MODULATION OF MITOCHONDRIAL MEDIATED APOPTOSIS BY PURIFIED FRACTIONS OF Calliandra portoricensis BENTH

Abstract

Apoptosis is downregulated in all forms of cancers. The mitochondrion has been implicated in the apoptotic process and, recently has been targeted in cancer therapy because of its role in cancer progression. Medicinal plants are used in the treatment of cancer, in particular, Calandra portoricensis (CP) in the management of urinary obstruction in elderly males. There is paucity of Information to justify the use of the plant in ethnomedicine. This study was designed to investigate the modulatory effects of CP on mitochondrial-mediated apoptosis in cancer cells and rats. Root barks of CP (UIH-22466) were extracted with methanol by cold maceration to obtain crude methanol extract (MECP), which was fractionated using vacuum liquid chromatography to obtain methanol fraction (MFCP). Inhibition of mitochondrial lipid peroxidation (mLP), mitochondrial ATPase (mATPase) activity and mitochondrial Permeability Transition (mPT) were determined in MFCP by spectrophotometry. Prostatic LNCaP, DU-145, lung adenocarcinoma and healthy VERO cells were used to assess cell proliferation. Cell cycle analysis was evaluated by flow cytometry. Levels of pro-apoptotic Bax, anti-apoptotic Bcl-2, Cytochrome C Release (CCR) and activation of caspases 3(C3) and 9 (C9) were determined by ELISA, while mitochondrial integrity was evaluated by Fluorescent intensity Ratio (FIR). Thirty-five male Wistar rats (70-80 g) were divided into five groups of seven animals and each group was orally administered distilled water (control), 25, 50, 100 and 200 mg/kg of MFCP, respectively for 30 days. Rat liver mitochondria were isolated by differential centrifugation and mPT evaluated spectrophotometrically. The MFCP was purified by thin layer and column chromatography. Proton NMR, ¹³C and liquid chromatography mass spectroscopy were carried out on the partially purified compound. Data were analysed using descriptive statistics and ANOVA at α0.05. The MECP and MFCP induced mPT pore opening at 10, 20, 40 and 60 ug/mL by 1.1, 2.8, 4.5, and 13.8 folds, and 2.7, 4.5, 6.8, 7.4 folds, respectively compared with control. The MFCP enhanced mATPase activity and inhibited mLP maximally at 100 ug/mL, and 120 ug/mL by 3.5 folds and 93.1%, respectively compared with control. Opening of mPT pore by MFCP was observed after treatment at 100 and 200 mg/kg by 2.6 and 3.3 folds, respectively. The MFCP inhibited proliferation of prostatic LNCaP, DU-145, lung adenocarcinoma and VERO cells with IC₅₀ values of 2.4±0.2, 3.3 ±0.2, 3.6±0.2 and 17.9± 1.6 ug/mL respectively. The growth inhibition by MFCP correlated with a 3-fold decreased expression of Bcl-2 and a 4-fold increase in Bax levels at 10 ug/mL in LNCaP cells. Furthermore, MFCP caused a 3.5-fold reduction in FIR at 10ug/mL and induced CCR by 4.2 folds at the same concentration relative to control. The MFCP-induced CCR is associated with activation of C3 and C9 at 10 ug/mL by 4.2 and 5.1 folds, respectively which prompted cancer cells to arrest at S phase (by up to 64.3%). The NMR data revealed the presence of gallic-acid and xanthone in MFCP. Studies have shown that xanthone inhibits growth of cancer cells. Methanol fraction of Calliandra portoricensis exhibited anticancer and antiproliferative activities via mitochondrial-mediated apoptosis.