Download PDF
Planta Med 2022; 88(13): 1233-1244
DOI: 10.1055/a-1676-4307
Natural Product Chemistry and Analytical Studies
Original Papers

Quantitative Proteomics Based on iTRAQ Reveal that Nitidine Chloride Induces Apoptosis by Activating JNK/c-Jun Signaling in Hepatocellular Carcinoma Cells

Shipeng Chen
1   School of Pharmaceutical Sciences, Guangxi Medical University, Nanning, Guangxi, China;
2   Department of Pharmacy, Liuzhou Peopleʼs Hospital, Liuzhou, Guangxi, China
,
Yinan Liao
1   School of Pharmaceutical Sciences, Guangxi Medical University, Nanning, Guangxi, China;
,
Jinyan Lv
1   School of Pharmaceutical Sciences, Guangxi Medical University, Nanning, Guangxi, China;
,
Huaxin Hou
1   School of Pharmaceutical Sciences, Guangxi Medical University, Nanning, Guangxi, China;
,
Jie Feng 
1   School of Pharmaceutical Sciences, Guangxi Medical University, Nanning, Guangxi, China;
› Author AffiliationsSupported by: National Natural Science Foundation of China 81960706
Supported by: Guangxi First-class Discipline Project for Pharmaceutical Sciences GXFCDP-PS-2018
Supported by: Natural Science Foundation of Guangxi Province of China 2017GXNSFAA198304
› Further Information
    Permissions and Reprints

Abstract

The aim of the present study was to investigate the cytotoxic effects and underlying molecular mechanisms of nitidine chloride (NC) in hepatocellular carcinoma cells via quantitative proteomics. MTT assays were used to detect the inhibitory effects of NC in Bel-7402 liver cancer cells, and the number of apoptotic cells was measured by flow cytometry. Quantitative proteomics technology based on iTRAQ was used to discover differential expressed proteins after NC treatment, and bioinformatic techniques were further used to screen potential targets of NC. Molecular docking was applied to evaluate the docking activity of NC with possible upstream proteins, and their expression was detected at the mRNA and protein levels by quantitative reverse transcription PCR and western blotting. NC inhibited the proliferation of Bel-7402 cells after 24 h of treatment and stimulated apoptosis in vitro. The proteomics experiment showed that NC triggers mitochondrial damage in HCC cells and transcription factor AP-1 (c-Jun) may be a potential target of NC (fold change = 4.36 ± 0.23). Molecular docking results revealed the highest docking score of NC with c-Jun N-terminal kinase (JNK), one of the upstream proteins of c-Jun. Moreover, the mRNA and protein expression of c-Jun and JNK were significantly increased after NC treatment (p < 0.05). These findings indicate that NC significantly induced mitochondrial damage in HCC cells, and induced apoptosis by activating JNK/c-Jun signaling.

Key words

Zanthoxylum nitidum - Rutaceae - nitidine chloride - apoptosis - hepatocellular carcinoma - quantitative proteomics

Supporting Information

    1H and 13C NMR data and spectra of NC (Fig. 1S and Fig. 2S), HR-ESI-MS spectrum of NC (Fig. 3S), distribution of NC in Bel-7402 cells (Fig. 4S), protein distributions detected by proteomics (Fig. 5S), repeated experiments performed for obtaining the CV distribution (Fig. 6S) and cytotoxic effects of NC on three HCC cell lines (Fig. 7S) are provided as Supporting Information.

  • Supporting Information


Publication History

Received: 06 June 2021

Accepted after revision: 28 September 2021

Article published online:
01 February 2022

© 2021. Thieme. All rights reserved.

Georg Thieme Verlag KG
Rüdigerstraße 14, 70469 Stuttgart, Germany

 

  • References

  • 1 Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, Bray F. Global cancer statistics 2020: Globocan estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 2021; 71: 209-249
    Google Scholar
  • 2 Lu Q, Ma R, Yang Y, Mo Z, Pu X, Li C. Zanthoxylum nitidum (Roxb.) DC: Traditional uses, phytochemistry, pharmacological activities and toxicology. J Ethnopharmacol 2020; 260: 112946
    CrossrefPubMedGoogle Scholar
  • 3 Wu Z, Raven PH, Garden MB. Flora of China. Beijing: Science Press; 2008
    Google Scholar
  • 4 Liu H, Feng J, Feng K, Lai M. Optimization of the extraction conditions and quantification by RP-LC analysis of three alkaloids in Zanthoxylum nitidum roots. Pharm Biol 2014; 52: 255-261
    CrossrefPubMedGoogle Scholar
  • 5 Yang N, Yue R, Ma J, Li W, Zhao Z, Li H, Shen Y, Hu Z, Lv C, Xu X, Yang Y, Dai X, Liu X, Yu Y, Zhang W. Nitidine chloride exerts anti-inflammatory action by targeting Topoisomerase I and enhancing IL-10 production. Pharmacol Res 2019; 148: 104368
    CrossrefPubMedGoogle Scholar
  • 6 Hu J, Shi X, Mao X, Chen J, Zhu L, Zhao Q. Antinociceptive activity of Rhoifoline A from the ethanol extract of Zanthoxylum nitidum in mice. J Ethnopharmacol 2013; 150: 828-834
    CrossrefPubMedGoogle Scholar
  • 7 Zhao LN, Guo XX, Liu S, Feng L, Bi QR, Wang Z, Tan NH. (±)-Zanthonitidine A, a pair of enantiomeric furoquinoline alkaloids from Zanthoxylum nitidum with antibacterial activity. Nat Prod Bioprospect 2018; 8: 361-367
    CrossrefPubMedGoogle Scholar
  • 8 Chen J, Wang J, Lin L, He L, Wu Y, Zhang L, Yi Z, Chen Y, Pang X, Liu M. Inhibition of STAT3 signaling pathway by nitidine chloride suppressed the angiogenesis and growth of human gastric cancer. Mol Cancer Ther 2012; 11: 277-287
    CrossrefPubMedGoogle Scholar
  • 9 Cui Y, Wu L, Cao R, Xu H, Xia J, Wang ZP, Ma J. Antitumor functions and mechanisms of nitidine chloride in human cancers. J Cancer 2020; 11: 1250-1256
    CrossrefPubMedGoogle Scholar
  • 10 Chen S, Yang L, Feng J. Nitidine chloride inhibits proliferation and induces apoptosis in ovarian cancer cells by activating the Fas signalling pathway. J Pharm Pharmacol 2018; 70: 778-786
    CrossrefPubMedGoogle Scholar
  • 11 Cheng Z, Guo Y, Yang Y, Kan J, Dai S, Helian M, Li B, Xu J, Liu C. Nitidine chloride suppresses epithelial-to-mesenchymal transition in osteosarcoma cell migration and invasion through Akt/GSK-3β/Snail signaling pathway. Oncol Rep 2016; 36: 1023-1029
    CrossrefPubMedGoogle Scholar
  • 12 Huang X, Hu M, Li K, Luo F, Zhu H. Nitidine chloride efficiently induces autophagy and apoptosis in melanoma cells via AMPK-mTOR signaling pathway. Pharmazie 2020; 75: 440-442
    PubMedGoogle Scholar
  • 13 Liao J, Xu T, Zheng JX, Lin JM, Cai QY, Yu DB, Peng J. Nitidine chloride inhibits hepatocellular carcinoma cell growth in vivo through the suppression of the JAK1/STAT3 signaling pathway. Int J Mol Med 2013; 32: 79-84
    CrossrefPubMedGoogle Scholar
  • 14 Ou X, Lu Y, Liao L, Li D, Liu L, Liu H, Xu H. Nitidine chloride induces apoptosis in human hepatocellular carcinoma cells through a pathway involving p53, p21, Bax and Bcl-2. Oncol Rep 2015; 33: 1264-1274
    CrossrefPubMedGoogle Scholar
  • 15 Lin J, Shen A, Chen H, Liao J, Xu T, Liu L, Lin J, Peng J. Nitidine chloride inhibits hepatic cancer growth via modulation of multiple signaling pathways. BMC Cancer 2014; 14: 729
    CrossrefPubMedGoogle Scholar
  • 16 Piao C, Zhang Q, Jin D, Wang L, Tang C, Zhang N, Lian F, Tong X. A study on the mechanism of milkvetch root in the treatment of diabetic nephropathy based on network pharmacology. Evid Based Complement Alternat Med 2020; 2020: 6754761
    CrossrefPubMedGoogle Scholar
  • 17 Hsin KY, Ghosh S, Kitano H. Combining machine learning systems and multiple docking simulation packages to improve docking prediction reliability for network pharmacology. PLoS One 2013; 8: e83922
    CrossrefPubMedGoogle Scholar
  • 18 Aslam B, Basit M, Nisar MA, Khurshid M, Rasool MH. Proteomics: technologies and their applications. J Chromatogr Sci 2017; 55: 182-196
    CrossrefPubMedGoogle Scholar
  • 19 Saei AA, Sabatier P, Tokat ÜG, Chernobrovkin A, Pirmoradian M, Zubarev RA. Comparative proteomics of dying and surviving cancer cells improves the identification of drug targets and sheds light on cell life/death decisions. Mol Cell Proteomics 2018; 17: 1144-1155
    CrossrefPubMedGoogle Scholar
  • 20 Cifani P, Kentsis A. Towards comprehensive and quantitative proteomics for diagnosis and therapy of human disease. Proteomics 2017; DOI: 10.1002/pmic.201600079.
    CrossrefPubMedGoogle Scholar
  • 21 Zhang X, Hou HT, Wang J, Liu XC, Yang Q, He GW. Plasma proteomic study in pulmonary arterial hypertension associated with congenital heart diseases. Cell Death Dis 2016; 6: 36541
    PubMedGoogle Scholar
  • 22 Zhang X, Wang L, Qiu K, Xu D, Yin J. Dynamic membrane proteome of adipogenic and myogenic precursors in skeletal muscle highlights EPHA2 may promote myogenic differentiation through ERK signaling. FASEB J 2019; 33: 5495-5509
    CrossrefPubMedGoogle Scholar
  • 23 Zhou N, Fan C, Liu S, Zhou J, Jin Y, Zheng X, Wang Q, Liu J, Yang H, Gu J, Zhou J. Cellular proteomic analysis of porcine circovirus type 2 and classical swine fever virus coinfection in porcine kidney-15 cells using isobaric tags for relative and absolute quantitation-coupled LC-MS/MS. Electrophoresis 2017; 38: 1276-1291
    CrossrefPubMedGoogle Scholar
  • 24 Ryan TA, Roper KM, Bond J, Bell SM, Sweeney ST, Morrison EE. A MAPK/c-Jun-mediated switch regulates the initial adaptive and cell death responses to mitochondrial damage in a neuronal cell model. Int J Biochem Cell B 2018; 104: 73-86
    CrossrefPubMedGoogle Scholar
  • 25 Trop-Steinberg S, Azar Y. AP-1 expression and its clinical relevance in immune disorders and cancer. Am J Med Sci 2017; 353: 474-483
    CrossrefPubMedGoogle Scholar
  • 26 Yadav S, Kalra N, Ganju L, Singh M. Activator protein-1 (AP-1): a bridge between life and death in lung epithelial (A549) cells under hypoxia. Mol Cell Biochem 2017; 436: 99-110
    CrossrefPubMedGoogle Scholar
  • 27 Cao X, Fu M, Bi R, Zheng X, Fu B, Tian S, Liu C, Li Q, Liu J. Cadmium induced BEAS-2B cells apoptosis and mitochondria damage via MAPK signaling pathway. Chemosphere 2021; 263: 128346
    CrossrefPubMedGoogle Scholar
  • 28 Zhang J, Wang L, Xie W, Hu S, Zhou H, Zhu P. Melatonin attenuates ER stress and mitochondrial damage in septic cardiomyopathy: A new mechanism involving BAP31 upregulation and MAPK-ERK pathway. J Cell Physiol 2020; 235: 2847-2856
    CrossrefPubMedGoogle Scholar
  • 29 Low HB, Zhang Y. Regulatory roles of MAPK phosphatases in cancer. Immune Netw 2016; 16: 85-98
    CrossrefPubMedGoogle Scholar
  • 30 Li L, Wang X, Sharvan R, Gao J, Qu S. Berberine could inhibit thyroid carcinoma cells by inducing mitochondrial apoptosis, G0/G1 cell cycle arrest and suppressing migration via PI3K-AKT and MAPK signaling pathways. Biomed Pharmacother 2017; 95: 1225-1231
    CrossrefPubMedGoogle Scholar
  • 31 Sui X, Kong N, Ye L, Han W, Zhou J, Zhang Q, He C, Pan H. p38 and JNK MAPK pathways control the balance of apoptosis and autophagy in response to chemotherapeutic agents. Cancer Lett 2014; 344: 174-179
    CrossrefPubMedGoogle Scholar
  • 32 Dai C, Zhang C, Sun X, Pan Q, Peng J, Shen J, Ma X. LukS-PV induces differentiation by activating the ERK signaling pathway and c-JUN/c-FOS in human acute myeloid leukemia cells. Int J Biochem Cell B 2016; 76: 107-114
    CrossrefPubMedGoogle Scholar
  • 33 Yang XG, Jiang BW, Jing QQ, Li WJ, Tan LP, Bao YL, Song ZB, Yu CL, Liu L, Liu YC, Li YX. Nitidine chloride induces S phase cell cycle arrest and mitochondria-dependent apoptosis in HaCaT cells and ameliorates skin lesions in psoriasis-like mouse models. Eur J Pharmacol 2019; 863: 172680
    CrossrefPubMedGoogle Scholar
  • 34 Jia M, Wang Y, Guo Y, Yu P, Sun Y, Song Y, Zhao L. Nitidine chloride suppresses epithelial-mesenchymal transition and stem cell-like properties in glioblastoma by regulating JAK2/STAT3 signaling. Cancer Med 2021; 10: 3113-3128
    CrossrefPubMedGoogle Scholar
  • 35 Liu LM, Xiong DD, Lin P, Yang H, Dang YW, Chen G. DNA topoisomerase 1 and 2A function as oncogenes in liver cancer and may be direct targets of nitidine chloride. Int J Oncol 2018; 53: 1897-1912
    PubMedGoogle Scholar
  • 36 Yang IH, Jung W, Kim LH, Shin JA, Cho NP, Hong SD, Hong KO, Cho SD. Nitidine chloride represses Mcl-1 protein via lysosomal degradation in oral squamous cell carcinoma. J Oral Pathol Med 2018; 47: 823-829
    CrossrefPubMedGoogle Scholar
  • 37 Fang Z, Tang Y, Jiao W, Xing Z, Guo Z, Wang W, Xu Z, Liu Z. Nitidine chloride induces apoptosis and inhibits tumor cell proliferation via suppressing ERK signaling pathway in renal cancer. Food Chem Toxicol 2014; 66: 210-216
    CrossrefPubMedGoogle Scholar
  • 38 Zhai H, Hu S, Liu T, Wang F, Wang X, Wu G, Zhang Y, Sui M, Liu H, Jiang L. Nitidine chloride inhibits proliferation and induces apoptosis in colorectal cancer cells by suppressing the ERK signaling pathway. Mol Med Rep 2016; 13: 2536-2542
    CrossrefPubMedGoogle Scholar
  • 39 Feng J, Yang XW, Huang RB, Zhang HY, He M, Huang QC. Development and validation of an LC-ESI-MS/MS method for the determination of nitidine chloride in rat plasma. J Chromatogr B Analyt Technol Biomed Life Sci 2012; 887 – 888: 43-47
    CrossrefPubMedGoogle Scholar
  • 40 Wen B, Zhou R, Feng Q, Wang Q, Wang J, Liu S. IQuant: An automated pipeline for quantitative proteomics based upon isobaric tags. Proteomics 2014; 14: 2280-2285
    CrossrefPubMedGoogle Scholar
  • 41 Yu G, Wang LG, Han Y, He QY. ClusterProfiler: An R package for comparing biological themes among gene clusters. OMICS 2012; 16: 284-287
    CrossrefPubMedGoogle Scholar
  • 42 Wu M, Shang X, Sun Y, Wu J, Liu G. Integrated analysis of lymphocyte infiltration-associated lncRNA for ovarian cancer via TCGA, GTEx and GEO datasets. PeerJ 2020; 8: e8961
    CrossrefPubMedGoogle Scholar
  • 43 Szklarczyk D, Gable AL, Lyon D, Junge A, Wyder S, Huerta-Cepas J, Simonovic M, Doncheva NT, Morris JH, Bork P, Jensen LJ, Mering CV. STRING v11: protein-protein association networks with increased coverage, supporting functional discovery in genome-wide experimental datasets. Nucleic Acids Res 2019; 47: D607-D613
    CrossrefPubMedGoogle Scholar
  • 44 Doncheva NT, Morris JH. Cytoscape StringApp: Network analysis and visualization of proteomics data. J Proteome Res 2019; 18: 623-632
    CrossrefPubMedGoogle Scholar
  • 45 Kielkopf CL, Bauer W, Urbatsch IL. Bradford assay for determining protein concentration. Cold Spring Harb Protoc 2020; 2020: 102269
    CrossrefPubMedGoogle Scholar