CYTOPROTECTION BY LEAF EXTRACT OF Tridax procumbens (LINN.) IN EMBRYONIC MOUSE, HEPG2 AND PANC-1 CELL LINES AND ARSENITEINDUCED TOXICITY IN RATS

Abstract

Pancreatic Ductal Adenocarcinoma (PDA) and Hepatocellular Carcinoma (HCC) are among the deadliest cancer types worldwide that are associated with arsenic intoxication. Current drugs used in cancer management show a lot of side effects. There is, therefore, an increased search for alternatives from medicinal plants with anticancer properties. Tridax procumbens (TP) is a medicinal plant rich in antioxidant phytochemicals with little information on its effects on cancer and arsenic toxicity. This study was designed to investigate the effect of Tridax procumbens in PDA, HCC and arsenite-induced toxicity using in vivo and in vitro models. The TP was authenticated (UIH-22542), air-dried, blended, soaked in ethanol, and concentrated to obtain the Crude Extract (CETP). The CETP was fractionated using Hexane, Dichloromethane, and Ethyl Acetate to obtain Fractions (HXF, DCMF, and EAF, respectively). Antioxidant assays of TP were performed. For in vivo study, 32 male Wistar rats (80-100g) were assigned into four groups (n=8), and treated as follows; A (control); 1 ml/kg body weight olive oil, B; 2.5 mg/kg Sodium Arsenite (SA), C; 50 mg/kg CETP, D; SA+CETP. Olive oil and CETP were administered once daily for 14 days, and SA twice (days 7 and 14). Histological examination of lungs and brain, and micronucleated Polychromatic Erythrocytes (mPCEs) frequency were carried out. The HCC and PDA cell lines; HepG2 and Panc-1, were maintained in a humidified incubator, and treated with (10, 20, 50, 100, and 250 𝜇g/ml) dimethyl sulfoxide (control), CETP and CETP-fractions for 24 and 48 hours. Similarly, embryonic mouse pancreas (E11.5d) were cultured for five days and treated with the test samples (20 𝜇g/ml) for two days. Cytotoxicity assays [3- (4,5-dimethylyhiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), Live/Dead] were carried out, and expression of proteins (cytokeratin-7, peanut agglutinin (PNA), α- fetoprotein, insulin, glucagon, amylase, catalase, alkaline phosphatase, Glutathione Stransferase-pi (GST-pi), caspase-3, Bcl-2, Adenomatous Polyposis Coli (APC), p53, p21Cip1/Wap-1, vimentin, Ki-67, Sox9, β-catenin, α1-antitrypsin, albumin, transferrin) were evaluated using immunofluorescence. Data were analysed using descriptive statistics and ANOVA at α0.05. The DCMF scavenged 2,2-diphenyl-1-picrylhydrazyl hydrate and nitric oxide radicals (IC50: 0.43 and 0.39 𝜇g/ml, respectively) relative to control (IC50: 0.85 and 0.14 𝜇g/ml, respectively). The CETP reduced arsenite-induced histological lesions in lungs and brain, and mPCEs [SA (21.00±3.33) to SA+CETP (3.80±0.58)] compared to control (0.60±0.40). The DCMF elicited cell death (IC50=23.1 𝜇g/ml) compared to CETP (IC50=114.2 𝜇g/ml). Relative to control, phenotype morphogenesis was observed in DCMF-treated embryonic pancreas with immunopositivity for insulin, vimentin and amylase (1.3-fold), cytokeratin- 7 (1.5-fold), PNA (1.9-fold), and glucagon (2.8-fold). There were significant elevations in transferrin (1.5-fold), albumin (1.8-fold), p53 (2.8-fold), caspase-3 (2.9-fold), catalase (4.0-fold), p21Cip1/Wap-1 (4.4-fold), and alkaline phosphatase (5.0-fold) in DCMF-treated cells compared to control. Conversely, there were significant reductions in β-catenin (1.3- fold), α1-antitrypsin and cytokeratin-7 (2.0-fold), PNA and GST-pi (2.3-fold), α- fetoprotein and Ki-67 (2.7-fold), and vimentin (10.8-fold) in DCMF-treated cells relative to control. The DCMF induced Bcl-2 punctate nuclear staining, and cytoplasmic translocation of APC, Sox9, and β-catenin in treated cells. Leaf extract of Tridax procumbens exhibited antiproliferative activities via the decrease in biomarkers of cancer induction and increase cellular antioxidant defence system.