Pomegranate Seeds and Peel Ethanolic Extracts Anticancer Potentials and Related Genetic, Histological, Immunohistochemical, Apoptotic and Oxidative Stress Profiles: In vitro Study

Abstract

Introduction: Owing to their great quantity of hydrolyzable anthocyanins and tannins, the peel and seeds of pomegranate are edible and possess potent anti-oxidant and anti-inflammatory characteristics. This work aims to trace the pomegranate seed and peel ethanolic extracts' anticancer activity against liver cancer cell line, namely HepG2 and related histopathological, immunohistochemical, genetic and oxidative stress profile.

Methods: In vitro study for both seed and peel extract showed the prevalence of phenols, polyphenols and acids, those have anti-proliferative potential against liver cancer cell line (HepG2) with 50% inhibitory concentration (IC50) of seed significantly reduced that of peel. Toxicity of test extracts was concentration dependent and accompanied with cell cycle arrest and cell death at theG0/G1 and S phases but not at the G2/M phase. Cell arrest was supplemented with raised ROS, MDA and decreased SOD, GSH and Catalase.

Results and discussion: Apoptosis-related genes showed significant up-expression of pro-apoptotic gene (P53), Cy-C, Bax, and casp-3 and down expression of anti-apoptotic gene (Bcl-2). Also, Casp-3 and P53 proteins were substantially expressed under the effect of test extracts. Histopathological study demonstrated that the untreated cells (control group) were regular cells with nuclear pleomorphism and hyperchromatic nuclei, while seed and peel extracts-treated cells showed necrosis, mixed euchromatin and heterochromatin, intra-nuclear eosinophilic structures, burst cell membranes, and the shrunken apoptotic cells with nuclear membranes and irregular cells. Finally, PCNA gene detected by immunohistochemistry was down regulated significantly under the effect of seed extract treatment than in case of cell medication with peel extract.

Keywords: HepG2 cells; apoptosis; cell viability; immunohistochemistry; oxidative stress; pomegranate.

Figure 1

Evaluation of HepG2 viability % post cell treatment with pomegranate seed and peel ethanolic extract relative to concentration.

Figure 2

Evaluation of Inhibitory concentration (IC50) of both pomegranate seed and peel ethanolic extract.

Figure 3

Apoptotic phases: evaluation of cell cycle profile and cell arrest phases and apoptosis profile post HepG2 cell treatment with the IC₅₀ of pomegranate seed and peel ethanolic extract.

Figure 4

Assessment of pro-apoptotic and anti-apoptotic genes post HepG2 cells therapy with the IC₅₀ values of pomegranate seed and peel extracts using rt-PCR.

Figure 5

(A) Photomicrograph demonstrated regular cells with nuclear pleomorphism and hyperchromatic nuclei. (B) A photomicrograph showed necrotic cells with combined euchromatin and heterochromatin (red arrows), Intranuclear eosinophilic structures (orange arrows), Ruptured cell membranes (green arrows), Shrunken apoptotic cells with nuclear membranes and irregular cell (black arrows) and (C) A photomicrograph demonstrating necrotic cells with combined euchromatin and heterochromatin (red arrows), Intranuclear eosinophilic structures (orange arrows), Ruptured cell membranes (green arrows), Shrunken apoptotic cells with nuclear membranes and irregular cell (black arrows) (H and E, Original magnification 100×, Oil).

Figure 6

Evaluation of apoptotic genes using Western blot assay post HepG2cells treatment with pomegranate seed and peel extracts.

Figure 7

Evaluation of PCNA protein immunohistochemical expression in HepG2 cell post treatment showing downregulation of the staining (from A to C).