Abstract
Calendula officinalis extracts have been known to possess anti-tumor properties, but questions regarding their mechanisms of action still need to be answered. Therefore, the present study aims to investigate the selective cytotoxicity, the biochemical profile and the corresponding molecular effects of two extracts of C. officinalis: flowers and leaves, against several breast cancer cell lines in vitro. Dry flowers and leaves were subjected to ultrasonication assisted extraction in methanol 70%. The phenolic and volatile profiles of the extracts, determined by HPLC-MS and nontargeted GC-MS, revealed high levels of specific phenolic acids, flavonols and coumarin and several volatile compounds, including mono- and sesquiterpenes, ketones, aldehydes and esters. Both extracts proved to possess selective cytotoxic activities against tumor cells in comparison to healthy endothelial cells, according to the MTT assay. The flower extract was superior in terms of both cytotoxicity and selectivity when compared to the leaf extract, in accordance to their biochemical profiles. The gene expression pattern for 10 genes of interest was evaluated by RT-qPCR. The expression level of several genes involved in apoptosis (BCL2, BAX, BBC3, ZMAT3), and cell cycle progression (NFkB, CCND1, STAT3) was modulated by the treatment with both extracts. Therefore, C. officinalis extracts proved to be rich in compounds characterized by cancer-related cytotoxicity and are capable of inducing selective cytotoxicity on breast cancer cell lines.
Highlights
MethodsPlant material and culture conditions C. officinalis seeds were collected from Covasna County, Romania, in October 2016
The flower extract was superior in terms of both cytotoxicity and selectivity when compared to the leaf extract, in accordance to their biochemical profiles
A number of 28 volatile compounds were identified in C. officinalis extracts, 24 of them being present in the flower extract and 16 appeared as leaf extract constituents
Summary
Plant material and culture conditions C. officinalis seeds were collected from Covasna County, Romania, in October 2016. Authentication of the plant species was done at the CL Herbarium, Babes-Bolyai University, Romania (specimen voucher 668431). Plant flowers and leaves were collected separately, dried in a dark room and powdered for further analysis. Cell lines and culture conditions Four human cell lines were used in this study: MCF7 (luminal breast cancer cells), MDA-MB-231, Hs578T (triple negative breast cancer cells) and HUVEC (human umbilical vein endothelial cells); cell lines were obtained from the European Collection of Authenticated Cell Cultures. MCF7 cells were cultured in standard conditions in MEM, MDA-MB-231 in RPMI-1640, Hs578T in DMEM and HUVEC in ECM growth media, supplemented with 10% fetal bovine serum (FBS), 1% glutamine and 1% penicillinstreptomycin. All cell culture reagents were obtained from Sigma-Aldrich (München, Germany)
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