【文2】STAT3 抑制剂Niclosamide（1）

STAT3 抑制剂氯硝柳胺，在人结肠癌细胞中，与厄洛替尼发挥协同作用
Niclosamide inhibition of STAT3 synergizes with erlotinib in human colon cancer

Abstract

Niclosamide, an anthelmintic drug approved by the US Food and Drug Administration against cestodes, is used to treat tapeworm infection. In this study, we show that niclosamide can potentially inhibit signal transducer and activator of transcription 3 (STAT3) in colon cancer cell lines. Combined inhibition of epidermal growth factor receptor and STAT3 by erlotinib and niclosamide synergistically induces apoptosis and antiproliferation in colon cancer cell lines. Our findings suggest that erlotinib and niclosamide combination provides an effective therapeutic approach to improving the prognosis of colon cancer.

摘要

尼氯沙明是美国食品药品监督管理局批准的一种驱虫药物，用于治疗绦虫感染。在本研究中，我们发现尼氯沙明可能抑制结肠癌细胞系中的信号转导子和转录激活子3（STAT3）。厄洛替尼和尼氯沙明联合使用，可抑制表皮生长因子受体和STAT3，可协同诱导结肠癌细胞凋亡和抗增殖。我们的研究结果表明，厄洛替尼和尼氯沙明联合应用可有效改善结肠癌的预后。

词汇：

1、Abstract

Niclosamide. [药] 氯硝柳胺；[药] 灭绦灵

synergize 英 ['sɪnədʒaɪz] vi. 起增效剂作用 vt. 协同加强...的活动

anthelmintic [,ænθ(ə)l'mɪntɪk] adj. 驱除肠内寄生虫的 n. 驱虫剂；打虫药

cestode ['sestəʊd] n. 绦虫 adj. 绦虫的

tapeworm ['teɪpwɜːm] n. [基医] 绦虫

signal transducer and activator of transcription 3（STAT3）信号转导转录激活因子

epidermal growth factor receptor(EGFR) 表皮生长因子受体

induces apoptosis [,æpə(p)'təʊsɪs] 诱导凋亡

antiproliferation [pro,lɪfə'reʃən] 抗增殖

prognosis [prɒg'nəʊsɪs] n. [医] 预后；预知

Introduction

Colon cancer is the third most common cancer worldwide and the second leading cause of cancer death in the US.1The mortality rate of patients with colon cancer has decreased owing to improved detection, but the incidence of colon cancer continues to increase.2Considerable progress in the development of chemotherapy for advanced colon cancer has been observed in the recent decade.3The epidermal growth factor receptor (EGFR) has been reported as a critical therapeutic target for the treatment of colon cancer.4However, an anti-EGFR agent used to treat colon cancer has exhibited moderate efficacy.The mechanisms of this resistance have yet to be elucidated. Nevertheless, the potential of EGFR inhibitors to induce activation of the EGFR-independent pathway is under consideration.6,7

结肠癌是世界上第三大最常见的癌症，也是美国第二大癌症死亡原因。1由于检测水平的提高，结肠癌患者的死亡率有所下降，但结肠癌的发病率仍在继续上升。2近十年来，在晚期结肠癌化疗的发展方面，已取得可预见性的进展。表皮生长因子受体（EGFR）已被报道为治疗结肠癌的关键治疗靶点。4然而，用于治疗结肠癌的抗egfr药物已显示出中等疗效，且耐药机制仍未阐明。然而，EGFR抑制剂诱导EGFR通路激活的潜力仍在研究之中。

Signal transducer and activator of transcription (STAT) proteins significantly influence diverse biological processes, including cell proliferation, differentiation, survival, and inflammatory response.8,9One of the well-studied STAT members, STAT3, has been shown to be an important molecule in various malignancies and verified as an effective target for cancer therapy, including endometrial, cervical, breast, brain, prostate, and colon cancer.10–13A number of studies have indicated that aberrant STAT3 activation contributes to cell proliferation, differentiation, migration, and survival.14,15

信号转导子和转录激活子（stat）蛋白显著影响多种生物学过程，包括细胞增殖、分化、存活和炎症反应。8,9 Stat成员之一Stat3在各种恶性肿瘤中被证明是一种重要的分子，并被证实是一种有效的治疗肿瘤的药物。癌症治疗的目标，包括子宫内膜癌、宫颈癌、乳腺癌、脑癌、前列腺癌和结肠癌。10-13许多研究表明，异常的STAT3激活有助于细胞增殖、分化、迁移和存活。14,15

Niclosamide, particularly effective against cestodes, has been used to treat tapeworm infection for approximately 50 years.16Finding a new use for traditional medicines is considerably easier than inventing a new drug because traditional medicines have known pharmacokinetics and have often been approved for human treatments.17In the past 5 years, niclosamide has been identified as a potential anticancer agent targeting multiple signaling pathways (eg, NF-κB, ROS, Notch, Wnt/b-catenin, and mTORc1).18–21Recently, several studies have reported that niclosamide exhibits antiproliferative activity in head and neck, ovarian, breast, and hematologic cancer.22–25Till date, no studies demonstrating the therapeutic efficacy of niclosamide in colon cancer are available. Taking this background into account, the present study aimed to investigate the effects of niclosamide on colon cancer and the molecular mechanisms underlying its therapeutic action.

尼氯沙明，特别是对绦虫有效的，已经被用于治疗绦虫感染大约50年。16发现传统药物的新用途比发明一种新药物要容易得多，因为传统药物已经知道药代动力学，并且经常被批准用于人类治疗。17在过去5年中尼氯沙明被认为是一种潜在的抗癌药物，靶向多种信号途径（如NF-κB、ROS、Notch、Wnt/B-连环蛋白和mtorc1）。18-21最近，一些研究报告尼氯沙明在头颈癌、卵巢癌、乳腺癌和血液癌中表现出抗增殖活性。22-25到目前为止，还没有研究表明对尼氯沙明治疗结肠癌的疗效进行了评价。考虑到这一背景，本研究旨在研究尼氯沙明对结肠癌的作用及其治疗作用的分子机制。

Our outcomes demonstrated that niclosamide acted as a potent STAT3 inhibitor in colon cancer cell lines. The combination of niclosamide and erlotinib induced apoptosis and antiproliferation in colon cancer cell lines as well as sensitization of colon cancer cells to erlotinib. On the basis of our findings, we suggest that combination of niclosamide and erlotinib can potentially improve the prognosis of colon cancer.

我们的研究结果表明，尼氯沙明在结肠癌细胞系中作为一种有效的STAT3抑制剂发挥作用。尼氯沙明和厄洛替尼联合应用可诱导结肠癌细胞株的凋亡和增殖，并使结肠癌细胞对厄洛替尼敏感。根据我们的研究结果，我们认为尼氯沙明和厄洛替尼联合应用可能改善结肠癌的预后。

词汇：

2、Introduction

mortality [mɔː'tælɪtɪ] n. 死亡数，死亡率

moderate ['mɒd(ə)rət] adj. 稳健的，温和的；vi. 变缓和，变弱vt. 节制；减轻

elucidated [ɪ'l(j)uːsɪdeɪt] ·· vt. 阐明；说明

endometrial [ɛndʌ'mɛtrɪəl] adj. [解剖] 子宫内膜的

cervical ['sɜːvɪk(ə)l] adj. 颈的；子宫颈的

prostate ['prɒsteɪt] adj. 前列腺的 n. [解剖] 前列腺

aberrant [ə'ber(ə)nt] adj. 异常的；畸变的；

differentiation [,dɪfərenʃɪ'eɪʃn] n. 变异，[生物] 分化

approximate [ə'prɒksɪmət] vt. 近似；粗略估计vi. 接近于adj. [数] 近似的

pharmacokinetics [,fɑ:məkəuki'netiks, -kai-] n. 药物（代谢）动力学

hematologic 血液学的

hematology [,hemə'tɒlədʒɪ] 血液学

ovarian [əʊ'veərɪən] adj. [解剖] 卵巢的；子房的

sensitization [,sɛnsɪtɪ'zeʃən] n. 敏化作用；促进感受性；感光度之增强

Materials and reagents

Cell culture and antibodies

Human colon cancer cell lines (HCT116, SW620, and HT29) were obtained from Cell Resources center of the Shanghai Institutes for Biological Sciences (Chinese Academy of Sciences, Shanghai, People’s Republic of China). The cells were routinely cultured in RPMI-1640 (Gibco/BRL; Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT, USA) and 1% of antibiotic solution (100 units/mL penicillin and 100 µg/mL streptomycin) in a humidified atmosphere of 5% CO2at 37°C.

Antibodies such as anti-BCL-2, anti-Bax, anti-cleaved PARP, anti-GAPDH, horseradish peroxidase (HRP)- conjugated goat anti-mouse IgG, and HRP-conjugated donkey anti-rabbit IgG were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Antibodies against Phospho-STAT3 and STAT3 were purchased from Cell Signaling Technology (Danvers, MA, USA).

Inhibitor drugs

Nifuroxazide, niclosamide, cryptotanshinone, and alantolactone were purchased from Selleck Chemicals (Houston, TX, USA). Nifuroxazide is an oral nitrofuran antibiotic not currently approved for use in the USA but is used elsewhere as an antidiarrheal agent.26Niclosamide has been used to treat tapeworm infection and also as a molluscicide for water treatment in schistosomiasis control programs.27Cryptotanshinone, a major lipophilic component isolated from Salvia miltiorrhiza Bunge, has been shown to possess chemotherapeutic properties against various types of cancer cells.28Alantolactone is used as an insect repellent, antibacterial, and anticancer agent.29The drug compounds were dissolved in sterile dimethyl sulfoxide (DMSO) to produce a 20 mM stock solution, which was then stored at −20°C and further diluted freshly with cell culture medium.

MTT assay

Cells were seeded into wells of a 96-well plate at 5×103cells per well in 100 µL of the corresponding medium. The cells were then treated with drugs at different concentrations for 72 h. Subsequently, they were treated with a fresh solution of MTT (5 mg/mL) for 4 h at 37°C. The purple formazan crystals were finally solubilized with DMSO solution, and absorbance was recorded using a multi-well plate reader at 490 nm.

Western blot analysis

Cells were lysed in a lysis buffer containing a phosphatase inhibitor, and the lysates were clarified by centrifugation (12,000 rpm) at 4°C for 10 min. The supernatant was run on 10% and 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane. After being blocked with 5% nonfat dry milk in Tris Buffer Solution Tween for 1.5 h, membranes were incubated with a specific primary antibody of 1:1,000 dilution overnight and a HRP-conjugated secondary antibody of 1:3,000 dilution for 1 h. Immunoreactive bands were visualized using enhanced chemiluminescence reagent.

Molecular docking

As one of the most widely used computational approaches for structure-based drug design, molecular docking study was used to predict the binding pose of compound in STAT3 SH2-binding site by using the software AutoDock (version 4.2.6).30The crystallographic coordinate for human STAT3 SH2 (Protein Data Bank [PDB] ID: 1BG1) was obtained from the PDB.31Prior to docking, protein structures were prepared by removing water molecules and other ligands using PyMol software.32A grid box size of 60×60×60 dimensions with a spacing of 0.375 Å between the grid points was implemented and covered almost the entire SH2-binding site. The grid parameter files were created setting up the map files directly. The Lamarckian genetic algorithm was applied to deal with the interactions of protein and inhibitors. The number of individuals in population was set to 300, and trials of 100 dockings and maximum number of energy evaluations were set as default along with other settings. AutoDockTools version 1.5.6 and PyMol were used to analyze the docking results.

Clonogenic assay

A total of 500 cells per well were seeded into a 6-well plate with 2 mL of RPMI-1640 and incubated overnight. The cells were then pretreated with nifuroxazide and erlotinib or DMSO for 8–12 h. After treatment, the cells were washed with phosphate buffer saline (PBS) twice and transferred to a fresh medium to grow for 7 days. Colonies were washed with PBS and then fixed with 4% methanol for 15 min at room temperature. The cells were washed with PBS twice and stained with 1% crystal violet (25% methanol) for 10 min at room temperature. Each experiment was conducted thrice.

Analysis of cell apoptosis

Cells (3×105) were seeded in 6-well plates and incubated overnight and then treated with nifuroxazide and erlotinib for 24 h. After treatment, the cells were harvested with trypsin and then washed with cold PBS twice. The cells were stained with Annexin V for 10 min under dark conditions and then with propidium iodide (PI) for 5 min. Apoptotic cells were counted using the FACS Calibur flow cytometer and quantified by flow cytometric analysis.

Statistical analyses

Data are represented as mean ± standard error of the mean of 3 independent experiments. Student’st-test was performed to determine the statistical significance between 2 groups by using GraphPad Prism 6.0 (GraphPad Software, Inc., La Jolla, CA, USA). Differences between groups were analyzed by the log-rank test using GraphPad Prism 6.0.P<0.05 was considered statistically significant.

词汇：
3、Materials and reagents

supplement ['sʌplɪm(ə)nt] vt. 增补，补充 n. 补充；补充物；增刊，副刊

fetal ['fiːt(ə)l] adj. 胎的，胎儿的

bovine ['bəʊvaɪn] adj. 牛的；似牛的；迟钝的 n. 牛科动物

serum ['sɪərəm] n. 血清；免疫血清；精华液

nifuroxazide 硝呋齐特

cryptotanshinone 隐丹参醌,隐丹参酮

crypto ['krɪptəʊ] n. 秘密赞同者；秘密党员

tanshinone 丹参醌,丹参酮

humidify [hjʊ'mɪdɪfaɪ] vt. 使潮湿；使湿润

antibiotic [,æntɪbaɪ'ɒtɪk] adj. 抗生的；抗菌的 n. 抗生素，抗菌素

horseradish ['hɔːsrædɪʃ] n. 【植物】辣根

nitrofuran [,naɪtrəʊ'fjʊəræn] n. [药] 硝基呋喃

antidiarrheal ['æntidaiə'ri:l] n. 止泻剂adj. 止泻的

molluscicid 杀软体动物剂

schistosomiasis [,ʃɪstə(ʊ)sə'maɪəsɪs] n. [内科] 血吸虫病

Salvia miltiorrhiza Bunge 丹参

Alantolactone 超氧自由基

sterile ['steraɪl] adj. 不育的；无菌的；贫瘠的；不毛的；枯燥乏味的

dimethyl sulfoxide (DMSO) [daɪˈmiːθaɪl] [sʌlf'ɒksaɪd] 二甲基亚砜

formazan ['fɔ:mə,zæn] n. 甲瓒；甲腊；有色甲

solubilize ['sɑljəbə,laɪz] vt. 使溶解；使增溶 vi. 溶解

absorbance [əb'zɔːb(ə)ns] n. [物化] 吸光度；吸收率

lyse [laɪz] vi. 病状渐退；细胞溶解 vt. 溶化；溶解

phosphatase ['fɒsfəteɪz] n. [生化] 磷酸酶

clarify ['klærɪfaɪ] vt. 澄清；阐明 vi. 得到澄清；变得明晰；得到净化

centrifugation [sen,trɪfjʊ'ɡeɪʃən] n. 离心分离

supernatant [,suːpə'neɪt(ə)nt-] adj. 浮在表面的；上层的n. 浮层；上层清液

sodium dodecyl sulfate-polyacrylamide gel electrophoresis

十二烷基硫酸钠-聚丙烯酰胺凝胶电泳

sodium ['səʊdɪəm] n. [化学] 钠（11号元素，符号 Na）

dodecyl ['dodə,sɪl] n. 十二烷基

sulfate ['sʌlfeɪt] n. [无化] 硫酸盐 vt. 使成硫酸盐 vi. 硫酸盐化

polyacrylamide [,pɒlɪə'krɪləmaɪd] n. [高分子] 聚丙烯酰胺

polyvinylidene fluoride membrane (PVDF) PVDF膜

electrophoresis [ɪ,lektrə(ʊ)fə'riːsɪs] n. [化学] 电泳

polyvinylidene [,pɒlɪvaɪ'nɪlə,diːn] n. 聚乙二烯

fluoride ['flʊəraɪd] n. 氟化物

dilution [daɪ'luːʃn] n. 稀释，冲淡

Immunoreactive [,ɪmjʊnəʊrɪ'æktɪv] adj. 免疫反应性的

chemiluminescence [,kemɪ,l(j)uːmɪ'nes(ə)ns] n. [化学] 化学发光，化合光

computational [kɒmpjʊ'teɪʃənl adj. 计算的

binding pose ？

crystallographic [,krɪstəlɑ'ɡræfɪk] adj. 结晶的

coordinate [kəʊ'ɔ:dɪneɪt] n. 坐标；同等的人或物

adj. 并列的；同等的 vt. 调整；整合 vi. 协调

grid [grɪd] n. 网格；栅格

Clonogenic assay

Analysis of cell apoptosis

Statistical analyses

4、Results

Antiproliferative effects of niclosamide in human colon cancer cells

Nifuroxazide acts as a potent inhibitor of STAT3 signaling pathway in breast cancer cells, though it has little effect on cells lacking STAT3 activation.33 Niclosamide has recently been identified to target multiple signaling pathways (eg, NF-κB, ROS, Notch, and STAT3).16 Cryptotanshinone has previously been observed to possess the most powerful antibacterial, anti-inflammatory, and antitumor effect.34 Alantolactone has an inhibitory effect on cancer cells migration, invasion, adhesion, and colony formation.35 Therefore, we screened the antiproliferative effects of these 4 compounds in human colon cancer cells by MTT assay.After the cells were treated with nifuroxazide, niclosamide, cryptotanshinone, and alantolactone for 72 h, MTT assay was employed. As shown in Figure 1, administration of these 4 compounds in a dose-dependent manner reduced viability in SW620 (Figure 1A), HCT116 (Figure 1B), and HT29 cells (Figure 1C). Among these compounds, niclosamide exhibited the most potent antiproliferative effect against all tested human colon cancer cell lines. After exposure of the cells to niclosamide for 72 h, the half maximal inhibitory concentration (IC50) in SW620, HCT116, and HT29 cell lines was found to be 2.9, 0.4, and 8.1 µM, respectively (Figure 1D). This suggests that niclosamide can inhibit the proliferation of human colon cancer cells.

硝呋沙嗪是乳腺癌细胞中STAT3信号通路的一种有效抑制剂，尽管它对缺乏STAT3激活的细胞几乎没有作用。33尼氯沙明最近被鉴定为靶向多种信号通路（如NF-κB、ROS、Notch和STAT3）。16隐丹参酮以前被观察到具有最强的信号通路。抗菌、抗炎和抗肿瘤作用。34-丙妥拉通对癌细胞迁移、侵袭、粘附和菌落形成有抑制作用。35因此，我们用MTT法筛选了这4种化合物在人结肠癌细胞中的抗增殖作用。在用硝呋唑嗪、尼氯沙明、地塞米松治疗后，我们检测了这4种化合物在人结肠癌细胞中的抗增殖作用。奥坦辛酮、丙氨酸妥拉酮72小时，MTT测定。如图1所示，以剂量依赖性方式投与这4种化合物可降低SW620（图1a）、HCT116（图1b）和HT29细胞（图1c）的存活率。在这些化合物中，尼氯沙明对所有试验的人结肠癌细胞株显示出最有效的抗增殖作用。将细胞暴露于尼氯沙明72小时后，发现SW620、HCT116和HT29细胞系中的半最大抑制浓度（IC50）分别为2.9、0.4和8.1μm（图1d）。这表明尼氯沙明可以抑制人结肠癌细胞的增殖。

Molecular docking between STAT3 and niclosamide

STAT3, a member of the STAT family of transcription factors, has recently been verified as an attractive therapeutic target and shown to be an important molecule in cancer therapy, including colon cancer.36 As the STAT3 SH2 domain is critical for the activation and biological function of STAT3,37 we investigated interactions between the compound and the SH2 domain of STAT3 by molecular docking analysis of the active compound niclosamide (Figure 2A). We applied PyMol software to remove water molecules and other ligands to prepare the STAT3 SH2 domain protein structures and used the software AutoDock (version 4.2.6) to predict the binding pose of compound in STAT3 SH2-binding site to identify whether compound niclosamide acts as a STAT3 inhibitor that targets the STAT3 SH2 domain.38 The molecular docking results indicated that niclosamide formed 4 hydrogen bonds with the side chain of ARG-595, SER-636, GLU-594, and LYS-591 as shown in Figure 2B. It was predicted that niclosamide could fit into the 2 major binding sites, the pTyr705 and the side pocket site, and so it could inhibit STAT3 phosphorylation. In summary, critical hydrogen bond interaction between niclosamide and STAT3 SH2 domain were predicted by a rational method combined with molecular docking. These methods clarified the interaction of niclosamide with STAT3. Further verification of co-crystallization of STAT3 and niclosamide is in progress.

Stat3是Stat转录因子家族的一员，最近被证实是一个有吸引力的治疗靶点，并被证明是包括结肠癌在内的癌症治疗中的一个重要分子。36由于Stat3 sh2结构域对Stat3的激活和生物学功能至关重要，37我们研究了化合物a和b之间的相互作用。通过活性化合物尼氯沙明的分子对接分析，得到了STAT3的SH2结构域（图2a）。我们应用pymol软件去除水分子和其他配体，以制备stat3 sh2结构域蛋白结构，并使用autodock软件（4.2.6版）预测stat3 sh2结合位点化合物的结合位置，以确定化合物尼氯沙明是否作为靶向stat3 sh2结构域的stat3抑制剂。38分子结构AR对接结果表明，尼氯沙明与arg-595、ser-636、glu-594、lys-591侧链形成4个氢键，如图2b所示，预计尼氯沙明可与2个主要结合位点ptyr705和侧口袋位点结合，从而抑制Stat3磷酸化。综上所述，采用合理的方法结合分子对接，预测了尼氯沙明与STAT3-SH2结构域的临界氢键相互作用。这些方法阐明了尼氯沙明与STAT3的相互作用。STAT3和尼氯沙明共结晶的进一步验证正在进行中。

Inhibition of STAT3 phosphorylation by niclosamide in colon cancer cells HCT116 and SW620

We performed Western blot analysis on colon cancer cell lines to examine whether niclosamide could inhibit STAT3. Western blot analysis indicated that niclosamide inhibited STAT3 phosphorylation (Figure 3). Notably, niclosamide inhibited STAT3 phosphorylation in a time- and dose-dependent manner in HCT116 and SW620 cell lines.

尼氯沙明对结肠癌细胞HCT116和SW620中STAT3磷酸化的抑制作用

我们对结肠癌细胞株进行了Western blot分析，以检测尼氯沙明是否能抑制STAT3。Western blot分析表明，尼氯沙明抑制Stat3磷酸化（图3）。值得注意的是，在HCT116和SW620细胞系中，尼氯沙明以时间和剂量依赖性方式抑制Stat3磷酸化。

Sensitization of colon cancer cells to erlotinib by treatment of cells with niclosamide

Erlotinib or gefitinib, EGFR tyrosine kinase inhibitors, have been shown to benefit patients with non-small-cell lung cancer and pancreatic cancer. However, almost all patients develop a progressive disease during therapy. As shown in Figure 4A, after treatment of cells with erlotinib and gefitinib for 72 h, the IC50 in SW620, HCT116, and HT29 cells exceeded 60 µM. These results indicate that erlotinib and gefitinib could not inhibit the proliferation of SW620, HCT116, and HT29 cell lines. We thus examined the anti-proliferative effects of erlotinib as well as erlotinib combined with niclosamide by MTT assay. As shown in Figure 4B, treatment of cells with niclosamide sensitized colon cancer cells to erlotinib, while niclosamide had no effect on SW620, HCT116, and HT29 cells at 0.5 µM and 1 µM concentrations (Figure 4C). To verify the anticancer properties of the combination, we performed colony-forming experiments. SW620 cells (5000) were seeded in a 6-well plate overnight and then treated with erlotinib (10 µM) or/and niclosamide (2.5 µM) for 1 week. We then removed the drugs and washed the cells with PBS twice. The cell colonies were stained with crystal violet and then counted. The combination of niclosamide and erlotinib suppressed the colony formation of colon cancer cells, as shown in Figure 4D and E. Colony formation assay demonstrated that the synergistic effect of niclosamide and erlotinib efficiently inhibits the growth of colon cancer cells.

尼氯沙明对结肠癌细胞的致敏作用

Erlotinib或吉非替尼（egfr酪氨酸激酶抑制剂）已被证明对非小细胞肺癌和胰腺癌患者有益。然而，几乎所有的病人在治疗过程中都会发展成一种进行性疾病。如图4a所示，用厄洛替尼和吉非替尼处理细胞72小时后，SW620、HCT116和HT29细胞中的IC50超过60μm。这些结果表明厄洛替尼和吉非替尼不能抑制SW620、HCT116和HT29细胞系的增殖。因此，我们用MTT法检测了厄洛替尼、厄洛替尼联合尼氯沙明的抗增殖作用。如图4b所示，在0.5μm和1μm浓度下，用尼氯沙明致敏的结肠癌细胞治疗埃洛替尼，而尼氯沙明对SW620、HCT116和HT29细胞无影响（图4c）。为了验证该组合的抗癌性能，我们进行了集落形成实验。将SW620细胞（5000）接种于6孔板中过夜，然后用厄洛替尼（10μm）或/和尼氯沙明（2.5μm）处理1周。然后我们取出药物，用PBS清洗细胞两次。细胞集落用结晶紫染色后计数。如图4d和E所示，尼氯沙明和厄洛替尼的联合抑制了结肠癌细胞的集落形成。集落形成分析表明，尼氯沙明和厄洛替尼的协同作用可有效抑制结肠癌细胞的生长。

Induction of apoptosis in colon cancer cells with combined niclosamide and erlotinib

Compared with single-agent treatment, the combination of erlotinib and niclosamide enhanced the inhibition of cell viability in all tested cell lines (Figure 4B). We sought to investigate whether the combination of erlotinib and niclosamide could also induce cell apoptosis. We first employed Western blot analysis. HCT116 and SW620 cells were treated with erlotinib and niclosamide alone, or with both for 24 h. The combination significantly increased cleaved PARP and BAX, as shown in Figure 5A and B. BCL-2 expression was markedly decreased.

We also used flow cytometry to examine whether the combination induced cell apoptosis in SW620 cells. SW620 cells were treated with erlotinib (10 µM) or/and niclosamide (2.5 µM) for 24 h and then stained with Annexin V and PI for apoptosis analysis. The combination resulted in a significant increase in early (Annexin V+, PI−) and late apoptotic cells (Annexin V+, PI+), as shown in Figure 5C and D.

尼氯沙明与厄洛替尼联合诱导结肠癌细胞凋亡

与单剂处理相比，厄洛替尼和尼氯沙明的组合增强了对所有被测细胞系细胞活力的抑制（图4b）。我们试图研究厄洛替尼和尼氯沙明联合应用是否也能诱导细胞凋亡。我们首先采用蛋白质印迹分析。HCT116和SW620细胞单独或同时用二者治疗24小时。如图5a和b所示，联合治疗显著增加了切割后的parp和bax。bcl-2表达显著降低。

我们还用流式细胞仪检测了联合用药是否诱导SW620细胞凋亡。SW620细胞用厄洛替尼（10μm）或/和尼氯沙明（2.5μm）处理24小时，然后用膜联蛋白V和PI染色进行凋亡分析。这种组合导致早期（膜联蛋白V+，pi-）和晚期凋亡细胞（膜联蛋白V+，pi+）显著增加，如图5C和D所示。

5、Discussion

Colon cancer is one of the most prevalent tumors worldwide.39 Despite conventional therapies such as surgery, radiation, and chemotherapy, the overall survival rate for colon cancer has not significantly improved.40 The human EGFR is overexpressed in various gastrointestinal cancer types, including 60%–80% of colon cancers, correlating with poor prognosis and early disease progression.41 In addition, cancer cell also upregulates other important downstream genes such as STAT3, which contributes to cell proliferation, cell survival, and angiogenesis in colon cancer.42 Therefore, more effective approaches need to be developed to improve the prognosis of colon cancer. Here, our results showed that niclosamide reduces cell viability in a dose-dependent manner. This finding suggests that niclosamide could be effective against colon cancer. Several groups have independently revealed the activity of niclosamide against cancer cells;16,27,43 however, the precise mechanism underlying this antitumor activity has yet to be elucidated. Therefore, we first used molecular docking study to verify whether there is interaction between niclosamide

and STAT3. In our study, we found critical hydrogen bond interaction between niclosamide and STAT3 SH2 domain. We further investigated the correlation between niclosamide and STAT3 in SW620 and HCT116 cell lines, and the results showed that niclosamide suppressed STAT3 phosphorylation in a time- and dose-dependent manner. Several studies have revealed that inhibition of EGFR by erlotinib induces activation of STAT3, which contributes to erlotinib resistance.44,45 Our data showed that when erlotinib and niclosamide were combined, they synergistically induced apoptosis of colon cancer cells. It has been reported that multiple signal transduction pathway inhibitors, including the mitogen-activated protein kinase, fibroblast growth factor receptor inhibitors, and chemotherapy drugs, can induce activation of STAT3 survival signaling pathway, leading to resistance.38,46 It is possible that, in addition to erlotinib, niclosamide may also sensitize these target drugs. Based on our findings, we conclude that niclosamide may represent a novel and more effective drug for colon cancer. Drug development process from initial lead discovery to final medication is costly, lengthy, and incremental.33 Finding new uses for old or natural products is much easier and more economical than inventing a new drug from scratch. The present study demonstrates that niclosamide can inhibit the cell

结肠癌是世界上最普遍的肿瘤之一。39尽管传统的治疗方法如手术、放疗和化疗，结肠癌的总生存率并没有显著提高。40人类的表皮生长因子受体在各种类型的胃肠道癌中过度表达，包括60%-80%的结肠癌，与穷人相关。预后和早期疾病进展。41此外，癌细胞还上调了其他重要的下游基因，如有助于结肠癌细胞增殖、细胞存活和血管生成的STAT3。42因此，需要开发更有效的方法来改善结肠癌的预后。在这里，我们的研究结果表明，尼氯沙明以剂量依赖性的方式降低细胞活力。这一发现表明，尼氯沙明可以有效地治疗结肠癌。几个小组独立地揭示了尼氯沙明对癌细胞的活性；然而，16，27，43，这种抗肿瘤活性的确切机制尚未阐明。因此，我们首先使用分子对接研究来验证尼氯沙明之间是否存在相互作用和状态3。在我们的研究中，我们发现了尼氯沙明和Stat3sh2结构域之间的临界氢键相互作用。我们进一步研究了在SW620和HCT116细胞系中，尼氯沙明与STAT3的相关性，结果表明，尼氯沙明以时间和剂量依赖性的方式抑制了STAT3的磷酸化。有几项研究表明，埃洛替尼抑制表皮生长因子受体（egfr）可诱导Stat3的活化，这有助于埃洛替尼的耐药性。44,45我们的数据表明，当埃洛替尼和尼氯沙明联合使用时，它们可协同诱导结肠癌细胞凋亡。据报道，包括丝裂原活化蛋白激酶、成纤维细胞生长因子受体抑制剂和化疗药物在内的多种信号转导途径抑制剂可诱导STAT3生存信号通路的活化，从而导致耐药性。38,46除了厄洛替尼外，尼氯沙明还可能导致耐药。使这些靶向药物增敏。根据我们的发现，我们得出结论，尼氯沙明可能是一种新的和更有效的治疗结肠癌的药物。从最初的铅发现到最终的药物开发过程是昂贵的、漫长的和渐进的。33发现旧产品或天然产品的新用途比从头发明新药物更容易和更经济。本研究表明，尼氯沙明对细胞有抑制作用。通过抑制STAT3磷酸化来生长结肠癌。我们还研究了埃洛替尼和尼氯沙明协同抑制结肠癌对EGFR和STAT3的联合抑制作用。本研究仅在体外进行，一些化合物在体外可能表现出有效的抗肿瘤活性，但在体内没有显示出抗肿瘤活性。因此，有必要在临床前和临床环境中进行进一步的分子研究，以协同方式评估抗癌潜力，以证实尼氯沙明是一种有效的结肠癌治疗候选药物。