature. The next day a number of colonies were counted which were visible to the well treated and the results were compared to the colonies formed in well controls that were not given radioactive iodine.

Statistical Analysis
Data analyzed by statistic with t-test difference two group and using ANOVA for more than two group with significance level p<0.05.

RESULTS AND DISCUSSIONS
The confluent cells were incubated with radioiodine for an uptake and a cell proliferation rate analysis as shown in Figure 1. The mean of radioiodine uptake was 194±50 cpm/106 cells. The average cell proliferation after 131 I exposure was 54%±5% and control 99%±2%, with p=0.005 (Figure 2).  I-131 is widely used for diagnosis and treatment of benign thyroid diseases. 10 Rates of thyroid cancer in women with a history of breast cancer are higher than expected. Similarly, rates of breast cancer in those with a history of thyroid cancer are increased. Explanations for these associations include detection bias, shared hormonal risk factors, treatment effect, and genetic susceptibility. 11 Thyroid and breast cancer are two of the malignant diseases with highest incidence in females. Based on clinical experience, breast and thyroid cancer often occur synchronously. Therefore, thyroid and breast cancer might share some common etiological factors. 12 Vol. 43 No. 1 2020 I-131 is an effective and low-risk therapy modality in well-differentiated thyroid cancer patients post near-total thyroidectomy. Extra thyroidal tumors such as breast cancer are known to be able to uptake 131 I. 13 HER2+ is one of the most studied tumor-associated antigens for cancer immunotherapy. It is overexpressed in various human cancers, such as breast. 14 Up to 70-90% breast cancer cell expressed NIS included HER2+ subtype. 6,15-17 NIS is a co-transporter of iodine to crossed cell membrane. In this study, SKBR-3 cell which represent HER2+ subtypes taken up of radioiodine. The exposure of radioiodine reduced cell proliferation significantly when is compared to the control cell (p=0.005). This finding is in line with previous study. 18 I-131 is caused DNA damage and inducing secondary immuno-reactivity. The effects of 131 I on malignant cells appears to be depended on absorbed dose. 131 I-mediated cellular damage, and some studies observed a necrotic cellular death following high-dose 131 I treatment. 19 The exact mechanisms of cellular death of SKBR3 cell have not yet been fully elucidated. Further studies are needed to uncover this issue.

CONCLUSSIONS
Radioiodine was taken by SKBR3 cells. Its exposure, reducing of cellular proliferation rate significantly. It may open an opportunity for 131 I as an alternative therapy for breast cancer. However, cell death mechanism that activated after 131 I exposure should be explored further in order to fully delineate the cell response.