A novel methuosis inducer DZ-514 possesses antitumor activity via activation of ROS-MKK4-p38 axis in triple negative breast cancer 一种新型的甲磺酸诱导剂 DZ-514 通过激活 ROS-MKK4-p38 轴在三阴性乳腺癌中具有抗肿瘤活性
Luzhen Wang , Dazhao , Jinhui , Wenjing Liu , Yi Zhang , Chunyan Wang , 陆振旺，大照，金辉，刘文静，张毅，王春燕Yihua Chen , Ceshi Chen 陈一华，陈测试a School of Life Science, University of Science & Technology of China, Hefei, 230027, Anhui, China 中国科学技术大学生命科学学院，中国安徽省合肥市 230027 Key Laboratory of Animal Models and Human Disease Mechanisms of the Chinese Academy of Sciences and Yunnan Province, Kunming Institute of Zoology, Chinese 中国科学院和云南省动物模型与人类疾病机制重点实验室，昆明动物研究所Academy of Sciences, Kunming, 650201, China 中国昆明科学院，邮编 650201' Shanghai Frontiers Science Center of Genome Editing and Cell Therapy, Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of 生物医学科学研究院和生物调控生物学上海市重点实验室，上海基因编辑和细胞治疗前沿科学中心Life Sciences, East China Normal University, Shanghai, 200241, China 华东师范大学生命科学学院，中国上海 200241 The First Hospital of Hunan University of Chinese Medicine, Changsha, 410007, Hunan, China 湖南中医药大学第一附属医院，中国湖南省长沙市 410007e The Third Affiliated Hospital, Kunming Medical University, Kunming, 650118, China 昆明医科大学第三附属医院，中国昆明市，邮编 650118 Department of Breast and Thyroid Surgery, Southwest Hospital, The First Affiliated Hospital of the Army Military Medical University, Chongqing, 400038, China 中国陆军军医大学第一附属医院西南医院乳腺和甲状腺外科部 Department of the Pathology, First Affiliated Hospital of Kunming Medical University, Kunming, Yunnan, 650032, China 昆明医科大学第一附属医院病理科，中国云南省昆明市 650032 号 Academy of Biomedical Engineering, Kunming Medical University, Kunming, 650500, China 昆明医科大学生物医学工程学院，中国昆明 650500
A R T I C L E I N F O 文章信息
Keywords: 关键词：
Methuosis
Triple-negative breast cancer 三阴性乳腺癌
BCL6
Reactive oxygen species 活性氧物种
MKK4
p38
Abstract 摘要
A B S T R A C T Triple-negative breast cancer (TNBC) is one of the most malignant tumors with poor prognosis. Methuosis is a new type of nonapoptotic cell death characterized by the accumulation of cytoplasmic vacuoles. In this study, we synthesized and screened a series of -phenyl-4-pyrimidinediamine derivatives in TNBC cells, finding that DZ514 was the best compound with high toxicity independent of the inhibition of BCL6. DZ-514 decreased cell viability, inhibited cell cycle progression, and induced caspase-independent cell death in TNBC cells. Interestingly, DZ-514 induced cytoplasm vacuolation, which could be blocked by Baf A1, the V-ATPase inhibitor. Furthermore, we found that DZ-514-induced vacuoles were derived from macropinosomes rather than autophagosomes. Most importantly, methuosis induced by DZ-514 was partially mediated by activating the ROSMKK4-p38 axis. Finally, we demonstrated that DZ-514 significantly inhibited tumor growth in an HCC1806 xenograft mouse model. These findings revealed that the novel methuosis inducer DZ-514 could be developed for TNBC treatment. 摘要 三阴性乳腺癌（TNBC）是一种预后不良的恶性肿瘤之一。Methuosis 是一种新型的非凋亡细胞死亡方式，其特征是细胞质空泡的积累。在本研究中，我们合成并筛选了一系列 TNBC 细胞中的 -苯基-4-嘧啶二胺衍生物，发现 DZ514 是一种具有高毒性的最佳化合物，独立于对 BCL6 的抑制作用。DZ-514 降低了细胞存活率，抑制了细胞周期进展，并在 TNBC 细胞中诱导了非半胱天冬氨酸蛋白酶依赖的细胞死亡。有趣的是，DZ-514 诱导了细胞质空泡化，这可以被 V-ATPase 抑制剂 Baf A1 阻断。此外，我们发现 DZ-514 诱导的空泡来自巨噬泡，而不是自噬泡。最重要的是，DZ-514 诱导的 methuosis 部分通过激活 ROS-MKK4-p38 轴介导。最后，我们证明 DZ-514 显著抑制了 HCC1806 异种移植小鼠模型中的肿瘤生长。这些发现揭示了新型 methuosis 诱导剂 DZ-514 可用于 TNBC 治疗。
1. Introduction 介绍
Cancer remains a major global unresolved public health problem, with breast cancer accounting for about 30% of cancers in women [1]. The age of onset of breast cancer is gradually decreasing, which seriously endangers women's physical and mental health worldwide. Breast cancer is heterogeneous, and clinical immunohistochemistry can be divided into three subtypes, among which TNBC accounts for of breast cancer cases, with poor prognosis, high recurrence rate, strong metastasis, and poor overall survival, which is a major clinical challenge [2-4]. Chemotherapy has been the primary first-line treatment option for TNBC patients for decades, but it inevitably leads to drug resistance, relapse, and metastasis [5]. Recently, targeted agents for TNBC have also made some progress, such as PARP inhibitors (Talazoparib or Olaparib) as treatment options for patients with germinal BRCA1 or BRCA2 mutations, but there has been no significant improvement in overall 癌症仍然是一个全球未解决的重大公共卫生问题，其中乳腺癌在女性中占约 30％的癌症[1]。乳腺癌的发病年龄逐渐降低，严重危害着全球妇女的身心健康。乳腺癌是异质性的，临床免疫组化可以分为三个亚型，其中三阴性乳腺癌（TNBC）占乳腺癌病例的 ，预后差，复发率高，转移能力强，总体生存率差，是一个重大的临床挑战[2-4]。化疗长期以来一直是 TNBC 患者的一线治疗选择，但不可避免地导致药物耐药、复发和转移[5]。最近，针对 TNBC 的靶向药物也取得了一些进展，例如 PARP 抑制剂（Talazoparib 或 Olaparib）作为具有胚系 BRCA1 或 BRCA2 突变的患者的治疗选择，但总体生存率没有显著改善。
Abbreviations: TNBC, Triple-negative breast cancer; OS, Overall survival; BCL6, B-cell lymphoma 6; ROS, Reactive oxygen species; STR, Short Tandem Repeat; SRB, Sulforhodamine B solution; TEM, Transmission electron microscopy; Baf A1, Bafilomycin A1; HCQ, Hydroxychloroquine; 3-MA, 3-Methyladenine; LY, Lucifer yellow; HTRF, Homogeneous time-resolved fluorescence; Nec-1, Necrostatin-1. TNBC，三阴性乳腺癌；OS，总生存期；BCL6，B 细胞淋巴瘤 6；ROS，活性氧物质；STR，短串联重复；SRB，硫罗丹明 B 溶液；TEM，透射电子显微镜；Baf A1，巴菲霉素 A1；HCQ，羟氯喹；3-MA，3-甲基腺嘌呤；LY，荧光素黄；HTRF，均相时间分辨荧光；Nec-1，坏死素-1。
Corresponding author. Academy of Biomedical Engineering, Kunming Medical University, Kunming, 650500, China. 通讯作者。昆明医科大学生物医学工程学院，中国昆明 650500。
Received 10 November 2022; Received in revised form 25 December 2022; Accepted 27 December 2022 2022 年 11 月 10 日收到；2022 年 12 月 25 日收到修订稿；2022 年 12 月 27 日接受
Available online 3 January 2023 2023 年 1 月 3 日起可在线使用
0304-3835/C 2023 Elsevier B.V. All rights reserved. 0304-3835/C 2023 Elsevier B.V. 版权所有。
survival (OS) in patients with metastatic disease [6,7]. The recently approved antibody-coupled drug sacituzumab govitecan is another new targeted agent for patients with metastatic TNBC, with a median survival of only 12.1 months despite improved OS [8]. Pembrolizumab, an anti-PD-1 monoclonal antibody, is approved for the treatment of patients with metastases or locally recurrent unresectable TNBC whose tumors express PD-L1, but is not applicable to all patients [9]. It is urgent to identify new targets or new effective small molecule inhibitors in the treatment of TNBC. 转移性疾病患者的生存期（OS）[6,7]。最近批准的抗体偶联药物 sacituzumab govitecan 是另一种针对转移性三阴性乳腺癌患者的新型靶向药物，尽管 OS 有所改善，但中位生存期仅为 12.1 个月[8]。Pembrolizumab 是一种抗 PD-1 单克隆抗体，已获批用于治疗 PD-L1 阳性的转移性或局部复发无法手术切除的三阴性乳腺癌患者，但并非适用于所有患者[9]。在治疗三阴性乳腺癌中，迫切需要确定新的靶点或新的有效小分子抑制剂。
BCL6 (B-cell lymphoma 6) is a transcriptional repressor of the BTB/ POZ family and has been identified for its involvement in Non-Hodgkin lymphoma-associated chromosomes translocation[10,11]. BCL6 also plays a vital role in the development of breast cancer. Poor prognosis for breast cancer is associated with high expression of BCL6, and BCL6 inhibitors can enhance the efficacy of radiotherapy and chemotherapy for breast cancer [12,13]. We previously synthesized a series of -phenyl-4-pyrimidine diamine derivatives and validated that inhibited lymphoma in vitro and in vivo via reactivating the BCL6 downstream target gene [14]. BCL6（B 细胞淋巴瘤 6）是 BTB/POZ 家族的转录抑制因子，已被发现与非霍奇金淋巴瘤相关的染色体易位有关[10,11]。BCL6 在乳腺癌的发展中也起着重要作用。乳腺癌的预后不良与 BCL6 的高表达有关，而 BCL6 抑制剂可以增强乳腺癌放疗和化疗的疗效[12,13]。我们先前合成了一系列苯基-4-嘧啶二胺衍生物，并验证了 通过重新激活 BCL6 下游靶基因在体外和体内抑制淋巴瘤的作用[14]。
In 1986, the researchers described a phenomenon in which overexpression of -Ras can induce membrane folds and pinocytosis in rat embryonic fibroblasts [15]. In 1999, Chi et al. further found that Ras can induce cell death of glioblastoma cells characterized by cytoplasmic vacuoles, which is different from classical apoptosis but is consistent with the morphological characteristics of autophagy [16]. In 2018, Jean et al. proved that large vacuoles expressing activated -Ras accumulated in glioblastoma cells and were actually derived from macropinosomes. This new form of the cell death process is termed methuosis [17]. To date, many small molecule inducers of methuosis have been discovered and designed. For example, MIPP (3-(2-methyl-1H-indol-3-yl)-1-(4-pyridinyl)-2-propen-1-one) disrupts the normal process of macrocytosis by disrupting the cycle of Rab5, promoting extreme intracellular vacuolization, inducing methuosis [18]; MOMIPP, a compound derived from MIPP, can induce glioblastoma methuosis by targeting PIKFYVE and activating JNK [19-21]; ursolic acid-derived 17 was demonstrated to be a methuosis inducer in HeLa cells [22]; epimedokoreanin C partly induced methuosis in lung cancer cells by regulating Rac1 and Arf6 [23]; CX-4945 ultimately promoted methuosis associated with massive vacuolation via inhibiting AKT by binding to CK2 in colorectal cancer [24]; and 12A activated the MAPK/JNK signaling pathway and induced methuosis in breast and cervical cancer [25]. 1986 年，研究人员描述了一种现象，即过度表达 -Ras 可以诱导大鼠胚胎成纤维细胞产生膜褶和胞吞作用[15]。1999 年，Chi 等人进一步发现 Ras 可以诱导胶质母细胞瘤细胞的细胞死亡，其特征是细胞质空泡，这与经典的凋亡不同，但与自噬的形态特征一致[16]。2018 年，Jean 等人证明，在胶质母细胞瘤细胞中，表达活化的 -Ras 的大空泡实际上是来自巨噬泡。这种新型的细胞死亡过程被称为 methuosis[17]。迄今为止，已经发现和设计了许多诱导 methuosis 的小分子物质。 例如，MIPP（3-(2-甲基-1H-吲哚-3-基)-1-(4-吡啶基)-2-丙烯-1-酮）通过干扰 Rab5 的循环，破坏巨噬细胞过程的正常进行，促进细胞内极端空泡化，诱导甲状腺肿瘤[18]；MOMIPP，一种由 MIPP 衍生的化合物，通过靶向 PIKFYVE 并激活 JNK 来诱导胶质母细胞瘤甲状腺肿瘤[19-21]；已证明来源于熊果酸的 17 号化合物能够在 HeLa 细胞中诱导甲状腺肿瘤[22]；Epimedokoreanin C 通过调节 Rac1 和 Arf6 在肺癌细胞中部分诱导甲状腺肿瘤[23]；CX-4945 通过与 CK2 结合抑制 AKT，最终促进结直肠癌中与大量空泡化相关的甲状腺肿瘤[24]；12A 激活 MAPK/JNK 信号通路，在乳腺癌和宫颈癌中诱导甲状腺肿瘤[25]。
, an evolutionarily conserved serine/threonine protein kinase, contains four members: , and [26-29]. p38 can regulate cell proliferation, differentiation, and apoptosis. MKK3, MKK4, and MKK6 are upstream kinases for p38 [30-32]. Various inflammatory cytokines and some external environmental stresses, including tumor necrosis factor, oxidative stress, and ultraviolet radiation, can strongly activate p38. Reactive oxygen species (ROS) can activate p38 and block cell proliferation [33]. In TNBC, several compounds can induce cell death and inhibit cell proliferation by activating the p38 pathway [34,35]. In this study, p38 refers specifically to . 是一个在进化中保守的丝氨酸/苏氨酸蛋白激酶，包括四个成员： ，和 [26-29]。p38 可以调节细胞增殖、分化和凋亡。MKK3、MKK4 和 MKK6 是 p38 的上游激酶[30-32]。各种炎症因子和一些外部环境压力，包括肿瘤坏死因子、氧化应激和紫外线辐射，都可以强烈激活 p38。活性氧（ROS）可以激活 p38 并阻断细胞增殖[33]。在 TNBC 中，几种化合物可以通过激活 p38 通路诱导细胞死亡和抑制细胞增殖[34,35]。在本研究中，p38 特指 。
In this study, we demonstrated that compound DZ-514 was the most cytotoxic -phenyl-4-pyrimidinediamine derivative among the investigated ones in TNBC cells. However, DZ-514 did not function through the inhibition of BCL6. In addition, we found that DZ-514 induced caspaseindependent cell death and inhibited cell proliferation. Importantly, we discovered that DZ-514 induced extreme cytoplasmic vacuolization in a dose- and time- dependent manner and found that vacuoles were derived from macropinosomes with the characteristics of methuosis. We further found that DZ-514 induced methuosis through the ROS-MKK4p38 signaling pathway. DZ-514 showed antitumor activity in a mouse xenograft model of TNBC. 在这项研究中，我们证明化合物 DZ-514 是在 TNBC 细胞中最具细胞毒性的 -苯基-4-嘧啶二胺衍生物之一。然而，DZ-514 并不通过抑制 BCL6 来发挥作用。此外，我们发现 DZ-514 诱导非半胱天冬酶依赖的细胞死亡并抑制细胞增殖。重要的是，我们发现 DZ-514 以剂量和时间依赖的方式诱导细胞质极端空泡化，并发现这些空泡来自具有甲状突症特征的巨噬细胞吞噬体。我们进一步发现 DZ-514 通过 ROS-MKK4p38 信号通路诱导甲状突症。DZ-514 在 TNBC 小鼠异种移植模型中显示出抗肿瘤活性。
2. Materials and methods 材料和方法
2.1. Compound library and DZ-514 化合物库和 DZ-514
The library of screening -phenyl-4-pyrimidinediamine derivatives was derived from our previously reported work [14]. The detailed synthesis method and characterization of the candidate DZ-514, which is mainly mentioned here, was described in supporting information. 筛选库中的 -苯基-4-嘧啶二胺衍生物是基于我们先前报道的工作[14]。候选药物 DZ-514 的详细合成方法和表征在支持信息中描述。
2.2. Cell lines and cell culture 2.2. 细胞系和细胞培养
In this paper, all of the cells mentioned were obtained from the American Type Culture Collection (ATCC, Manassas, Virginia, USA) and were identified by Short Tandem Repeat (STR). Different cells were cultured in different media, the specific formula is listed in Table S1. 在本文中，提到的所有细胞均来自美国类型培养物收藏中心（ATCC，美国弗吉尼亚州马纳萨斯）并通过短串联重复（STR）鉴定。不同的细胞在不同的培养基中培养，具体配方见表 S1。
2.3. Cell viability assays 2.3. 细胞存活率测定
All breast cancer cells were cultured in 96-well plates at a density of 6000-8000 cells/well. The cells were cultured for a certain time and incubated with various concentrations of DZ-514 (24 or ) according to the requirements of the experiment. Then, fixed the cells with TCA overnight at , washed excess TCA with (deionized water) and air dry. Next, stain the fixed cells using sulforhodamine B solution (SRB) in acetic acid for , and washed times with acetic acid. Tris-base was used to dissolve SRB dyes that bind specifically to cells. Automated spectrophotometric was used to read the optical results at . 所有乳腺癌细胞以 6000-8000 个细胞/孔的密度培养在 96 孔板中。根据实验要求，细胞在一定时间内培养，并与不同浓度的 DZ-514（24 或 ）共同孵育。然后，用 TCA 固定细胞过夜在 ，用 （去离子水）洗去多余的 TCA 并风干。接下来，使用硫酸罗丹明 B（SRB）溶液在 醋酸中染色固定细胞 ，并用 次 醋酸洗涤。三氢基甲基胺用于溶解与细胞特异性结合的 SRB 染料。使用自动分光光度计在 读取光学结果。
2.4. Western blotting 2.4. 蛋白印迹
Breast cancer cells were planted in 6-well plates at . After incubated with various concentrations of DZ-514 for specified times, the protein was extracted via RIPA Buffer, including protease inhibitors and phosphatase inhibitors for at . Cell lysates were collected into EP tubes, centrifuged and collected the supernatant, and protein lysates were quantified using Pierce BCA Protein Assay Kitt, then added loading buffer to the supernatant and boiled for at . The special protein was insolated via SDS-PAG gel, and detailed experimental methods were described in our previous study [36]. Details of the specified primary antibodies are listed in Table S2. 乳腺癌细胞被植入 6 孔板中，以 浓度孵育。在指定时间内，用 DZ-514 处理细胞，用 RIPA 缓冲液提取蛋白质，包括蛋白酶抑制剂和磷酸酶抑制剂，以 浓度进行处理。细胞裂解液被收集到 EP 管中，离心并收集上清液，用 Pierce BCA 蛋白质测定试剂盒定量蛋白质裂解液，然后向上清液中加入加载缓冲液，在 温度下煮沸。通过 SDS-PAG 凝胶分离特定的蛋白质，详细的实验方法在我们之前的研究中已经描述[36]。指定的一抗详细信息列在表 S2 中。
2.5. siRNA transfection 2.5. siRNA 转染
In this study, all siRNAs were purchased from RIBOBIO (Guangzhou, China). Opti-MEM and Lipofectamine (Invitrogen, Carlsbad California, USA) were used for transfection, and the specific procedure was carried out according to the manufacturer's instructions. The details of siRNA sequences are listed in Table S4. 在这项研究中，所有的 siRNA 都是从 RIBOBIO（中国广州）购买的。转染使用 Opti-MEM 和 Lipofectamine （Invitrogen，美国加利福尼亚州卡尔斯巴德），具体操作按照制造商的说明进行。siRNA 序列的详细信息列在表 S4 中。
2.6. Stable overexpression and knockdown of BCL6 2.6. BCL6 的稳定过表达和沉默
The full-length fragment of the BCL6 gene was cloned from the cDNA of MDA-MB-468 cells by PCR. Then, the full-length BCL6 gene was cloned into pCDH vectors. The shRNA and Scramble were cloned into pLKO. 1 vectors. HEK-293T cells were used to package the Lentiviruses and then transfected it into different cells. WB was carried out to validate the stable knockdown and overexpression effects. The primers and shBCL6 sequences are shown in Table S5. 从 MDA-MB-468 细胞的 cDNA 中，通过 PCR 克隆了 BCL6 基因的全长片段。然后，将全长 BCL6 基因克隆到 pCDH 载体中。shRNA 和 Scramble 被克隆到 pLKO.1 载体中。使用 HEK-293T 细胞包装 Lentiviruses，然后转染到不同的细胞中。通过 WB 验证稳定的沉默和过表达效果。引物和 shBCL6 序列见表 S5。
2.7. Tumorigenesis 2.7. 肿瘤发生
The animal experiments in this study have been approved by the Ethics Committee of the Kunming Institute of Zoology, Chinese Academy of Sciences. Fifteen nude mice (about 6 weeks) were raised in an SPF animal facility, obtained from SJA Lab Animal Co., Ltd. (Changsha, 该研究中的动物实验已获得中国科学院昆明动物研究所伦理委员会的批准。15 只裸鼠（约 6 周大）在 SPF 动物实验室中饲养，由 SJA 实验动物有限公司（长沙）提供。
Fig. 1. DZ-514 is the most cytotoxic compound independent of BCL6 图 1. DZ-514 是最具细胞毒性的化合物，与 BCL6 无关。
(A) Western blotting for BCL6 protein level in 10 breast cancer cell lines and two immortalized breast epithelial cell lines. （A）在 10 种乳腺癌细胞系和两种免疫化的乳腺上皮细胞系中检测 BCL6 蛋白水平的免疫印迹。
(B) DZ-514 showed the strongest anticancer activity in HCC1806 among 55 different -phenyl-4-pyrimidinediamine derivatives. SRB experiments were performed to detect the cell viability of HCC1806 after treatment with 55 compounds(24 h). （B）DZ-514 在 55 种不同的苯基-4-嘧啶二胺衍生物中对 HCC1806 显示出最强的抗癌活性。在处理 55 种化合物（24 小时）后，使用 SRB 实验检测 HCC1806 的细胞存活能力。
(C) The chemical structure of DZ-514. (C) DZ-514 的化学结构。
(D) The IC 50 values of DZ-514 in different breast cell lines and two immortalized breast epithelial cell lines. (D) DZ-514 在不同的乳腺细胞系和两种免疫乳腺上皮细胞系中的 IC 50 值。
(E) Knockdown of BCL6 did not affect DZ-514-induced cell death in HCC1806 cells. The knockdown effect was assessed by Western blotting (up). Scramble, shBCL6 1#, shBCL6 2# HCC1806 cells viability were measured via SRB assay after treatment with indicated concentrations of DZ-514 (24 h) (bottom). BCL6 的敲低对 HCC1806 细胞中 DZ-514 诱导的细胞死亡没有影响。敲低效果通过 Western blotting 进行评估（上）。在接受指定浓度的 DZ-514 处理（24 小时）后，通过 SRB 测定法测量 Scramble、shBCL6 1#、shBCL6 2# HCC1806 细胞的存活率（下）。
China). 15 female nude mice were injected with HCC1806 cells cells/spot) by in-situ injection of fat pads on the left and right sides. When all mice tumors grew to , mice were randomly distributed into control and experimental groups (DZ-514 or DZ-514 10 ), and DZ-514 was treated by intraperitoneal injection every two days until 18 days. Vernier calipers were used to measure the size of tumors every two days. After the experiment was terminated, tumors were harvested for analysis. 中国）。15 只雌性裸鼠被注射 HCC1806 细胞（每个点位 个细胞/点位），通过在左右侧脂肪垫处进行原位注射。当所有裸鼠肿瘤生长到 时，随机分为对照组和实验组（DZ-514 或 DZ-514 10 ），并且 DZ-514 每两天进行一次腹腔注射治疗，直到 18 天。游标卡尺用于每两天测量肿瘤的大小。实验结束后，收集肿瘤进行分析。
2.8. Live cell imaging 2.8. 活细胞成像
Lucifer yellow (LY) was purchased from Sigma-Aldrich (St Louis, USA). TNBC cells were inoculated in confocal dishes and exposed to DZ-514 for . Then, was used to incubate the cells for at , washed twice with PBS, and finally cultured for 30-60 min with MitoTracker (Beyotime, Shanghai, China), LysoTracker (Beyotime, Shanghai, China) and ERtracker (Invitrogen , Carlsbad California, USA) at . The Carl Zeiss (LSM880) microscope was used to photograph the phase-contrast and fluorescence of the living cells. Lucifer yellow (LY)从 Sigma-Aldrich（美国圣路易斯）购买。TNBC 细胞接种在共聚焦培养皿中，并暴露于 DZ-514 。然后，使用 孵育细胞 ，用 PBS 洗涤两次，最后在 使用 MitoTracker（Beyotime，中国上海）、LysoTracker（Beyotime，中国上海）和 ERtracker（Invitrogen，美国加利福尼亚州卡尔斯巴德）培养 30-60 分钟。使用 Carl Zeiss（LSM880）显微镜拍摄活细胞的相差和荧光图像。
C
50к 150к 50 千 150 千
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Fig. 2. DZ-514 inhibits cell proliferation and induces Caspase-independent cell death in TNBC. 图 2. DZ-514 抑制三阴性乳腺癌细胞增殖并诱导非 Caspase 依赖的细胞死亡。
(A) Representative pictures of colony formation in HCC1806 and MAD-MB-468 cells, incubating with indicated concentrations of DZ-514 for 15 days. （A）HCC1806 和 MAD-MB-468 细胞在指定浓度的 DZ-514 下孵育 15 天后的菌落形成的代表性图片。
(B). DZ-514 inhibited the colony formation of TNBC cells. The graph shows the statistics of colony formation. （B）DZ-514 抑制了 TNBC 细胞的集落形成。图表显示了集落形成的统计数据。
(C). DZ-514 increased the cells in the G2/M phase. TNBC cells were dyed with PI and RNase and analyzed with flow cytometry after incubation with DZ-514 (24 h). （C）DZ-514 增加了 G2/M 期的细胞。TNBC 细胞在与 DZ-514 孵育（24 小时）后，用 PI 和 RNase 染色，并通过流式细胞术进行分析。
(D). After DZ-514 treatment compared to the control group, the proportion of each stage of the cell cycle changes were shown. （D）与对照组相比，经过 DZ-514 处理后，细胞周期各阶段的比例发生了变化。
(E). DZ-514 regulated cell cycle-related proteins. WB assay was performed to detect cell cycle-related proteins, including cyclin B1, cyclin D1, CDK4/6, p21, and p27. TNBC cells were incubated with indicated concentrations of DZ-514 (24 h). （E）DZ-514 调节细胞周期相关蛋白。进行 WB 检测细胞周期相关蛋白，包括 cyclin B1、cyclin D1、CDK4/6、p21 和 p27。TNBC 细胞与指定浓度的 DZ-514 孵育（24 小时）。
(F) Statistics showed that DZ-514 did not affect the positive proportion of Annexin-V and PI. 统计数据显示，DZ-514 对 Annexin-V 和 PI 的阳性比例没有影响。
(G). DZ-514 did not cause alterations of indicated apoptosis-associated proteins. The cells were incubated with DZ-514 and DDP for 24 h. Cell lysates were gathered for WB to detect the cleavage of PARP and Caspases. DDP served as a positive control. (G). DZ-514 对所示的凋亡相关蛋白没有引起改变。细胞与 DZ-514 和 DDP 共同孵育 24 小时。细胞裂解物被收集用于 Western Blot 检测 PARP 和 Caspases 的裂解。DDP 作为阳性对照。
(A). The phase contrast diagram showed that the cytoplasmic vacuolation of DZ-514 (0.5-1.5 treated TNBC cells were affected in a dose-dependent manner. (A). 相位对比图显示，DZ-514（0.5-1.5 处理的 TNBC 细胞的胞质空泡化受剂量依赖的影响。
(B). DZ-514-induced vacuolization was counted. 100 cells were counted for each area. （B）计算 DZ-514 引起的液泡化。每个区域计数 100 个细胞。
(C). MDA-MB-468 and HCC1806 cells were treated with DZ-514 (2 for different times, and phase-contrast images were obtained. (C). MDA-MB-468 和 HCC1806 细胞被处理了 DZ-514（2 不同时间，并获得了相差对比图像。
(D). DZ-514 significantly induced vacuolization in a time-dependent manner. 100 cells were counted for each area. （D）DZ-514 在时间依赖性的方式下显著诱导了液泡化。每个区域计数了 100 个细胞。
(E). TEM image of MDA-MB-468 cells after exposed to DZ-514 for . (E). 经过 DZ-514 处理后，MDA-MB-468 细胞的透射电子显微镜图像。
(F). DZ-514 did not induce vacuolization in normal breast epithelial cells. 184B5 cells were incubated with indicated concentrations of DZ-514 (24 h), and phasecontrast images were obtained. （F）。DZ-514 在正常乳腺上皮细胞中没有引起空泡化。184B5 细胞与指定浓度的 DZ-514（24 小时）孵育，获取相差显微图像。
(G) The graph showed a quantitative plot of vacuole cells. 100 cells were counted for each area. 图表显示了液泡细胞的定量图。每个区域计数了 100 个细胞。
2.9. Immunofluorescence 2.9. 免疫荧光
MDA-MB-468 and HCC1806 cells were planted in 24 plates containing cover glasses, cultured for a certain time and treated with DZ514 (24 h). The paraformaldehyde was used to fix cells at RT for 20 min, PBS washed. Then, Triton X-100/PBS permeable was used to permeabilize at RT for , PBS washed. The cells were blocked with MDA-MB-468 和 HCC1806 细胞被种植在含有盖玻片的 24 孔板中，培养一段时间后用 DZ514 处理（24 小时）。用甲醛在室温下固定细胞 20 分钟，PBS 洗涤。然后，使用 Triton X-100/PBS 渗透剂在室温下渗透 ，PBS 洗涤。细胞被阻断
5% BSA/PBST for at RT before being incubated overnight with Rab7 and LAMP1 primary antibodies at (specific antibodies are listed in Table S2). The next day, the cells were washed twice with PBS, and incubated with secondary antibody (the specific secondary antibodies are listed in Table S2) for at RT in the dark. After washing the cells twice with PBS, we labeled the nuclei with DAPI and then sealed the cells with a tablet. Photographs were taken using a high-resolution 5% BSA/PBST 在室温下用于 之前的孵育，随后与 Rab7 和 LAMP1 一抗在 过夜孵育（具体抗体详见表 S2）。第二天，细胞用 PBS 洗涤两次，然后在室温下暗处孵育 与二抗（具体二抗详见表 S2）。细胞用 PBS 洗涤两次后，我们用 DAPI 标记细胞核，然后用片剂密封细胞。使用高分辨率相机拍摄照片。
fluorescence microscope. 荧光显微镜。
2.10. Measurement of ROS levels 2.10. 测量 ROS 水平
MDA-MB-468 and HCC1806 cells were exposed to indicated concentrations of DZ-514 (8 h). The DCFH-DA (Beyotime, Shanghai, China) was used to incubate cells for at in the dark. Then, the cells were collected into EP tubes, and washed three times with serumfree medium, and 10,000 cells were collected with flow cytometry to detect the average fluorescence intensity. MDA-MB-468 和 HCC1806 细胞暴露于指定浓度的 DZ-514（8 小时）。DCFH-DA（上海百一生物科技有限公司）用于在黑暗中孵育细胞 。然后，将细胞收集到 EP 管中，用无血清培养基洗涤三次，用流式细胞术收集 10,000 个细胞以检测平均荧光强度。
2.11. Transmission electron microscopy (TEM) 透射电子显微镜（TEM）
The cells were fixed, dehydrated, and embedded in resin after of treatment with DZ-514. Pictures were obtained from the Public Technology Center, Kunming Institute of Zoology, Chinese Academy of Sciences. TEM was performed with a JEM 1400 Plus electron microscope. 细胞经过 DZ-514 处理后，固定、脱水并嵌入树脂。图片由中国科学院昆明动物研究所公共技术中心获取。使用 JEM 1400 Plus 电子显微镜进行了透射电子显微镜检查。
2.12. Statistical analysis 2.12. 统计分析
In this study, the statistical analysis of the data involved was completed by SPSS software. The drawing and composition of the images were completed with the help of ImageJ, ZEN.2, Photoshop, FlowJo, and other software. All data without special annotation were analyzed by Student's -test. ns for no significant difference, * for for for . 在这项研究中，所涉及的数据的统计分析是通过 SPSS 软件完成的。图像的绘制和组合是在 ImageJ、ZEN.2、Photoshop、FlowJo 等软件的帮助下完成的。所有没有特殊注释的数据都是通过学生 t 检验进行分析的。ns 表示无显著差异，*表示显著差异， 表示显著差异， 表示显著差异， 表示显著差异， 表示显著差异。
3. Results 3. 结果
3.1. Discovery of a small molecule inhibitor DZ-514 that inhibits TNBC growth 发现一种抑制三阴性乳腺癌生长的小分子抑制剂 DZ-514。
Increasing publications indicate that BCL6 is highly related to the occurrence and development of breast cancer and contributes to the therapeutic resistance [13,39]. Therefore, BCL6 was regarded as a potential therapeutic target for breast cancer treatment. We tested the endogenous expression of BCL6 in several subtypes of breast cancer cell lines and found that BCL6 is highly expressed in most TNBC cell lines (Fig. 1A). In order to investigate the therapeutic potential of BCL6 inhibitors in TNBC, we performed phenotypic screening of our previously reported a series of -phenyl-4-pyrimidinediamine derivatives in TNBC cells [14]. We used the TNBC cell line HCC1806 to screen these -phenyl-4-pyrimidine diamine derivatives at a fixed concentration (1 ) to identify compounds capable of suppressing the proliferation of HCC1806 cells. This strategy identified DZ-514, which exhibited the strongest cytotoxicity (Fig. 1B). 增加的出版物表明 BCL6 与乳腺癌的发生和发展密切相关，并且对治疗抵抗有贡献。因此，BCL6 被视为乳腺癌治疗的潜在治疗靶点。我们测试了几种亚型的乳腺癌细胞系中 BCL6 的内源性表达，并发现 BCL6 在大多数 TNBC 细胞系中高表达（图 1A）。为了研究 BCL6 抑制剂在 TNBC 中的治疗潜力，我们对我们先前报道的一系列苯基-4-嘧啶二胺衍生物在 TNBC 细胞中进行了表型筛选。我们使用 TNBC 细胞系 HCC1806 以固定浓度（1）筛选这些苯基-4-嘧啶二胺衍生物，以确定能够抑制 HCC1806 细胞增殖的化合物。这种策略鉴定出 DZ-514，其显示出最强的细胞毒性（图 1B）。
3.2. DZ-514 induces TNBC cell death in a BCL6-independent manner 3.2. DZ-514 以一种与 BCL6 无关的方式诱导 TNBC 细胞死亡。
To test whether DZ-514 suppresses breast cancer by targeting BCL6, we analyzed whether the expression level of BCL6 in the breast cancer cell line was correlated with of DZ-514. We selected 2 immortalized breast epithelial cell lines and 7 different subtypes of breast cancer cell lines for the SRB assay. Interestingly, we found that MCF7 cells, which have a low level of endogenous BCL6, were very sensitive to DZ-514 (Fig. 1A and D), suggesting that the expression of BCL6 and the IC 50 of DZ-514 were not correlated. Then, we established BCL6 stable knockdown HCC1806 and MDA-MB-468 cells and incubated them with indicated concentrations of DZ-514. As a result, BCL6 knockdown did not affect DZ-514 sensitivity (Fig. 1E and S1B). Moreover, we stably constructed BCL6-overexpressing HCC1806 and HCC1937 cells (Figs. S1D-E) and performed SRB assays to detect their cell viability after DZ-514 treatment for . Our data suggested that the values of DZ-514 were not significantly different between BCL6-overexpressing cells and their control cells. These results indicate that DZ-514 inhibited TNBC independent of BCL6 (Figs. S1D-E). 为了测试 DZ-514 是否通过靶向 BCL6 来抑制乳腺癌，我们分析了乳腺癌细胞系中 BCL6 的表达水平是否与 DZ-514 的 IC50 相关。我们选择了 2 种免疫化的乳腺上皮细胞系和 7 种不同亚型的乳腺癌细胞系进行 SRB 实验。有趣的是，我们发现内源性 BCL6 水平较低的 MCF7 细胞对 DZ-514 非常敏感（图 1A 和 D），这表明 BCL6 的表达与 DZ-514 的 IC50 无关。然后，我们建立了 BCL6 稳定敲低的 HCC1806 和 MDA-MB-468 细胞，并将它们与指定浓度的 DZ-514 一起培养。结果显示，BCL6 敲低不影响 DZ-514 的敏感性（图 1E 和 S1B）。此外，我们稳定构建了 BCL6 过表达的 HCC1806 和 HCC1937 细胞（图 S1D-E），并进行 SRB 实验以检测它们在 DZ-514 处理后的细胞存活率。我们的数据表明，DZ-514 的 IC50 值在 BCL6 过表达细胞和对照细胞之间没有显著差异。这些结果表明，DZ-514 独立于 BCL6 抑制 TNBC（图 S1D-E）。
3.3. DZ-514 suppresses colony formation and cell cycle progression in TNBC cells 3.3. DZ-514 抑制 TNBC 细胞的集落形成和细胞周期进展。
As introduced above, DZ-514 significantly decreased cell viability in TNBC. We further confirmed its anticancer activity by colony formation assays. This is shown in Fig. 2A-B, DZ-514 significantly decreased the colony formation of TNBC cells at a lower concentration. We further examined cell cycle progression by PI staining and found that the cells were arrested in the G2/M phase after DZ-514 treatment (Fig. 2C-D). Consistently, DZ-514 decreased the level of Cyclin D1, Cyclin B1, CDK4, and CDK6 and upregulated the level of p21, but did not affect the expression of p27 (Fig. 2E). 如上所述，DZ-514 显著降低了 TNBC 细胞的存活率。我们通过集落形成实验进一步确认了其抗癌活性。如图 2A-B 所示，DZ-514 在较低浓度下显著降低了 TNBC 细胞的集落形成。我们进一步通过 PI 染色检查细胞周期进展，发现细胞在 DZ-514 处理后停滞在 G2/M 期（图 2C-D）。一致地，DZ-514 降低了 Cyclin D1、Cyclin B1、CDK4 和 CDK6 的水平，并上调了 p21 的水平，但不影响 p27 的表达（图 2E）。
3.4. DZ-514 does not induce apoptosis and necrosis in TNBC cells 3.4. DZ-514 在 TNBC 细胞中不会引发细胞凋亡和坏死。
Apoptosis is one of the classical approaches to programmed cell death [40]. We wondered whether DZ-514 induces apoptosis. We exposed TNBC cells to various concentrations of DZ-514 (24 h) and detected apoptosis using an Annexin V-FITC/PI double staining kit. As shown in Fig. 2F, S2A, DZ-514 did not significantly increase the percentage of Annexin V/PI-positive apoptotic cells, indicating that DZ-514-induced cell death may not be apoptosis. Furthermore, we further examined some markers of apoptosis and found that DZ-514 did not increase the cleavage of PARP or Caspase-9, -8, -7, or -3 (Fig. 2G). These results suggest that DZ-514-induced cell death is not apoptosis. 凋亡是程序性细胞死亡的经典途径之一[40]。我们想知道 DZ-514 是否诱导凋亡。我们将三阴性乳腺癌细胞暴露于不同浓度的 DZ-514（24 小时），并使用 Annexin V-FITC/PI 双染试剂盒检测凋亡。如图 2F、S2A 所示，DZ-514 并未显著增加 Annexin V/PI 阳性凋亡细胞的百分比，表明 DZ-514 诱导的细胞死亡可能不是凋亡。此外，我们进一步检测了一些凋亡标志物，发现 DZ-514 并未增加 PARP 或 Caspase-9、-8、-7 或-3 的裂解（图 2G）。这些结果表明，DZ-514 诱导的细胞死亡不是凋亡。
Next, we asked whether DZ-514 induced necrosis. SRB assays were performed to detect whether Necrostatin-1(Nec-1, a specific inhibitor of necroptosis) could rescue DZ-514-induced HCC1806 and MDA-MB-468 cell death. Nec-1 did not rescue DZ-514-induced cell death (Fig. S2B). Therefore, DZ-514-induced cell death is not necrosis. 接下来，我们询问 DZ-514 是否引起了坏死。进行 SRB 测定以检测 Necrostatin-1（坏死程序化抑制剂）是否能够拯救 DZ-514 引起的 HCC1806 和 MDA-MB-468 细胞死亡。Nec-1 未能拯救 DZ-514 引起的细胞死亡（图 S2B）。因此，DZ-514 引起的细胞死亡不是坏死。
3.5. DZ-514 induces the accumulation of cytoplasmic vacuolation in TNBC cells 3.5. DZ-514 在 TNBC 细胞中引起细胞质空泡的积累
We noticed that DZ-514 induced extensive cellular vacuolization in a dose- and time-dependent manner in TNBC cells (Fig. 3A-D). DZ-514 induced cell vacuolization within . The number of vacuoles in each cell gradually increased, and the small vacuoles gradually fused with each other over time. When the area of vacuolation gradually increased, a vacuole occupying more than of the cell volume was formed (Fig. 3C). We wondered if DZ-514 induces vacuolation in other breast cancer cells. Therefore, we treated MCF7, T47D, and HCC1937 breast cancer cell lines with different concentrations of DZ-514. As expected, DZ-514 induced vacuolation in other breast cancer cells (Fig. S3), indicating that DZ-514 is a broad spectrum vacuolation inducer in breast cancer. Electron microscopy clearly showed that a large number of vacuoles formed in the cytoplasm of MDA-MB-468 cells incubated with DZ-514. The sizes of the vacuoles were between 0.5 and in diameter. Consistent with the results from Fig. 2F, there was no nuclear shrinkage, which is a biomarker of apoptosis (Fig. 3E). In contrast, DZ514 failed to induce vacuolation in the immortalized mammary epithelial cell line 184B5 at the same concentration (Fig. 3F-G). These observations substantially indicate that DZ-514 may be a cancer-specific vacuolation inducer. 我们注意到 DZ-514 以剂量和时间依赖的方式在 TNBC 细胞中引起了广泛的细胞空泡化（图 3A-D）。DZ-514 在 内诱导细胞空泡化。每个细胞中的空泡数量逐渐增加，小的空泡随着时间的推移逐渐融合。当空泡化区域逐渐增大时，形成了占细胞体积 以上的空泡（图 3C）。我们想知道 DZ-514 是否会在其他乳腺癌细胞中引起空泡化。因此，我们用不同浓度的 DZ-514 处理了 MCF7、T47D 和 HCC1937 乳腺癌细胞系。如预期，DZ-514 在其他乳腺癌细胞中引起了空泡化（图 S3），表明 DZ-514 是一种广谱的乳腺癌空泡化诱导剂。电子显微镜清楚地显示，在与 DZ-514 孵育的 MDA-MB-468 细胞的细胞质中形成了大量的空泡。空泡的大小在 0.5 到 之间。与图 2F 的结果一致，没有核收缩，这是凋亡的生物标志物（图 3E）。 相比之下，DZ514 在相同浓度下未能引起 184B5 乳腺上皮细胞系的空泡形成（图 3F-G）。这些观察结果明显表明 DZ-514 可能是一种特异性的癌症空泡形成诱导剂。
3.6. DZ-514-induced cell death is distinct from autophagy 3.6. DZ-514 诱导的细胞死亡与自噬不同。
To test whether DZ-514 induces cell vacuolization through autophagy, we detected the expression of LC3 and p62, which are autophagy-related markers. Western blotting results showed that both LC3-II and p62 accumulated upon treatment with DZ-514, while p62 degradation and LC3-II accumulation were induced by rapamycin, an autophagy inducer (Fig. S4A), indicating that autophagy was actually inhibited. The autophagy inhibitor hydroxychloroquine (HCQ) is well known to inhibit the fusion of autophagosomes and lysosomes [41] and the principle of 3-methyladenine (3-MA) inhibiting autophagy is to 为了测试 DZ-514 是否通过自噬诱导细胞空泡化，我们检测了与自噬相关的标志物 LC3 和 p62 的表达。Western blotting 结果显示，在 DZ-514 处理后，LC3-II 和 p62 都积累增加，而自噬诱导剂雷帕霉素诱导了 p62 的降解和 LC3-II 的积累（图 S4A），表明实际上抑制了自噬。自噬抑制剂羟氯喹（HCQ）被广泛认为是抑制自噬体与溶酶体融合的药物[41]，而 3-甲基腺嘌呤（3-MA）抑制自噬的原理是
Fig. 4. DZ-514 induces methuosis by stimulating catastrophic macropinocytosis in TNBC cells 图 4. DZ-514 通过刺激三阴性乳腺癌细胞的灾难性巨噬作用诱导甲状噬作用。
(A). The vacuolization induced by DZ-514 was blocked by Baf A1. TNBC cells were incubated with DZ-514 and Baf A1 alone or in combination for , and phase-contrast images were obtained. (A). DZ-514 引起的液泡化作用被 Baf A1 阻断。TNBC 细胞分别与 DZ-514 和 Baf A1 单独或联合孵育 ，并获取相差显微镜图像。
(B). Baf A1 completely blocked the loss of cell viability induced by DZ-514. The SRB assay was performed to detect cell viability. TNBC cells were incubated with 1.5 DZ-514 and Baf A1 alone or in combination for . (B). Baf A1 完全阻断了 DZ-514 引起的细胞存活率下降。使用 SRB 试验检测细胞存活率。TNBC 细胞分别与 1.5 DZ-514 和 Baf A1 单独或联合孵育 。
(C). LY accumulated in the phase vacuoles induced by DZ-514. Phase contrast and fluorescent images of the living cells were obtained using an LSM880 highresolution fluorescence microscopy system. The cells were treated with DZ-514 or DMSO for and incubated with LY (1 for at . (D). HCC1806 and MDA-MB-468 cells were treated with DZ-514 for and incubated with LY for at . The cells were washed with PBS and incubated with MitoTracker, LysoTracker, and ERtracker for 30-60 min at . Fluorescent images were captured by confocal microscopy. （C）DZ-514 诱导的相位液泡中积累了 LY。使用 LSM880 高分辨率荧光显微镜系统获取了活细胞的相差相位和荧光图像。细胞经过 DZ-514 或 DMSO 处理 ，并与 LY（1 ）孵育 在 。 （D）HCC1806 和 MDA-MB-468 细胞经过 DZ-514 处理 ，并与 LY 孵育 在 。细胞用 PBS 洗涤，并与 MitoTracker、LysoTracker 和 ERtracker 在 孵育 30-60 分钟。荧光图像由共聚焦显微镜捕获。
(E). The protein expression levels of LAMP1 and Rab7 were increased by DZ-514. TNBC cells were incubated with DZ-514 (24 h), and cell lysates were collected for WB to evaluate LAMP1 and Rab7. (E). DZ-514 增加了 LAMP1 和 Rab7 的蛋白表达水平。TNBC 细胞与 DZ-514 孵育（24 小时），并收集细胞裂解液进行 WB 评估 LAMP1 和 Rab7。
(F, G). DZ-514 increased the levels of LAMP1 in TNBC cells, as detected by immunofluorescence staining. The cells were treated with DZ-514 ( ). The quantitative results are shown below. The average immunofluorescence intensity of each field was calculated using ImageJ software, and 4 fields were selected for each treatment （F，G）。DZ-514 增加了 TNBC 细胞中 LAMP1 的水平，通过免疫荧光染色检测。细胞经过 DZ-514 处理（ ）。定量结果如下所示。使用 ImageJ 软件计算了每个视野的平均免疫荧光强度，并为每个处理选择了 4 个视野。
(H, I). DZ-514 increased the levels of Rab7 in TNBC cells, as detected by immunofluorescence staining. The cells were incubated with DZ-514 (2 M, 24 h), the quantitative results are shown below. （H，I）。DZ-514 增加了 TNBC 细胞中 Rab7 的水平，通过免疫荧光染色检测。细胞与 DZ-514（2 M，24 小时）孵育，定量结果如下所示。
prevent the formation of autophagosomes by inhibiting PI3K [42]. Consistently, neither HCQ nor 3-MA rescued DZ-514-induced vacuolization and cell death in TNBC cells (Figs. S4B-E). These results indicate that DZ-514-induced cell vacuolization is independent of autophagy. 通过抑制 PI3K 来阻止自噬体的形成[42]。一致地，无论是 HCQ 还是 3-MA 都不能拯救 DZ-514 诱导的 TNBC 细胞空泡化和细胞死亡（图 S4B-E）。这些结果表明，DZ-514 诱导的细胞空泡化与自噬无关。
3.7. DZ-514 induces methuosis in TNBC cells 3.7. DZ-514 在三阴性乳腺癌细胞中引发甲状腺溶解现象。
Bafilomycin A1 (Baf A1) has been reported to be a vacuolation ATPase inhibitor, which effectively inhibit lysosome acidification, the formation of macropinosomes, vacuolization of late endosomes, and endocytic protein degradation [43,44]. We found that Baf A1 completely blocks DZ-514-induced cytoplasmic vacuolation and cell death after treating TNBC cells in combination or separately with DZ-514 or Baf A1 (Fig. 4A-B). It is also recognized that macropinocytosis can lead to the uptake of particles and fluids [45,46]. Lucifer yellow (LY) is a tracer of liquid phase pinocytosis, which cannot penetrate the cell membrane of living cells [47]. To validate whether DZ-514 induces macropinocytosis, we treated TNBC cells with DZ-514 (24 h) and incubated the cells with LY at for . We observed that most of the intracellular vacuoles were LY-positive in the DZ-514 treatment group, suggesting that the vacuoles were macropinosomes (Fig. 4C). We further labeled several cellular organelles with Lysotracker, Mitotracker, and ERtracker. We found that DZ-514-induced vacuoles did not overlap with mitochondria, lysosomes, or the endoplasmic reticulum, suggesting that vacuoles were not derived from mitochondria, lysosomes, or the endoplasmic reticulum (Fig. 4D). Vacuoles in methuosis have been reported to have characteristics of late endosomes (Rab7-and LAMP1-positive) [48]. Therefore, the protein levels of Rab7 and LAMP1 were examined by WB. As expected, DZ-514 increased the protein levels of Rab7 and LAMP1 in a dose-dependent manner (Fig. 4E). We also performed immunofluorescence staining with specific antibodies to detect Rab7 and LAMP1. High-resolution microscopy showed that the expression levels of intracellular LAMP1 and Rab7 were upregulated by DZ-514 (Fig. 4F-I). These data collectively support that DZ-514 promotes methuosis by inducing dysfunctional micropinocytosis. Bafilomycin A1（Baf A1）被报道为一种液泡 ATP 酶抑制剂，能有效抑制溶酶体酸化、巨噬体形成、晚期内体的液泡化和内吞蛋白降解[43,44]。我们发现，在与 DZ-514 或 Baf A1 单独或联合处理 TNBC 细胞后，Baf A1 完全阻断了 DZ-514 诱导的细胞质液泡化和细胞死亡（图 4A-B）。同时，巨噬体内吞作用可以导致颗粒和液体的摄取[45,46]。Lucifer yellow（LY）是液相内吞的示踪剂，不能穿透活细胞的细胞膜[47]。为了验证 DZ-514 是否诱导巨噬体内吞作用，我们将 TNBC 细胞与 DZ-514 处理（24 小时），并在 的条件下与 LY 共同孵育 。我们观察到，在 DZ-514 处理组中，大部分细胞内液泡为 LY 阳性，表明这些液泡为巨噬体（图 4C）。我们进一步使用 Lysotracker、Mitotracker 和 ERtracker 标记了几个细胞器。 我们发现 DZ-514 引起的液泡与线粒体、溶酶体或内质网没有重叠，这表明液泡不是来自于线粒体、溶酶体或内质网（图 4D）。已有报道称，甲状腺素引起的液泡具有晚期内体的特征（Rab7 和 LAMP1 阳性）[48]。因此，我们通过 WB 检测了 Rab7 和 LAMP1 的蛋白水平。如预期，DZ-514 以剂量依赖的方式增加了 Rab7 和 LAMP1 的蛋白水平（图 4E）。我们还使用特异性抗体进行了免疫荧光染色以检测 Rab7 和 LAMP1。高分辨率显微镜显示，DZ-514 上调了细胞内 LAMP1 和 Rab7 的表达水平（图 4F-I）。这些数据共同支持 DZ-514 通过诱导功能失调的微吞噬作用来促进甲状腺素作用。
To better characterize the efficacy of DZ-514 to induce methuosis, we selected well-established methuosis inducers MOMIPP and PP242 as positive control . Our results showed that DZ-514 had the similar vacuolation-inducing ability as MOMIPP in MDA-MB-468 cells and HCC1806 cells (Fig. S5A). We also tested the activity of MOMIPP in two types of TNBC cells, and the results showed that although MOMIPP could induce the vacuolation of cells at low concentrations, the induced cell membrane rupture required a higher concentrations (Fig. S5B). In addition, our results show that DZ-514 is more potent than the methuosis inducer PP242 (Figs. S5C-D). 为了更好地表征 DZ-514 诱导甲状腺肿瘤的功效，我们选择了已经被确认的甲状腺肿瘤诱导剂 MOMIPP 和 PP242 作为阳性对照。我们的结果显示，DZ-514 在 MDA-MB-468 细胞和 HCC1806 细胞中具有与 MOMIPP 相似的形成空泡的能力（图 S5A）。我们还测试了 MOMIPP 在两种三阴性乳腺癌细胞中的活性，结果显示，尽管 MOMIPP 可以在低浓度下诱导细胞形成空泡，但诱导细胞膜破裂需要更高的浓度（图 S5B）。此外，我们的结果表明，DZ-514 比甲状腺肿瘤诱导剂 PP242 更有效（图 S5C-D）。
3.8. DZ-514-induced methuosis depends on the ROS-MKK4-p38 pathway 3.8. DZ-514 诱导的 methuosis 依赖于 ROS-MKK4-p38 通路
It has been shown that overexpression of Ras in glioblastoma cells can induce methuosis through Rac1-dependent signaling pathways [17]. To test whether Rac1 is involved in DZ-514-induced methuosis, we knocked down Rac1 (Fig. S6A). Rac1 knockdown by three different siRNAs did not block cytoplasmic vacuolation induced by DZ-514 (Fig. S6B). In addition, the Rac1 inhibitor NSC23766 was unable to prevent DZ-514-induced cytoplasmic vacuoles (Fig. S6C). These results indicate that DZ-514-induced methuosis is independent of Rac1 activation. 已经证明，在胶质母细胞瘤细胞中过表达 Ras 可以通过 Rac1 依赖的信号通路诱导甲状腺肿。为了测试 Rac1 是否参与 DZ-514 诱导的甲状腺肿，我们敲除了 Rac1（图 S6A）。通过三种不同的 siRNA 敲除 Rac1 并不能阻断 DZ-514 诱导的细胞质空泡形成（图 S6B）。此外，Rac1 抑制剂 NSC23766 也不能阻止 DZ-514 诱导的细胞质空泡形成（图 S6C）。这些结果表明，DZ-514 诱导的甲状腺肿与 Rac1 的激活无关。
It has been reported that 5-((4-(pyridin-3-yl)pyrimidin-2-yl)amino)1H-Indole-2-Carbohydrazide derivatives induced methuosis via regulating the MAPK/JNK signaling pathway in breast and cervical cancer [25]. Activation of the JNK signaling pathway is the first step in MOMIPP-induced glioblastoma methuosis [20]. To test whether the MAPK/JNK signaling pathway participated in DZ-514-induced methuosis, we performed WB to detect the activation of ERK/JNK and found that DZ-514 indeed increased ERK and JNK phosphorylation (Fig. S7A). However, the knockdown of ERK and JNK did not block DZ-514-induced vacuolation in TNBC cells (Figs. S7B-G). We conclude that the ERK/JNK signaling pathway may not be involved in DZ-514-induced methuosis. 据报道，5-((4-(吡啶-3-基)嘧啶-2-基)氨基)1H-吲哚-2-羧酰肼衍生物通过调节 MAPK/JNK 信号通路在乳腺癌和宫颈癌中诱导了甲状腺肿。JNK 信号通路的激活是 MOMIPP 诱导的胶质母细胞瘤甲状腺肿的第一步。为了测试 MAPK/JNK 信号通路是否参与了 DZ-514 诱导的甲状腺肿，我们进行了 WB 检测 ERK/JNK 的激活，并发现 DZ-514 确实增加了 ERK 和 JNK 的磷酸化（图 S7A）。然而，ERK 和 JNK 的沉默并没有阻断 DZ-514 诱导的 TNBC 细胞的空泡形成（图 S7B-G）。我们得出结论，ERK/JNK 信号通路可能不参与 DZ-514 诱导的甲状腺肿。
MAPKs are composed of three major families: ERK, JNK, and p38 [50]. We wondered whether DZ-514 activates p38 to promote methuosis. Indeed, DZ-514 promoted the phosphorylation of p38 in a dose-dependent manner in TNBC cells (Fig. 5A). Importantly, p38 knockdown significantly blocked cytoplasmic vacuolation and cell death induced by DZ-514 (Fig. 5B-E). To further demonstrate that p38 was involved in DZ-514-induced methuosis, we used the p38-specific inhibitor SR-318 to block p38 activation. As expected, SR-318 significantly inhibited DZ-514-induced vacuolization in TNBC cells (Fig. 5F-H). We found that SR-318 significantly rescued DZ-514-induced cell death (Fig. 5I). These observations suggest that p38 plays an important role in the methuosis induced by DZ-514. MAPKs 由三个主要家族组成：ERK、JNK 和 p38 [50]。我们想知道 DZ-514 是否激活 p38 来促进甲状腺乳头状癌。事实上，DZ-514 以剂量依赖的方式促进了 TNBC 细胞中 p38 的磷酸化（图 5A）。重要的是，p38 的沉默显著阻断了 DZ-514 引起的细胞质空泡化和细胞死亡（图 5B-E）。为了进一步证明 p38 参与了 DZ-514 诱导的甲状腺乳头状癌，我们使用了 p38 特异性抑制剂 SR-318 来阻断 p38 的激活。如预期，SR-318 显著抑制了 TNBC 细胞中 DZ-514 引起的空泡化（图 5F-H）。我们发现 SR-318 显著挽救了 DZ-514 引起的细胞死亡（图 5I）。这些观察结果表明 p38 在 DZ-514 诱导的甲状腺乳头状癌中起着重要作用。
It has been reported that the mTOR inhibitors OSI-027 and PP242 induce dysfunctional macropinocytosis by activating the ROS-MKK4 axis [49] and that MKK4 can activate p38 under conditions of oxidative stress [30]. We wondered whether DZ-514 induced methuosis death by activating ROS-MKK4. We performed WB to test whether DZ-514 activates the phosphorylation of MKK4 and found that DZ-514 can activate both -MKK4 and p-p38 (Fig. 6A). When we knocked down MKK4, the activation of p-p38 by DZ-514 was blocked (Fig. 6B), indicating that DZ-514 induces phosphorylation of p38 by activating MKK4. To further explore whether DZ-514 induces methuosis through MKK4, we knocked down MKK4 in TNBC cells and detected the amount of vacuolation and cell death induced by DZ-514. Our results show that the knockdown of MKK4 partially blocks DZ-514-induced cell vacuolization and cell death (Fig. 6C-E and S8). 据报道，mTOR 抑制剂 OSI-027 和 PP242 通过激活 ROS-MKK4 轴诱导功能失调的巨噬细胞吞噬作用[49]，而 MKK4 在氧化应激条件下可以激活 p38[30]。我们想知道 DZ-514 是否通过激活 ROS-MKK4 诱导甲状噬死亡。我们进行了 WB 实验，测试 DZ-514 是否激活 MKK4 的磷酸化，并发现 DZ-514 可以同时激活 -MKK4 和 p-p38（图 6A）。当我们敲除 MKK4 时，DZ-514 对 p-p38 的激活被阻断（图 6B），表明 DZ-514 通过激活 MKK4 诱导 p38 的磷酸化。为进一步探究 DZ-514 是否通过 MKK4 诱导甲状噬死亡，我们在 TNBC 细胞中敲除了 MKK4，并检测了 DZ-514 诱导的液泡形成和细胞死亡的程度。我们的结果显示，MKK4 的敲除部分阻断了 DZ-514 诱导的细胞液泡形成和细胞死亡（图 6C-E 和 S8）。
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Fig. 6. DZ-514 induces methuosis through the ROS-MKK4-p38 pathway 图 6. DZ-514 通过 ROS-MKK4-p38 途径诱导甲磺酸化作用。
(A). DZ-514 significantly increased the phosphorylation of p38 and MKK4. These two cell lines lysates were gathered for WB after treatment with DZ-514 for 24 h. (B). Knockdown of MKK4 partially blocked DZ-514-induced phosphorylated MKK4 and p38. The cells were transfected with three siRNAs and then stimulated with DZ-514 ( , and the protein lysates were gathered for WB to examine the levels of p-p38 and p-MKK4. （A）DZ-514 显著增加了 p38 和 MKK4 的磷酸化。在用 DZ-514 处理 24 小时后，这两种细胞系的裂解液被收集用于 WB 检测。（B）MKK4 的沉默部分阻断了 DZ-514 诱导的 MKK4 和 p38 的磷酸化。细胞转染了三种 siRNA，然后用 DZ-514 刺激（ ，并收集蛋白质裂解液进行 WB 检测 p-p38 和 p-MKK4 的水平。
(C). Phase-contrast images show that silence of MKK4 significantly decreased DZ-514-induced vacuolization in MDA-MB-468 cells. (C). 相位对比图像显示，MKK4 的沉默显著减少了 DZ-514 诱导的 MDA-MB-468 细胞的液泡化。
(D). Quantification of vacuolated cells in MKK4 knockdown and control MDA-MB-468 cells treated with DZ-514. The proportion of vacuolated cells in seven fields was counted for each treatment. (D). 对 MKK4 敲低和对照的 MDA-MB-468 细胞在 DZ-514 处理下的液泡细胞进行定量。对每种处理的七个视野中的液泡细胞比例进行计数。
(E). Knockdown of MKK4 in MDA-MB-468 cells partially rescued DZ-514-induced cell death. The cells were transfected with three siRNAs for 36-48 h and then exposed to DZ-514 for . （E）在 MDA-MB-468 细胞中敲除 MKK4 部分逆转了 DZ-514 诱导的细胞死亡。细胞转染了三种 siRNA，经过 36-48 小时后，暴露于 DZ-514 中。
(F). DZ-514 significantly increased the accumulation of intracellular ROS levels. HCC1806 and MDA-MB-468 cells were incubated with DZ-514 (8 h), and DCFDA staining was used to analyze the ROS levels by flow cytometry. （F）。DZ-514 显著增加了细胞内 ROS 水平的积累。HCC1806 和 MDA-MB-468 细胞与 DZ-514 共培养（8 小时），使用 DCFDA 染色通过流式细胞术分析 ROS 水平。
(G). DZ-514 increased the protein levels of HO-1 and NRF2. (G). DZ-514 增加了 HO-1 和 NRF2 的蛋白水平。
(H). NAC significantly blocked DZ-514-induced phosphorylation of p38 and MKK4. HCC1806 and MDA-MB-468 cells were pretreated with NAC (2 mM, 2 h) and then incubated with DZ-514 for . Then, the cells were collected for WB. (H). NAC 显著阻断了 DZ-514 引起的 p38 和 MKK4 的磷酸化。HCC1806 和 MDA-MB-468 细胞预先用 NAC（2 mM，2 h）处理，然后与 DZ-514 一起孵育 。然后，收集细胞进行 WB。
(I, J). NAC partially blocked DZ-514-induced cell vacuolization. Phase contrast images of TNBC cells pretreated for in the presence and absence of NAC ( ). (K). NAC partially rescued DZ-514-induced cell death. The cells were pretreated for in the presence and absence of NAC before DZ-514 treatment for 6 or . The SRB assay was used to measure cell viability. （I，J）。NAC 部分阻断了 DZ-514 引起的细胞空泡化。在 NAC 存在和不存在的情况下，对 TNBC 细胞进行预处理，相差显微镜下的相位对比图像（ ）。（K）。NAC 部分挽救了 DZ-514 引起的细胞死亡。在 DZ-514 处理前，细胞在存在和不存在 NAC 的情况下进行预处理，持续时间为 ，然后进行 6 或 的 DZ-514 处理。使用 SRB 测定法测量细胞存活率。
Fig. 7. DZ-514 suppresses HCC1806 tumor growth in vivo 图 7. DZ-514 在体内抑制 HCC1806 肿瘤生长
(A). Images of the tumors collected after 18 days of DZ-514 treatment. DMSO was used as a negative control. （A）DZ-514 治疗 18 天后收集的肿瘤图像。DMSO 被用作阴性对照。
(B). DZ-514 significantly suppressed tumor growth in nude mice. When the tumor volume grew than 50 , DZ-514 (5 and ) was injected intraperitoneally every two days for 18 days. These data were analyzed by ANOVA, *** for . （B）DZ-514 显著抑制了裸鼠体内的肿瘤生长。当肿瘤体积增长到 50 以上时，每隔两天腹腔注射 DZ-514（5 和 ）持续 18 天。这些数据通过 ANOVA 进行分析，***表示 。
(C). DZ-514 significantly decreased the tumor weights. After three weeks of continuous administration, mice were sacrificed as ethically permitted, then tumors were isolated and weighed. （C）DZ-514 显著降低了肿瘤重量。连续给药三周后，按照伦理要求，小鼠被牺牲，然后分离并称重肿瘤。
(D). The expression levels of LAMP1 and Rab7 in HCC1806-derived tumor tissue sections were detected by immunofluorescence. （D）使用免疫荧光法检测 HCC1806 源性肿瘤组织切片中 LAMP1 和 Rab7 的表达水平。
Subsequently, we found that DZ-514 induced the accumulation of ROS in TNBC cells in a dose-dependent manner (Fig. and S9). Consistently, DZ-514 increased the levels of the oxidative stress-related proteins HO-1 and Nrf2 (Fig. 6G). These results indicate that DZ-514 induced oxidative stress in TNBC. To explore whether DZ-514 activates p38 and MKK4 by inducing ROS, we pretreated TNBC cells with NAC for before DZ-514 treatment and found that NAC could block the phosphorylation of MKK4 and p38 (Fig. 6H). The phase-contrast images showed that NAC reduced the number of DZ-514-induced cellular vacuolation (Fig. 6I-J). SRB assays showed that NAC rescued DZ-514-induced cell death (Fig. 6K). In conclusion, DZ-514 induces methuosis via the ROS-MKK4-p38 axis in TNBC cells.重试错误原因
We wondered whether DZ-514 also has good anti-cancer activity in vivo, so we established a xenograft tumor model of HCC1806 in nude mice and administered DZ-514 to the mice by intraperitoneal injection. Mice were sacrificed and tumors were isolated after treatment with DZ514 (18 days) (Fig. 7A). DZ-514 (5-10 mg/kg) treatment group significantly inhibited tumor growth compared with the DMSO group according to the tumor volume (Fig. 7B). Consistently, the weight significantly decreased in tumors compared to the DMSO group after treatment with DZ-514 (Fig. 7C). Furthermore, immunofluorescence analysis showed that the protein levels of Rab7 and LAMP1 were increased in DZ-514-treated HCC1806 xenografts (Fig. 7D). In addition, DZ-514 did not significantly decrease the body weight of the mice (Fig. S10A), nor did it cause liver and kidney damage (Figs. S10B-D). 我们想知道 DZ-514 在体内是否也具有良好的抗癌活性，因此我们在裸鼠中建立了 HCC1806 异种移植瘤模型，并通过腹腔注射给小鼠注射 DZ-514。在 DZ-514 治疗后（18 天），小鼠被牺牲，肿瘤被分离（图 7A）。与 DMSO 组相比，DZ-514（5-10 mg/kg）治疗组根据肿瘤体积显著抑制了肿瘤生长（图 7B）。一致地，与 DMSO 组相比，肿瘤的重量在 DZ-514 治疗后显著减少（图 7C）。此外，免疫荧光分析显示，DZ-514 处理的 HCC1806 异种移植瘤中 Rab7 和 LAMP1 的蛋白水平增加（图 7D）。此外，DZ-514 没有显著降低小鼠的体重（图 S10A），也没有引起肝脏和肾脏损伤（图 S10B-D）。
4. Discussion 讨论
TNBC is currently a pathological classification with a poor prognosis in breast cancer treatment due to its high heterogeneity, strong aggressiveness, and high metastatic [4]. BCL6 is an oncogenic transcription factor and therapeutic target in lymphoma and solid tumors, including breast cancer [51-53]. High BCL6 expression is associated with reduced progression-free survival in TNBC [13]. In this study, we initially evaluated the effect of our previously reported -phenyl-4-pyrimidinediamine derivatives on the cell viability of HCC1806 cells and identified DZ-514 as the most potent candidate. However, in our previously established homogeneous time-resolved fluorescence (HTRF) assay, DZ-514 only exhibited limited inhibition of BCL6 function [14]. At the same time, we also demonstrated the antiproliferative effect of DZ-514 was not correlated with the expression level of BCL6 in breast cancer cell lines. As a result, we speculated that the antiproliferative effect of DZ-514 against TNBC cells was independent of the BCL6 pathway. To understand the underlying mechanism of action, we TNBC 目前是乳腺癌治疗中预后不良的病理分类，由于其高度异质性、强烈侵袭性和高转移性[4]。BCL6 是一个致癌转录因子，也是淋巴瘤和实体肿瘤（包括乳腺癌）的治疗靶点[51-53]。高 BCL6 表达与 TNBC 的无进展生存期缩短相关[13]。在本研究中，我们首先评估了我们先前报道的 -苯基-4-嘧啶二胺衍生物对 HCC1806 细胞的细胞存活率的影响，并确定 DZ-514 为最有效的候选药物。然而，在我们先前建立的均一时间分辨荧光（HTRF）测定中，DZ-514 只表现出对 BCL6 功能的有限抑制[14]。同时，我们还证明了 DZ-514 的抗增殖作用与乳腺癌细胞系中 BCL6 的表达水平无关。因此，我们推测 DZ-514 对 TNBC 细胞的抗增殖作用与 BCL6 通路无关。为了了解其作用机制，我们...
performed a series of experiments to confirm the ability of DZ-514 to induce methuosis in TNBCs. Further assessments indicated that DZ-514 significantly inhibited cell cycle progression, colony formation, and cell proliferation in vitro and suppressed tumor growth in nude mice. 进行了一系列实验以确认 DZ-514 诱导三阴性乳腺癌细胞发生甲状腺溶解作用的能力。进一步评估表明，DZ-514 在体外显著抑制了细胞周期进展、集落形成和细胞增殖，并且在裸鼠体内抑制了肿瘤生长。
Interestingly, DZ-514 induced caspase-independent death. We ruled out that DZ-514-induced cell death was apoptosis, necrosis, and autophagy. We demonstrated that DZ-514 induced methuosis in TNBC cells. First, DZ-514 caused cytoplasmic vacuolation in multiple breast cancer cells. Further studies showed that DZ-514-induced vacuoles were derived from macropinosomes rather than autophagosomes because the induced vacuoles were positive for LY, Rab7, and LAMP1. Meanwhile, Baf A1 but not HCQ could block DZ-514-induced vacuole formation and cell death. 有趣的是，DZ-514 引发了非半胱天冬酶依赖的细胞死亡。我们排除了 DZ-514 引发的细胞死亡是凋亡、坏死和自噬。我们证明了 DZ-514 在三阴性乳腺癌细胞中引发了甲状噬细胞现象。首先，DZ-514 导致多种乳腺癌细胞的细胞质空泡化。进一步的研究表明，DZ-514 引发的空泡来自巨噬体而不是自噬体，因为引发的空泡对 LY、Rab7 和 LAMP1 呈阳性。同时，Baf A1 而不是 HCQ 可以阻断 DZ-514 引发的空泡形成和细胞死亡。
Methuosis is considered a dysfunctional micropinocytosis that has harmful effects on cell proliferation. In normal macropinocytosis, external stimuli promote the formation of the ruffle membrane/lamellipodia, the beginning of macropinocytosis [48]. The activation of Ras [54], RhoC [55], Rab5 [56], PAK1 [57], Arf6 [58], Rac1 [59], and JNK [20] has been reported to promote macropinocytosis. After the macropinocytosis cup is closed, the macropinocytosis corpuscle forms. After the giant macropinocytosis corpuscle matures, vacuoles become increasingly dense, and Rab5 is replaced by activated Rab7 [60]. Mature macropinosomes undergo two processes: recycling and degradation. The WASH (WASP and SCAR homologs) complex [61] and two-pore channel (TRC) [62] mediate the recycling of macropinosomes; PIKfyve [63] and TRPML1 [64] are involved in the degradation of macropinosomes. Methuosis 被认为是一种对细胞增殖有害的功能失调的微吞噬作用。在正常的巨吞噬作用中，外部刺激促进了褶皱膜/薄片的形成，标志着巨吞噬作用的开始[48]。已报道 Ras [54]、RhoC [55]、Rab5 [56]、PAK1 [57]、Arf6 [58]、Rac1 [59]和 JNK [20]的激活促进了巨吞噬作用。巨吞噬作用杯闭合后，形成巨吞噬作用小体。巨吞噬作用小体成熟后，液泡变得越来越密集，Rab5 被活化的 Rab7 取代[60]。成熟的巨吞噬体经历两个过程：回收和降解。WASH（WASP 和 SCAR 同源物）复合物[61]和双孔通道（TRC）[62]介导巨吞噬体的回收；PIKfyve [63]和 TRPML1 [64]参与巨吞噬体的降解。
In hyperactivated macropinocytosis, macropinocytosis is neither cycling nor degradation, and vacuoles rapidly mature and interact to form giant vacuoles characteristic of late endosomes (LAMP1- and Rab7positive), eventually leading to plasma membrane rupture and cell death [48]. DZ-514 significantly induced cytoplasmic vacuolation and macropinosomes. A recent study reported that the mTORC1/mTORC2 inhibitor OSI-027 induces ROS production, which activates MKK4 to promote macropinocytosis [49]. Our results suggest that DZ-514-induced methuosis is involved in the activation of the MKK4-p38 axis by accumulating ROS. However, the specific mechanism by which MKK4-p38 promotes macropinocytosis needs further investigation in the future. 在高度活化的巨噬细胞吞噬作用中，巨噬细胞吞噬作用既不是循环也不是降解，液泡迅速成熟并相互作用形成晚期内体的巨大液泡（LAMP1 和 Rab7 阳性），最终导致细胞膜破裂和细胞死亡[48]。DZ-514 显著诱导细胞质液泡化和巨噬细胞吞噬作用。最近的研究报道了 mTORC1/mTORC2 抑制剂 OSI-027 诱导 ROS 产生，激活 MKK4 促进巨噬细胞吞噬作用[49]。我们的结果表明，DZ-514 诱导的甲状噬细胞病涉及通过积累 ROS 激活 MKK4-p38 轴。然而，MKK4-p38 促进巨噬细胞吞噬作用的具体机制需要进一步研究。
In summary, we identified DZ-514, an -phenyl-4-pyrimidinediamine derivative, as a novel methuosis inducer in TNBC, showed potent anticancer activity in vitro and in vivo. DZ-514 induces methuosis by activating the ROS-MKK4-p38 axis. These results may provide new treatment opportunities for TNBC and other cancers. 总之，我们确定了 DZ-514，一种 -苯基-4-嘧啶二胺衍生物，作为 TNBC 中的一种新型甲状腺功能亢进诱导剂，在体外和体内显示出强大的抗癌活性。DZ-514 通过激活 ROS-MKK4-p38 轴诱导甲状腺功能亢进。这些结果可能为 TNBC 和其他癌症提供新的治疗机会。
The authors have no conflicts of interest to declare 作者们声明没有利益冲突
Acknowledgments 致谢
This work was supported by the National Key R&D Program of China (No.2020YFA0112300), the National Natural Science Foundation of China (N0.82160461, 81830087, U2102203, and 81973160), the Yunnan Fundamental Research Projects (No.202101AS070050), the 该工作得到了中国国家重点研发计划（No.2020YFA0112300）、中国国家自然科学基金（No.82160461、81830087、U2102203 和 81973160）、云南省基础研究项目（No.202101AS070050）的支持。
Major Projects for Fundamental Research of Yunnan Province (202201BC070002), and the Hunan Provincial Department of Education Fund (No.20A373, China). 云南省基础研究重点项目（202201BC070002），以及湖南省教育厅基金（编号 20A373，中国）。
Appendix A. Supplementary data 附录 A. 补充数据
Supplementary data to this article can be found online at https://doi. org/10.1016/j.canlet.2022.216049. 本文的补充数据可在 https://doi.org/10.1016/j.canlet.2022.216049 上找到。
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