Correlation of pendrin expression with 131Iodine uptake in post-Nal treated thyroid cancer animal model Gholve C.S., Shete Y.H., Rakshit S., Basu S., Kulkarni S.P.*, Baghel Nawab Singh Radiation Medicine Centre, BARC, C/o TMH Annexe, Parel, Mumbai-400 012, India *Address for Correspondence Kulkarni S.P., Head, TIID, MCF & RP Section, Radiation Medicine Centre, BARC, C/o TMH Annexe, Parel, Mumbai-400 012, E-mail: savitapk@barc.gov.in
Online Published on 27 March, 2024. Abstract Radioactive iodine (RAI) refractoriness develops in the background of a loss of thyroid differentiation features representing major therapeutic challenges in thyroid cancer management. Several studies have reported decreased or even loss of pendrin (SLC26A4 gene) expression in thyroid cancers, signifying the role of pendrin in the impaired ability of thyroid cancer cells for uptake and concentration of iodine. As acute iodide treatment has been shown to increase SLC26A4 mRNA, the study of pendrin expression in malignant tissues is of interest. Hence, we focused on evaluating the pendrin expression and 131I uptake in an N-bis-(2-hydroxypropyl) nitrosamine (DHPN) induced thyroid cancer model in Wistar rats followed by a single dose of excess sodium iodide (NaI) treatment to see if any change occurs in the RAI uptake. 131I uptake by scintigraphy showed thyroid standard uptake values of 10.16±1.6 and 13.13±0.115 in DHPN-treated and control groups respectively at 24 h. However, post-NaI treatment decreasing patterns at 24 h, 48 h and 72 h were observed in a few animals similar to the control. However, the rising pattern of 131I uptake was also observed in a few DHPN-induced thyroid cancer animals compared to the control. In conclusion, further extensive research is warranted to corroborate the role of NaI in the expression of pendrin in conjunction with animal species and age, along with the dose and exposure time of NaI. In addition, the expression and quantification of pendrin at the molecular level could be potentially used to enhance its utility in the management of RAI refractory human thyroid cancer patients. Top Keywords Follicular cells, Immunohistochemistry, 131Iodine uptake, N-bis-(2-hydroxypropyl) nitrosamine, Pathology, Pendrin, Radioactive iodine, Sodium iodide, Thyroid cancer, Wistar rat. Top |
Introduction Sodium iodide symporter (NIS) and pendrin, functioning as iodide (I-) transporters, are proteins found principally, but not exclusively, in the thyroid tissue. Pendrin, a 110 kDa glycoprotein, encoded by the Pendred syndrome gene (PDS), is a member of the SLC26A4 gene family. It is an anion transporter that is predominantly expressed in the inner ear, thyroid and kidney. In thyroid cells, pendrin is involved in apical iodide efflux1. The occurrence and level of pendrin expression and iodide efflux are regulated by TTF 1, TSH and thyroglobulin, while iodide itself does not have a major effect on pendrin gene expression2. On the contrary, Calil-Silveira et al.3 showed that acute iodide treatment increased SLC26A4 mRNA content, in both in vitro and in vivo models. Although pendrin is suggested to be an apical iodide transporter in the thyroid, the expression and localization of pendrin in diseased thyroids have not been adequately investigated4. The diagnostic and therapeutic uses of radioiodine in the management of thyroid cancers, however, are often hindered by the decreased ability of the thyroid cancer cells to uptake and concentrate iodine. This represents a major therapeutic challenge in thyroid cancer management. Hence, presently treatment tactics for radioactive refractory (RAI-R) thyroid cancer focus on novel approaches to re-differentiate thyroid cancer cells to restore the responsiveness to radioiodine administration by increasing the 131I uptake. In this direction, various clinical trials are being conducted using different redifferentiating compounds in the preclinical experimental models of thyroid cancer as well as RAI-R DTC5. |
Several studies have reported decreased or even absent expression of SLC26A4 in many thyroid tumors6-10 demonstrating a pathological role of pendrin in the impairment of the iodide-concentrating mechanism of thyroid cancer cells. Therefore, the study of pendrin expression in pathological thyroid tissues, particularly in cancerous tissues, is of interest due to its iodide translocating activity, which serves as one of the iodide suppliers for organification processes. Our earlier animal studies have revealed the important role of pendrin in the uptake of 131I in the thyroid follicles11. As acute iodide treatment has been shown to increase SLC26A4 mRNA3, the study of pendrin expression in malignant thyroid tissues is of interest. The present work is a preclinical study that attempts to evaluate the expression of pendrin in a chemically induced thyroid cancer animal model in Wistar rats and to observe if any change in 131I uptake, post-Nal treatment used for redifferentiation. |
Top Materials and Methods Treatments All the animal experiments were carried out as per the protocols/guidelines previously approved by the Committee for Control and Supervision of Experimental Animals (CPCSEA), India. The project was approved by Bhabha Atomic Research Centre, Animal Ethics Committee, Mumbai (Approval No. BAEC/27/18). A total of 18 male (270-280 g) 8-week-old healthy inbred Wistar strain rats were kept on ad libitum of commercial pelleted feed and filtered drinking water and maintained at a controlled temperature of 23±1OC, humidity of 55±5%, in a 12h light/12h dark cycle. The animals were grouped into 2 groups based on their body weights. Group 1 (treatment group animals, n=12) and Group 2 (control animals, n=6) animals. Group 1 (treatment group) was injected with DHPN obtained from MedChem, UK (catalog number 53609-64-6) at a dose rate of 83 mg/animal by i/p route, fortnightly, for 4 months, whereas Group 2 (control group) animals received only saline. Post-treatment animals were subjected to tumor development analysis. Thyroid scintigraphy After a period of 16 weeks, Group 1 (DHPN treatment, n=12) and Group 2 (control, n=6) animals were subjected to the analysis of metabolic alteration in the thyroid gland by giving 296 KBq 131I (8 pCi) orally and thyroid uptake and imaging was performed at 24h followed by excess Nal (2 mg Nal in 0.5 ml NaCl 0.9%) treatment with 2h, 24h, 48h and 72h uptake. The analysis of the uptake was done by a camera-based method with reference to the 131Iodine standards source. Animal imaging was performed under Ketamine (80 mg/kg body weight) and Xylazine (5 mg/kg body weight) by i/p route for further studies. Ketamine and Xylazine are used as mild sedatives and muscle relaxants during imaging which was calculated and administered to the animals during the imaging considering further survival important in these animals to carry out the studies. Postimaging the animals recover to normal state very fast. The uptake analysis was done with the help of software for the evaluation of disease initiation and its propagation12. All the radioactivity-related experiments were carried out under the supervision of the Radiation Safety Officer. Histopathology of thyroid sections At the end of 20 weeks, the terminal sacrifice of the animals was done by exsanguination, under ether anesthesia and thyroid tissues along with the other vital organs were collected for histopathology and immunohistochemistry. The bilateral thyroid lobes were excised and fixed in 10% neutral buffer formaldehyde and routinely embedded in paraffin. Further, tissue sections of 3-5pm thickness were prepared and stained with hematoxylin and eosin (H&E) stain. The histopathological changes in the thyroid gland were observed by light microscopic analysis. As per the published standard guidelines, focal proliferative lesions of follicular epithelial cells were classified in H&E-stained sections as focal hyperplasia, adenomas, intrathyroidal carcinomas, and invasive carcinomas to the thyroid capsule or adjacent tissues13. Immunohistochemical staining of thyroid sections Immunohistochemistry of the thyroid tissue section was performed for the evaluation of the pendrin expression. Specific purified primary polyclonal anti-pendrin antibodies raised in rabbits (ready-to-use) were purchased from Thermo Fisher Scientific (Cat. no. PA5-42060 SLC26A4). The localization and expression of pendrin in thyroid follicular cells were done by immunohistochemistry using an indirect method of staining4 using fluorescent dye. The microscopic evaluation was performed using a fluorescent microscope (Inverted Leica DMi8 S). Statistical analysis As the study was initiated as a pilot experiment, the number of animals was decided and sanctioned by the Institutional Animal Ethics Committee, Bhabha Atomic Research Centre (BARC) for the project. All data are reported as Means ± SD. Student's t-test was used to determine the difference between the treatment group (DHPN) and the control group. The significance level was set at 5% (p < 0.05). Top Results Scintigraphy imaging of the thyroid gland At 24h, 131I uptake by scintigraphy imaging showed thyroid standard uptake values of 13.13±0.115 and 10.16±1.6 in the control (Fig. 1) and DHPN-treated groups respectively (Fig. 2). However, post-NaI treatment decreasing patterns at 24h, 48h and 72h were observed in a few DHPN-treated animals similar to the control (Fig. 3). Nonetheless, the increasing trend of 131I uptake was also observed in a few DHPN-induced thyroid cancer animals compared to the control group (Fig. 4). Histopathological observations of thyroid sections The histological examination of H&E-stained thyroid sections in the healthy control animals showed a normal pattern of follicles of different sizes with colloidal secretion (Fig. 5) in the light microscope. New blood vessel formations and invasion were extended between thyroid follicles (Fig. 5). Overall, no gross or histopathological abnormalities were recorded in the healthy control. Further, in the DHPN-treated animals, the modifications in the epithelial lining of the thyroid follicles were observed with hyperplastic changes in the multifocal area (Fig. 6) which was observed with lung metastasis (Fig. 9) in one case. The presence of dilated congested blood vessels and congested infiltrating capillaries was noted (Fig. 6). Immunohistochemical evaluation of thyroid sections In the control group, immunohistochemical expression of thyrocytes was characterized by normal expression of the protein pendrin at the apical membrane (Fig. 7). On the contrary, in the DHPN-treated animals, the immunohistochemical evaluation showed reduced expression of the pendrin protein at the apical membrane of thyroid follicular cells as compared to the control animals (Fig. 8). Top Discussion The incidence of thyroid cancer has increased over time. The application of gold standard treatment of 131I has been extensively used for the treatment of thyroid cancer disease in humans. However, the pattern of disease has been varying with the patients and the incidence of 131I resistance has risen in about 5-15% of patients with DTC and approximately 50% of metastatic DTCs are refractory to RAI treatment. Iodide uptake and organification are the key features seen in RAI-refractory cancer cells due to the loss of thyroid differentiation features as a result of altered molecular protein signaling pathways that hinder the uptake of 131Iodine in thyroid cancer patients14. Several clinical trials with redifferentiating compounds are being carried out for RAI refractivity and the management of RAI-refractive metastatic, recurrent thyroid cancer5, 14. Considering the overall scenario, we have focused on the expression of pendrin in a chemically induced thyroid cancer model in Wistar rats followed by excess NaI treatment for redifferentiation thereby improving the efficacy of 131I uptake. |
Preclinical models have been used for investigating genetic and epigenetic alterations occurring in thyroid neoplasia5. Hence, in the present study, animals in experimental group 1 (n=12) were initiated with a single i/p DHPN injection for a period of 3 months whereas the control group (n=6) received saline only. Further, all the animals were put for 131I treatment. The 131I uptake by scintigraphy showed thyroid standard uptake values of 10.16±1.6 and 13.13±0.115 in DHPN-treated and control groups respectively at 24h. The decreased uptake of 131I in thyroid cancer indicated cell proliferation with alteration in cell metabolism that hampered in 131I uptake pattern as compared to control animals. |
Further to have more understanding of the reversal effect at the cellular level studies were done with NaI treatment. The NaI is known and tested by Calil-Silveira et al., who demonstrated that acute iodide treatment increased SLC26A4 mRNA content, in both in vitro and in vivo models3, 15. Thus, these earlier research findings were considered and extended in our experiments for in vivo studies using 131I supported by nuclear imaging techniques to study thyroid uptake in NaI-treated Wistar rats. |
Redifferentiation using various epigenetic drugs could imply a valid substitute for targeted therapy in the subgroup of patients with absent iodine uptake by restoring responsiveness to 131I therapy. However, sufficient validation is required for their effectiveness in therapeutic use5. In the present study, the experiments were conducted with a single dose of NaI by intraperitoneal route. Post-NaI treatment showed decreasing patterns at 24h, 48h and 72h in a few animals similar to the control. |
Nonetheless, this could be attributed to an autoregulatory phenomenon known as the 'Wolff-Chaikoff effect' which results from acute iodide excess that transiently impairs thyroid hormone synthesis15. These results do not concur with the excess NaI treated in thyroid cancer animals. This could perhaps be due to the inadequate dose of NaI that was planned and used for the redifferentiation purpose. Hence, it is proposed to optimize the dose of NaI for carrying out in vivo studies in Wistar rats used as thyroid cancer animal models. Overall, it is observed that the probable reason would be variation in animal species, oxidative stress induced by the chemicals, NaI-induced altered response at the cellular level or changes in thyroid hormone levels. Hence, there are multifactorial processes involved that resolute in the variation in the uptake form seen in animals. However, the rising pattern of 131I uptake was also observed in a few DHPN-induced thyroid cancer animals compared to the control, which could perhaps be related to the effect of iodide excess treatment influencing the expression and functionality of pendrin15. |
The alteration in the thyroid gland was further confirmed with histology indicative of a multifocal area of hyperplastic changes in the epithelial lining of the thyroid follicles which was observed in one case with lung metastasis. Furthermore, the immunohistochemical evaluation showed reduced expression of the pendrin at the apical membrane of thyroid follicular cells in DHPN-treated animals compared to the control animals. |
In conclusion, present findings indicate that further research is warranted to verify the role of NaI in the expression of pendrin in conjunction with animal species and age, along with the dose and exposure time of NaI. In addition, the expression and quantification of pendrin at the molecular level could be potentially useful to enhance its utility in the management of RAI refractory human thyroid cancer patients since they represent the main cause of thyroid cancer-related death. Thus, with an optimized dose of NaI, it can serve as a cheap and unique redifferentiation tool in these classes of patients. In this way, redifferentiation would offer a major benefit by avoiding, or at least suspending, long-term systemic treatment in patients who could be readdressed to RAI therapy. Nevertheless, additional studies with sufficient validation are required to confirm the suitability of this approach for therapeutic use in patients. |
Top Figures | Fig. 1:: 131Iodine-scintigraphy scan of the thyroid gland in the control group (Group II) with normal uptake at 24 hours
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| | Fig. 2.: 131I-scin-tigraphy scan of the thyroid gland in the DHPN-treated group (Group I) with reduced uptake at 24 hours.
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| | Fig. 3:: Decreasing 131I uptake in NAI-treated rats at different time points.
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| | Fig. 4:: Increasing 131I uptake in NAI-treated rats at different time points.
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| | Fig. 5:: Representative photomicrograph showing histopathology of the thyrocytes of a male Wistar rat in the control (Group II) with normal follicles (F) with colloidal secretion (C) and lining epithelium (arrow), with H&E (400X). Blood capillaries (zigzag arrow) are seen between thyroid follicles
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| | Fig. 6.: Representative photomicrograph showing thyrocytes of a male Wistar rat in the DHPN-treated group (Group I) with follicular pattern (arrow) of the thyroid cell (F), stained with H&E (400X). Note the infiltration of follicles by congested capillaries (zigzag arrows)
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| | Fig. 7.: Representative photomicrograph showing immunohistochemical expression of thyrocytes with normal pendrin expression (arrow) at the apical membrane in the control group (Group II) (200X)
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| | Fig. 8.: Representative photomicrograph showing immunohistochemical expression of thyrocytes with reduced pendrin expression (arrow) at the apical membrane in the DHPN-treated group (Group I) (200X).
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| | Fig. 9:: Representative photomicrograph showing lung metastasis of thyroid cells (arrow) with infiltration of inflammatory cells and adjacent alveoli (A) (200X).
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