Effets anticancéreux de l’extrait aqueux des écorces de Anthonotha macrophylla P. Beauv (Caesalpiniaceae) : études in vitro et in vivo

Abstract

Breast cancer is the most common malignant neoplasia and the leading cause of cancer death in women worldwide. The treatments developed by modern medicine have been associated with many inconveniences, hence the need to find alternatives to these treatments. This work was undertaken with the aim to evaluate in vitro and in vivo anticancer effects of the aqueous extract of barks of Anthonotha macrophylla. The chemopreventive effects of the aqueous extract of A. macrophylla were evaluated on the breast cancer model induced by 7,12 dimethylbenz [a] anthracene (DMBA) in rats. The antioxidant activity was done by assaying oxidative stress parameters such as malondialdehyde (MDA) and reduced glutathione (GSH) and the anti-free radical (DPPH and ABTS) and chelating activities of the extract were also evaluated. A quantitative phytochemical study and GC-MS were performed. The A. macrophylla extract mechanisms of action were determined by in vitro tests such as: cytotoxicity using the alamar blue (resazurin) assay on five tumors (MCF-7, MDA-MB-231, 4T1, SF-295 and SK-MEL-28) and two non-tumor cell lines (MCR-5 and HUVEC), cell growth and proliferation, inhibition of clone formation, apoptosis, impact on the cell cycle and the effect of the extract on adhesion and the cell migration respectively. The antiestrogenic effects of the aqueous extract of A. macrophylla were evaluated using the MCF-7 cell proliferation assay and following a 3-days uterotrophic test. The acute toxicity of the extract was also evaluated. The results obtained from the evaluation of the chemopreventive effects showed that after 28 weeks of treatment, the extract (75 and 150 mg/kg) significantly reduced tumor incidence (p < 0.01 and p < 0.05), tumor burden and tumor volume (p < 0.001 and p < 0.01) respectively. No signs of hyperplasia were observed in the mammary glands of animals treated with the extract of A. macrophylla at dose of 75 mg/kg compared to the DMBA group. The extract of A. macrophylla induced in vitro a non-selective cytotoxic effect on all cancer cell lines with a significant effect on rodent mammary cancer cells (4T1) and inhibition of the growth of all cell types. After 24 h and 48 h, the extract induced a significant decrease (p < 0.05) in cell proliferation at 100 µg/mL. The extract also induced a significant decrease (p < 0.01 and p < 0.001) in the number of MDA-MB-231 cell clones at the concentrations of 50 µg/mL and 100 µg/mL respectively; apoptosis of MDA-MB 231 cells at 100 µg/mL after 24 h; inhibited cell cycle progression in S and G2/M phases at concentrations of 50 and 100 µg/mL after 24 h and 48 h; decreased the anchorage of cells to collagen and fibronectin and significantly inhibited (p < 0.05 and p < 0.01) cell migration at concentrations of 50 and 100 µg/mL respectively. The results of the evaluation of antiestrogenic activity in vitro showed that ABSTRACTxxvii the proliferation of MCF-7 cells stimulated by estradiol was inhibited (p < 0.001) by the aqueous extract of A. macrophylla at all concentrations tested. The chemopreventive effects of A. macrophylla would be due to its antiestrogenic activity and its ability to increase the levels of GHS at the doses of 37.5 mg/kg (p < 0.05) and of 75 mg/kg (p <0.05), and to decrease MDA levels at the doses of 37.5 mg/ kg (p < 0.01) and of 150 mg/kg (p < 0.01)] in the mammary gland. In addition, this extract may be endowed with anti-free radical activities (EC50 = 198.7 μg/mL DPPH and EC50 = 198.4 μg/mL ABTS) and a metal chelating activity. These beneficial effects of A. macrophylla would be due to polyphenolic compounds (10.24 ± 0.55 mg eq of quercetin/g of dry extract) especially flavonoids (9.76 ± 0.99 mg eq of quercetin) and hexadecenoic methyl ester and 9-octadecenoic (Z) methyl ester acids present in this extract. In addition, the toxicity study showed that the aqueous extract of A. macrophylla is weakly toxic. Its LD50 has been estimated to be greater than 2000 mg/kg BW. The results obtained would justify the use of A. macrophylla by traditional healers of the Mungo department in the management of breast cancer in women.