Conjugation of Diclofenac with Novel Oleanolic Acid Derivatives Modulate Nrf2 and NF-κB Activity in Hepatic Cancer Cells and Normal Hepatocytes Leading to Enhancement of Its Therapeutic and Chemopreventive Potential.

Maria Narożna,Violetta Krajka-Kuźniak,Hanna Piotrowska-Kempisty,Robert Kleszcz,Małgorzata Kucińska,Jacek Kujawski,Barbara Bednarczyk-Cwynar,Wanda Baer-Dubowska,Marek Murias,Adam Plewiński

doi:10.3390/ph14070688

Maria Narożna, Violetta Krajka-Kuźniak + Show 8 more

Open Access

https://doi.org/10.3390/ph14070688

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Abstract

Combining NSAIDs with conventional therapeutics was recently explored as a new strategy in cancer therapy. Our earlier studies showed that novel oleanolic acid oximes (OAO) conjugated with aspirin or indomethacin may enhance their anti-cancer potential through modulation of the Nrf2 and NF-κB signaling pathways. This study focused on the synthesis and biological evaluation of four diclofenac (DCL)–OAO derivative conjugates in the context of these pathways’ modification and hepatic cells survival. Treatment with the conjugates 4d, 3-diclofenacoxyiminoolean-12-en-28-oic acid morpholide, and 4c, 3-diclofenacoxyiminoolean-12-en-28-oic acid benzyl ester significantly reduced cell viability in comparison to the DCL alone. In THLE-2, immortalized normal hepatocytes treated with these conjugates resulted in the activation of Nrf2 and increased expression in SOD-1 and NQO1, while the opposite effect was observed in the HepG2 hepatoma cells. In both cell lines, reduced activation of the NF-κB and COX-2 expression was observed. In HepG2 cells, conjugates increased ROS production resulting from a reduced antioxidant defense, induced apoptosis, and inhibited cell proliferation. In addition, the OAO morpholide derivative and its DCL hybrid reduced the tumor volume in mice bearing xenografts. In conclusion, our study demonstrated that conjugating diclofenac with the OAO morpholide and a benzyl ester might enhance its anti-cancer activity in HCC.

Highlights

Alone tended to increase Nrf2 transcript levels and decrease both NF-kB subunits, but the observed differences in comparison with untreated control cells were not statistically significant. These results indicate that DCL–oleanolic acid oximes (OAO) conjugates, with the benzyl and morpholide groups at the C-17 position, diminished the activation of key signaling pathways involved in Hepatocellular carcinoma (HCC) development and affected the expression of the transcription factors participating in the final stages of these pathways
Our study focused on the evaluation of synthetic hybrids of DCL with OAO derivatives in the context of modifications to signaling pathways critical for cell survival
The results of the molecular docking performed in this study demonstrated that the compound (4d) is able to bind to Kelch-like ECH-associated protein-1 (Keap1) and this suggests that in normal cells, Nrf2 activation may occur through the stabilization of Keap1

Summary

Introduction

Hepatocellular carcinoma (HCC) remains the most frequent primary liver cancer and is a leading cause of cancer-related deaths. Chronic inflammation resulting mainly from viral hepatitis or metabolic disorders is considered the major risk factor of HCC [1]. In the case of advanced forms of HCC, make necessary the development of new therapeutic and/or preventive strategies [2]. The key roles in the induction of inflammation involve two signaling pathways: Nrf2-ARE and NF-κB. While activation of the latter leads to transcription of genes encoding pro-inflammatory proteins, including COX-2 and iNOS enzymes, Nrf is responsible for cell cytoprotection and is the major cellular defense against reactive oxygen (ROS) and electrophilic species [3,4]

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