ANTICANCER RESEARCH 34: 4857-4868 (2014)

Tunicamycin Induces Paraptosis Potentiated by Inhibition of BRAFV600E in FRO Anaplastic Thyroid Carcinoma Cells

SI HYOUNG KIM1, HAE-YOUNG SHIN2, YONG-SUN KIM2,3, JUN GOO KANG1, CHUL SIK KIM1, SUNG-HEE IHM1, MOON GI CHOI1, HYUNG JOON YOO1 and SEONG JIN LEE1

1Division of Endocrinology and Metabolism, Department of Internal Medicine, College of Medicine, Hallym University, Chuncheon, Republic of Korea; 2Ilsong Institute of Life Science, Gyeonggi-Do, Republic of Korea; 3Department of Microbiology, College of Medicine, Hallym University, Chuncheon, Republic of Korea

Abstract. Background/Aim: The aim of the present study Programmed cell includes , , was to elucidate whether tunicamycin (TM) induces paraptosis and mitotic catastrophe (3). Paraptosis has paraptosis as a subroutine in anaplastic thyroid characteristic features including extensive cytoplasmic carcinoma (ATC) cells. Materials and Methods: 8505C, vacuolation derived from (ER) and/or CAL62 and FRO cells were used. After treatment of TM, cell mitochondrial swelling without detection of apoptotic survival and morphology were investigated. The effect of the hallmarks such as activation, plasma membrane BRAFV600E inhibitor PLX4032 in combination with TM was blebbing and apoptotic bodies (3). Epidermal growth factor, evaluated. Results: In FRO cells, TM induced paraptosis glucocorticoid and calcium influx induce paraptosis in many characteristic of cytoplasmic vacuolation and endoplasmic cells (4-7). Recently, it was reported that γ-tocotrienol reticulum (ER) swelling, which was not associated with induces paraptosis in colon cells (8). However, caspase activation and ER stress. TM-induced paraptosis whether paraptosis is defined as cell death subroutine in was ameliorated by pre-treatment with the translation thyroid cancer has not been investigated. inhibitor cycloheximide, while it was accelerated by pre- Tunicamycin (TM), a naturally-occurring antibiotic, treatment with the inhibitor MG132. PLX4032 blocks the biosynthesis of N-linked oligosaccharides in cells augmented TM-induced paraptosis. Conclusion: TM induces (9). TM induces ER stress through accumulation of paraptosis relevant to de novo protein synthesis and misfolded proteins by interfering with protein folding within proteasomal activity, and inhibition of BRAFV600E the ER lumen (9). Although TM results in apoptosis and potentiates TM-induced paraptosis in FRO cells harboring autophagy, whether TM leads to paraptosis has not been BRAFV600E. evaluated (10, 11). BRAF, an isoform of RAF kinases, plays a pivotal role in Anaplastic thyroid carcinoma (ATC) is a high-grade regulating survival, proliferation and differentiation in malignant neoplasm of the thyroid gland owing to response to various stimuli, and BRAF mutation frequently extrathyroidal invasion and distant metastasis (1, 2). occurs in thyroid cancer (12, 13). The most common BRAF Clinically substantial improvement in the survival rate of mutation is the T1799A transversion causing a glutamic acid ATC has not been achieved despite multi-modal strategies for valine (BRAFV600E), and the average frequency of and new therapeutic agents are under exploration (1, 2). BRAFV600E was reported to be 44% in papillary thyroid carcinoma and 24% in ATC (13, 14). In this regard, it was reported that PLX4032 selectively inhibits proliferation, migration and invasion of human ATC cells harboring mutant Correspondence to: Professor Seong Jin Lee, M.D., Ph.D., Division BRAFV600E (15). of Endocrinology and Metabolism, Department of Internal In the present study, our results demonstrate, for the first Medicine, College of Medicine, Hallym University, Chuncheon time, that TM induces paraptosis characteristic of cytoplasmic 200-704, Republic of Korea. Tel: +82 313803700, Fax: +82 vacuolation, ER swelling, retention of plasma membrane 313833768, e-mail: [email protected] integrity, lack of apoptotic bodies and independence of caspase Key Words: Anaplastic thyroid carcinoma, paraptosis, tunicamycin, in FRO cells, a subtype of ATC cells. Moreover, our results BRAF. indicate that inhibition of BRAFV600E augments TM-induced

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paraptosis in FRO cells harboring BRAFV600E. Considering that raised against uncleaved and cleaved caspase-3, binding ATC is resistant to most therapeutic drugs, an understanding of immunoglobulin protein (Bip), CCAAT/enhancer-binding protein- TM-induced paraptosis may be significant for the development homologous protein (CHOP), phospho-PERK (Thr-980) and IRE1 were purchased from Cell Signaling Technology (Danvers, MA, USA), of ‘new-concept’ therapeutic agents in human ATC. while primary antibodies raised against β-actin, calnexin and ubiquitin were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All Materials and Methods other reagents were obtained from Sigma unless otherwise stated.

Materials. TM, z-VAD-fmk, cycloheximide (CHX) and MG132 were Cell culture. For experiments, ATC cell lines of 8505C, CAL62 and purchased from Sigma (St. Louis, MO, USA). PLX4032 was obtained FRO cells were used. 8505C and CAL62 cells were purchased from from Selleck Chemicals (Houston, TX, USA). Primary antibodies Deutsche Sammlung von Mikroorganismen und Zellkulturen

4858 Kim et al: Tunicamycin Induces Paraptosis in FRO Cells

Figure 1. continued

4859 ANTICANCER RESEARCH 34: 4857-4868 (2014)

Figure 1. The effect of TM on survival and morphology of ATC cells. A-C: 8505C, CAL62 and FRO cells were treated with TM at 1, 5 and 10 μg/ml for 24 and 48 h. Cell viability (A) and the percentages of dead (B) as well as vacuolated cells (C) were measured using the CCK-8 assay, trypan blue assay and light microscope, respectively. D: FRO cells were treated with TM at 10 μg/ml for 48 h and the ER was immunostained with the calnexin antibody. DAPI was used to label nucleus. E: The ultrastructure of FRO cells showing extensive cytoplasmic vacuolation after treatment with TM at 10 μg/ml for 48 h was observed by transmission electron microscopy. Arrows indicate the ribosomes aligned with the ER membrane in control cells and arrowheads indicate the swollen ER membrane without ribosome in TM-treated cells. All experiments were performed in triplicate. Data are expressed as mean±S.E. *p<0.05 vs. each matched control.

(DSMZ GmbH, Braunschweig, Germany). FRO cells, authenticated supplemented with 10% heat-inactivated FBS and 1% as shown previously, were provided by Professor Young Joo Park streptomycin/penicillin. Cells received fresh medium at regular (Division of Endocrinology and Metabolism, Seoul National intervals. Treatments and experiments were performed using cells University, Republic of Korea) (16). 8505C and FRO cells harbored that were 50 to 70% confluent. the mutated BRAFV600E and CAL62 cells harbored the wild-type BRAF. 8505C and CAL62 cells were grown in Dulbecco’s Modified Cell viability assay. Cell viability was determined by the cell Eagle’s Medium supplemented with 10% heat-inactivated fetal counting kit-8 (CCK-8) assay using the corresponding Kit (Dojindo bovine serum (FBS) and 1% streptomycin/penicillin. FRO cells laboratories, Kumamoto, Japan). Cells (5×103/100 μl) in each well were grown in Roswell Park Memorial Institute-1640 medium on 96-well plates were incubated for overnight and treated for an

4860 Kim et al: Tunicamycin Induces Paraptosis in FRO Cells

Figure 2. The dependence of TM-induced cell death on caspase activation in FRO cells. A-D: Cells were pre-treated with z-VAD-fmk (30 μM, 1 h) before treatment of TM at 10 μg/ml for 48 h. Uncleaved (inactivated, 30 kDa) and cleaved (activated, 17/19 kDa) caspase-3 protein levels (A), cell viability (B) and the percentages of dead (C) as well as vacuolated cells (D) were measured. All experiments were performed in triplicate. The blots are representative of independent experiments. Data are expressed as mean±S.E. *p<0.05 vs. control.

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4862 Kim et al: Tunicamycin Induces Paraptosis in FRO Cells

Figure 3. The relationship of ER stress with TM-induced paraptosis in FRO cells. A: Cells were treated with TM at 1, 5 and 10 μg/ml for 48 h and at 10 μg/ml for 24 and 48 h. The mRNA levels of BIP and CHOP and the protein levels of Bip, CHOP, phospho-PERK and IRE1 were measured. B-D: Cells were transfected with CHOP siRNA prior to treatment of TM at 10 μg/ml for 48 h. Cell viability (B) and the percentages of dead (C) as well as vacuolated cells (D) were measured. All experiments were performed in triplicate. The blots are representative of independent experiments. Data are expressed as mean±S.E. *p<0.05 vs. control.

additional 4 h at 37˚C. Absorbance was measured at 450 nm using phosphate-buffered saline (PBS) on ice for 2 h. The cell pellets were a spectrophotometer (Molecular Devices, Palo Alto, CA, USA). fixed on ice for 1.5 h with 1% osmium tetroxide buffered with 0.1 M PBS and rinsed three times with 0.1 M PBS. The fixed cell Trypan blue assay. Cells (1×104/500 μl) in each well on 12-well pellets were dehydrated by successive treatments at increasing plates were incubated and mixed with trypan blue dye at 37˚C. concentrations of ethanol and propylene oxide for 15 min and Stained cells were counted using a hemocytometer. finally embedded in Epon 812 (Electron Microscopy Sciences, Hatfield, PA, USA). Ultra-thin sections (75 nm) were cut in an Vacuolated cell counting. Vacuolated cells were counted using a RMC MTXL ultramicrotome (Tucson, Arizona, USA). The sections method similar to the previous study (17). Light micrographs were were collected on grids, double stained with uranyl acetate and lead obtained from different fields and number of vacuolated cells having citrate and examined with a transmission electron microscope (JEM- the total -occupying area more than 50% of cytoplasmic 1011, JEOL, Japan). area was counted in at least 300 cells for each condition. Western blotting. Cells were lysed in lysis buffer containing 1ⅹ Immunofluorescence. Cells were fixed, permeabilized with acetone protease inhibitor cocktail and 1ⅹ phophatase inhibitor cocktail set V for 10 min at room temperature and blocked. Cells were incubated (Calbiochem, La Jolla, CA, USA). Western blotting was performed with the calnexin antibody overnight at 4˚C and then with FITC- using specific primary antibodies and horseradish peroxidase- conjugated IgG secondary antibody for 1 h at room temperature. conjugated anti-rabbit or anti-mouse secondary antibodies. Bands The secondary antibody was purchased from Vector Laboratories were detected using ECL or ECL Plus Western Blotting Detection (Burlingame, CA, USA). Cells were mounted and identified by System (Amersham Biosciences, Buckinghamshire, UK). β-actin was flexible confocal microscope LSM 700 (Carl Zeiss MicroImaging used as positive control. GmbH, Oberkochen, Germany). Reverse transcription-polymerase chain reaction (RT-PCR). Total RNA Transmission electron microscopy. Cells were fixed in 4% was isolated using TRI Reagent (Molecular Research Center, paraformaldehyde and 2.5% gluteraldehyde buffered with 0.1 M Cincinnati, OH, USA) according to the manufacturer’s protocol.

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Figure 4. continued

cDNA was synthesized using ImProm-II Reverse Transcription System thermal cycler. The number of cycles (optimized in a preliminary study (Promega, Madison, WI, USA). Amplification of the resulting cDNA the aim of which was to determine the exponential range of was conducted. The following primers were used: BIP, 5’- amplification for each gene) was 22 for BIP, CHOP and β-ACTIN. The ATGAGGACCTGCAAGAG-3’ and 5’-TCCTCCTCAGTCAGCC-3’; amplified products were analyzed by 2% agarose gel electrophoresis. CHOP, 5’-GCACCTCCCAGAGCCCTCACTCTCC-3’ and 5’- The mRNA expression of BIP and CHOP was compared to the mRNA GTCTACTCCAAGCCTTCCCCCTGCG-3’; β-ACTIN, 5’-CAAGA expression of β-actin (normalization control). GTGGCCACGGCTGCT-3’ and 5’-TCCTTCGCATCCTG TCGGCA- 3’. Reactions were successively incubated at 95˚C for 15 s, at 60˚C Transfection of small interfering RNA (siRNA). The human CHOP for 15 s and at 72˚C for 15 s using a GeneAmp PCR System 9700 siRNA and the scramble nonsense siRNA (Scramble) were

4864 Kim et al: Tunicamycin Induces Paraptosis in FRO Cells

Figure 4. The relevance of protein synthesis and proteasomal activity to TM-induced paraptosis in FRO cells. A-C: Cells were pre-treated with CHX (100 μM, 1 h) before treatment of TM at 10 μg/ml for 48 h. Cell viability (A) and the percentages of dead (B) as well as vacuolated cells (C) were measured. D-G: Cells were pre-treated with MG132 (10 μM, 1 h) and/or CHX (100 μM, 1 h) prior to treatment of TM at 10 μg/ml for 48 h. The ubiquitinated protein levels (D), cell viability (E) and the percentages of dead (F) as well as vacuolated cells (G) were measured. All experiments were performed in triplicate. The blots are representative of independent experiments. Data are expressed as mean±S.E. *p<0.05 vs. control. **p<0.05 vs. TM alone-treated cells. ***p<0.05 vs. MG132-pretreated and TM-treated cells without CHX.

purchased from Cell Signaling Biotechnology and Bioneer Statistical analysis. All data are expressed as mean±standard error (Daejeon, Republic of Korea). Cells were transfected with siRNA (S.E.). Data were analyzed by unpaired Student’s t-test or ANOVA using Lipofectamine 2000 Reagent (Invitrogen, Carlsbad, CA, USA) as appropriate. A p-value less than 0.05 was considered to be according to manufacturer’s protocol. Transfection efficiency was statistically significant. All analyses were performed using the SPSS tested by western blotting. version 21.0 (SPSS, Chicago, IL, USA).

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Figure 5. The influence of inhibition of BRAFV600E on TM-induced paraptosis in FRO cells. A-C: Cells were co-treated with TM at 10 μg/ml and PLX4032 at 10 μM for 48 h. Cell viability (A) and the percentages of dead (B) as well as vacuolated cells (C) were measured. All experiments were performed in triplicate. Data are expressed as mean±S.E. *p<0.05 vs. control. **p<0.05 vs. TM alone-treated cells.

Results 1B). The percentage of cytoplasmic vacuoles-containing cells greatly increased in FRO cells in a time- and concentration- TM causes cytoplasmic vacuolation cell death in FRO cells. dependent manner, while cytoplasmic vacuoles were very In 8505C, CAL62 and FRO cells, to evaluate the effect of rarely found in 8505C and CAL62 cells exposed to TM, even TM on cell survival, cells were treated with TM at 1, 5 and at 10 μg/ml (Figure 1C). 10 μg/ml for 24 and 48 h. After treatment, the viability of In TM-treated FRO cells showing extensive cytoplasmic FRO cells decreased to a lesser degree than that of 8505C vacuolation, the ER was immunostained with the ER-specific and CAL62 cells in a time- and concentration-dependent marker calnexin and the cytoplasmic vacuoles were seen manner (Figure 1A). The percentage of dead cells increased through the dilated ERs (Figure 1D). The inflated cells with in FRO cells less compared to that of 8505C and CAL62 cytoplasmic vacuoles and dilated ERs were detected by cells in a time- and concentration-dependent manner (Figure transmission electron microscopy. The cytoplasmic vacuoles

4866 Kim et al: Tunicamycin Induces Paraptosis in FRO Cells appeared to be swollen ER cisternae and pushed the inhibitor MG132 prior to treatment of TM at 10 μg/ml for and other cellular organelles closer to the nucleus (Figure 1E). 48 h. In control experiments, MG132 blocked proteasomal Ribosomes were aligned with the ER membrane in control activity thereby elevating ubiquitinated protein levels, cells, but shedding of ribosomes on the swollen ER membrane which was mitigated by CHX (Figure 4D). In MG132-pre- was exhibited in TM-treated cells. Plasma membrane integrity treated and TM-treated cells, compared with TM alone- was retained and the apoptotic body was not visible. Overall, treated cells, cell viability was further diminished (Figure these findings demonstrate that cytoplasmic vacuoles induced 4E) and the percentages of dead (Figure 4F) as well as by TM are derived from dilated ER cisternae in FRO cells. vacuolated cells (Figure 4G) were further enhanced, an action attenuated by CHX. Cytoplasmic vacuolation cell death resulting from TM is paraptosis independent of caspase activation in FRO cells. In Hindrance of BRAFV600E potentiates TM-induced paraptosis this study, since TM led to cell death accompanied by in FRO cells harboring BRAFV600E. FRO cells harbor the cytoplasmic vacuolation and ER swelling only in FRO cells, mutated BRAFV600E and thus we documented the role of not 8505C and CAL62 cells, all subsequent experiments were BRAFV600E in TM-induced paraptosis. First, when cells were performed in FRO cells. To explore whether TM-induced treated with the BRAFV600E inhibitor PLX4032 at 10 μM for cytoplasmic vacuolation cell death depends on caspase 48 h, cell viability decreased (Figure 5A) and the percentage activation, cells were pre-treated with the broad-spectrum of dead cells increased (Figure 5B). However, the percentage apoptosis inhibitor z-VAD-fmk before treatment of TM at of vacuolated cells was not changed (Figure 5C). Next, cells 10 μg/ml for 48 h. In TM-treated cells, cleaved (activated) were co-treated with TM at 10 μg/ml and PLX4032 at 10 μM caspase-3 protein levels were not detected regardless of pre- for 48 h. In cells co-treated with TM and PLX4032, compared treatment with z-VAD-fmk (Figure 2A). Moreover, cell with cells treated with TM alone, cell viability further viability (Figure 2B) and the percentages of dead (Figure 2C) decreased (Figure 5A), while the percentages of dead (Figure as well as vacuolated cells (Figure 2D) were not changed by 5B) and vacuolated cells (Figure 5C) further increased. pre-treatment with z-VAD-fmk, indicating caspase-independent cell death. Taken together, our results suggest that TM induces Discussion paraptosis characteristic of cytoplasmic vacuolation, ER swelling, retention of plasma membrane integrity, lack of TM causes cell death by activating intrinsic apoptosis and apoptotic bodies and independence of caspase in FRO cells. enhances autophagy-mediated cell death (10, 11). Recently, it was reported that γ-tocotrienol induces paraptosis in colon TM-induced paraptosis is not related to ER stress in FRO cancer cells (8). However, whether paraptosis is defined as a cells. Since TM is an ER stress inducer, we examined the cell death sub-routine in thyroid cancer has not been elucidated. relationship of ER stress with TM-induced paraptosis (9). The present study demonstrated for the first time that TM First, when cells were treated with TM at 1, 5 and 10 μg/ml induces paraptosis characteristic of cytoplasmic vacuolation, for 48 h and at 10 μg/ml for 24 and 48 h, the mRNA levels ER swelling, retention of plasma membrane integrity, lack of of BIP and CHOP and the protein levels of Bip, CHOP, apoptotic body and independence of caspase in FRO cells, a phospho-PERK and IRE1 were multiplied (Figure 3A). subtype of ATC cells. In view of therapeutic implications, our However, as cells were transfected with CHOP siRNA prior results may offer a ‘new-concept’ of paraptosis as a novel to treatment of TM at 10 μg/ml for 48 h (Figure 3B), cell tumor-killing process for the treatment of human ATC, which is viability (Figure 3C) and the percentages of dead (Figure refractory to conventional therapies. However, the development 3D) as well as vacuolated cells (Figure 3E) were not altered of a TM derivative that is able to effectively activate paraptosis by pre-treatment with CHOP siRNA. without serious side effect should be warranted. ER stress leads to ER swelling that is capable of TM-induced paraptosis is relevant to de novo protein producing cytoplasmic vacuolation. Cytoplasmic vacuolation synthesis and proteasomal activity in FRO cells. To clarify without ER stress is able to up-regulate the expression of ER whether protein synthesis participates in TM-induced stress-related genes, insisting that both cytoplasmic paraptosis, cells were pre-treated with the translation vacuolation and the overexpression of ER stress-related inhibitor CHX before treatment of TM at 10 μg/ml for 48 h. genes are not specific findings (9, 18). On this regard, it was In CHX-pre-treated and TM-treated cells, compared with TM reported that results in ER stress as well as alone-treated cells, cell viability was elevated as shown in cytoplasmic vacuolation and ER stress does not participate Figure 4A, while the percentages of dead (Figure 4B) and in the formation of cytoplasmic vacuoles by paclitaxel (18). vacuolated cells (Figure 4C) were reduced. TM is a well-known ER stress inducer and thus we examined To verify whether proteasomal activity takes part in TM- the relationship of ER stress with TM-induced paraptosis (9). induced paraptosis, cells were pre-treated with the proteasome As expected, after treatment of TM, the expression of ER

4867 ANTICANCER RESEARCH 34: 4857-4868 (2014) stress markers increased, while knockdown of CHOP, an 5 Fombonne J, Reix S, Rasolonjanahary R, Danty E, Thirion S, effector of ER stress, did not alter cell viability and the Laforge-Anglade G, Bosler O, Mehlen P, Enjalbert A and percentages of dead as well as vacuolated cells. These results Krantic S: Epidermal growth factor triggers an original, caspase- independent pituitary cell death with heterogeneous phenotype. suggest that ER stress is not the major event mediated in Mol Biol Cell 15: 4938-4948, 2004. TM-induced paraptosis in FRO cells. 6 Fombonne J, Padrón L, Enjalbert A, Krantic S and Torriglia A: A Paraptosis requires de novo protein synthesis and the novel paraptosis pathway involving LEI/L-DNasell for EGF- ubiquitin-proteasome system, responsible for protein induced cell death in somato-lactotrope pituitary cells. Apoptosis degradation, can be activated during paraptosis (18, 19). In 11: 367-375, 2006. the present study, the translation inhibitor CHX ameliorated 7 Valamanesh F, Torriglia A, Savoldelli M, Gandolphe C, Jeanny the changes in cell viability and the percentages of dead as JC, BenEzra D and Behar-Cohen F: Glucocorticoids induce retinal toxicity through mechanisms mainly associated with well as vacuolated cells under condition of TM with or paraptosis. Mol Vis 13: 1746-1757, 2007. without the proteasome inhibitor MG132, indicating that 8 Zhang JS, Li DM, Ma Y, He N, Gu Q, Wang FS, Jiang SQ, Chen TM-induced paraptosis is relevant to de novo protein BQ and Liu JR: γ-Tocotrienol induces paraptosis-like cell death in synthesis and proteasomal activity. human colon carcinoma SW620 cells. PLoS One 8: e57779, 2013. FRO cells bear the mutated BRAFV600E gene and thus we 9 Ron D: Translational control in the endoplasmic reticulum stress documented the role of BRAFV600E in TM-induced paraptosis. response. J Clin Invest 110: 1383-1388, 2002. Intriguingly, the BRAFV600E inhibitor PLX4032 potentiated 10 Yorimitsu T, Nair U, Yang Z and Klionsky DJ: Endoplasmic reticulum stress triggers autophagy. J Biol Chem 281: 30299- TM-induced paraptosis, manifesting that inhibition of V600E 30304, 2006. BRAF augments TM-induced paraptosis in FRO cells. In 11 Szegezdi E, Logue SE, Gorman AM and Samali A: Mediators of view of therapeutic implications, TM may be valuable in endoplasmic reticulum stress-induced apoptosis. EMBO Rep 7: overcoming resistance to BRAFV600E inhibitor in the 880-885, 2006. subtype(s) of ATC cells harboring the mutated BRAFV600E gene. 12 Mercer KE and Pritchard CA: Raf proteins and cancer: B-Raf is In conclusion, our results suggest that TM induces identified as a mutational target. Biochim Biophys Acta 1653: paraptosis relevant to de novo protein synthesis and 25-40, 2003. 13 Xing M: BRAF mutation in thyroid cancer. Endocr Relat Cancer proteasomal activity in FRO cells. Moreover, considering that V600E 12: 245-262, 2005. the BRAF inhibitor potentiates the antitumor effect of 14 Caronia LM, Phay JE and Shah MH: Role of BRAF in thyroid V600E TM on FRO cells harboring BRAF , strategies targeting oncogenesis. Clin Cancer Res 17: 7511-7517, 2011. paraptosis in combination with inhibition of BRAFV600E may 15 Nucera C, Nehs MA, Nagarkatti SS, Sadow PM, Mekel M, provide future directions for therapeutic development in the Fischer AH, Lin PS, Bollag GE, Lawler J, Hodin RA and subtype(s) of ATC cells harboring BRAFV600E. Parangi S: Targeting BRAFV600E with PLX4720 displays potent antimigratory and anti-invasive activity in preclinical models of human thyroid cancer. Oncologist 16: 296-309, 2011. Acknowledgements 16 Schweppe RE, Klopper JP, Korch C, Pugazhenthi U, Benezra M, Knauf JA, Fagin JA, Marlow LA, Copland JA, Smallridge RC This research was supported by the Basic Science Research and Haugen BR: Deoxyribonucleic acid profiling analysis of 40 Program through the National Research Foundation of Korea (NRF) human thyroid cancer cell lines reveals cross-contamination funded by the Ministry of Education (2012R1A1A2008786) to S.J. resulting in cell line redundancy and misidentification. J Clin Lee, Republic of Korea, and also was supported by Hallym Endocrinol Metab 93: 4331-4341, 2008. University Research Fund to S.J. Lee, Republic of Korea. 17 Wang WB, Feng LX, Yue QX, Wu WY, Guan SH, Jiang BH, Yang M, Liu X and Guo DA: Paraptosis accompanied by References autophagy and apoptosis was induced by celastrol, a natural compound with influence on proteasome, ER stress and Hsp90. J Cell Physiol 227: 2196-2206, 2012. 1 Onoda N, Kashiwagi S, Noda S, Kawajiri H, Takashima T, 18 Wang C and Chen T: Intratumoral injection of taxol in vivo Ishikawa T and Hirakawa K: High efficacy of chemoradiation suppresses A549 tumor showing cytoplasmic vacuolization. J therapy sensitized by weekly docetaxel for anaplastic thyroid Cell Biochem 113: 1397-1406, 2012. cancer. Anticancer Res 33: 3445-3448, 2013. 19 Yoon MJ, Kang YJ, Lee JA, Kim IY, Kim MA, Lee YS, Park JH, 2 Smallridge RC, Marlow LA and Copland JA: Anaplastic thyroid Lee BY, Kim IA, Kim HS, Kim S-A, Yoon A-R, Yun C-O, Kim E- cancer: molecular pathogenesis and emerging therapies. Endocr Y, Lee K and Choi KS: Dimethoxycurcumin stronger proteasomal Relat Cancer 16: 17-44, 2009. inhibition and higher CHOP induction are responsible for more 3 Bröker LE, Kruyt FA and Giaccone G: Cell death independent of effective induction of paraptosis by dimethoxycurcumin than : a review. Clin Cancer Res 11: 3155-3162, 2005. curcumin. Cell Death Differ doi:10.1038/cddis.2014.85, 2014. 4 Jambrina E, Alonso R, Alcalde M, Rodríguez MD, Serrano A, Martínez AC, García-Sancho J and Izquierdo M: Calcium influx through receptor-operated channel induces mitochondria- Received June 2, 2014 triggered paraptotic cell death. J Biol Chem 278: 14134-14145, Revised July 1, 2014 2003. Accepted July 2, 2014

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