Activité antiproliférative et mécanismes d’action de quelques plantes médicinales camerounaises

Abstract

Fruits of Fagara leprieuri Guill. & Perr (F. leprieuri, Rutaceae) and Fagara xanthoxyloides Waterm. (F. xanthoxyloides, Rutaceae), almonds of Monodora myristica (Gaertn.) Dunal (M. myristica, Annonaceae), leaves of Leea guineensis G. Don (L.guineensis, Leeaceae) and cloves of Xylopia aethiopica Dunal A. Rich (X. aethiopica, Annonaceae) are used as spices in traditional local foods (except L. guineensis) and in traditional medicine to treat various diseases and infections. The present work aimed to study the antiproliferative properties of the 70% ethanol plants extracts on several cancer cell lines by various methods and identify the compound responsible for the most important antiproliferative activity. To achieve this objective, the study was organized in four main points. Firstly, the screening of antiproliferative activity of plants extracts was done on the one hand by the cell proliferation assay of human osteosarcoma (U2OS), colorectal cancer cells (HCT 116), breast carcinoma cells (Sum PT 159), pancreas adenocarcinoma cells (Capan-2 PC) and of leukemia cells KG1a and U937, and on the other hand using clonogenic test and cell viability tests (using WST-1 and sulforhodamine B reagents). Secondly, the mechanism of the antiproliferative activity of extracts was investigated through evaluation of the activity of caspase-3, DNA condensation and mitochondria functions (membrane potential and oxygen consumption). In the third part of the study, antioxidant properties of different extracts was evaluated by determining their antioxidant contents and activities. Finally, X. aethiopica extract was analyzed by HPLC coupled to mass spectrometry and the activity of different collected fractions analyzed using the cell viability test using the WST-1 reagent. The most active fraction was identified and characterized. For the antiproliferative activity, using the cell proliferation assay, X. aethiopica extract inhibited the growth of the different cell lines with a highly significant effect on pancreatic cells Capan 2PC (1 ± 0.5 % of the control) and U 937 (1 ± 0 % of the control) which showed no growth upon treatment with that extract.The clonogenic test and the cell viability test revealed significant cytotoxic effects of X. aethiopica on HCT 116 (7.38 ±3.01 % of colonies at 3.1μg/ml and 12.03 ± 4.76% at 25 μg/ml). F. leprieuri extract also showed an antiproliferative activity mainly on HCT 116 (69.70 ± 17.52% of the control) and U2OS (72.85 ± 6.72% of the control), while F. xanthoxyloides inhibited growth of breast cancer cell lines MDA-MB 231 and MCF 7. Antioxidant analyses revealed X.aethiopica as having the highest antioxidant content (phenols: 68.62 ± 0.16 mg equivalent catechin/g of powder for methanol 1% HCl extract, α and β-carotene : 2.74 ± 0.13 et 2.85 ± 0.22 ng/mg of powder respectively, γ-tocopherols : 7.05 ± 0.16 ng/mg of powder, lutein :20.18 ± 0.70 ng/mg) and significant free radical scavenging activity (90.04 ± 1.07 % DPPH˚ inhibition for methanol 1% HCl extract) and ferric reducing power (375.67 ± 0.23 mM FeO4/g of dry material). The study of the mechanism of the antiproliferative activity showed time dependent activation of caspase 3 and DNA fragmentation by the extracts. In addition, the extracts disrupted the mitochondria membrane potential and increased respiration non-coupled to ATP synthesis. This finding indicates a stimulatory effect of the extracts on the opening of mitochondria transition pore followed by the release out of the mitochondria apoptotic activating compounds. Characterization of X. aethiopica 70% ethanol extract showed the induction of DNA fragmentation and cell cycle arrest in S phase. Four fractions were isolated through HPLC analysis of X. aethiopica extract and their effects on HCT 116 cells viability showed the compound present in the peak 1 as the most active. The latter caused more DNA fragmentation (in 37.5% of cells after 8 h of treatment) than the total extract and induced cell arrest in G1 phase. It was identified as terpenoid, ent-15-oxokaur-16-en-19-oic acid (EOKA, C20H28O3). These findings demonstrate the significant antiproliferative activities and antioxidant properties of X. aethiopica, F. leprieuri and F. xanthoxyloides extracts. Furthermore, a pure compound was isolated from X. aethiopica extract and its antiproliferative mechanism delineated. Taken together our study confirms spices and plants as sources of anticancer drugs.