All-trans-retinoic Acid Promotes Iodine Uptake Via Up-regulating the Sodium Iodide Symporter in Medullary Thyroid Cancer Stem Cells

MTC accounts around 5%-10% of thyroid cancer, and the death rate is more than 10%. It has a poor capability of iodine uptake which limits the way of therapy. Furthermore, many people believe that MTCSCs may play significant roles in metastasis and recurrence of MTC. Cancer stem cells (CSCs) have been reported in several solid tumors and in cancer cell lines (Reya et al., 2001; Reya et al., 2001; Ailles and Weissman, 2007; Lobo et al., 2007). Recently, people believed that only a few cells of CSCs have the capacity to sustain the growth of tumor. And this subpopulation of cells are capable of self-renew, proliferate and differentiate into other tumor cells (Jiang et al., 2012; Tabarestani and Ghafouri-Fard, 2012; Francipane et al., 2013). It also expresses high levels of ABCG2 and OCT4, which may cause their resistance to the conventional therapies of cancer (Tabarestani and Ghafouri-Fard, 2012; Zhang et al., 2012). Thus, if we find a way to eliminate the CSCs, we may cure the cancer. All-trans-retinoic acid (ATRA) has the capacity of inducing differentiation, inhibiting proliferation and promoting apoptosis of many tumors and CSCs. It can induce differentiation of acute promyelocytic leukemia stem cells, and increase 5-year survival rate with arsenic trioxide (Cunha De Santis et al., 2007; Tomita et al., 2013). The sodium iodide symporter (NIS), as the first step in thyroid hormone biosynthesis, mediated thyroidal Itransport from the bloodstream to the colloid (Schmutzler et al., 1997; Foujilas et al., 2009; Damle et al., 2011). This


Introduction
MTC accounts around 5%-10% of thyroid cancer, and the death rate is more than 10%.It has a poor capability of iodine uptake which limits the way of therapy.Furthermore, many people believe that MTCSCs may play significant roles in metastasis and recurrence of MTC.
Cancer stem cells (CSCs) have been reported in several solid tumors and in cancer cell lines (Reya et al., 2001;Reya et al., 2001;Ailles and Weissman, 2007;Lobo et al., 2007).Recently, people believed that only a few cells of CSCs have the capacity to sustain the growth of tumor.And this subpopulation of cells are capable of self-renew, proliferate and differentiate into other tumor cells (Jiang et al., 2012;Tabarestani and Ghafouri-Fard, 2012;Francipane et al., 2013).It also expresses high levels of ABCG2 and OCT4, which may cause their resistance to the conventional therapies of cancer (Tabarestani and Ghafouri-Fard, 2012;Zhang et al., 2012).Thus, if we find a way to eliminate the CSCs, we may cure the cancer.
All-trans-retinoic acid (ATRA) has the capacity of inducing differentiation, inhibiting proliferation and promoting apoptosis of many tumors and CSCs.It can induce differentiation of acute promyelocytic leukemia stem cells, and increase 5-year survival rate with arsenic trioxide (Cunha De Santis et al., 2007;Tomita et al., 2013).
In this study, we try to increase the capability of iodine uptake of MTCSCs through ATRA, which may provide new ideas for the clinical treatment of the MTC, even of thyroid cancer.

Colony formation analysis
To evaluate the proliferation of TT cells and sphere cells, a colony formation assay was performed.TT cells and sphere cells were planted to 6-well, with 10 3 of each well and medium being changed every 3 days.Two weeks later, washed it with ice-clod PBS and fixed it for 5 minutes with methanol, stained with Giemsa dye, and observed under light microscope (n=500 cells).

FCM analysis
The cells include TT and sphere cells were incubated with saturating amounts of OCT3/4-PE or ABCG2-PE, which came from santa cruz (USA), for 20 min at 4℃ in the dark.Then, cells were washed and analyzed on FCM.

Nude mouse xenograft model
This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National standard of China.The protocol was approved by the Committee on the Ethics of Animal Experiments of the Chongqing medical university (Reference Number: CQMU 2013005).All surgery was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering.
For in vivo tumorigenesis assay, 10 3 , 10 4 , 10 5 and 10 6 cells of the TT and TT sphere cells were athymic nude mice (NOD/SCID, Beijing HFK Bioscience, LTD, China), respectively.In this study, every group had 3 nude mice. 2 weeks later, measured the diameter of tumors, which exceeds 5 mm.
All of the cells were used to measure the inhibition ratio by the methyl-thiazolyl-tetrazolium (MTT) test.Briefly, the medium containing 5mg/ml MTT was added to each well and incubated at 37 ℃ for 6 hours.The product was dissolved in Dimethyl sulfoxide (DMSO) for 10 min and the plates were read at 490 nm using a plate reader.The inhibition ratio was expressed as 1-experimental/control and calculated the half maximal inhibitory concentration of a substance (IC50) with the inhibition ratio.

Western blot analysis
Collecting the TT cells and MTCSCs with or without ATRA for 24 hours, the protein extracts were prepared to use a nuclear extraction kit.Twenty microliters of sample were boiled at 95°C for 5 minutes and centrifuged at 12000 r.p.m. for 5 minutes at 4 ℃.The sample was separated in a 10% SDS-polyacrylamide gel electrophoresis gel and transferred to Polyvinylidene Fluoride (PVDF) membranes by a wet transfer system.The membranes were washed by TBS containing 0.1% Tween (TBST) twice.And incubated with the primary antibodies were used: mouse anti-human ABCG2, mouse anti-human OCT4, mouse anti-human NIS and mouse anti-human GAPDH (Abcam, UK) for overnight at 4 ℃.The membranes were incubated with Peroxidase-Conjugated AffiniPure Goat Anti-Mouse IgG (ZSGB-BIO, China) for 1 hours after washing with TBST 3 times.At last, ECL detection system (Thermo) was used to detect the four proteins.

I uptake assay
To determine whether the ATRA can influence radioactive iodide uptake, iodide uptake assay was performed as described by Weiss et al. (1984) with some modifications.The TT cells and MTCSCs, which were cultured 24 hours with ATRA, packed it in centrifuge tubes which have 1 ml of Hanks' balanced salt solution (HBSS) with 10 5 cells.Added 37 kBq of 131 I to each tube and washed with ice-cold HBSS 2 hours later in 37 ℃.Their radioactivity of the cells was measured with a gamma counter.The counts were normalized the number of viable cells.

Statistical analysis
Statistical analysis was performed with Graphpad Prism 5 software.Numerical data were expressed as means ± SD.Significance was concluded at p<0.05.

Characterization of TT cells and TT sphere cells
To determine the cells which were separated by serumfree culturing had some stem-like feature, the colony formation analysis, xenograft tumor model and FCM were performed.As revealed by colony formations assay, we found that the PE of TT sphere cells was 41.69%±4.45%and 6.65%±0.69% of TT (Figure 1A).It means that the TT sphere cells had more proliferation than TT cells.Addition, when detected the maker by FCM, the expression of ABCG2 and OCT4 were 74.98% and 67.15% in TT sphere cells respectively, while them were 11.06% and 7.66% in TT cells (Figure 1B).
Moreover, in the tumorigenesis assay, we found that all the group of TT sphere cells generated xenograft   tumor model, and the tumorigenesis only 10 6 cells of TT (Figure 1C).The xenograft tumor model was considered successful formed while the tumor was more than 5mm in diameter.

Expression of OCT4, ABCG2 and NIS
In order to prove the proteins of TT cells and TT sphere cells are different, the western blot was used.We assayed the expression of OCT4 and ABCG2 of TT cells and TT sphere cells (Figure 2A), In TT sphere cells, both of them had a higher expression levels than it in TT cells (Figure 2A).In summary, the TT sphere cells, which had the ability to self-renew, proliferate, formed xenograft tumor and expressed the stem-like maker, were considered as medullary thyroid cancer stem cells (MTCSCs).Interestingly, when we detected the NIS, the expression levels of TT sphere cells were lower than TT cells (Figure 2B).

Treatment of ATRA
The plate reader showed that inhibition ratio was positively correlated with the concentration.It was proved that the capacity of inhibition would be enhanced as the concentration increased (Figure 3).And the IC50 of MTCSCs was 1.06×10 -6 M, 1.10×10 -6 M of TT cells (Table 1).
Addition, the western blot showed that OCT4 and ABCG2 of MTCSCs were down-regulated the same as TT cells, after treated by ATRA with 1*10 -6 M (Figure 2A).But, NIS was up-regulated as the same treated (Figure 2B).

Capability of iodine uptake
The capability of iodine uptake of MTCSCs and TT cells had changed after being treated by ATRA with difference concentration.The capability of MTCSCs and TT cells was increased as the concentration of ATRA increased.And at the concentration of IC50, the capability of MTCSCs increased 8.30 times and 4.27 of TT cells (Figure 4).

Discussion
Cancer stem cells (CSCs) are thought to represent a small sub-population of cells presented in most tumors, possess the ability to self-renew, proliferate and produce progenitor cells that can reconstitute and sustain tumor growth (Collins, 2002;Christina Zechel, 2005;Rashidi et al., 2013).Moreover, CSCs also play a key role in cancer metastasis, cancer recurrence, and cancer drug resistance (Hadnagy, 2006;Jordan et al., 2006;Lin, 2011).
In this study, we have demonstrated that MTCSCs could be isolated by serum-deprivation, to acquire some characterized of stem-like cells.In this population of cells, we found that the MTCSC had high expression levels of ABCG2 and OCT4, observed by FCM.And the ABCG2, which was believed as a gene of drug resistance, of MTCSCs have a high level.These may explain why CSCs are resistant to chemotherapy and radiotherapy.
The colony formations test showed that the capacity of MTCSCs, namely self-renewal and proliferation, were more powerful than that of TT cells.And the level of stemness protein (OCT4), which is considered key genes for the production of murine and human induced pluripotent stem cell (Bao et al., 2006;Martin et al., 2008), was higher in MTCSCs than TT cells.Moreover, the tumorigenesis of MTCSCs can cause the tumor when planted in nude mice only 10 3 , as previous reported (Baumann et al., 2008).We found difference concentration of ATRA could cause the different effects in TT cells and MTCSCs.At present, the regulation and function of NIS have been well know in thyroid tissue.This may make a better understanding of thyroid diseases and therapy using iodine (Doha´n and Carrasco, 2003).The NIS, as the first step in thyroid hormone biosynthesis, mediated thyroidal I-transport from the bloodstream to the colloid (Segalla et al., 2003;Takahashi and Yamanaka, 2006;Yu et al., 2007;Tseng et al., 2012).It can provide the basis for the effective therapeutic management of thyroid cancer with radioiodine.In this study, we find that the NIS was up-regulated by ATRA.Its ability to mediate iodine uptake is demonstrated by the test of iodine uptake.With the ATRA administrated, the capability of iodine uptake was increased.
Overall, in this study, we certificated that ATRA can affect the capacity of iodine uptake through up-regulating the expression of NIS (Biermann et al., 2011;Elola et al., 2011).Next, we will demonstrate that ATRA can promote the eradication of MTCSC by iodine in vivo.In conclusion, in this study, we used the ATRA to treat the MTCSCs and TT cells, revealing that the ATRA can induce the differentiation of MTCSCs and TT cells, and also increase the capacity of iodine uptake, which will be helpful to treat MTC for clinic.

Figure 1 .Figure 2 .Figure 3 .
Figure 1.Characterization of TT Cells and MTCSCs.(A) colony formation analysis, the ratio of colony formations, it showed that the ratio of MTCSCs was 41.69%±4.45%with 6.65%±0.69% of TT.The results are presented as x(-)±s of eight independent experiments, n=8.*p<0.05.(B) the maker of stemlike were detected by FCM, (C) The nude mouse xenograft model

Figure 4 .
Figure 4. Ioiodine Uptake.The TT and MTCSCs cells were administrated by ATRA, and CPM were detected by the the gamma counter.All the results are presented as x(-)±s of fifteen independent experiments, n=15.*p<0.05