Basic Study
Copyright ©The Author(s) 2019. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastrointest Oncol. Aug 15, 2019; 11(8): 599-621
Published online Aug 15, 2019. doi: 10.4251/wjgo.v11.i8.599
KMT2D deficiency enhances the anti-cancer activity of L48H37 in pancreatic ductal adenocarcinoma
Si-Si Li, Wei-Liang Jiang, Wen-Qin Xiao, Kai Li, Ye-Fei Zhang, Xing-Ya Guo, Yi-Qi Dai, Qiu-Yan Zhao, Ming-Jie Jiang, Zhan-Jun Lu, Rong Wan
Si-Si Li, Wei-Liang Jiang, Wen-Qin Xiao, Kai Li, Ye-Fei Zhang, Xing-Ya Guo, Yi-Qi Dai, Qiu-Yan Zhao, Zhan-Jun Lu, Rong Wan, Department of Gastroenterology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 201620, China
Si-Si Li, Qiu-Yan Zhao, Ming-Jie Jiang, Rong Wan, Shanghai Key Laboratory of Pancreatic Diseases, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 201620, China

Author contributions: Li SS and Wan R contributed equally to this work; Li SS and Wan R designed the research; Li SS, Jiang WL, Xiao WQ, Zhang YF, Guo XY and Dai YQ performed the research; Li SS, Jiang WL, Li K and Xiao WQ contributed to the analytic tools; Zhao QY, Jiang MJ and Lu ZJ provided the clinical specimen and reagents; Li SS wrote the paper.
Institutional review board statement: All patients participated in this study gave their informed consent. Institutional review board approval of our hospital was obtained for this study. Colleague Ming-Jie Jiang provided human pancreatic cancer tissue specimens in our study.
Institutional animal care and use committee statement: All animal experiments were in line with the Shanghai Jiao Tong University's Policy on the Care and Use of Laboratory Animals.
Conflict-of-interest statement: There is no conflict of interest.
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Corresponding author: Rong Wan, PhD, Chief Doctor, Department of Gastroenterology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, No. 100 Haining Road, Hongkou District, Shanghai 201620, China. doctorwanrong1970@126.com
Telephone: +86-21-63240090 Fax: +86-21-63241377
Received: October 22, 2018
Peer-review started: October 23, 2018
First decision: November 27, 2018
Revised: January 23, 2019
Accepted: February 27, 2019
Article in press: February 27, 2019
Published online: August 15, 2019
Processing time: 297 Days and 20.8 Hours
Abstract
BACKGROUND

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).

AIM

To determine the anti-cancer effects of L48H37 in PDAC, and the role of KMT2D on its therapeutic efficacy.

METHODS

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. In vitro 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. In vivo tumor xenografts were established by injecting nude mice with PDAC cells. Bioinformatics analyses were also conducted using gene expression databases and TCGA.

RESULTS

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.

CONCLUSION

L48H37 exerts a potent anti-cancer effect in PDAC, which is augmented by KMT2D deficiency.

Keywords: Pancreatic neoplasms; Curcumin analog; Histone methyltransferase 2D; Therapeutic effects; Bioinformatics

Core tip: We are the first to report an anti-tumor effect of L48H37 in pancreatic cancer, and ascertain that KMT2D deficiency contributes significantly to the therapeutic effect, in part through the protein kinase RNA-like endoplasmic reticulum kinase/eukaryotic initiation factor 2α/activating transcription factor 4/CHOP signaling pathway. It is worth noting that the relationship between the KMT2D expression pattern and treatment efficacy in clinical practice has yet to be further explored.