Li Si-Si, Jiang Wei-Liang, Xiao Wen-Qin, Li Kai, Zhang Ye-Fei, Guo Xing-Ya, Dai Yi-Qi, Zhao Qiu-Yan, Jiang Ming-Jie, Lu Zhan-Jun, Wan Rong
Department of Gastroenterology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 201620, China.
Shanghai Key Laboratory of Pancreatic Diseases, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 201620, China .
World J Gastrointest Oncol. 2019 Aug 15;11(8):599-621. doi: 10.4251/wjgo.v11.i8.599.
Novel therapeutic strategies are urgently needed for patients with a delayed diagnosis of pancreatic ductal adenocarcinoma (PDAC) in order to improve their chances of survival. Recent studies have shown potent anti-neoplastic effects of curcumin and its analogues. In addition, the role of histone methyltransferases on cancer therapeutics has also been elucidated. However, the relationship between these two factors in the treatment of pancreatic cancer remains unknown. Our working hypothesis was that L48H37, a novel curcumin analog, has better efficacy in pancreatic cancer cell growth inhibition in the absence of histone-lysine N-methyltransferase 2D (KMT2D).
To determine the anti-cancer effects of L48H37 in PDAC, and the role of KMT2D on its therapeutic efficacy.
The viability and proliferation of primary (PANC-1 and MIA PaCa-2) and metastatic (SW1990 and ASPC-1) PDAC cell lines treated with L48H37 was determined by CCK8 and colony formation assay. Apoptosis, mitochondrial membrane potential (MMP), reactive oxygen species (ROS) levels, and cell cycle profile were determined by staining the cells with Annexin-V/7-AAD, JC-1, DCFH-DA, and PI respectively, as well as flow cytometric acquisition. migration was assessed by the wound healing assay. The protein and mRNA levels of relevant factors were analyzed using Western blotting, immunofluorescence and real time-quantitative PCR. The in situ expression of KMT2D in both human PDAC and paired adjacent normal tissues was determined by immunohistochemistry. tumor xenografts were established by injecting nude mice with PDAC cells. Bioinformatics analyses were also conducted using gene expression databases and TCGA.
L48H37 inhibited the proliferation and induced apoptosis in SW1990 and ASPC-1 cells in a dose- and time-dependent manner, while also reducing MMP, increasing ROS levels, arresting cell cycle at the G2/M stages and activating the endoplasmic reticulum (ER) stress-associated protein kinase RNA-like endoplasmic reticulum kinase/eukaryotic initiation factor 2α/activating transcription factor 4 (ATF4)/CHOP signaling pathway. Knocking down ATF4 significantly upregulated KMT2D in PDAC cells, and also decreased L48H37-induced apoptosis. Furthermore, silencing KMT2D in L48H37-treated cells significantly augmented apoptosis and the ER stress pathway, indicating that KMT2D depletion is essential for the anti-neoplastic effects of L48H37. Administering L48H37 to mice bearing tumors derived from control or KMT2D-knockdown PDAC cells significantly decreased the tumor burden. We also identified several differentially expressed genes in PDAC cell lines expressing very low levels of KMT2D that were functionally categorized into the extrinsic apoptotic signaling pathway. The KMT2D high- and low-expressing PDAC patients from the TCGA database showed similar survival rates,but higher KMT2D expression was associated with poor tumor grade in clinical and pathological analyses.
L48H37 exerts a potent anti-cancer effect in PDAC, which is augmented by KMT2D deficiency.
对于胰腺导管腺癌（PDAC）诊断延迟的患者，迫切需要新的治疗策略以提高其生存几率。近期研究显示姜黄素及其类似物具有强大的抗肿瘤作用。此外，组蛋白甲基转移酶在癌症治疗中的作用也已阐明。然而，这两个因素在胰腺癌治疗中的关系仍不清楚。我们的工作假设是，新型姜黄素类似物L48H37在缺乏组蛋白赖氨酸N -甲基转移酶2D（KMT2D）的情况下，对胰腺癌细胞生长抑制具有更好的疗效。
确定L48H37对PDAC的抗癌作用以及KMT2D对其治疗效果的作用。
通过CCK8和集落形成试验测定用L48H37处理的原发性（PANC - 1和MIA PaCa - 2）和转移性（SW1990和ASPC - 1）PDAC细胞系的活力和增殖。分别用膜联蛋白V/7 -氨基放线菌素D、JC - 1、二氯荧光素二乙酸酯和碘化丙啶对细胞进行染色，并通过流式细胞术检测凋亡、线粒体膜电位（MMP）、活性氧（ROS）水平和细胞周期分布。通过伤口愈合试验评估迁移情况。使用蛋白质印迹、免疫荧光和实时定量PCR分析相关因子的蛋白质和mRNA水平。通过免疫组织化学确定KMT2D在人PDAC和配对的相邻正常组织中的原位表达。通过向裸鼠注射PDAC细胞建立肿瘤异种移植模型。还使用基因表达数据库和TCGA进行生物信息学分析。
L48H37以剂量和时间依赖性方式抑制SW1990和ASPC - 1细胞的增殖并诱导凋亡，同时还降低MMP、增加ROS水平、使细胞周期停滞在G2/M期并激活内质网（ER）应激相关蛋白激酶RNA样内质网激酶/真核起始因子2α/激活转录因子4（ATF4）/CHOP信号通路。敲低ATF4可显著上调PDAC细胞中的KMT2D，并且还降低L48H37诱导的凋亡。此外，在L48H37处理的细胞中沉默KMT2D可显著增强凋亡和ER应激途径，表明KMT2D缺失对于L48H37的抗肿瘤作用至关重要。向携带源自对照或KMT2D敲低的PDAC细胞的肿瘤的小鼠施用L48H37可显著降低肿瘤负担。我们还在表达极低水平KMT2D的PDAC细胞系中鉴定了几个差异表达基因，其功能分类为外在凋亡信号通路。来自TCGA数据库的KMT2D高表达和低表达的PDAC患者显示出相似的生存率，但在临床和病理分析中，较高的KMT2D表达与较差的肿瘤分级相关。
L48H37在PDAC中发挥强大的抗癌作用，KMT2D缺乏可增强该作用。
