NMR Profiling of Ononis diffusa Identifies Cytotoxic Compounds against Cetuximab-Resistant Colon Cancer Cell Lines.

Vittoria Graziani,Nicoletta Potenza,Teresa Troiani,Brigida D’Abrosca,Monica Scognamiglio,Antonio Fiorentino,Stefania Napolitano

doi:10.3390/molecules26113266

Vittoria Graziani, Nicoletta Potenza + Show 5 more

Open Access

https://doi.org/10.3390/molecules26113266

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Abstract

In the search of new natural products to be explored as possible anticancer drugs, two plant species, namely Ononis diffusa and Ononis variegata, were screened against colorectal cancer cell lines. The cytotoxic activity of the crude extracts was tested on a panel of colon cancer cell models including cetuximab-sensitive (Caco-2, GEO, SW48), intrinsic (HT-29 and HCT-116), and acquired (GEO-CR, SW48-CR) cetuximab-resistant cell lines. Ononis diffusa showed remarkable cytotoxic activity, especially on the cetuximab-resistant cell lines. The active extract composition was determined by NMR analysis. Given its complexity, a partial purification was then carried out. The fractions obtained were again tested for their biological activity and their metabolite content was determined by 1D and 2D NMR analysis. The study led to the identification of a fraction enriched in oxylipins that showed a 92% growth inhibition of the HT-29 cell line at a concentration of 50 µg/mL.

Highlights

Natural products play a crucial role in the discovery of anticancer compounds even today, when new technologies are available to obtain a wide range of drug candidates
In the search of new natural products to be explored as possible anticancer drugs, two plant species, namely Ononis diffusa and Ononis variegata, were screened against colorectal cancer cell lines
The cytotoxic activity of the crude extracts was tested on a panel of colon cancer cell models including cetuximab-sensitive (Caco-2, GEO, SW48), intrinsic (HT-29 and HCT-116), and acquired (GEO-CR, SW48-CR) cetuximab-resistant cell lines

Summary

Introduction

Natural products play a crucial role in the discovery of anticancer compounds even today, when new technologies are available to obtain a wide range of drug candidates. The need for anticancer compounds has become pressing and, in this context, colorectal cancer is definitely under the spotlight. The outcomes of patients with metastatic colorectal cancer (mCRC) has improved in recent years [8], new challenges are on the horizon Nowadays, resistance to both chemotherapy and molecularly targeted therapies represents a major problem for setting up effective treatments. Extracts from the two species which are the objects of this study were first screened for their antiproliferative activity on colorectal cancer cell lines (Caco-2, HT-29, and HCT-116) characterized by different genetic profiles. The Caco-2 cell line is sensitive to cetuximab as it has no genetic defects associated with anti-EGFR therapy resistance. O. diffusa crude extract was further tested on cetuximab-sensitive (GEO and SW48) and on acquired cetuximab-resistant (GEO-CR and SW48-CR) human colon cancer cell lines. A partial purification, always paired with the NMR analysis, and biological tests led to the identification of a mixture of oxylipins as putative bioactive compounds responsible for the antiproliferative properties of the crude extract

Results

NMR Profiling of the Extracts

Partial Purification of the Extract and Biological Activity

Plant Sampling

Proliferation Assay