MODULATORY EFFECTS OF Cajanus cajan LINN. LEAVES ON MITOCHONDRIAL-MEDIATED APOPTOSIS IN EXPERIMENTAL MURINE MODELS

Abstract

The opening of the Mitochondrial Membrane Permeability Transition (MMPT) pore causes cytochrome C release (CCR) which is a point of no return for apoptosis to take place. Some medicinal plants are known to induce MMPT pore opening and may be useful in the management of cancer. Cajanus cajan (CC) is used traditionally to treat breast cancer, however there is no scientific basis for this claim. This study was designed to assess the effects of CC on MMPT, mitochondrial ATPase (mATPase) activity and CCR in experimental murine models.

The leaves of CC were obtained from Bode market in Ibadan and authenticated at the Department of Botany, University of Ibadan (UIH22500). They were extracted with methanol to obtain methanol extract of CC (MECC). Fractionation of MECC by Vacuum Liquid Chromatography yielded chloroform fraction (CFCC) and ethyl acetate fraction (EACC). The EACC was purified on silica gel and sephadex LH 20 column chromatography and structure of compounds determined using Fourier transform infrared (FTIR), Ultraviolet (UV) spectroscopy and Gas chromatography mass spectrometry (GC-MS). The MECC, CFCC and EACC at different concentrations: 10, 30, 50 and 70 µg/mL were used for in vitro assays. In the in vivo studies, animals were treated with MECC and EACC.

Eighteen male mice (25±0.04 g) were assigned into three groups of six animals each and orally treated with corn oil (control), EACC (100 mg/kg) and EACC (200 mg/kg) separately. The mice were treated daily for fourteen consecutive days. Similar treatment was repeated in male Wistar rats (150±2.00 g) and animals were sacrificed by cervical dislocation.

The MMPT pore opening, mATPase activity and CCR (Rat and Mice) were determined spectrophotometrically. Caspase 3(C3), caspase 9(C9) and CCR were determined immunohistochemically. Formalin-fixed, H&E stained tissues were examined under light microscope. Data were subjected to descriptive statistics using ANOVA at α 0.05

In vitro, the MECC at 10, 30 µg/mL had no effect on MMPT pore, while 50 and 70 µg/mL induced pore opening by 7 and 13 folds. Similarly, enhancement of ATPase activities was by 3, 3, 4 and 4 folds at 10, 30, 50 and 70 µg/mL, respectively. The EACC induced MMPT pore opening at the same concentrations by 19, 20, 21 and 23 folds, and enhanced ATPase activities by 9, 11, 13 and 15 folds respectively. In the in vivo studies; the MECC had no effect on the MMPT pore at 100 mg/kg but induced opening at 200 mg/kg by 8 folds and activated C3, C9 and CCR significantly.

The EACC induced MMPT pore opening in vivo by 0.136±0.03 and 0.229±0.03nm relative to control 0.023±0.002 nm and stimulated CCR at 100 and 200 mg/kg by 15.19±0.3 and 19.14±0.3 ng/mL relative to control 10.15±0.1 ng/mL, respectively. Histological changes revealed perivascular infiltration by inflammatory cells in hepatic tissue. The column fractionation yielded a partially pure compound; 2-Methyl-z, z, 3, 13 Octadecadienol.

The ethyl acetate fraction of Cajanus cajan induced mitochondrial-mediated apoptosis via the induction of mitochondrial membrane permeability transition and therefore could be useful in conditions of downregulated apoptosis such as cancer.