STAT3 抑制劑氯硝柳胺，在人結腸癌細胞中，與厄洛替尼發揮協同作用
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
尼氯沙明是美國食品藥品監督管理局批準的一種驅蟲藥物，用于治療絳蟲感染。在本研究中，我們發現尼氯沙明可能抑制結腸癌細胞系中的信號轉導子和轉錄激活子3（STAT3）。厄洛替尼和尼氯沙明聯合使用，可抑制表皮生長因子受體和STAT3，可協同誘導結腸癌細胞凋亡和抗增殖。我們的研究結果表明，厄洛替尼和尼氯沙明聯合應用可有效改善結腸癌的預后。
詞匯：
1、Abstract
????Niclosamide.? ?[藥] 氯硝柳胺；[藥] 滅絳靈?
????synergize? ? ? ? 英?['s?n?d?a?z]?vi. 起增效劑作用 vt. 協同加強...的活動
????anthelmintic? ? ? ??[,?nθ(?)l'm?nt?k]?adj. 驅除腸內寄生蟲的 n. 驅蟲劑；打蟲藥
? ?? cestode? ? ? ? ? ? ['sest??d]?n. 絳蟲? adj. 絳蟲的
????tapeworm?????????['te?pw??m]?n. [基醫] 絳蟲
? ??signal transducer and activator of transcription 3（STAT3）? ? 信號轉導轉錄激活因子
????epidermal growth factor receptor(EGFR)? ? 表皮生長因子受體?
? ?? induces apoptosis [,?p?(p)'t??s?s]? ? ? ? ? ? 誘導凋亡
? ??antiproliferation?[pro,l?f?'re??n] ? ? ? 抗增殖
? ??prognosis [pr?g'n??s?s]? ? ? ? ? ? ? ?n. [醫] 預后；預知
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
結腸癌是世界上第三大最常見的癌癥，也是美國第二大癌癥死亡原因。1由于檢測水平的提高，結腸癌患者的死亡率有所下降，但結腸癌的發病率仍在繼續上升。2近十年來，在晚期結腸癌化療的發展方面，已取得可預見性的進展。表皮生長因子受體（EGFR）已被報道為治療結腸癌的關鍵治療靶點。4然而，用于治療結腸癌的抗egfr藥物已顯示出中等療效，且耐藥機制仍未闡明。然而，EGFR抑制劑誘導EGFR通路激活的潛力仍在研究之中。
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
信號轉導子和轉錄激活子（stat）蛋白顯著影響多種生物學過程，包括細胞增殖、分化、存活和炎癥反應。8,9 Stat成員之一Stat3在各種惡性腫瘤中被證明是一種重要的分子，并被證實是一種有效的治療腫瘤的藥物。癌癥治療的目標，包括子宮內膜癌、宮頸癌、乳腺癌、腦癌、前列腺癌和結腸癌。10-13許多研究表明，異常的STAT3激活有助于細胞增殖、分化、遷移和存活。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，特別是對絳蟲有效的，已經被用于治療絳蟲感染大約50年。16發現傳統藥物的新用途比發明一種新藥物要容易得多，因為傳統藥物已經知道藥代動力學，并且經常被批準用于人類治療。17在過去5年中尼氯沙明被認為是一種潛在的抗癌藥物，靶向多種信號途徑（如NF-κB、ROS、Notch、Wnt/B-連環蛋白和mtorc1）。18-21最近，一些研究報告尼氯沙明在頭頸癌、卵巢癌、乳腺癌和血液癌中表現出抗增殖活性。22-25到目前為止，還沒有研究表明對尼氯沙明治療結腸癌的療效進行了評價。考慮到這一背景，本研究旨在研究尼氯沙明對結腸癌的作用及其治療作用的分子機制。
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.
我們的研究結果表明，尼氯沙明在結腸癌細胞系中作為一種有效的STAT3抑制劑發揮作用。尼氯沙明和厄洛替尼聯合應用可誘導結腸癌細胞株的凋亡和增殖，并使結腸癌細胞對厄洛替尼敏感。根據我們的研究結果，我們認為尼氯沙明和厄洛替尼聯合應用可能改善結腸癌的預后。
詞匯：
2、Introduction
?????mortality? [m??'t?l?t?]? ? ? ? ? ? ? ? ? ?n. 死亡數，死亡率
?????moderate??['m?d(?)r?t]????????????? adj. 穩健的，溫和的；vi. 變緩和，變弱vt. 節制；減輕
? ???elucidated?[?'l(j)u?s?de?t]? ? ? ··? ?vt. 闡明；說明
? ??endometrial?[?nd?'m?tr??l]?????????adj. [解剖] 子宮內膜的
? ???cervical??['s??v?k(?)l]?????????????????adj. 頸的；子宮頸的
?????prostate? ['pr?ste?t]?????????????????adj. 前列腺的 n. [解剖] 前列腺
? ??aberrant?[?'ber(?)nt]?????????????????adj. 異常的；畸變的；
? ??differentiation?[,d?f?ren??'e??n]??n. 變異，[生物] 分化 ? ? ? ? ? ? ? ?
? ??approximate?[?'pr?ks?m?t]?????????vt. 近似；粗略估計vi. 接近于adj. [數] 近似的
?????pharmacokinetics?[,fɑ:m?k?uki'netiks, -kai-]?n. 藥物（代謝）動力學
????hematologic? ? ? ? ? ? ? ? ? ? ? ? ? ? ?血液學的
? ??hematology?[,hem?'t?l?d??]? ? ?血液學
? ??ovarian?[??'ve?r??n]? ? ? ? ? ? ? ? ?adj. [解剖] 卵巢的；子房的
? ??sensitization?[,s?ns?t?'ze??n]? ? ?n. 敏化作用；促進感受性；感光度之增強
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 ? 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.
詞匯：
3、Materials and reagents
? ??supplement?['s?pl?m(?)nt]?? ? ? ?vt. 增補，補充 n. 補充；補充物；增刊，副刊
? ??fetal?['fi?t(?)l]????????????????????????????adj. 胎的，胎兒的
????bovine?['b??va?n]????????????????????adj. 牛的；似牛的；遲鈍的 n. 牛科動物
????serum?['s??r?m]????????????????????????n. 血清；免疫血清；精華液
? ??nifuroxazide????????????????????????????硝呋齊特
? ? cryptotanshinone????????????????????隱丹參醌,隱丹參酮
? ? crypto??['kr?pt??]????????????????????n. 秘密贊同者；秘密黨員
? ? ?tanshinone????????????????????????????丹參醌,丹參酮
? ???humidify [hj?'m?d?fa?]? ? ? ? ? vt. 使潮濕；使濕潤
? ??antibiotic??[,?nt?ba?'?t?k]? ? ? ?adj. 抗生的；抗菌的 n. 抗生素，抗菌素
? ??horseradish?['h??sr?d??]? ? ? ??n. 【植物】辣根?
? ? ?nitrofuran?[,na?tr??'fj??r?n]?n. [藥] 硝基呋喃?????
? ??antidiarrheal?['?ntidai?'ri:l]? ? ?n. 止瀉劑adj. 止瀉的
? ??molluscicid?????????????????????????????殺軟體動物劑
? ? ?schistosomiasis?[,??st?(?)s?'ma??s?s]?n. [內科] 血吸蟲病
? ??Salvia miltiorrhiza?Bunge? ? ? ??丹參
? ??Alantolactone? ? ? ? ? ? ? ? ? ? ? ? ? ?超氧自由基
? ?sterile?['stera?l]????????????????????????adj. 不育的；無菌的；貧瘠的；不毛的；枯燥乏味的
????dimethyl sulfoxide (DMSO)??[da??mi?θa?l]?[s?lf'?ksa?d]? 二甲基亞砜
? ? ?formazan?['f?:m?,z?n]? ? ? ? ? ?n. 甲瓚；甲臘；有色甲
? ??solubilize ['sɑlj?b?,la?z]? ? ? ? ? ??vt. 使溶解；使增溶 vi. 溶解
? ??absorbance?[?b'z??b(?)ns]????????n. [物化] 吸光度；吸收率
?????lyse [la?z]????????????????????????????????vi. 病狀漸退；細胞溶解? vt. 溶化；溶解
? ??phosphatase?['f?sf?te?z]? ? ? ? ? n. [生化] 磷酸酶
? ??clarify ['kl?r?fa?]????????????????????????vt. 澄清；闡明 vi. 得到澄清；變得明晰；得到凈化
? ??centrifugation?[sen,tr?fj?'ɡe???n]?n. 離心分離
? ??supernatant [,su?p?'ne?t(?)nt-]? ? adj. 浮在表面的；上層的n. 浮層；上層清液
? ??sodium dodecyl sulfate-polyacrylamide gel electrophoresis?
????十二烷基硫酸鈉-聚丙烯酰胺凝膠電泳
????sodium?['s??d??m]?????????????????????n. [化學] 鈉（11號元素，符號 Na）
? ??dodecyl?['dod?,s?l]? ? ? ? ? ? ? ? ? ? ?n. 十二烷基
????sulfate? ?['s?lfe?t]?????????????????????????n. [無化] 硫酸鹽 vt. 使成硫酸鹽? vi. 硫酸鹽化
? ??polyacrylamide?[,p?l??'kr?l?ma?d]????????n. [高分子] 聚丙烯酰胺? ? ? ? ? ? ?
????polyvinylidene fluoride membrane (PVDF)? PVDF膜
????electrophoresis?[?,lektr?(?)f?'ri?s?s]? ? ? ?n. [化學] 電泳
? ??polyvinylidene?[,p?l?va?'n?l?,di?n]?? ? ? ? ?n. 聚乙二烯
????fluoride?['fl??ra?d]? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ?n. 氟化物
????dilution?[da?'lu??n]????????????????????????????????????n. 稀釋，沖淡
????Immunoreactive?[,?mj?n??r?'?kt?v]? ? ? ??adj. 免疫反應性的
????chemiluminescence?[,kem?,l(j)u?m?'nes(?)ns]?n. [化學] 化學發光，化合光
? ??computational?[k?mpj?'te???nl?????????????????adj. 計算的
? ???binding pose? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ？
? ??crystallographic?[,kr?st?lɑ'ɡr?f?k]? ? ? ? ? ? ?adj. 結晶的
? ??coordinate? [k??'?:d?ne?t]?? ? ? ? ? ? ? ? ? ? ? n. 坐標；同等的人或物
????????????????????????????????????????????????????????????????????adj. 并列的；同等的 vt. 調整；整合 vi. 協調
? ??grid?[gr?d]????????????????????????????????????????????????n. 網格；柵格
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 ? 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.
? ??
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.? ?
硝呋沙嗪是乳腺癌細胞中STAT3信號通路的一種有效抑制劑，盡管它對缺乏STAT3激活的細胞幾乎沒有作用。33尼氯沙明最近被鑒定為靶向多種信號通路（如NF-κB、ROS、Notch和STAT3）。16隱丹參酮以前被觀察到具有最強的信號通路。抗菌、抗炎和抗腫瘤作用。34-丙妥拉通對癌細胞遷移、侵襲、粘附和菌落形成有抑制作用。35因此，我們用MTT法篩選了這4種化合物在人結腸癌細胞中的抗增殖作用。在用硝呋唑嗪、尼氯沙明、地塞米松治療后，我們檢測了這4種化合物在人結腸癌細胞中的抗增殖作用。奧坦辛酮、丙氨酸妥拉酮72小時，MTT測定。如圖1所示，以劑量依賴性方式投與這4種化合物可降低SW620（圖1a）、HCT116（圖1b）和HT29細胞（圖1c）的存活率。在這些化合物中，尼氯沙明對所有試驗的人結腸癌細胞株顯示出最有效的抗增殖作用。將細胞暴露于尼氯沙明72小時后，發現SW620、HCT116和HT29細胞系中的半最大抑制濃度（IC50）分別為2.9、0.4和8.1μm（圖1d）。這表明尼氯沙明可以抑制人結腸癌細胞的增殖。
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轉錄因子家族的一員，最近被證實是一個有吸引力的治療靶點，并被證明是包括結腸癌在內的癌癥治療中的一個重要分子。36由于Stat3 sh2結構域對Stat3的激活和生物學功能至關重要，37我們研究了化合物a和b之間的相互作用。通過活性化合物尼氯沙明的分子對接分析，得到了STAT3的SH2結構域（圖2a）。我們應用pymol軟件去除水分子和其他配體，以制備stat3 sh2結構域蛋白結構，并使用autodock軟件（4.2.6版）預測stat3 sh2結合位點化合物的結合位置，以確定化合物尼氯沙明是否作為靶向stat3 sh2結構域的stat3抑制劑。38分子結構AR對接結果表明，尼氯沙明與arg-595、ser-636、glu-594、lys-591側鏈形成4個氫鍵，如圖2b所示，預計尼氯沙明可與2個主要結合位點ptyr705和側口袋位點結合，從而抑制Stat3磷酸化。綜上所述，采用合理的方法結合分子對接，預測了尼氯沙明與STAT3-SH2結構域的臨界氫鍵相互作用。這些方法闡明了尼氯沙明與STAT3的相互作用。STAT3和尼氯沙明共結晶的進一步驗證正在進行中。
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.
尼氯沙明對結腸癌細胞HCT116和SW620中STAT3磷酸化的抑制作用
我們對結腸癌細胞株進行了Western blot分析，以檢測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（圖3）。值得注意的是，在HCT116和SW620細胞系中，尼氯沙明以時間和劑量依賴性方式抑制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.
尼氯沙明對結腸癌細胞的致敏作用
Erlotinib或吉非替尼（egfr酪氨酸激酶抑制劑）已被證明對非小細胞肺癌和胰腺癌患者有益。然而，幾乎所有的病人在治療過程中都會發展成一種進行性疾病。如圖4a所示，用厄洛替尼和吉非替尼處理細胞72小時后，SW620、HCT116和HT29細胞中的IC50超過60μm。這些結果表明厄洛替尼和吉非替尼不能抑制SW620、HCT116和HT29細胞系的增殖。因此，我們用MTT法檢測了厄洛替尼、厄洛替尼聯合尼氯沙明的抗增殖作用。如圖4b所示，在0.5μm和1μm濃度下，用尼氯沙明致敏的結腸癌細胞治療埃洛替尼，而尼氯沙明對SW620、HCT116和HT29細胞無影響（圖4c）。為了驗證該組合的抗癌性能，我們進行了集落形成實驗。將SW620細胞（5000）接種于6孔板中過夜，然后用厄洛替尼（10μm）或/和尼氯沙明（2.5μm）處理1周。然后我們取出藥物，用PBS清洗細胞兩次。細胞集落用結晶紫染色后計數。如圖4d和E所示，尼氯沙明和厄洛替尼的聯合抑制了結腸癌細胞的集落形成。集落形成分析表明，尼氯沙明和厄洛替尼的協同作用可有效抑制結腸癌細胞的生長。
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.
尼氯沙明與厄洛替尼聯合誘導結腸癌細胞凋亡
與單劑處理相比，厄洛替尼和尼氯沙明的組合增強了對所有被測細胞系細胞活力的抑制（圖4b）。我們試圖研究厄洛替尼和尼氯沙明聯合應用是否也能誘導細胞凋亡。我們首先采用蛋白質印跡分析。HCT116和SW620細胞單獨或同時用二者治療24小時。如圖5a和b所示，聯合治療顯著增加了切割后的parp和bax。bcl-2表達顯著降低。
我們還用流式細胞儀檢測了聯合用藥是否誘導SW620細胞凋亡。SW620細胞用厄洛替尼（10μm）或/和尼氯沙明（2.5μm）處理24小時，然后用膜聯蛋白V和PI染色進行凋亡分析。這種組合導致早期（膜聯蛋白V+，pi-）和晚期凋亡細胞（膜聯蛋白V+，pi+）顯著增加，如圖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
結腸癌是世界上最普遍的腫瘤之一。39盡管傳統的治療方法如手術、放療和化療，結腸癌的總生存率并沒有顯著提高。40人類的表皮生長因子受體在各種類型的胃腸道癌中過度表達，包括60%-80%的結腸癌，與窮人相關。預后和早期疾病進展。41此外，癌細胞還上調了其他重要的下游基因，如有助于結腸癌細胞增殖、細胞存活和血管生成的STAT3。42因此，需要開發更有效的方法來改善結腸癌的預后。在這里，我們的研究結果表明，尼氯沙明以劑量依賴性的方式降低細胞活力。這一發現表明，尼氯沙明可以有效地治療結腸癌。幾個小組獨立地揭示了尼氯沙明對癌細胞的活性；然而，16，27，43，這種抗腫瘤活性的確切機制尚未闡明。因此，我們首先使用分子對接研究來驗證尼氯沙明之間是否存在相互作用和狀態3。在我們的研究中，我們發現了尼氯沙明和Stat3sh2結構域之間的臨界氫鍵相互作用。我們進一步研究了在SW620和HCT116細胞系中，尼氯沙明與STAT3的相關性，結果表明，尼氯沙明以時間和劑量依賴性的方式抑制了STAT3的磷酸化。有幾項研究表明，埃洛替尼抑制表皮生長因子受體（egfr）可誘導Stat3的活化，這有助于埃洛替尼的耐藥性。44,45我們的數據表明，當埃洛替尼和尼氯沙明聯合使用時，它們可協同誘導結腸癌細胞凋亡。據報道，包括絲裂原活化蛋白激酶、成纖維細胞生長因子受體抑制劑和化療藥物在內的多種信號轉導途徑抑制劑可誘導STAT3生存信號通路的活化，從而導致耐藥性。38,46除了厄洛替尼外，尼氯沙明還可能導致耐藥。使這些靶向藥物增敏。根據我們的發現，我們得出結論，尼氯沙明可能是一種新的和更有效的治療結腸癌的藥物。從最初的鉛發現到最終的藥物開發過程是昂貴的、漫長的和漸進的。33發現舊產品或天然產品的新用途比從頭發明新藥物更容易和更經濟。本研究表明，尼氯沙明對細胞有抑制作用。通過抑制STAT3磷酸化來生長結腸癌。我們還研究了埃洛替尼和尼氯沙明協同抑制結腸癌對EGFR和STAT3的聯合抑制作用。本研究僅在體外進行，一些化合物在體外可能表現出有效的抗腫瘤活性，但在體內沒有顯示出抗腫瘤活性。因此，有必要在臨床前和臨床環境中進行進一步的分子研究，以協同方式評估抗癌潛力，以證實尼氯沙明是一種有效的結腸癌治療候選藥物。
