MLN2238 exerts its anti-tumor effects via regulating ROS/JNK/mitochondrial signaling pathways in intrahepatic cholangiocarcinoma

Hao Xu; Guangyu Xu; Qianhui Xu; Chang Xu; Xiaohu Zhou; Yang Bai; Lu Yin; Yuan Ding; Weilin Wang

doi:10.3389/fphar.2022.1040847

MLN2238 exerts its anti-tumor effects via regulating ROS/JNK/mitochondrial signaling pathways in intrahepatic cholangiocarcinoma

Front Pharmacol. 2022 Oct 31:13:1040847. doi: 10.3389/fphar.2022.1040847. eCollection 2022.

Authors

Hao Xu^{1

2

3

4

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6}, Guangyu Xu^{1

2

3

4

5

6}, Qianhui Xu^{1

2

3

4

5

6}, Chang Xu^{1

2

3

4

5

6}, Xiaohu Zhou¹, Yang Bai^{1

2

3

4

5

6}, Lu Yin⁷, Yuan Ding^{1

2

3

4

5

6}, Weilin Wang^{1

2

3

4

5

6}

Affiliations

¹ Department of Hepatobiliary and Pancreatic Surgery, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China.
² Key Laboratory of Precision Diagnosis and Treatment for Hepatobiliary and Pancreatic Tumor of Zhejiang Province, Hangzhou, China.
³ Research Center of Diagnosis and Treatment Technology for Hepatocellular Carcinoma of Zhejiang Province, Hangzhou, China.
⁴ Clinical Medicine Innovation Center of Precision Diagnosis and Treatment for Hepatobiliary and Pancreatic Disease of Zhejiang University, Hangzhou, China.
⁵ Clinical Research Center of Hepatobiliary and Pancreatic Diseases of Zhejiang Province, Hangzhou, China.
⁶ Cancer Center, Zhejiang University, Hangzhou, China.
⁷ Department of Pathology, Affiliated Hangzhou First People's Hospital, Zhejiang University School of Medicine, Hangzhou, China.

Abstract

Background: Intrahepatic Cholangiocarcinoma (iCCA) is a highly malignant tumor with limited treatment options that contributes largely to cancer-related deaths worldwide. Compared with traditional transcriptomic analysis, single-cell RNA sequencing (scRNA-seq) is emerging as a more advanced and popular tool for the in-depth exploration of cellular diversity and molecular complexity. As a next-generation proteasome inhibitor, MLN2238 presents better pharmacodynamics, pharmacokinetics, and therapeutic responses in various cancers. However, its effects and mechanisms of action in iCCA remain unknown. Methods: iCCA tumor heterogeneity was determined based on 4,239 qualified scRNA-seq data from 10 iCCA samples. The potential biological roles of proteasome-related genes in iCCA were investigated using a pseudo-trajectory reconstruction. The effect of MLN2238 on iCCA cell proliferation was estimated using the CCK-8, EdU, and clone formation assays. Flow cytometry was used to examine the effect of added MLN2238 on cell cycle and apoptosis levels. Autophagic flux was detected using AdPlus-mCherry-GFP-LC3B cells. ROS levels and mitochondrial membrane potential were determined using DCFH-DA probing and JC-1 staining. JNK activation and mitochondrial apoptosis were observed using western blotting and immunofluorescence microscopy, respectively. Finally, we used a tumor-bearing mouse model to validate its efficacy in vivo for iCCA treatment. Results: Proteasome-related genes were dysregulated in iCCA progression and expressed at higher levels in tumor tissues. MLN2238 suppressed cell proliferation, blocked the cell cycle in the G2/M phase, promoted apoptosis, and induced cytoprotective autophagy in iCCA cells. Furthermore, MLN2238 increased ROS levels and activated the JNK signaling pathway. Inhibition of ROS and JNK activation by NAC and SP600125 significantly reversed MLN2238-induced apoptosis. MLN2238 also suppressed the growth of iCCA tumors in vivo. Conclusion: Proteasome-related genes play pivotal roles in iCCA development. MLN2238, as a proteasome inhibitor, induces apoptosis in iCCA cells through ROS/JNK/mitochondrial signaling pathways, and hence, making MLN2238 a potential therapeutic choice for iCCA.

Keywords: ROS/JNK/mitochondrial signaling pathway; apoptosis; autophagy; intrahepatic cholangiocarcinoma; proteasome inhibitors.