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Introduction

Thyroid cancer is the most common type of malignant tumor in the endocrine system. I-131 therapy has been used to treat differentiated thyroid cancer (DTC) (1). With demonstrable curative effects, this therapy has been recognized by clinical practice (2). However, the iodine uptake capacities of tumor nidi in ~10% of patients are decreased or depleted entirely, which causes a reduction of treatment efficacy, resulting in a higher severity of anaplastic thyroid carcinoma (ATC) (3). Furthermore, with short median survival time and high mortality rates, these patients are less sensitive to various traditional therapies (for example, surgery and endocrinotherapy) (4).

DTC and ATC describe two possible endpoints of thyroid cancer (5). In fact, changes from DTC to ATC do not occur at once. As far as differentiated degrees, cell morphology and biological tumor behavior are concerned, there is a progression from DTC to ATC, i.e., from differentiated to poorly differentiated and then to dedifferentiated (6). The World Health Organization considers poorly differential thyroid carcinoma (PDTC) to be an intermediate between DTC and ATC (7).

With a length of <10 amino acids, cell-penetrating peptide is a micromolecular polypeptide that is able to penetrate through the cytomembrane into cytoplasm or the cell nucleus without damaging structures of the cell membrane (8). Studies in recent years have shown that cell-penetrating peptides may carry a series of substances with biological activities into living cells, including proteins, polypeptides, nucleic acids and oligonucleotides (9,10). Hairpin-shaped cell-penetrating peptide is a recently identified transmembrane small peptide (11). Its hairpin structure can construct numerous protease sites, which may provide novel approaches for molecular targeted diagnosis and therapy.

As a glycoprotein, the Na+/I-symporter (NIS), also known as an iodine pump, is located in the membrane of epithelial cells of the thyroid follicle (12). NIS participates in the iodine uptake process and plays a rate-limiting role in iodine transportation (13). For normal human bodies, expression levels of NIS in thyroid tissues are higher than those in other tissues (14). However, it has been hypothesized that due to obstructions to NIS positioning and reductions in NIS protein expression levels, iodine uptake capacities for certain patients with thyroid cancer are reduced or depleted entirely, which may influence the efficacy of I-131 treatment and worsen prognosis (15). By improving functional position expression levels of NIS, iodine uptake capacities of thyroid cancer tissues may be increased (16). Thus, in the present study we investigated whether the cell penetrating peptide of NIS affects the efficacy of I-131 radiotherapy in human thyroid cancer cells.

Materials and methods

Cell lines and cell culture

Human thyroid carcinoma TPC-1 cells were purchased from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). TPC-1 cells were maintained in RPMI-1640 medium (Invitrogen; Thermo Fisher Scientific, Inc., Carlsbad, CA, USA) and 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc., Grand Island, NY, USA), 100 U/ml penicillin and 50 µg/ml streptomycin (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), and incubated at 37°C in a humidified atmosphere of 5% CO2.

dTAT nanoparticle (NP)-NIS transfection

dTAT NP-NIS and the negative control (NC) plasmids were purchased from Shanghai GenePharma, Co., Ltd. (Shanghai, China). TPC-1 cells were incubated in six-well plates at a density of 1–2×106 cells/well. Subsequently, 100 nmol/l plasmids were transfected Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions and transfected into TPC-1 cells.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis of dTAT NP-NIS

Following transfection, TPC-1 cells were seeded six-well plates at a density of 1–2×106 cells/well for 24 h. Total RNA from the TPC-1 cells was extracted using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Following treatment with DNase (Takara Biotechnology Co., Ltd., Dalian, China) a TaqMan miRNA Reverse Transcription kit (Applied Biosystems; Thermo Fisher Scientific, Inc., Waltham, MA, USA) was used to synthesize cDNA using 1 µg RNA. Subsequently, a 7500 Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) was used to conduct RT-qPCR using SYBR Premix Ex Taq (Takara Biotechnology Co., Ltd.). The sequences were as follows: NIS forward, 5′-ACSCACTGGAAGCACGGCGG-3′ and reverse, 5′-GTGGMRCCGTGCAKRTTGG-3′; β-actin forward, 5′-AGGCACCAGGGCGTGAT-3′ and reverse, 5′-TGCTCCCAGTTGGTGACGAT-3′. The cycling conditions used were 95°C for 4 min, 95°C for 15 sec and 60°C for 30 sec for 40 cycles. The RT-qPCR experiment was conducted 6 times. Relative expression of mRNA was quantified using the 2−∆∆CT method (17).

Western blot analysis

TPC-1 cell total protein was extracted using a radioimmunoprecipitation assay lysis buffer (Sigma-Aldrich; Merck KGaA). Protein concentrations were determined using a Bradford assay (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Equal quantities of total protein (50 µg) were resolved using 10–12% SDS-PAGE (Bio-Rad Laboratories, Inc.) and transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories, Inc.). The membranes were blocked in 5% non-fat milk for 2 h at room temperature and then incubated with rabbit anti-human NIS (1:500; sc-134515), caspase-3 (1:500; sc-98785), Akt (1:200; sc-8312) and phosphorylated-Akt (1:500; sc-33437) and PTEN primary antibodies (1:500; sc-9145; all Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) overnight at 4°C. The membranes were washed with Tris-buffered saline containing Tween 20 (Sigma-Aldrich; Merck KGaA) and then incubated with horseradish peroxidase-conjugated secondary goat anti-rabbit immunoglobulin G antibodies (1:1,000; sc-2922; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) for 1 h at room temperature. The membranes were visualized using an Immobilon Western chemiluminescent HRP substrate (EMD Millipore, Billerica, CA, USA), and the blots were densitometrically analyzed using Image Lab 3.0 software (Bio-Rad Laboratories, Inc.).

MTT assay

TPC-1 cells were seeded onto 96-well plates at a density of 1–2×103 cells/well for 12, 24 and 48 h, and subsequently incubated with 20 µl MTT (5 mg/ml; Sigma-Aldrich; Merck KGaA) for 4 h. Then, 150 µl DMSO (Invitrogen; Thermo Fisher Scientific, Inc.) was added to each well and shaken for 15 min after the medium was removed. The optical density (OD) of each well was detected at 490 nm using a microplate reader (BioTek Instruments, Inc., Winooski, VT, USA).

Flow cytometry for evaluation of apoptosis

TPC-1 cells were seeded onto six-well plates at a density of 1–2×106 cells/well for 24 h and washed twice using phosphate-buffered saline (PBS). TPC-1 cells were resuspended using buffer solution (from a FITC Annexin V Apoptosis Detection kit; BD Biosciences, Franklin Lakes, NJ, USA), and incubated with 5 µl Annexin V-fluorescein isothiocyanate and 10 µl propidium iodide for 30 min at 4°C in the dark. Flow cytometry (FACSCalibur; BD Biosciences) was used to measure cell apoptosis rates of the TPC-1 cells.

DAPI staining assay

TPC-1 cells were seeded onto six-well plates at a density of 1–2×106 cells/well for 24 h and washed twice using PBS. TPC-1 cell was fixed using 4% paraformaldehyde for 30 min at 4°C. Then, fixed TPC-1 cells were washed twice using PBS and incubated with sodium citrate (0.1%) containing 0.1% Triton X-100 (Beyotime Institute of Biotechnology, Haimen, China) for 5 min at 4°C. TPC-1 cells were dyed by DAPI staining and incubated for 10 min at 4°C in the dark, then activated using ultraviolet. DAPI staining was observed and images captured using a fluorescence microscope (CKX41; Olympus Corporation, Tokyo, Japan) at 340 nm.

Statistical analysis

All values were expressed as the mean ± standard deviation. Statistical analyses were conducted using SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA). Data were analyzed using the Student's t-test. P<0.05 was considered to indicate a statistically significant difference between values.

Results

Gene expression level of NIS in TPC-1 cells

To evaluate the gene expression of NIS in TPC-1 cells transfected with dTAT NP and negative control, RT-qPCR was performed. As shown in Fig. 1, there was a significant increase in the relative gene expression of NIS of dTAT NP group, compared with the NC group (P<0.05).

Figure 1. mRNA expression level of NIS in TPC-1 cells. ##P<0.05 vs. negative control group. NIS, sodium-iodide symporter; dTAT NP, dTAT nanoparticle.

Protein expression level of NIS in TPC-1 cells

In order to characterize the dTAT NP-NIS and NC plasmid influence on the protein expression of NIS, western blot analysis was performed. The expression of NIS protein was significantly higher than that of the NC group (P<0.05; Fig. 2).

Figure 2. Protein expression level of NIS in TPC-1 cells. (A) Protein expression of NIS was evaluated using western blot analysis. (B) Statistical analysis of NIS protein expression in TPC-1 cells. ##P<0.05 vs. negative control group. NIS, sodium-iodide symporter; dTAT NP, dTAT nanoparticle.

Effect of NIS expression on cell growth in TPC-1 cells

We clarified the overexpression of NIS influence on cell growth of TPC-1 cell. The results from MTT assay showed the cell growth of TPC-1 cell was significantly suppressed after dTAT NP-NIS transfection at 24 and 48 h, compared with the NC group (P<0.05; Fig. 3).

Figure 3. Effect of NIS expression on the cell growth of TPC-1 cells. ##P<0.05 vs. negative control group. dTAT NP-NIS, dTAT nanoparticle-sodium-iodide symporter.

Effect of NIS expression on apoptosis rates in TPC-1 cells

To determine the effect of the overexpression of NIS apoptosis rates in TPC-1 cells, cellular apoptosis was observed in TPC-1 cells using flow cytometry. As shown in Fig. 4, cellular apoptosis of dTAT NP-NIS transfection was significantly higher than that of the NC group, after dTAT NP-NIS transfection for 24 h (P<0.05).

Figure 4. Effect of NIS expression on the apoptosis rate of TPC-1 cells. ##P<0.05 vs. negative control group. dTAT NP-NIS, dTAT nanoparticle-sodium-iodide symporter.

Effect of NIS expression on cell nucleus apoptosis in TPC-1 cells

To validate the overexpression of NIS influence on cell nucleus apoptosis of TPC-1 cell, TPC-1 cells were stained with DAPI. After dTAT NP-NIS transfection at 24 or 48 h, there was a visible increase in the nuclear apoptosis rate of TPC-1 cell-transfected with dTAT NP-NIS compared with the NC group (Fig. 5).

Figure 5. Effect of NIS expression on cell nuclear apoptosis rates of TPC-1 cells at (A) 12, (B) 24 and (C) 48 h (DAPI staining; magnification, ×400). ##P<0.05 vs. negative control group. dTAT NP-NIS, dTAT nanoparticle-sodium-iodide symporter.

Effect of NIS expression on caspase-3 expression in TPC-1 cells

To further clarify whether the effect of NIS expression on caspase-3 protein expression of TPC-1 cell, caspase-3 protein expression was analyzed using western blot analysis in TPC-1 cell. Western blot analysis displayed that caspase-3 protein expression was significantly activated in the dTAT NP-NIS transfected cells at 24 h, compared with the NC group (Fig. 6).

Figure 6. Effect of NIS expression on caspase-3 protein expression of TPC-1 cell. (A) Western blot analysis and (B) statistical analysis of caspase-3 protein expression in TPC-1 cells. ##P<0.05 vs. negative control group. dTAT NP-NIS, dTAT nanoparticle-sodium-iodide symporter.

Effect of NIS expression on Akt expression in TPC-1 cells

To further validate the effect of NIS expression on Akt signaling in TPC-1 cells, Akt and p-Akt protein expression levels were detected. Compared with the NC group, the relative Akt/p-Akt rate was significantly reduced by overexpression of NIS (Fig. 7).

Figure 7. Effect of NIS expression on Akt protein expression of TPC-1 cell. (A) Western blot and (B) statistical analysis of p-Akt/Akt protein expression in TPC-1 cells. ##P<0.05 vs. negative control group. dTAT NP-NIS, dTAT nanoparticle-sodium-iodide symporter.

Effect of NIS expression on PTEN expression in TPC-1 cells

To further evaluate the effects of NIS expression on PTEN signaling in TPC-1 cells, PTEN protein expression was evaluated. As shown in Fig. 8, relative PTEN protein expression was significantly elevated compared with the NC group (Fig. 8).

Figure 8. Effect of NIS expression on PTEN protein expression of TPC-1 cell. (A) Western blot and(B) statistical analysis of PTEN protein expression in TPC-1 cells. ##P<0.05 vs. negative control group. dTAT NP-NIS, dTAT nanoparticle-sodium-iodide symporter.

Discussion

Thyroid cancer is among the most common diseases affecting the thyroid. Iodine therapy following surgery may evidently lower recurrence rates (18). However, during the treatment process, sensitivity of tumor nidi or metastatic tumor tissue to iodine is decreased or lost entirely, which may be caused by de-differentiation (19). In cases of the greatest severity with the worst prognosis, methods as surgery, radiotherapy and chemotherapy may not be sufficient to successfully treat PDHC (20). Previous studies have investigated the pathogenesis, therapeutic strategies and drug development associated with PDHC (16,21). The present results showed that dTAT NP-NIS inhibits cell growth and induces cellular apoptosis in TPC-1 cells.

At present, a comprehensive therapeutic method for PTC involves surgery assisted by endocrinotherapy and radioactive I-131 treatment (22). Located on the epithelial cell membrane, NIS is a type of glycoprotein, whose genes are on the short arm of chromosome 19 (23). NIS carries iodine into cytoplasm using a sodium potassium pump, and is important for the maintenance of iodine uptake capacities of thyroids, while also participating in the synthesis of thyroid hormones (24). Furthermore, NIS may be expressed in other locations, such as mammary and prostate glands (25). The present results suggest that the anti-cancer effect of dTAT NP-NIS may be associated with the activation of caspase-3 pathway in TPC-1 cells.

Radioactive I-131 therapy is essential in diagnosis and treatment of ATC and metastasis. The presupposition of the lethal effects of radioactive I-131 to tumor cells is that tumor cells possess increased iodine uptake capacities (26). PTEN/PI3K/AKT signal transduction pathways play an important part in regulating NIS protein synthesis (27). Overactivity of the PTEN/PI3K/AKT signal transduction pathways may affect locations of NIS proteins in cells. The present results suggests that there was significant suppression of PI3K/AKT signaling, which may be associated with the anti-cancer effects of dTAT NP-NIS on TPC-1 cells.

The gene for PTEN protein lie in chromosome 10q23.3 area. PTEN protein is expressed in numerous early embryonic tissues, such as kidney, stomach, central and peripheral nervous system tissue (28). The expression levels of PTEN in the early stage of thyroid tissue growth and in adults are relatively high. In normal human cells, PTEN participates in the regulation of numerous signal transduction pathways, including PTEN/PI3K/AKT, FAK/PI30C and MAPK (28). Through these pathways, PTEN is involved in the maintenance of normal and regulatory substance metabolism and stabilization of the internal environment (29). Low expression levels of PTEN protein in cancer tissues are closely associated with the genesis and progression of tumors (30). The present results suggest that the anti-cancer effects of dTAT NP-NIS increased the protein expression levels of PTEN in TPC-1 cells.

In conclusion, to our knowledge the present study is the first to demonstrate that dTAT NP-NIS inhibits growth and induces the apoptosis of TPC-1 cells, possibly via the caspase-3 and PTEN/PI3K/AKT pathways. Our data provide a preclinical proof-of-concept for a novel gene delivery system that efficiently delivers NIS to the targeted cancer cells and presents a satisfactory efficacy. This may offer an effective strategy for improving thyroid cancer gene therapy, and warrants further investigation.

Acknowledgements

This study was partially supported by Guangdong Provincial Natural Science Foundation (grant no. S2013010015733).

References