Basic Study
Copyright ©The Author(s) 2020. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Dec 7, 2020; 26(45): 7131-7152
Published online Dec 7, 2020. doi: 10.3748/wjg.v26.i45.7131
Nimbolide inhibits tumor growth by restoring hepatic tight junction protein expression and reduced inflammation in an experimental hepatocarcinogenesis
Amit Kumar Ram, Balasubramaniyan Vairappan, BH Srinivas
Amit Kumar Ram, Balasubramaniyan Vairappan, Liver Diseases Research Lab,Department of Biochemistry, Jawaharlal Institute of Postgraduate Medical Education and Research (JIPMER), Dhanvantari Nagar, Puducherry 605006, India
BH Srinivas, Department of Pathology, Jawaharlal Institute of Postgraduate Medical Education and Research (JIPMER), Dhanvantari Nagar, Puducherry 605006, India
Author contributions: Ram AK and Vairappan B conceived and designed the study, analyzed the data statistically; Ram AK performed the experiments and wrote the manuscript; Vairappan B critically reviewed the manuscript; Srinivas BH interpreted histology and immunohistochemical findings.
Supported by JIPMER intramural research grant; Indian Council of Medical Research (ICMR), New Delhi, India, No. 3/1/3 J.R.F.-2016/LS/HRD; and Department of Biotechnology, Government of India, No. 102/IFD/SAN/22/2013-14.
Institutional review board statement: This study was reviewed and approved by the JIPMER scientific advisory committee (JSAC).
Institutional animal care and use committee statement: This study was reviewed and approved by JIPMER Institute Animal Ethics Committee (IAEC). All institutional and national guidelines for the care and use of laboratory animals were followed.
Conflict-of-interest statement: The authors declare that there is no conflict of interest related to this study.
ARRIVE guidelines statement: The authors have read the ARRIVE guidelines, and the manuscript was prepared and revised according to the ARRIVE guidelines.
Open-Access: This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (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: Balasubramaniyan Vairappan, PhD, Associate Professor, Liver Diseases Research Lab, Department of Biochemistry, Jawaharlal Institute of Postgraduate Medical Education and Research (JIPMER), Dhanvantari Nagar, Puducherry 605006, India. balasubramaniyan.v@jipmer.edu.in
Received: July 25, 2020
Peer-review started: July 25, 2020
First decision: September 14, 2020
Revised: September 28, 2020
Accepted: November 12, 2020
Article in press: November 12, 2020
Published online: December 7, 2020
Processing time: 131 Days and 20.6 Hours
Abstract
BACKGROUND

Altered tight junction (TJ) proteins are correlated with carcinogenesis and tumor development. Nimbolide is a tetranotriterpenoid that has been shown to have antioxidant and anti-proliferative properties; however, its anticancer effects and molecular mechanism in hepatocellular carcinoma (HCC) remains obscure.

AIM

To investigate the effect of nimbolide on TJ proteins, cell cycle progression, and hepatic inflammation in a mouse model of HCC.

METHODS

HCC was induced in male Swiss albino mice (CD-1 strain) by a single intraperitoneal injection of 100 mg/kg diethylnitrosamine (DEN) followed by 80 ppm N-nitrosomorpholine (NMOR) in drinking water for 28 wk. After 28 wk, nimbolide (6 mg/kg) was given orally for four consecutive weeks in DEN/NMOR induced HCC mice. At the end of the 32nd week, all the mice were sacrificed and blood and liver samples were collected for various analyses. Macroscopic examinations of hepatic nodules were assessed. Liver histology and HCC tumor markers such as alpha-fetoprotein (AFP) and glypican-3 were measured. Expression of TJ proteins, cell proliferation, and cell cycle markers, inflammatory markers, and oxidative stress markers were analyzed. In silico analysis was performed to confirm the binding and modulatory effect of nimbolide on zonula occludens 1 (ZO-1), nuclear factor of kappa light polypeptide gene enhancer in B-cells (NF-κB), and tumor necrosis factor alpha (TNF-α).

RESULTS

We found nimbolide treatment at a concentration of 6 mg/kg to HCC mice reduced hepatic tumor size by 52.08% and tumor volume (P < 0.01), and delayed tumor growth in HCC mice with a concomitant reduction in tumor markers such as AFP levels (P < 0.01) and glypican-3 expression (P < 0.05). Furthermore, nimbolide treatment increased tight junction proteins such as ZO-1 and occludin expression (P < 0.05, respectively) and reduced ZO-1 associated nucleic acid binding protein expression (P < 0.001) in HCC mice liver. Nimbolide treatment to HCC mice also inhibited cell proliferation and suppressed cell cycle progression by attenuating proliferating cell nuclear antigen (P < 0.01), cyclin dependent kinase (P < 0.05), and CyclinD1 (P < 0.05) expression. In addition, nimbolide treatment to HCC mice ameliorated hepatic inflammation by reducing NF-κB, interleukin 1 beta and TNF-α expression (P < 0.05, respectively) and abrogated oxidative stress by attenuating 4-hydroxynonenal expression (P < 0.01). Molecular docking studies further confirmed that nimbolide interacts with ZO-1, NF-κB, and TNF-α.

CONCLUSION

Our current study showed for the first time that nimbolide exhibits anticancer effect by reducing tumor size, tumor burden and by suppressing cell cycle progression in HCC mice. Furthermore, nimbolide treatment to HCC mice ameliorated inflammation and oxidative stress, and improved TJ proteins expression. Consequently, nimbolide could be potentially used as a natural therapeutic agent for HCC treatment, however further human studies are warranted.

Keywords: Hepatocellular carcinoma; Nimbolide; Tight junction; Inflammation; Oxidative stress; Zonula occludens 1 associated nucleic acid binding protein

Core Tip: The effects of nimbolide on tight junction (TJ) proteins, cell cycle progression, and hepatic inflammation in diethylnitrosamine (DEN) and N-nitrosomorpholine (NMOR) induced hepatocellular carcinoma (HCC) mouse model is unknown. This study revealed for the first time that nimbolide suppresses tumor growth by restoring hepatic TJ proteins, inhibiting cell proliferation and cell cycle progression, and attenuating inflammation and oxidative stress in HCC mice. Moreover, nimbolide showed a modulatory effect on zonula occludens 1, nuclear factor of kappa light polypeptide gene enhancer in B-cells and tumor necrosis factor alpha proteins confirmed by in silico analysis. All the above results clarified that nimbolide abolished DEN and NMOR induced hepatocarcinogenesis and suggested that nimbolide could be a future potential drug candidate for the treatment of human HCC.