Pro-apoptotic and cytotoxic effects of enriched fraction of Elytranthe parasitica (L.) Danser against HepG2 Hepatocellular carcinoma

Abstract

Background: Hepatocellular carcinoma (HCC), the most common type of liver cancer accounts for more than one million deaths worldwide. Current treatment modality for HCC is marginally effective. Plants belonging to Mistletoe family (Loranthaceae) have been used in chemotherapy for many years. The present study was aimed at exploring the anti-proliferative, pro-oxidant and pro-apoptotic potential of stem of Elytranthe parasitica (L.) Danser (EP), a parasitic shrub belonging to Loranthaceae.

Methods: Elytranthe parasitica (L.) Danser, a climbing parasitic shrub was investigated for its cytotoxic activity against HepG2, a hepatocellular carcinoma cell line by Sulforhodamine B (SRB) assay. Further, pro-oxidant activity of EP extract/fractions was studied using copper phenanthroline assay. To understand the mechanism of cell death, the pro-apoptotic effects of Hep-G2 cells treated with EP extract/fractions were visualized by dual staining using acridine orange and ethidium bromide, a morphological marker of apoptosis. Phytochemical profiling of EP was explored by estimating the phenol, flavonoid and tannin content in its various fractions and extract. The occurrence of gallic acid, a principal polyphenol in EP extract and fractions was detected and further quantified using HPTLC (High Performance Thin Layer Chromatography) fingerprinting.

Result: Active fraction of Elytranthe parasitica, EP.DEE exhibited potent cytotoxic activity in a dose dependent manner against HepG2 hepatocellular carcinoma cell line with an IC50 of 56.7 ± 7.8 μg/mL. Dual staining with acridine orange and ethidium bromide revealed that HepG2 cells treated with EP active fractions underwent cell death chiefly by apoptosis. Highest phenol, flavonoid and tannin content were observed in active fractions, EP.EA (Ethyl acetate fraction) and EP.DEE (Diethyl ether fraction). Gallic acid was identified and quantified in EP extract and active fractions, EP.DEE and EP.EA.

Conclusion: Our findings indicate EP active fraction could be a promising contender in the treatment of hepatocellular carcinoma.

Keywords: Anti-proliferative; Apoptosis; Elytranthe parasitica; Gallic acid; HepG2; Hepatocellular carcinoma; Phytochemical.

Fig. 1

Effect of EP extract/fractions on cell viability and growth of (a) human HepG2 hepatocellular carcinoma cells and (b) Vero cell line. HepG2 and Vero cells were seeded onto 96 well plate at a density of 5,000 cells/well and subsequently treated with EP extract/fractions for 48 h. Data is expressed as IC50 (mean ± SD) of three individual experiments. Cell viability was estimated by Sulforhodamine B assay. EP. DEE- Diethyl ether fraction, EP.EA- Ethyl Acetate fraction, EP.M- Methanol extract

Fig. 2

% Inhibitory effect of EP extract/fractions in hydroxyl radical mediated DNA degradation assay. The test compounds were screened at concentrations of 10, 100, 500 and 1000 μg/mL (from left to right). Ascorbic acid was employed as standard at the same concentrations. Results are expressed as % inhibitory capacity (mean ± SD) of three individual experiments. Statistical Analysis was performed by 2 way ANOVA followed by Bonferroni’s post hoc test of significance. Columns with similar superscripts did not differ significantly (P > 0.05). EP. M- Methanol extract, EP.PE -Petroleum ether fraction, EP. DEE- Diethyl ether fraction, EP.EA- Ethyl Acetate fraction, EP. But - Butanol fraction, EP.Aq- Aqueous fraction

Fig. 3

Visualization of apoptosis in HepG2 hepatocellular carcinoma cells after treatment (AO/EB dual staining) HepG2 cells were seeded onto 6 well plates at a density of 50,000 cells/well, treated with test compounds and stained with AO/EB dye after 48 h of treatment. The treatment groups are as follows: (a) Control or untreated HepG2 cells (b) Curcumin reference group (4 μg/mL) (c) EP. DEE (15 μg/mL) (d) EP. DEE (30 μg/mL). Arrows in the figure are directed towards the apoptotic bodies, that exhibited bright yellowish-green fluorescence. Condensed chromatin was observed in all treated groups. Control group cells showed normal morphological features with undamaged cell membrane. e Graph depicting number of apoptotic cells in different treatment groups. *P value > 0.05 when compared to control group. Columns with different superscripts differ significantly (P > 0.05). EP. DEE- Diethyl ether fraction

Fig. 4

HPTLC fingerprint profile of EP extract and fractions (a) EP.M, Methanol extract (b) EP.PE, Petroleum ether fraction (c) EP. DEE, Diethyl ether fraction (d) EP.EA, Ethyl acetate fraction (e) EP. But, Butanol fraction (f) EP.Aq, Water fraction fraction (g) Gallic acid (reference standard)

Fig. 5

Overlay of UV absorption spectra of reference gallic acid and corresponding band in EP extract/fractions [Absorbance, AU vs. Wavelength, nm] (a) Gallic acid and EP.M extract (b) Gallic acid and EP. DEE (c) Gallic acid and EP.EA