Dichloroacetate Inhibits the Proliferation of a Human Anaplastic Thyroid Cancer Cell Line Via a P53-Independent Pathway

ISSN (Print) 1225-9918 ISSN (Online) 2287-3406 Journal of Life Science 2018 Vol. 28. No. 12. 1469~1476 DOI : https://doi.org/10.5352/JLS.2018.28.12.1469

Dichloroacetate Inhibits the Proliferation of a Human Anaplastic Thyroid Cancer Cell Line via a p53-independent Pathway

Yam Bahadur KC1, Sunil Poudel2, Eon Ju Jeon3, Ho Sang Shon3, Sung June Byun4 and Nam Ho Jeoung2* 1Department of Biomedical Science, Daegu Catholic University, Gyeongsan 38430, Korea 2Department of Pharmaceutical Science and Technology, Daegu Catholic University, Gyeongsan 38430, Korea 3Department of Internal Medicine, Daegu Catholic University School of Medicine, Daegu 42472, Korea 4Animal Biotechnology Division, National Institute of Animal Science (NIAS), RDA, Wanju-gun 55365, Korea Received November 3, 2018 /Revised November 11, 2018 /Accepted November 13, 2018

Occurrence of the Warburg effect in solid tumors causes resistance to cancer chemotherapy, and tar- geting energy metabolisms such as aerobic glycolysis is a potential strategy for alternative treatment. Dichloroacetate (DCA), an inhibitor of pyruvate dehydrogenase kinase (PDK), shifts glucose metabolism from aerobic glycolysis to oxidative phosphorylation (OxPhos) in many cancers. In this study, we investigated the anticancer effect of DCA on a human anaplastic thyroid cancer (ATC) cell line, 8505C. We found that DCA selectively inhibits cell proliferation of the 8505C line but not of a normal thyroid line. In 8505C, the cell cycle was arrested at the G1/S phase with DCA treatment as a result of decreased antiapoptotic proteins such as HIF1α, PDK1, and Bcl-2 and increased proapoptotic proteins such as Bax and p21. DCA treatment enhanced the production of reactive oxygen species which consequently induced cell cycle arrest and apoptosis. Interestingly, DCA treatment not only reduced lactate production but also increased the expression of sodium-iodine symporter, indicating that it re- stores the OxPhos of glucose metabolism and the iodine metabolism of the ATC. Taken together, our findings suggest that PDK inhibitors such as DCA could be useful anticancer drugs for the treatment of ATC and may also be helpful in combination with chemotherapy and radiotherapy.

Key words : Anaplastic thyroid cancer, apoptosis, cell cycle arrest, dichloroacetate, Warburg effect

Introduction ylation (OxPhos) to the aerobic glycolysis. Therefore glucose metabolism shifting from OxPhos to aerobic glycolysis in- Among all endocrine malignancies, thyroid cancer is the creases malignancy of thyroid cancer [6]. most common and accounts for 3.8% of new cancer cases The mitochondrial pyruvate dehydrogenase complex in the United States [22]. The frequently found genetic alter- (PDC) is one of major regulatory enzymes for glucose metab- ations in anaplastic thyroid cancer (ATC) are mutations seen olism and its activity should be tightly regulated. Pyruvate in the TP53 gene, a tumor suppressor gene, in more than dehydrogenase kinases (PDKs) are responsible for the phos- 70% of ATC [14, 29]. There have been reported that p53-defi- phorylation of PDC [10]. So far, four PDK isoenzymes cient primary cells increases the aerobic glycolysis, a (PDK1, PDK2, PDK3, and PDK4) have been identified in Warburg effect, via activation of IKK-NFκB pathway [13] mammalian tissues [4]. In thyroid cancer and other many and the BRAF(V600E) mutant cancer cells significantly in- cancers the expressions of the PDK1 as well as several other crease the glucose uptake compared to that of wild type glycolytic enzymes are up-regulated by c-Myc and HIF-1α BRAF harboring cancer cells [7], suggesting that ATC may [14, 26]. Inhibition of PDKs stimulates the glucose OxPhos also alter the glucose metabolism from oxidative phosphor- rather than aerobic glycolysis, which increases the mitochondrial reactive oxygen species (ROS) which induces the apop- *Corresponding author tosis of cancer cells [3, 16]. Dichloroacetate (DCA) is *Tel : +82-53-850-2563, Fax : +82-53-359-7431 well-known as a PDKs inhibitor for long time [31, 32, 34]. *E-mail : [email protected] This is an Open-Access article distributed under the terms of DCA has engrossed attention as a potentially simple and the Creative Commons Attribution Non-Commercial License economical means to target malignant tumors [3, 12, 31]. (http://creativecommons.org/licenses/by-nc/3.0) which permits Na+/I- symporter (NIS) facilitates the active transport of unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. iodine into the thyroid follicular cells as the crucial first step 1470 생명과학회지 2018, Vol. 28. No. 12 for thyroid hormone synthesis [5, 20]. Since NIS expression (DCA) and 10 mM (NAC). DCA stock solutions were diluted is reduced in thyroid tumors [30], ATC is resistant to radio- in dH2O and NAC stock solutions were diluted in 1 M so- iodine therapy [1, 33]. For the effectiveness of patient treat- dium bicarbonate (pH 7.0). ment, NIS can be a useful marker as better prognosis of thyroid cancer treatment [35]. MTT assay In this study, we examined whether DCA inhibits pro- Cell viability was determined by MTT assay. Briefly, cells liferation of a human anaplastic thyroid cancer, 8505C cell were seeded in 96-well plates at 6×103 cells/well and were line, bearing p53 gene mutation [11]. Using promoter analy- treated with DCA (0, 5, 10, 15, 25, and 30 mM) for various sis and western blot analysis we observed that DCA induces time periods (24, 48, and 72 hr). After treatment, 25 μl MTT the cell cycle arrest and apoptosis via p21 up-regulation, re- assay solution was added at a final concentration of 1 sulted from inhibition of Warburg effect. The results of this mg/ml to each well, and plates were incubated for 2-4 hr study indicate that PDKs are a good pharmacological candi- at 37℃. Thereafter, 100 μl of lysis buffer [50% DMF (N,N-di- date for anaplastic thyroid cancer and PDK inhibitor might methyl formamide), containing 20% SDS] was added to each improve the efficacy of radiotherapy as well. well and incubated overnight at 37℃. The absorbance at 570 nm was measured using a microplate reader. Three in- Materials and Methods dependent experiments were performed.

Cells and cell culture Cell cycle assay N-Thy-ori, a normal thyroid cell line, and 8505C, an ana- Cell cycle analysis was carried out by measuring DNA plastic thyroid cell line, were purchased from ATCC and content of cells using flow cytometry. Cells were incubated cultured in a humidified environment with 5% CO2 at 37℃ with DCA (25 mM) for 24 hr. After treatment, cells were in RPMI-1640 containing 25 mmol/l HEPES (pH 7.4) supple- collected, washed with PBS containing 2% FBS and fixed mented with 10% fetal bovine serum (FBS) (Hyclone) and with cold absolute ethanol for overnight at 4℃. After wash- 1% penicillin and streptomycin. ing twice with ice cold PBS, the cells were incubated with 1 ml PI staining solution. Then, 50 μl of RNase A solution Chemicals, drugs, and antibodies (100 mg/ml RNase A) was added and incubated for 30 min Propidium iodide (PI), dimethylsulfoxide (DMSO), [3-(4,5- at room temperature in the dark. The DNA content of cells dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT), and cell-cycle distribution were analyzed by flow cytometry N-acetyl-L-cysteine (NAC), and sodium dichloroacetate (DCA) (Calibur, BD, USA.) were purchased from Sigma-Aldrich (St. Louis, MO, USA). FBS (Hyclone, lot AYK173682), RPMI-1640 (lot 1643134), Western blot analysis penicillin and streptomycin (lot 1546524) were purchased Cells were treated with DCA for various time periods, from Gibco-Life Technologies. Mouse polyclonal anti-human washed twice with PBS, and lysed for 30 min on ice using Bcl-2 (#2876), rabbit polyclonal anti-human Bax (#2772), cy- WIP cell-lysis buffer (150 mM NaCl, 50 mM Tris (pH 8.0), tochrome c (#4272), cyclin D1 (#2978), caspase-3 (#9661), containing 1% Triton X-100, 1 mM Na2EDTA, 1 mM EGTA, HIF-1α (NB100-123), p-P38 (#9215), and p-Rb (39308) anti- 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, bodies were purchased from Cell Signaling Technology 1 mM Na3VO4, and 1 mg/ml leupeptin). The insoluble cell (Beverly, MA, USA). p21 antibody (#556431) was purchased debris was removed by centrifugation at the 10,000× g for from BD Pharmingen. Antibodies specific to β-actin, GAPDH, 15 min at 4℃. The protein concentrations were determined and horseradish peroxidase-conjugated secondary antibodies using Bradford assay. Proteins were separated by 15% (goat-anti-rabbit IgG, goat anti-mouse IgG) were purchased SDS-PAGE and transferred to a PVDF membrane. After from Santa Cruz Biotechnology (Santa Cruz, CA, USA). blocking with 5% (w/v) non-fat milk and washing with TBST buffer (Tris-buffered saline-containing 0.05% Tween-20), the Drug treatment membranes were incubated overnight at 4℃ with specific For treatments, each drug was added directly to complete primary antibodies (1:1,000), followed by anti-rabbit IgG cell culture media to achieve final concentrations of 25 mM (1:2,000) or anti-mouse IgG (1:2,000) secondary antibodies for Journal of Life Science 2018, Vol. 28. No. 12 1471

1 h at room temperature and then washed again three times DCA treatment (Fig. 1C). We observed that a low dose (10 with TBST buffer. The membranes were incubated with en- mM) of DCA significantly inhibited the growth rate of 8505C hanced chemiluminescence substrate solution (Millipore) for cells within 72 hr of treatment whereas growth inhibitory 2 min according to the manufacturer’s instructions and vi- effect of DCA was further increased at a higher dose (25 sualized (DNR, MicroChemi systems, Jerusalem). mM) (Fig. 1D). These findings suggest that DCA is a strong candidate as a therapeutic drug for human anaplastic thy- p21 promoter assay roid cancer. Cell were seeded in 6-well plates (2×103 cells/well) prior to co-transfection with 1 μg of p21 promoter reporter vector DCA induces the cell cycle at G1 phase in ATC cells and 0.01 μg of the p-CMV-β-galactosidase plasmid vector To elucidate the effect of DCA on the cell cycle in thyroid as an internal control, using lipofectamine 2000 (Invitrogen). cancer cells, we performed flow cytometry analysis of Cells were transfected for 5 hr and waited for 12 hr before DCA-treated 8505C cells using PI staining. DCA (25 mM) the drug treatment. After 24 hr of treatment, cells were lysed significantly enriched the cell populations in G1 and S phas- with passive lysis buffer (Promega). Cell lysates were then es, but reduced the number of cells in G2/M phase (Fig. analyzed with the luciferase reporter assay system following 2A, Fig. 2B). the standard protocol using AB-2270 LuminescencerOcta As shown in Fig. 2C, HIF-1α was down regulated by DCA (ATTO Corporation Japan). Luciferase activities were nor- treatment up to 24 hr. Likewise, PDK1 was also down regu- malized to β-galactosidase activity of the co-transfected lated by DCA. Furthermore Bcl-2, an anti-apoptotic marker, vector. All transfection experiments were repeated in- was down regulated by DCA treatment after 12 hr. Bax, a dependently at least three times. pro-apoptotic protein, was up-regulated by DCA treatment at an early time point. These results suggested that DCA Lactate measurement could alter glucose metabolism and cell survival via HIF-1a. 8505C cells were seeded in 60-mm dishes (2×105 cells/ Similarly, DCA treatment increased the expression of p21 plate) 24 hr prior to the start of the experiment. The cells within 3 hr which persisted up to 24 hr (Fig. 2D). The ex- were washed with HBSS and incubated in RPMI-1640 me- pression of cyclin D1 and phosphorylation of Rb (p-Rb) were dium without FBS, containing the drug (25 mM DCA), for markedly reduced by DCA treatment (Fig. 2D). Taken to- 24 hr. After treatment, 1 ml medium was used for lactate gether, these results indicated that DCA induces cell cycle measurements as described previously [9]. arrest as well as apoptosis in human ATC cells.

Statistical analysis DCA inhibits 8505C cell proliferation by ROS- Results are expressed as mean ± SEM. Statistical sig- mediated cell cycle arrest nificances were evaluated by Student’s t-test. p<0.05 was con- DCA was found to switch the glucose metabolism from sidered significant. aerobic glycolysis to OxPhos in cancer cells, which induces the ROS production [12, 17]. Next, we examined the possi- Results bility that the effect of DCA on cell cycle arrest is dependent on ROS production. One hour pretreatment with N-acetyl Dichloroacetate (DCA) inhibits the proliferation of cysteine (NAC), a ROS scavenger [2], increased the HIF-1a anaplastic thyroid cancer cells expression reduced by DCA treatment (Fig. 3A). The ex- To examine the effect of DCA on different thyroid cell pression of PDK1 and Bcl-2 was enhanced by NAC treat- lines, we checked the growth pattern of 8505C and N-thy-ori ment compared to DCA alone treated cells (Fig. 3A). In addi- cells where 8505C showed an aggressive behavior, as shown tion, cell cycle markers, p21 and cyclin D1, were restored by its rapid growth compared to N-thy-ori (Fig. 1A). The by NAC treatment (Fig. 3B). cell proliferation rate of 8505C was decreased by DCA treat- We also examined whether NAC could release the cell ment in a dose-dependent manner while N-thy-ori was not cycle arrest caused by DCA in 8505C cells. As expected, the (Fig. 1B). MTT assay showed the decreased viability of can- cell cycle arrest by DCA was inhibited by NAC pretreatment cer cells rapidly as compared to the N-thy-ori cells upon (Fig. 3C, Fig. 3D). Phosphorylation of PDHE1α (p-PDHE1α) 1472 생명과학회지 2018, Vol. 28. No. 12

A B

C D

Fig. 1. Growth of thyroid cancer cells and effect of DCA on cell viability. (A) Growth of normal and anaplastic thyroid cancer cells. Cells were grown in 35 mm culture dishes and counted at various time points. *p˂0.05, **p˂0.01 normal thyroid cells vs. 8505C cells (n=3). (B) Cells were treated with indicated concentrations of DCA and counted after 24 hr. *p˂0.05, **p˂0.01 untreated- vs. treated-8505C cells; #p˂0.05, #p˂0.05 untreated- vs. treated- N-Thy-ori cells (n=3). (C) MTT assay was performed in 96-well plates. *p˂0.05, **p˂0.01 treated N-Thy-ori cells vs. treated 8505C cells (n=3). (D) Concentration and time dependent effects of DCA on 8505C cells. Cells survive was measured by MTT analysis. *p˂0.05, **p˂0.01 untreated- vs. treated-8505C cells (n=3).

A B

C D

Fig. 2. Effect of DCA on cell cycle profile and expression of various genes. (A) Representative flow cytometry images (n=3) after PI staining. (B) Quantitative analysis. *p˂0.05 untreated- vs. treated-8505C cells. (C & D) Effect of DCA treatment on expression of genes. The cells were treated with 25 mM DCA for the indicated time points. The expression of proteins was analyzed by Western blotting. Journal of Life Science 2018, Vol. 28. No. 12 1473 was reduced by DCA treatment, which was not rescued by the JNK pathway is not involved in DCA-induced cell cycle NAC pretreatment (Fig. 3E). BRAF (V600E) mutation-in- arrest, at least in 8505C cells (Fig. 3E). duced MAP kinases play a pivotal role in cell proliferation Next, we examined whether DCA-induced ROS could in- of cancers such as thyroid and breast cancers [5]. duce the mitochondrial apoptotic pathway in 8505C cells. Phosphorylation of ERK (p-ERK1/2) was reduced by DCA DCA treatment increased the cytosolic cytochrome C and treatment but was fully recovered by NAC pretreatment cleavage of caspase-3, a pivotal marker of apoptosis (Fig. (Fig. 3E). However, phosphorylation of p38, an induction 3F). NAC pretreatment inhibited the cytochrome C release marker of cell cycle arrest, was dramatically increased by and activation of caspase-3 induced by DCA (Fig. 3F). treatment of DCA. NAC pretreatment partially prevented the DCA-induced p38 activation (Fig. 3E). Phosphorylation p21 promoter activity, lactate production, and NIS of JNK was not altered by DCA treatment, indicating that expression

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C D

E F

Fig. 3. NAC pretreatment reverses the effect of DCA. (A and B) Cells were pre-treated with NAC before one hour of DCA treatment. Protein analysis was done by Western blot for cell signaling (A) and the cell cycle (B). Effect of NAC pre-treatment on cell cycle analysis: representative flow cytometric image (C) and quantitative analysis (D). *p˂0.05 untreated- vs. treated-8505C cells, #p˂0.05 DCA treated- vs. NAC+DCA treated-8505C cells. Effect of NAC pretreatment on DCA-treated cells on MAPK panel (E) and caspases cascade (F). GAPDH was used as a loading control. 1474 생명과학회지 2018, Vol. 28. No. 12

A B C

Fig. 4. DCA effect on p21 promoter activity, lactate production, and NIS expression. (A) The p21 promoter activity was measured after 24 hr of DCA treatment. β-gal assay was used as a control. *p˂0.01 untreated- vs. treated-8505C cells (n=3). (B) Lactate production in cultured medium was measured after DCA treatment for 24 hr. *p˂0.05 untreated- vs. treated-8505C cells (n=3). (C) NIS expression was measured by real time PCR after treatment with DCA for 24 hr. GAPDH was used as a control. *p˂0.01 untreated- vs. treated-8505C cells (n=3).

It has been reported that activation of p53 induces cell PDC is one of the major ROS sources in mitochondria [21]. cycle arrest and apoptosis via activation of p21 and p27 [24]. Mitochondrial ROS induces the intrinsic apoptosis via the However, most ATC cells have a mutant p53 which is not Bax-cyt C-caspase 3 signaling pathway [25]. Similar to these involved in the cell cycle arrest [23]. We examined whether findings DCA treatment increased Bax and cyt C as well DCA could directly increase p21 expression using transient as cleaved caspase 3 levels (Fig. 3). Moreover, pre-treatment transfection. Upon treatment with DCA after transient trans- with NAC, a ROS scavenger, inhibited Bax expression and fection of a p21-luciferase vector into 8505C cells, the pro- caspase 3 cleavage induced by DCA treatment, indicating moter activity of p21 was increased by two folds (Fig. 4A), the effect of DCA occurs in an ROS-dependent manner. suggesting that DCA treatment increased the expression of Earlier studies have shown that p21 is one of the major p21 via a p53-independent manner. cyclin dependent kinase inhibitors (CKIs), which directly in- Subsequently, DCA treatment significantly reduced the hibits the activity of CDKs, thereby leading to cell cycle ar- lactate production in human 8505C cells (Fig. 4B). In addi- rest in the G1 phase [8, 37]. Even though p53 is mutated tion, DCA treatment also increased the expression of NIS in 8505C cells, we observed that DCA treatment increased mRNA by two folds (Fig. 4C). These results suggested that p21expression and suppressed cyclin D1 and p-Rb ex-

DCA altered glucose and iodine metabolism of ATC cells pression (Fig. 2D), which induced cell cycle arrest in the G1 towards that of normal thyroid cells. phase. In addition, DCA treatment directly increased the p21 promoter activity (Fig. 4A). These results indicate that DCA Discussion could induce cell cycle arrest in a p53-independent manner in 8505C cells, which agrees to previous reports [18, 28]. Since the radio-iodine uptake is limited in ATC due to DCA treatment increased the phosphorylation of p38 and reduced NIS levels, radiotherapy is an uneffective treatment inhibited the phosphorylation of ERK, while the phosphor- of ATC [20, 33]. Therefore it is required to develop of an ylation of JNK was not affected (Fig. 3E), indicating that effective method to treat ATC. Activation of glycolytic genes MAPK signaling pathway might be regulated by oxidative by HIF-1a is considered critical for metabolic adaptation in stress in DCA treated 8505C cells. cancer cells to hypoxia through increased Warburg effect. Lactate is the by-product of rapid glycolysis, which occurs PDK inhibition therefore might be an ideal target to treat due to the Warburg effect [19, 36]. Lactate production is as- the malignant tumor by restoration of glucose metabolic sociated with the proliferation and aggressiveness of cancer pathways [12, 26]. ATC treated with DCA showed a decrease cells and poor prognosis [27]. In this study, DCA treatment in HIF-1a expression, leading suppression of the PDK1 ex- significantly reduced the lactate production in 8505C cells pression (Fig. 2C), and also reduced the phosphorylation of (Fig. 4B). In addition, we found that DCA treatment in- PDH-E1α (Fig. 3E), indicating increased PDC activity. Active creased the NIS expression in 8505C cells, suggesting that Journal of Life Science 2018, Vol. 28. No. 12 1475 restoration of OxPhos glucose metabolism could also re- moto, K., Tsuyama, N., Nakamura, N. and Akiyama, M. stored the iodine metabolism in ATC. Conclusively, PDK can 1994. Establishment of 2 human thyroid-carcinoma cell-lines (8305c, 8505c) bearing p53 gene-mutations. Int. J. Oncol. 4, be used as a good target for development of anti-ATC drug 583-586. by restoration of glucose metabolism as well as radio-iodine 12. Jeoung, N. H. 2015. Pyruvate dehydrogenase kinases: therapy for anaplastic thyroid cancers. Therapeutic targets for diabetes and cancers. Diabetes Metab. J. 39, 188-197. Acknowledgement 13. Kawauchi, K., Araki, K., Tobiume, K. and Tanaka, N. 2008. p53 regulates glucose metabolism through an IKK-NF- kappaB pathway and inhibits cell transformation. Nat. Cell This research was partially supported by the Basic Science Biol. 10, 611-618. Research Program through the National Research Founda- 14. Kim, J. W., Tchernyshyov, I., Semenza, G. L. and Dang, C. tion of Korea, which is funded by the Ministry of Education V. 2006. HIF-1-mediated expression of pyruvate dehydroge- (NRF-2014R1A1A2056130) (N.H.J.). nase kinase: a metabolic switch required for cellular adaptation to hypoxia. Cell Metab. 3, 177-185. 15. Manzella, L., Stella, S., Pennisi, M. S., Tirrò, E., Massimino, References M., Romano, C., Puma, A., Tavarelli, M. and Vigneri, P. 2017. New insights in thyroid cancer and p53 family 1. Are, C. and Shaha, A. R. 2006. Anaplastic thyroid carcinoma: proteins. Int. J. Mol. Sci. 18, 1325. biology, pathogenesis, prognostic factors, and treatment 16. Lee, M. and Yoon, J. H. 2015. Metabolic interplay between approaches. Ann. Surg. Oncol. 13, 453-464. glycolysis and mitochondrial oxidation: The reverse 2. Atkuri, K. R., Mantovani, J. J., Herzenberg, L. A. and Her- Warburg effect and its therapeutic implication. World J. Biol. zenberg, L. A. 2007. N-Acetylcysteine-a safe antidote for cys- Chem. 6, 148-161. teine/glutathione deficiency. Curr. Opin. Pharmacol. 7, 355- 17. Liou, G. Y. and Storz, P. 2010. Reactive oxygen species in 359. cancer. Free Radic. Res. 44, 479-496. 3. Bonnet, S., Archer, S. L., Allalunis-Turner, J., Haromy, A., 18. Lodygin, D., Menssen, A. and Hermeking, H. 2002. Induction Beaulieu, C., Thompson, R., Lee, C. T., Lopaschuk, G. D., of the Cdk inhibitor p21 by LY83583 inhibits tumor cell pro- Puttagunta, L., Bonnet, S., Harry, G., Hashimoto, K., Porter, liferation in a p53-independent manner. J. Clin. Invest. 110, C. J., Andrade, M. A., Thebaud, B. and Michelakis, E. D. 1717-1727. 2007. A mitochondria-K+ channel axis is suppressed in can- 19. Lopez-Lazaro, M. 2008. The warburg effect: why and how cer and its normalization promotes apoptosis and inhibits do cancer cells activate glycolysis in the presence of oxygen? cancer growth. Cancer Cell 11, 37-51. Anticancer Agents Med. Chem. 8, 305-312. 4. Bowker-Kinley, M. M., Davis, W. I., Wu, P., Harris, R. A. 20. Mian, C., Lacroix, L., Alzieu, L., Nocera, M., Talbot, M., and Popov, K. M. 1998. Evidence for existence of tissue-spe- Bidart, J. M., Schlumberger, M. and Caillou, B. 2001. Sodium cific regulation of the mammalian pyruvate dehydrogenase iodide symporter and pendrin expression in human thyroid complex. Biochem. J. 329 (Pt 1), 191-196. tissues. Thyroid 11, 825-830. 5. Carrasco, N. 1993. Iodide transport in the thyroid gland. 21. Muntean, D. M., Sturza, A., Danila, M. D., Borza, C., Duicu, Biochim. Biophys. Acta. 1154, 65-82. O. M. and Mornos, C. 2016. The role of mitochondrial re- 6. Coelho, R. G., Fortunato, R. S. and Carvalho, D. P. 2018. active oxygen species in cardiovascular injury and pro- Metabolic reprogramming in thyroid carcinoma. Front. tective strategies. Oxid. Med. Cell. Longev. 2016, 8254942. Oncol. 8, 82 22. Nguyen, Q. T., Lee, E. J., Huang, M. G., Park, Y. I., Khullar, 7. Falck Miniotis, M., Arunan, V., Eykyn, T. R., Marais, R., A. and Plodkowski, R. A. 2015. Diagnosis and treatment of Workman, P., Leach, M. O. and Beloueche-Babari, M. 2013. patients with thyroid cancer. Am. Health. Drug Benefits 8, MEK1/2 inhibition decreases lactate in BRAF-driven human 30-40. cancer cells. Cancer Res. 73, 4039-4049. 23. Nikiforov, Y. E. 2004. Genetic alterations involved in the 8. Gartel, A. L. and Tyner, A. L. 2002. The role of the cyclin-de- transition from well-differentiated to poorly differentiated pendent kinase inhibitor p21 in apoptosis. Mol. Cancer Ther. and anaplastic thyroid carcinomas. Endocr. Pathol. 15, 319- 1, 639-649. 327. 9. Gutmann, I. and Wahlefeld, A. W. 1974. Lactate determi- 24. Oren, M. 2003. Decision making by p53: life, death and nation with lactate dehydrogenase and NAD, pp. 1416-1468. cancer. Cell Death. Differ. 10, 431-442. In: Methods of Enzymatic Analysis, Bergmeyer HU (2ed.), 25. Pang, Y., Qin, G., Wu, L., Wang, X. and Chen, T. 2016. Arte- Academic Press, Inc.: New York, USA. sunate induces ROS-dependent apoptosis via a Bax-medi- 10. Harris, R. A., Bowker-Kinley, M. M., Huang, B. and Wu, ated intrinsic pathway in Huh-7 and Hep3B cells. Exp. Cell. P. 2002. Regulation of the activity of the pyruvate de- Res. 347, 251-260. hydrogenase complex. Adv. Enzyme Regul. 42, 249-259. 26. Papandreou, I., Cairns, R. A., Fontana, L., Lim, A. L. and 11. Ito, T., Seyama, T., Hayashi, Y., Dohi, K., Mizuno, T., Iwa- Denko, N. C. 2006. HIF-1 mediates adaptation to hypoxia 1476 생명과학회지 2018, Vol. 28. No. 12

by actively downregulating mitochondrial oxygen con- ple myeloma cells and increases sensitivity to bortezomib. sumption. Cell Metab. 3, 187-197. Br. J. Cancer 108, 1624-1633. 27. Pertega-Gomes, N., Felisbino, S., Massie, C. E., Vizcaino, J. 32.Shahrzad, S., Lacombe, K., Adamcic, U., Minhas, K. and R., Coelho, R., Sandi, C., Simoes-Sousa, S., Jurmeister, S., Coomber, B. L. 2010. Sodium dichloroacetate (DCA) reduces Ramos-Montoya, A., Asim, M., Tran, M., Oliveira, E., Lobo apoptosis in colorectal tumor hypoxia. Cancer Lett. 297, 75- da Cunha, A., Maximo, V., Baltazar, F., Neal, D. E. and 83. Fryer, L. G. 2015. A glycolytic phenotype is associated with 33. Spitzweg, C., Harrington, K. J., Pinke, L. A., Vile, R. G. and prostate cancer progression and aggressiveness: a role for Morris, J. C. 2001. Clinical review 132: The sodium iodide monocarboxylate transporters as metabolic targets for symporter and its potential role in cancer therapy. J. Clin. therapy. J. Pathol. 236, 517-530. Endocrinol. Metab. 86, 3327-3335. 28. Qiu, X., Forman, H. J., Schonthal, A. H. and Cadenas, E. 34. Stacpoole, P. W., Kurtz, T. L., Han, Z. and Langaee, T. 2008. 1996. Induction of p21 mediated by reactive oxygen species Role of dichloroacetate in the treatment of genetic mitochon- formed during the metabolism of aziridinylbenzoquinones drial diseases. Adv. Drug Deliv. Rev. 60, 1478-1487. by HCT116 cells. J. Biol. Chem. 271, 31915-31921. 35. Tavares, C., Coelho, M. J., Eloy, C., Melo, M., da Rocha, A. 29. Quiros, R. M., Ding, H. G., Gattuso, P., Prinz, R. A. and G., Pestana, A., Batista, R., Ferreira, L. B., Rios, E., Selmi- Xu, X. 2005. Evidence that one subset of anaplastic thyroid Ruby, S., Cavadas, B., Pereira, L., Sobrinho-Simões, M. and carcinomas are derived from papillary carcinomas due to Soares, P. 2018. NIS expression in thyroid tumors, relation BRAF and p53 mutations. Cancer 103, 2261-2268. with prognosis clinicopathological and molecular features. 30. Ringel, M. D., Anderson, J., Souza, S. L., Burch, H. B., Tam- Endocr. Connect. 7, 78-90. bascia, M., Shriver, C. D. and Tuttle, R. M. 2001. Expression 36. Warburg, O. 1956. On the origin of cancer cells. Science 123, of the sodium iodide symporter and thyroglobulin genes 309-314. are reduced in papillary thyroid cancer. Mod. Pathol. 14, 289- 37. Zhang, Z., He, H., Chen, F., Huang, C. and Shi, X. 2002. 296. MAPKs mediate S phase arrest induced by vanadate 31. Sanchez, W. Y., McGee, S. L., Connor, T., Mottram, B., through a p53-dependent pathway in mouse epidermal Wilkinson, A., Whitehead, J. P., Vuckovic, S. and Catley, C141 cells. Chem. Res. Toxicol. 15, 950-956. L. 2013. Dichloroacetate inhibits aerobic glycolysis in multi-

초록：Dichloroacetate의 p53 비의존적 경로를 통한 인간 역분화 갑상선 암세포주의 성장억제 효과

얌 바하더 케이씨1․수닐 포우델2․전언주3․손호상3․변승준4․정남호2* (1대구가톨릭대학교 의생명과학과, 2대구가톨릭대학교 제약산업공학과, 3대구가톨릭대학교 의과대학 내과, 4농촌 진흥청 국립축산과학원 동물바이오공학과)

Warburg 효과의 발생은 고형암에서 화학적 항암제의 내성을 발생시킨다. 따라서 호기성 해당과정과 같은 에너 지 대사과정은 암 치료의 중요한 표적으로 알려져 있다. Pyruvate dehydrogenase kinase (PDK) 활성 억제물질로 알려진 dichloroacetate (DCA)는 많은 암세포에서 포도당의 호기성 해당과정을 산화적인산화 과정으로 전환시킬 수 있음이 보고되었다. 이 연구는 치료가 매우 어렵다고 알려진 인간 역분화 갑상선 암세포주인 8505C의 성장에 미치는 DCA효과를 조사하였다. DCA는 정상 갑상선 세포주의 성장에는 영향을 주지 않은 반면 8505C 세포주의 성장을 특이적으로 저해하였다. DCA의 처리에 의해 8505C 세포주는 HIF1α, PDK1, Bcl-2와 같은 항-세포자살 관 련 단백질들의 발현이 감소하고, Bax와 p21과 같은 세포자살 유도 단백질과 세포주기 억제 단백질의 증가로 인하 여 세포주기 정지와 세포자살 유도에 의해 성장이 억제되었다. 이런 세포의 변화는 DCA 처리에 의한 활성산소족 의 생산이 증가하고, 포도당 대사가 호기성 해당과정에서 산화적인산화 과정으로 전환되었기 때문이란 것을 확인 하였다. 흥미롭게도, DCA는 포도당 대사과정의 변화뿐만 아니라 sodium/iodine symporter (NIS)의 발현양도 증가시킨다는 것을 확인하였다. 이 연구의 결과로 PDK 활성 저해제는 치료하기 힘든 역분화 갑상선 암 치료제로 이용할 수 있고, 또한 역분화 갑상선 암에 대한 방사능 치료의 효능을 높일 수 있을 것으로 기대된다.