Induction of Apoptosis in Human Papillary-Thyroid-Carcinoma BCPAP

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Bioactive Constituents, Metabolites, and Functions

Diallyl trisulfide induces apoptosis of human papillary thyroid carcinoma BCPAP cells through activating MAPK signaling pathway Jie Pan, Li Zhang, Shichen Xu, Xian Cheng, Huixin Yu, Jiandong Bao, and Rong-Rong Lu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b02243 • Publication Date (Web): 22 May 2018 Downloaded from http://pubs.acs.org on May 22, 2018

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Journal of Agricultural and Food Chemistry

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Diallyl trisulfide induces apoptosis of human papillary thyroid

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carcinoma BCPAP cells through activating MAPK signaling

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pathway

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Jie Pana,b,1, Li Zhangb,1, Shichen Xub, Xian Chengb, Huixin Yub, Jiandong Baob,

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Rongrong Lua, *

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a

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Wuxi, Jiangsu 214122, China

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b

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Ministry of Health, 20 Qian Rong Road, Wuxi, Jiangsu 214063, China

School of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue,

Jiangsu Institute of Nuclear Medicine, Key Laboratory of Nuclear Medicine,

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1

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*

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University, Wuxi 214122, China

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E-mail address: [email protected] (Rongrong Lu)

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Phone: 0510-85329061(O)

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Fax: + 86 510 85912155

These authors equally contributed in this work.

Correspondence author: School of Food Science and Technology, Jiangnan

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1

ACS Paragon Plus Environment


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Abstract

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This study aimed to elucidate the potential effects of diallyl trisulfide (DATS) on

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human papillary thyroid carcinoma BCPAP cells and its underling mechanisms. DATS

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is one of the organosulfur compounds which derived from garlic. In this study, we

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demonstrated that compared to the solvent control, DATS treatment at concentrations

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of 5, 10 and 20 µΜ decreased cell survival rate of BCPAP cells to 84.51 ± 2.67%,

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57.16 ± 1.18%, 41.22 ± 1.19% respectively. DATS also caused cell cycle arrest at

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G0/G1 phase and the proportion of cells arrested in G0/G1 phase rose from 68.8 ±

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8.38% to 80.4 ± 8.38%, which eventually resulted in cell apoptosis through a

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mitochondrial apoptotic pathway in BCPAP cells. Further evidences showed that

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DATS activated ERK, JNK and p38, the members of MAPK family. Moreover, ERK

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inhibitor and JNK inhibitor, partially reversed apoptosis in BCPAP cells induced by

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DATS treatment. Taken together, our results demonstrated that DATS exerted the

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apoptosis-inducing effect on papillary thyroid cancer cells via activating MAPK

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signaling pathway, which shed a light on a prospective therapeutic target for thyroid

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cancer treatment.

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Keywords: Diallyl trisulfide; Papillary thyroid carcinoma; Apoptosis; Cell cycle arrest;

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MAPK signaling pathway

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Introduction

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Diallyl trisulfide (DATS) is one of the organosulfur compounds (OSCs) with a strong

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pungent smell (Fig. 1). It mainly derives from alliaceae family such as onion and

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garlic. Garlic, a well-known functional food and its derivates have attracted more and

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more attentions recently due to their significant biological and pharmacological

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activities. It has been reported that garlic was used as a potent phytochemical

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medicine in traditional China, Egypt, Greece, Rome and India for a long history1.

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Accumulating reports have proved that DATS is the most efficient bioactive

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component among the three diallyl polysulfides: diallyl sulfide (DAS), diallyl

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disulfide (DADS) and diallyl trisulfide (DATS)2. The three sulfur atoms in the

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structure of DATS may substantially contribute to its stronger bioactivity compared

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with one sulfur atom in DAS and two sulfur atoms in DADS2, 3.

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The bioactivities of DATS contain two aspects. Firstly, DATS has a potent

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antioxidant activity in protecting normal cells from oxidative damage4. Secondly,

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previous studies showed that DATS could fight against many human diseases,

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including cardiac disease5, diabetes6, inflammation7 and cancers. It has been reported

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that DATS exerted anti-cancer effects against pancreatic cancer8, melanoma9 and

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breast cancer10. Moreover, the epidemiologic study in China has observed a close

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association between intake of raw garlic and lung cancer prevention11. In terms of

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thyroid cancers, previous study showed that DAS, another kind of OSCs, could

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induce apoptosis of anaplastic thyroid cancer cells12. In the present study, whether

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DATS has an anti-cancer effect on papillary thyroid carcinoma and its detailed

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mechanisms were further investigated.

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Thyroid cancer is one of the most common endocrine malignancy with a

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worldwide rising incidence rate13. According to its pathological histology, thyroid 3



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carcinoma can be classified into four subtypes, namely papillary thyroid carcinoma

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(PTC), follicular thyroid carcinoma (FTC), medullary thyroid carcinoma (MTC), and

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anaplastic thyroid carcinoma (ATC)14. PTC is the most common subtype accounting

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for about 80% of the thyroid cancer cases14. Currently, the major therapeutic

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modalities for thyroid cancer include surgical removal, radioiodine ablation and

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thyroid-stimulating hormone (TSH) suppression15. However, in some advanced

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papillary thyroid cancers harbouring BRAF and/or TERT mutations, the tumors will

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lose the radioiodine absorbing ability and subsequently become refractory to

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radioiodine treatment16. Thus, alternative systemic options are needed for these

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patients. Sorafenib, an oral multikinase inhibitor, was approved by the US Food and

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Drug Administration (FDA) for the treatment of 131I-refractory and metastatic thyroid

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cancer. Though sorafenib was currently considered as a first-line therapy for

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131

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after the treatment. Therefore, it is urgent to develop promising drugs or novel

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strategies to improve the therapeutic efficiency.

I-refractory metastatic PTC19, a proportion of patients were still in progress even

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Targeting apoptosis is a promising strategy for cancer therapy through regulating

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many intracellular signals including Nrf2/Akt17, NF-κB18 and MAPK signaling

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pathways17. Mitogen-active protein kinases (MAPKs) are serine/threonine kinases. In

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mammalian cells, this superfamily consists of three main members: the extracellular

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regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p3819. MAPK signaling

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pathway participates in various cell behaviors, including cell proliferation,

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differentiation and apoptosis20. The activation of JNK/p38 was a kind of apoptotic

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signal while the activation of ERK was generally a survival signal17. Moreover, many

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studies reported that ERK signaling pathway was activated in nearly 80% of PTC

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specimen21. Constitutive ERK activation is believed to be essential for the survival 4


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and development of thyroid cancer cells21. However, excessive activation of MAPK

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signaling induced by chemicals could also induce apoptosis in thyroid cancer cells22.

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Hence, MAPK pathway has a dual potential in the regulation of cell survival and

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apoptosis. Previous study demonstrated that DATS induced apoptosis in prostate

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cancer cells via activating MAPK/ERK/JNK signaling pathway23. Therefore, it

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prompted us to study the anti-cancer efficacy and the potential effects of DATS on

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MAPK signaling pathway in thyroid cancer cells.

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In the present study, we demonstrated that DATS inhibited cell proliferation,

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caused G0/G1 phase cell cycle arrest and subsequently elicited cell apoptosis in

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papillary thyroid cancer BCPAP cells. Our results provided evidences that excessive

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activation of MAPK signaling pathway as a central mechanism of DATS-induced

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growth inhibition and apoptosis in thyroid cancer cells. These results proposed a

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potential therapeutic target for PTC treatment.

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Materials and methods

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Chemicals, reagents and antibodies

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Diallyl trisulfide (DATS, HPLC≥98%) and Sulforhodamine B (SRB) were purchased

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from Sigma Aldrich (Saint Louis, Missouri, USA). Hoechst 33342 (C1022), PI

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(ST511), JC-1 (C2005), U0126 (S1901), SP600125 (S1876), SB203580 (S1863),

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reactive oxygen species (ROS) detection kit (S0033) and Dimethyl Sulfoxide (DMSO)

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were purchased from Beyotime Institute of Biotechnology (Nantong, China). The

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antibodies used in the present study were as follows: Anti-p-Rb (ab173289) was

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purchased from Abcam (Cambridge, UK). Anti-p-JNK (cst-4668), anti-Bax (cst-2772),

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anti-caspase-8 (cst-9746), anti-caspase-3 (cst-9665) were purchased from Cell

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Signaling Technology (Boston, Massachusetts, USA). Anti-PARP-1 (sc-7150),

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anti-β-actin (sc-47778), goat anti-mouse (sc-2005) or rabbit (sc-2004) IgG-HRP were 5



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purchased from Santa Cruz Biotechnology (Santa Cruz, California, USA).

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Anti-tubulin (AT819), anti-Cyclin D1 (AC853), anti-p27 (AP027), anti-Bcl-2

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(AB112), anti-Bcl-XL (AB126-1), anti-ERK (AM076), anti-p-ERK (AM071),

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anti-JNK (AJ518), anti-p-JNK (AJ516), anti-p38 (AM065), anti-p-p38 (AM063) were

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purchased from Beyotime Institute of Biotechnology (Nantong, China). Other

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chemicals were analytical grade and purchased from common source.

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Cell culture and drug treatment

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The human papillary thyroid carcinoma line BCPAP cells were obtained from the

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German Collection of Micro-organisms and Cell Cultures (Braunschweig, Germany).

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BCPAP cells were cultured in RPMI 1640 medium supplemented with 10% new born

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bovine serum (NBS), antibiotics (penicillin 100 U/ml and streptomycin 100 U/ml).

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Nthy-ori-3.1 cells, obtained from the German Collection of Micro-organisms and Cell

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Cultures (Braunschweig, Germany), were cultured in RPMI 1640 medium

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supplemented with 10% fetal bovine serum (FBS), antibiotics (penicillin 100 U/ml

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and streptomycin 100 U/ml). BCPAP cells and Nthy-ori-3.1 cells were both

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maintained at 37 °C in a standard humidified incubator containing 5% CO2. DATS

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was dissolved in DMSO at 20 mM as a stock solution and stored at -20 °C until

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diluting to indicated concentration before use. The solvent control contained an

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equivalent amount of DMSO corresponding to the highest used concentration of

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DATS and the final concentration of DMSO was less than or equal to 0.1%.

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Sulforhodamine B (SRB) assay

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The sulforhodamine B (SRB) assay was used to detect the cell number. Briefly,

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BCPAP or Nthy-ori-3.1 cells were seeded into 96-well plates at 7500 cells per well

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and cultured overnight. Cells were treated with various concentrations of DATS for

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indicated time and then fixed with 10% TCA (w/v) for 1 h at 4 °C, and stained with 6


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4% SRB for another 30 min at 37 °C. Then, tris base (10 mM) of 100 µl was added

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into each well and the plates were vibrated for 5 min to dissolve SRB completely. The

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optical density of the cell suspension was measured at 565 nm by a microplate reader

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(Epoch, Biotek). Cell survival was expressed as a percentage of SRB reduction,

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assuming that the absorbance of solvent control group was 100%.

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Colony formation assay

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BCPAP cells were seeded into 24-well plates at a density of 500 cells per well and

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cultured overnight. BCPAP cells were pretreated with different dosages of DATS

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(5-20 µΜ) for 24 h, then the medium was replaced by complete growth medium. After

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cultured for another 10 days, the cells were fixed with methanol and stained with

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crystal violet for 15 min. The visible colonies were photographed by a Bio-Rad

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Imager (SYSTEM GelDoc XR+).

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Cell cycle analysis

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Cell cycle distribution was determined by PI staining. Briefly, BCPAP cells were

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seeded into 6-well plates and cultured overnight. The cells were then treated with

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indicated concentrations of DATS for 24 h. After that, cells were harvested and fixed

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with 4% paraformaldehyde (PFA) at 4 °C for 1 h. Then the cells were washed by PBS

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and incubated with DNA staining reagent which contained 50 µg/ml RNase, 0.1%

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Triton X-100, 0.1 mM EDTA (pH 7.4), and 50 µg/ml PI in the dark for 30 min. The

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stained cells were subjected to a flow cytometer (FACSCalibur, Becton Dickinson,

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USA) for cell cycle distribution analysis.

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Hoechst 33342/PI staining

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Hoechst 33342/PI staining was conducted to detect cell apoptosis. Briefly, BCPAP

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cells were seeded into 12-well plates and cultured overnight. Then BCPAP cells were

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incubated with different concentrations of DATS (5-20 µΜ) for 24 h. Subsequently,

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the cells were stained with a mixture solution of Hoechst 33342 (10 µg/ml) and PI (10

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µg/ml) at 37 °C for 15 min. Cells were observed under a fluorescence microscope

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(Olympus, X51, Japan). Three independent fields per well were picked and cell

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Images were photographed.

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Mitochondrial membrane potential measurement

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JC-1, a kind of lipophilic cationic fluorescent dye, was used to measure the

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depolarization of mitochondria. BCPAP cells were treated with indicated

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concentrations of DATS for 24 h and subsequently stained with JC-1 (10 µg/ml) at

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37 °C for 30 min in the dark. After staining, cells were washed by PBS and

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immediately analyzed by flow cytometry (FACSCalibur, Becton Dickinson, USA) at

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FL1 and FL2 channels.

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Reactive oxygen species (ROS) detection

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ROS detection was performed according to the manufacturer’s protocol. After

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different concentrations of DATS treatment, the cells were washed with ice-cold PBS

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once, and then incubated with 20 µM of DCFH-DA at 37 °C for 20 min in the dark.

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The cells were then harvested and resuspended in ice-cold PBS. Cells were

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immediately analyzed by flow cytometry (FACSCalibur, Becton Dickinson, USA).

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Western blot

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Total cell lysates were prepared by RIPA lysis buffer containing protease inhibitor

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cocktail (1% v/v). The protein concentration was quantified by Bradford protein assay.

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Collected proteins were separated by 10% or 15% SDS polyacrylamide gel

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electrophoresis. The proteins were transferred to a nitrocellulose (NC) membrane and

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blocked with 5% skim milk dissolved in Tris-buffered saline containing 0.1% Tween

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20. Targeted proteins were detected by corresponding primary antibodies and

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subsequently incubated with horseradish peroxidase-conjugated secondary antibodies.

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The protein bands were visualized using an ECL Western blot kit (ABXBio). The

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β-actin or α-tubulin was used as an internal control for equal loading of total proteins.

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Statistical analysis

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Data were presented as mean ± S.D. and analyzed by GraphPad Prism 6 software.

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One-way ANOVA analysis followed by Dunnett's test for multiple comparisons were

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used to analyze the statistical significance and P < 0.05 was considered to be

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statistically significant.

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Results

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DATS inhibits clonogenic survival of human papillary thyroid carcinoma BCPAP

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cells

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Firstly, in order to investigate the potential cytotoxic effect of DATS on BCPAP cells,

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cellular morphological changes were observed after various concentrations of DATS

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treatment (5-20 µΜ). As illustrated in Fig. 2A, cells exposed to DATS became smaller

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in size and less adherent to culture plate compared to the solvent control group. Then,

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SRB assay was used to evaluate the effect of DATS on the growth of BCPAP cells.

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The results showed that DATS at a low concentration of 0.625 µM failed to inhibit the

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cell growth. However, when the concentration of DATS exceeded 1.25 µΜ, the cell

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number of each group decreased significantly (P < 0.05). The cell number of BCPAP

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cells reduced over 50% by the treatment of DATS at 20 µΜ (Fig. 2B). Next, in vitro

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colony formation assay was performed to further determine the growth-inhibition

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efficiency of DATS on BCPAP cells. As shown in Fig. 2C and 2D, DATS pretreatment

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dose-dependently decreased the clone numbers. SRB assay also demonstrated that

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DATS inhibited the cell growth of BCPAP cells in a time-dependent manner (Fig. 2E).

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Furthermore, the cytotoxic effect of DATS on the normal thyroid follicular epithelial

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cells was examined. As shown in Fig. 2F, SRB assay indicated that papillary thyroid 9



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carcinoma BCPAP cells were more sensitive to DATS treatment than normal thyroid

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follicular epithelial cell line Nthy-ori-3.1 cells did. This result suggested DATS

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selectively killed thyroid cancer cells rather than normal thyroid follicular epithelial

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cells. Taken together, these data indicated that DATS inhibited clonogenic survival of

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human papillary thyroid carcinoma BCPAP cells in dose- and time dependent

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manners.

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DATS arrests cell cycle at G0/G1 phase in BCPAP cells

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Cell cycle is abbrently fast in the malignant cells. It has been widely accepted that

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arresting cancer cells in a certain phase can inhibit tumor growth24. Therefore, we

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hypothesized that the growth-inhibitory effect of DATS was due to its cell cycle arrest

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ability. Next, the effects of DATS on cell cycle distribution were evaluated by

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propidium iodide (PI) staining. Compared to solvent control, the percentage of cell

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population distributing at G0/G1 phase was obviously increased after DATS treatment,

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while cells in S and G2/M phases showed a concomitant reduction (Fig. 3A). As

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shown in Fig. 3B, 68.8 ± 8.38% of the DATS-treated cells were arrested at G0/G1

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after 5 µΜ of DATS treatment, and this proportion was up to 77.3 ± 11.84% and

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80.4 ± 8.38% at the concentration of 10 µΜ and 20 µΜ, respectively. Moreover, the

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percentage of cell population at sub-G1, which served as a feature of apoptotic cells,

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also increased significantly in a dose-dependent manner. Moreover, the alternation of

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the cell cycle regulating proteins involved in G0/G1 phase such as cyclin D1, p-Rb

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and p2725 after DATS treatment were investigated. The results showed that DATS

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treatment at 5 to 20 µM dose-dependently decreased the expression of cyclin D1 and

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p-Rb. Accordingly, DATS treatment increased the protein level of p27 which is a CKI

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(cyclin kinase inhibitor) halting the cell cycle progression (Fig. 3C and 3D). Taken 10


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together, these data indicated that DATS inhibited cell growth and caused cell cycle

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arrest at G0/G1 phase in BCPAP cells.

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DATS induces BCPAP cells apoptosis through a mitochondrial apoptotic

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pathway

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The increasedly accumulating cells in the sub-G1 peak prompted us to investigate

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whether the growth inhibition effect of DATS on BCPAP cells was attributed to its

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apoptosis-inducing effect. Hence, Hoechst 33342/PI staining was performed to detecte

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the apoptotic phenotype of DATS-treated cells. As shown in Fig. 4A, DATS-treated

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BCPAP cells exihibited evident apoptotic features with increased cell membrane

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permeability (PI positive cells) and nuclear chromatin shrinkage (bright hoechst

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33342 staining cells). The declined mitochondrial membrane potential was believed to

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be an early event of apoptosis26. Thus, cell mitochondrial membrane potential was

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detected. The proportion of JC-1 momomer cells elevated remarkly with the reduction

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of JC-1 aggregates cells, which suggested that DATS certainly induced the

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depolarization of mitochondrial membrane potential in BCPAP cells (Fig. 4B and Fig.

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4C). In addition, the expressions of apoptosis-related proteins were detected by

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western blot. As illustrated in Fig. 5A and B, the expression of anti-apoptotic protein

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Bcl-2 and Bcl-XL was downregulated, while the expression of pro-apoptotic protein

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Bax was upregulated. It has been reported that the increased Bax bound to the outer

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mitochondrial membrane and Bcl-2 released from the mitochondria to initiate cell

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apoptosis27. Moreover, the cleaved caspase-3, -8 and the cleavage of downstream

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PARP were all increased (Fig. 5C). Besides, the decreased cell numbers induced by

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DATS treatment could be reversed by z-VAD-fmk, a pan-caspase inhibitor (Fig. 5D).

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Thus, we concluded that DATS treatment elicited caspase-mediated apoptosis through

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a mitochondrial apoptotic pathway in BCPAP cells.

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DATS-induced apoptosis is independent of ROS generation

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One of the well-known triggers of apoptosis is cellular ROS. Excess ROS, which is

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mainly produced in cell mitochondria, is reported as a typical characreristic of

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oxidative damage to mitochondrial membrane and permeability28. Indeed, the opening

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of mitochondrial permeability transition pore (MPTP) has been demonstrated to

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decrease the mitochondrial membrane potential29. Therefore, we next investigated

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whether the deareased cell mitochondrial membrane potential induced by DATS was

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triggered by ROS attack. Unexpectedly, DATS treatment did not change the

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intracellular ROS level, even at a higher concentration of 20 µΜ (Fig. 6). These data

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indicated that DATS-induced apoptosis was not dependent on the increased

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production of intracellular ROS.

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DATS induces apoptosis through activating MAPK signaling pathway in BCPAP

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cells

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Most of thyroid cancers harbour MAPK pathway activation, especially ERK1/2

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hyper-phosphorylation because of BRAF or RAS mutations,

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activation is considered to be a cell survival signaling31. Moreover, supression of ERK

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is able to attenuate the prolifitation and migration properties of thyroid cancer cells31.

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In contrast, it has been reported that excessive activation of ERK could also induce

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thyroid cancer cell apoptosis22. Hence, targeting this pathway maybe an effective

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strategy in thyroid cancer treatment. In order to investigate the potential role of

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MAPK signaling in the anti-cancer effect of DATS, we next determined both the total

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and phosphorylated levels of MAPKs in DATS-treated BCPAP cells. Interestingly, the

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ERK, which was constitutively actived in BCPAP cells, was further activated by

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DATS treatment. As illistrated in Fig. 7A, after BCPAP cells were exposed to DATS

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treatment, the phosphoryation of ERK increased in a dose-dependent manner, while

30

. In general, ERK

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the protein levle of the total ERK decreased. JNK and p38, the other two members of

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MAPK superfamily, were also significantly activated by DATS treatment. In order to

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further confirm the critial role of MAPK signaling pathway in DATS-induced cell

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apoptosis , BCPAP cells were pretreated with 20 µΜ of U0126 (ERK inhibitor) for 2

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h. As shown in Fig. 7B, U0126 pretreatment completely abolished the

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phosphorylation of ERK1/2 induced by DATS. Meanwhile, the protein levels of Bcl-2

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and Bax were partially reversed by U0126 in DATS-treated BCPAP cells (Fig. 7B and

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7C). Besides, JNK and p38 inhibiton were also performed to determine the role of

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JNK and p38 in cell apoptosis induced by DATS treatment. We found that pretreating

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with 20 µΜ of JNK inhibitor, SP600125, could rescue cell apoptosis induced by

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DATS treatment while it could not be restored by p38 inhibitor, SB203580 (Fig. 7D

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and 7E). Hence, the activation of ERK and JNK but not p38 were involved in the

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apoptosis induced by DATS. All these data confirmed that DATS elicited apoptosis in

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BCPAP cells through activating MAPK signaling pathway.

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Discussion

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The laboratorial and epidemiological studies demostrated that galic and its derivates

301

had an effective bioactivity against many diseases32. Recently, the functions of DATS

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has been tested in various disease models. Owing to its antioxidant property, DATS

303

has a cytoprotective effect on hepatocyte cells4. In addition, DATS was proved to play

304

a role in cancer prevention through different machanisms. Previous study

305

demonstrated that DATS could inhibit the proliferation as well as metastasis of breast

306

cancer cell33 and DATS also exhibited its anti-cancer character by sensitizing human

307

melanoma cancer cells to chemotherapy34. Most importantly, DATS could elicit

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apoptosis in many cancer cells such as lymphoma cells2 and gastric carcinoma cells35.

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In the present study, our results revealed that DATS induced apoptosis of thyroid 13



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cancer cells.

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The most common follicular cell-derived cancer is papillary thyroid carcinoma

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(PTC), which usually has a favorable prognosis after surgery, adjuvant radioactive

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iodine and thyroid-stimulating hormone suppression15. Radioactive iodine treatment is

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the first-line systemic treatment in patients with advanced thyroid cancer36.

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selectively causes radiation damages, such as DNA damage, membrane injury and

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protein oxidation to thyroid follicular epithelial cells including thyroid cancer cells,

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which eventually induces cell death of thyroid cancer cells37. However, a few patients

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will become refractory to radioactive iodine treatment. Therefore, it is essential to find

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an alternative strategy to inducing apoptosis of thyroid cancer cells as a

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supplementary option to radioactive iodine treatment. Our present results

321

demonstrated that DATS could significantly induce the apoptosis of thyroid cancer

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BCPAP cells.

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I

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In most cancer cells, unlimited cell proliferation is a consequence of the

324

dysregulated cell cycle24. Hence, the disturbance of cell cycle is a promising strategy

325

for cancer therapy24. Previous reports showed that DATS induced cell cycle arrest in

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different kinds of cancer cells. For example, DATS caused cell cycle arrest at G2/M

327

phase in DU145 human prostate cancer cell line38. Another research also showed that

328

DATS induced cell cycle arrest at G2/M phase in human gastric carcinoma cells via

329

upregulating cyclin B1 and cyclin-dependent kinase p2139. In line with previous

330

findings, we also demonstrated that DATS treatment induced cell cycle arrest at

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G0/G1 phase of BCPAP cells (Fig. 3), which may be the underlying mechanism of the

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growth-inhibitory effects of DATS on BCPAP cells.

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Cell cycle arrest spares time to repair impaired DNA and if fails, it will lead to

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cell apoptosis40. Apoptosis is termed as type I programmed cell death, and it can be

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mediated through two different pathways: intracellular and extracellular apoptotic

336

pathways41. The two pathways differ from the stimuli of cell apoptosis. Extrinsic

337

apoptotic pathway, also called the death receptor pathway, is triggered by

338

ligand-induced activation of death receptors such as Fas and TNF-related apoptosis

339

inducing ligand (TRAIL)34. The intracellular apoptotic pathway is also known as

340

mitochondrial apoptotic pathway, which is mainly induced by intracellular signals

341

such as DNA damage, growth factor starvation and oxidative stress27. The

342

mitochondrial apoptosis pathway is regulated by Bcl-2 family members41. It has been

343

proved that the expression of Bcl-2 and Bcl-XL, the anti-apoptotic protein, will

344

downregulate, accompanied with the upregulation of the expression of Bax, the

345

pro-apoptotic protein. Eventually, Bax directly induces mitochondrial outer membrane

346

permeabilization (MOMP) followed by cytochrome c releasing to cytoplasm, which

347

activates cascade of caspases41. Once activated, caspase family will trigger a chain of

348

intracellular events that elicit cell death41. Therefore, inducing cancer cell apoptosis is

349

an effective method for cancer chemoprevention and therapy. Consistent with these

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typical apoptotic features, our results showed that DATS-induced BCPAP cell

351

apoptosis depended on caspase cascade activation through a mitochondrial apoptotic

352

pathway (Fig. 4 and Fig. 5).

353

Mitochondria is the factory of ROS, and it is reported that excessive ROS causes

354

severe oxidative damage to intracellular molecules and eventually induces cell

355

apoptosis42. Indeed, DATS-induced apoptosis has been reported to be mediated by

356

excessive ROS production in human breast cancer cells43. Contrary to our expectation,

357

our data showed that DATS failed to altered the intracellular level of ROS in BCPAP

358

cells, indicating a non-involvement of ROS in DATS-induced apoptosis in thyroid

359

cancer cells (Fig. 6).

15



Page 16 of 31

360

Many genetic alterations have been identified in thyroid cancer which derived

361

from follicular cells. Among them, BRAF and RAS mutations are the most common

362

mutations in papillary thyroid cancer15. BRAF or RAS mutations always drive

363

constitutive activation of MAPK signaling pathway, which is generally believed to be

364

a survival signal in cancer cells44. Hence, targeting this signaling pathway is essential

365

for the treatment of thyroid cancer. In clinical practice, selumetinib, a kind of tyrosine

366

kinase inhibitor targeting MAPK signaling pathway, is used as a promising medicine

367

in thyroid cancer therapy45. Thomas C. Beadnell reported that inhibition of MEK or

368

ERK drove synergistic inhibition of growth and induction of apoptosis in thyroid

369

cancer46. In another study, local anesthetics was also found to be able to induce

370

apoptosis in thyroid cancer cells through attenuating extracellular signal-regulated

371

kinase 1/2 (ERK1/2) activity47. Yet this conclusion remained controversial, as some

372

other studies have highlighted the fact that transient activation but not inhibition of

373

ERK could also lead to the apoptosis in thyroid cancer cell lines22. This discrepancy

374

not only exists in thyroid cells, but also in other cancer type such as hepatocytes. M.

375

D. Abid et al. found that MAPK activation promoted hepatic cellular apoptosis48. On

376

the contrary, other reports showed that suppression of ERK could also induce

377

hepatocytes apoptosis49. These paradoxical conclusions imply that the survival or

378

death fate as a consequence of modulation of MAPK signaling pathway appears to be

379

highly cell type and context dependent. Of note, how long and to what extent does

380

MAPK signaling activate may affect different downstream targets, leading to a

381

different response to various stimuli. In our study, we confirmed that DATS induced

382

an excessive activation of MAPK signaling including ERK and JNK rather than p38,

383

which played an essential role in promoting apoptosis of thyroid cancer BCPAP cells

384

(Fig. 7). 16


Page 17 of 31


385

Concerning the absorption, distribution, metabolism and excretion (ADME)

386

characteristics of DATS, it has been proven that the absorption of DATS was excellent

387

and fast50. Additionally, DATS could rapidly distribute to blood, lung, liver, and it

388

metabolized instantly in vivo50, 51. Also, no noticeable toxicity of aged garlic extract

389

on human was found in preclinical trial52. Moreover, DATS with three sulfur atoms in

390

its structure is a donor of hydrogen sulfide, which is believed as the third gaseous

391

signal molecule in vivo53. It has been reported that exposure to exogenous hydrogen

392

sulfide increased the apoptotic percentage of oral cancer cells54. Whether hydrogen

393

sulfide is also attributed to DATS-induced cell apoptosis in thyroid cancer needs

394

further investigation.

395

In conclusion, to the best of our knowledge, our study demonstrated for the first

396

time that DATS significantly inhibited cell proliferation through causing G0/G1 phase

397

cell cycle arrest in thyroid cancer BCPAP cells. Additionally, we found that DATS

398

induced ROS-independent apoptosis via activating MAPK signaling pathway.

399

ABBREVIATIONS

400

DATS, diallyl trisulfide;

401

DAS, diallyl sulfide;

402

DADS, diallyl disulfide;

403

OSCs, organosulfur compounds;

404

ROS, reactive oxygen species;

405

∆Ψm, mitochondrial membrane potential;

406

Bax, Bcl-2-associated X protein;

407

Bcl-2, B-cell lymphoma 2;

408

PARP, poly(ADP-ribose) polymerase;

409

PI, propidium iodide; 17



Page 18 of 31

410

FBS, fetal bovine serum;

411

DMSO, dimethyl sulfoxide;

412

PBS, phosphate-buffered saline;

413

MAPK, mitogen-activated protein kinase;

414

JNK, c-Jun N-terminal kinase;

415

ERK, extracellular signal-regulated kinase;

416

PTC, papillary thyroid carcinoma;

417

Funding

418

This study was funded by the grants from the Science and Research Foundation of

419

Health Bureau of Jiangsu Province (No. H2017032), the National Natural Science

420

Foundation of China (Nos. 81602352 and 81602353), the Science and Research

421

Foundation of Health Bureau of Jiangsu Province (No. H2017032), the Natural

422

Science Foundation of Jiangsu Province (BK20171145 and BK20151119), and Wuxi

423

Municipal Commission of Health and Family Planning (Q201608).

424

Conflict of interest

425

The authors declare no competing financial interest.

426

18


Page 19 of 31


427

References

428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469

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810, pp 183-205. Ly, J. D.; Grubb, D. R.; Lawen, A., The mitochondrial membrane potential (deltapsi(m)) in apoptosis; an update. Apoptosis 2003, 8, 115-28. Son, H. Y.; Hwangbo, Y.; Yoo, S. K.; Im, S. W.; Yang, S. D.; Kwak, S. J.; Park, M. S.; Kwak, S. H.; Cho, S. W.; Ryu, J. S.; Kim, J.; Jung, Y. S.; Kim, T. H.; Kim, S. J.; Lee, K. E.; Park, D. J.; Cho, N. H.; Sung, J.; Seo, J. S.; Lee, E. K.; Park, Y. J.; Kim, J. I., Genome-wide association and expression quantitative trait loci studies identify multiple susceptibility loci for thyroid cancer. Nature communications 2017, 8, 15966. Zhou, Q. Y.; Chen, J.; Feng, J. L.; Xu, Y. N.; Zheng, W. J.; Wang, J. D., SOSTDC1 inhibits follicular thyroid cancer cell proliferation, migration, and EMT via suppressing PI3K/Akt and MAPK/Erk signaling pathways. Mol. Cell. Biochem. 2017, 435, 87-95. Zhang, L.; Cheng, X.; Gao, Y.; Bao, J.; Guan, H.; Lu, R.; Yu, H.; Xu, Q.; Sun, Y., Induction of ROS-independent DNA damage by curcumin leads to G2/M cell cycle arrest and apoptosis in human papillary thyroid carcinoma BCPAP cells. Food Funct. 2016, 7, 315-25. Chandra-Kuntal, K.; Lee, J.; Singh, S. V., Critical role for reactive oxygen species in apoptosis induction and cell migration inhibition by diallyl trisulfide, a cancer chemopreventive component of garlic. Breast Cancer Res. Treat. 2013, 138, 69-79. Murai, M.; Inoue, T.; Suzuki-Karasaki, M.; Ochiai, T.; Ra, C.; Nishida, S.; Suzuki-Karasaki, Y., Diallyl trisulfide sensitizes human melanoma cells to TRAIL-induced cell death by promoting endoplasmic reticulum-mediated apoptosis. Int. J. Oncol. 2012, 41, 2029-37. Choi, Y. H., Diallyl trisulfide induces apoptosis and mitotic arrest in AGS human gastric carcinoma cells through reactive oxygen species-mediated activation of AMP-activated protein kinase. Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie 2017, 94, 63-71. Cabanillas, M. E.; McFadden, D. G.; Durante, C., Thyroid cancer. Lancet 2016, 388, 2783-2795. Ryu, J., Diagnostic and Therapeutic Approaches to Radioactive Iodine Refractory Differentiated Thyroid Cancer. Medicine 2012, 55, 403. Herman-Antosiewicz, A.; Kim, Y.-A.; Kim, S.-H.; Xiao, D.; Singh, S. V., Diallyl Trisulfide-Induced G2/M Phase Cell Cycle Arrest in DU145 Cells Is Associated with Delayed Nuclear Translocation of Cyclin-Dependent Kinase 1. Pharm. Res. 2010, 27, 1072-1079. Shin, S. S.; Song, J. H.; Hwang, B.; Park, S. L.; Kim, W. T.; Park, S. S.; Kim, W. J.; Moon, S. K., Angiopoietin-like protein 4 potentiates DATS-induced inhibition of proliferation, migration, and invasion of bladder cancer EJ cells; involvement of G(2)/M-phase cell cycle arrest, signaling pathways, and transcription factors-mediated MMP-9 expression. Food Nutr. Res. 2017, 61, 1338918. Malumbres, M.; Barbacid, M., Cell cycle, CDKs and cancer: a changing paradigm. Nat. Rev. Cancer 2009, 9, 153-66. Radogna, F.; Dicato, M.; Diederich, M., Cancer-type-specific crosstalk between autophagy, necroptosis and apoptosis as a pharmacological target. Biochem. Pharmacol. 2015, 94, 1-11. 21



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42. Pierce, G. B.; Parchment, R. E.; Lewellyn, A. L., Hydrogen peroxide as a mediator of programmed cell death in the blastocyst. Differentiation 1991, 46, 181-6. 43. Na, H. K.; Kim, E. H.; Choi, M. A.; Park, J. M.; Kim, D. H.; Surh, Y. J., Diallyl trisulfide induces apoptosis in human breast cancer cells through ROS-mediated activation of JNK and AP-1. Biochem. Pharmacol. 2012, 84, 1241-50. 44. Xing, M., Molecular pathogenesis and mechanisms of thyroid cancer. Nat. Rev. Cancer 2013, 13, 184-99. 45. Hayes, D. N.; Lucas, A. S.; Tanvetyanon, T.; Krzyzanowska, M. K.; Chung, C. H.; Murphy, B. A.; Gilbert, J.; Mehra, R.; Moore, D. T.; Sheikh, A.; Hoskins, J.; Hayward, M. C.; Zhao, N.; O'Connor, W.; Weck, K. E.; Cohen, R. B.; Cohen, E. E., Phase II efficacy and pharmacogenomic study of Selumetinib (AZD6244; ARRY-142886) in iodine-131 refractory papillary thyroid carcinoma with or without follicular elements. Clin. Cancer Res. 2012, 18, 2056-65. 46. Beadnell, T. C.; Mishall, K. M.; Zhou, Q.; Riffert, S. M.; Wuensch, K. E.; Kessler, B. E.; Corpuz, M. L.; Jing, X.; Kim, J.; Wang, G.; Tan, A. C.; Schweppe, R. E., The Mitogen-Activated Protein Kinase Pathway Facilitates Resistance to the Src Inhibitor Dasatinib in Thyroid Cancer. Mol. Cancer Ther. 2016, 15, 1952-63. 47. Chang, Y. C.; Hsu, Y. C.; Liu, C. L.; Huang, S. Y.; Hu, M. C.; Cheng, S. P., Local anesthetics induce apoptosis in human thyroid cancer cells through the mitogen-activated protein kinase pathway. PLoS One 2014, 9, e89563. 48. Abid, M. D. N.; Chen, J.; Xiang, M.; Zhou, J.; Chen, X. P.; Gong, F. L., Khat (Catha edulis) generates reactive oxygen species and promotes hepatic cell apoptosis via MAPK activation. Int. J. Mol. Med. 2013, 32, 389-395. 49. Du, X. L.; Shi, Z.; Peng, Z. C.; Zhao, C. X.; Zhang, Y. M.; Wang, Z.; Li, X. B.; Liu, G. W.; Li, X. W., Acetoacetate induces hepatocytes apoptosis by the ROS-mediated MAPKs pathway in ketotic cows. J. Cell. Physiol. 2017, 232, 3296-3308. 50. Sun, X.; Guo, T.; He, J.; Zhao, M.; Yan, M.; Cui, F.; Deng, Y., Determination of the concentration of diallyl trisulfide in rat whole blood using gas chromatography with electron-capture detection and identification of its major metabolite with gas chromatography mass spectrometry. Yakugaku Zasshi Journal of the Pharmaceutical Society of Japan 2006, 126, 521-7. 51. Lawson, L. D.; Wang, Z. J., Allicin and allicin-derived garlic compounds increase breath acetone through allyl methyl sulfide: use in measuring allicin bioavailability. Journal of Agricultural & Food Chemistry 2005, 53, 1974-1983. 52. Tanaka, S.; Haruma, K.; Yoshihara, M.; Kajiyama, G.; Kira, K.; Amagase, H.; Chayama, K., Aged garlic extract has potential suppressive effect on colorectal adenomas in humans. J Nutr 2006, 136, 821S-826S. 53. Liang, D.; Wu, H.; Wong, M. W.; Huang, D., Diallyl Trisulfide Is a Fast H2S Donor, but Diallyl Disulfide Is a Slow One: The Reaction Pathways and Intermediates of Glutathione with Polysulfides. Org. Lett. 2015, 17, 4196-9. 54. Murata, T.; Sato, T.; Kamoda, T.; Moriyama, H.; Kumazawa, Y.; Hanada, N., Differential susceptibility to hydrogen sulfide-induced apoptosis between PHLDA1-overexpressing oral cancer cell lines and oral keratinocytes: role of PHLDA1 as an apoptosis suppressor. Exp. Cell Res. 2014, 320, 247-57. 22


Page 23 of 31


602

Figure Captions

603

Figure 1. The chemical structure of DATS.

604 605

Figure 2. DATS inhibits the colony formation ability of BCPAP cells. (A) DATS

606

treatment resulted in the morphological change of BCPAP cells. BCPAP cells were

607

exposed to different concentrations of DATS (5-20 µΜ) for 24 h and then

608

photographed with inverted microscope. Scale bar, 20 µm. (B) DATS inhibited cell

609

growth of BCPAP cells in a dose-dependent manner. Cells were treated with indicated

610

concentrations of DATS for 24 h and then the cell numbers were measured by SRB

611

assay. (C) DATS inhibited the colony formation ability of BCPAP cells. The cells

612

were incubated with various concentrations of DATS for 24 h and then stained with

613

crystal violet. Colonies consisting of more than 50 cells were counted for each group.

614

(D) Quantitative analysis of clone numbers in (C). (E) DATS inhibited the cell growth

615

of BCPAP cells in a time-dependent manner. Cells were treated with DATS at 20 µΜ

616

for indicated periods and then the cell numbers were measured by SRB assay. (F)

617

DATS exhibited selective cytotoxicity on thyroid cancer cells. BCPAP and

618

Nthy-ori-3.1 cells were treated with 5-20 µΜ of DATS for 24 h and the cell numbers

619

were determined by SRB assay. All data were represented as mean ± S.D. *P < 0.05,

620

**

621

Figure 3. DATS causes G0/G1 cell cycle arrest in BCPAP cells. (A) BCPAP cells

622

were treated with different concentrations of DATS for 24 h. Cell cycle distributions

623

were detected by flow cytometry and analyzed by FlowJo software. M1, M2, M3 and

624

M4 gates represented the sub-G1 phase, G0/G1 phase, S phase and G2/M phase,

625

respectively. (B) Cell cycle distributions after DATS treatment in BCPAP cells were

626

analyzed and the data were plotted in the bar chart. (C) After BCPAP cells were

P < 0.01, ***P < 0.001 vs. SC group. SC, solvent control.

23



Page 24 of 31

627

treated with various concentrations of DATS (5-20 µΜ), the protein levels of Cyclin

628

D1 and p-Rb and p27 were determined by western blot. Tubulin was used as an

629

internal control. (D) The bands quantification of (C) were determined from three

630

independent experiments (n = 3). *P < 0.05 vs. SC group of cyclin D1, #P < 0.05 and

631

##

632

Figure 4. DATS induces apoptosis of BCPAP cells. (A) BCPAP cells were exposed

633

to different concentrations of DATS for 24 h and then stained with Hoechst 33342 and

634

propidium iodide (PI). White arrows indicated the PI positive cells and green arrows

635

indicated the bright Hoechst 33342 staining cells. Images were taken under a

636

fluorescence microscope and representive image were shown. Scale bar, 20 µm. (B)

637

Cells were incubated with DATS for 24 h and subjected to mitochondrial membrane

638

potential analysis by flow cytometry. (C) The quatification of JC-1 monomer cells in

639

(B). The symbol ''*'' is compared with SC group in statistical analysis of JC-1

640

monomer cells. One-way ANOVA analysis followed by Dunnett's test for multiple

641

comparisons was used to analyze the statistical significance. *P < 0.05 vs. SC group.

642

SC, solvent control.

643

Fig. 5. DATS-induced apoptosis is mediated by caspase activation. (A) DATS

644

induced apoptosis of BCPAP cells in a mitochondria-mediated pathway. After BCPAP

645

cells were treated with various concentrations of DATS, the protein levels of Bcl-2,

646

Bax, Bcl-XL. Actin and tubulin were used as internal controls. (B) The bands

647

quantification of Bcl-XL, Bcl-2 and Bax were analyzed by Gel Image System of

648

Tanon. *P < 0.05 vs. SC group of Bcl-2. #P < 0.05 vs. SC group of Bcl-XL. &P < 0.05

649

vs. SC group of Bax. SC, solvent control. (C) BCPAP cells were treated with various

650

concentrations of DATS and then the protein levels of cleaved capase-3, -8 and PARP

651

were determined by western blot. C-PARP, C-Caspase-8 and C-Caspase-3 represented

P < 0.01 vs. SC group of p-Rb, &P < 0.05 vs. SC group of p27. SC, solvent control.

24


Page 25 of 31


652

their cleaved form respectively. Actin and tubulin were used as internal controls. The

653

symbol ''*'' represented the quantification of their cleaved form of caspase-8,caspase-3

654

and PARP. (D) DATS-induced decrease in cell survival were resuced by z-VAD-fmk.

655

BCPAP cells were co-treated with indicated concentrations of DATS (5-20 µΜ) and

656

z-VAD-fmk (10 µΜ or 20 µΜ) and then the cell survival was measured by SRB assay.

657

The symbol ''*'' is compared with the individual DATS treatment group represented as

658

a white column. *P < 0.05, ***P < 0.001 vs. SC group. SC, solvent control.

659

Figure 6. DATS-induced apoptosis is independent of ROS production. (A) BCPAP

660

cells were collected after different dosages of DATS treatment for 24 h. Then the cells

661

were stained with 10 µM of DCFH-DA at 37 °C for 20 min and analyzed by flow

662

cytometry. (B) Histogram indicated the quantification of mean fluorescent intensity

663

(MFI) in three independent experiments. N.S., no significant difference.

664

Figure 7. DATS activates MAPK signaling pathway in BCPAP cells. (A) BCPAP

665

cells were treated with indicated concentrations of DATS for 24 h. The protein levels

666

of the total and phosphorylated ERK, JNK and p38 were determined by western blot.

667

Actin and tubulin were used as internal controls. (B) BCPAP cells were pretreated

668

with U0126 (20 µΜ) for 2 h and then incubated with DATS (20 µΜ) for another 24 h.

669

The protein levels of Bax, Bcl-2, total and phosphorylated ERK were determined by

670

western blot. Actin was used as an internal control. (C) The quantification of Bcl-2

671

and Bax in (B). (D) BCPAP cell were pretreated with 20 µΜ of SP600125 for 2 h and

672

then the cells were co-treated with 20 µΜ of DATS and SP600125 for another 24 h.

673

Then the level of p-JNK and cleaved PARP were determined by western blot. The

674

symbol ''*'' represented the quantification of cleaved PARP. (E) BCPAP cells were

675

pretreated with 20 µΜ of SB203580 for 2 h and then the cells were co-treated with 20

676

µΜ of DATS and SB203580 for additional 24 h. Then the level of p-p38 and cleaved

25



Page 26 of 31

677

PARP were determined by western blot. Tubulin was used as an internal control. The

678

symbol ''*'' represented the quantification of cleaved form of PARP.

679

26


Page 27 of 31


680

Figure 1. The chemical structure of DATS.

681 682

Figure 2. DATS inhibits the colony formation ability of BCPAP cells.

683 27



684

Page 28 of 31

Figure 3. DATS causes G0/G1 cell cycle arrest in BCPAP cells.

685 686

Fig. 4 DATS induces apoptosis of BCPAP cells.

687 688

Fig. 5. DATS-induced apoptosis is mediated by caspase activation.

28


Page 29 of 31


689 690

Figure 6. DATS-induced apoptosis is independent of ROS production.

691 692

Figure 7. DATS activates MAPK signaling pathway in BCPAP cells.

29



Page 30 of 31

693 694

30


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695

Graphic for table of Contents

696

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Induction of Apoptosis in Human Papillary-Thyroid-Carcinoma BCPAP

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