Trigonella foenum (Fenugreek) Induced Apoptosis in Hepatocellular Carcinoma Cell Line, HepG2, Mediated by Upregulation of p53 and Proliferating Cell Nuclear Antigen

Abstract

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide and most current therapies are of limited efficacy. Trigonella foenum (Fenugreek) is a traditional herbal plant with antitumor activity, although the mechanisms of its activity remain unclear. Herein, a crude methanol extract was prepared from Fenugreek seeds (FCE) and its anticancer mechanism was evaluated, using HepG2 cell line. Growth-inhibitory effect and apoptosis induction of HepG2 cells were evidenced by MTT assay, cell morphology alteration, apoptosis enzyme-linked immunosorbent assay, flow cytometric analysis, caspase-3 activity, and expression of p53, proapoptotic protein, Bax, and proliferating cell nuclear antigen (PCNA) after (100 ∼ 500 μg/mL) FCE treatment for 48 h. Furthermore, FCE was analyzed by Chromatography-Mass Spectrometry (GC/MS). Our results revealed that FCE treatment for 48 h showed a cytotoxic effect and apoptosis induction in a dose-dependent manner that was mediated by upregulation of p53, Bax, PCNA, and caspase-3 activation in HepG2 cells. GC-MS analysis of FCE showed the presence of fourteen bioactive compounds such as Terpenoids and Flavonoids, including two main constituents with anticancer activity, Squalene and Naringenin (27.71% and 24.05%), respectively. Our data introduced FCE as a promising nontoxic herbal with therapeutic potential to induce apoptosis in HepG2 cells through p53, Bax, and PCNA upregulation in caspase-3 dependent manner.

Figure 1

Effect of Fenugreek seed crude extract (FCE) on cell viability of HepG2 cells. MTT assay was performed to detect the living cells as described under Section 2. Each data point is an average of results from three independent experiments performed in triplicate and presented as M ± SD.

Figure 2

Fenugreek seed crude extract (FCE) induced changes in HepG-2 cell line morphology. The results revealed that the control cells showed a typical polygonal and intact appearance, whereas the 100~500 μg/mL range of FCE-treated cells displayed morphological changes with preapoptotic characteristics. Representative data from three independent experiments are shown, using inverted phase contrast microscope at 200x magnification.

Figure 3

(a) Apoptosis enzyme-linked immunosorbent assay, ELISA. Apoptosis detection assay was applied to detect apoptosis induction in HepG2 cells after treatment with FCE. Cells were treated with (100~500 μg/mL range) FCE for 48 h. Cells without drug treatment were used as controls. ^∗ P < 0.05 as compared to the untreated controls, using the unpaired Student t-test. Each data point is an average of three independent experiments and is expressed as M ± SD. (b) Flow cytometric analysis of HepG2 cells after treatment with or without Fenugreek seed crude extract (FCE). Cell cycle analysis shows % of cell cycle phases of HepG2 cell line treated with or without 100~500 μg/mL range of FCE for 48 h. ^∗ P < 0.05 as compared to the untreated controls, using the unpaired Student t-test. Each data point was an average of results from three independent experiments performed in triplicate and presented as M ± SD.

Figure 4

Effect of Fenugreek seed crude extract (FCE) on caspase-3 activity (as indicated by (DEVD-pNA) cleavage) of HepG2 cells. Cells were treated with or without 100~500 μg/mL range of FCE for 48 h. Cells without drug treatment were used as controls. ^∗ P < 0.05 as compared to the untreated controls, using the unpaired Student t-test. Each data point is an average of three independent experiments and is expressed as M ± SD. More details are under Section 2.

Figure 5

(a) Fenugreek seed crude extract (FCE) upregulated p53 and PCNA expression levels in HepG-2 cells line, using western blot analysis. Cells were treated with or without (500 μg/mL) FCE for different time intervals. Our data showed a significant increase in p53 and PCNA protein expression, respectively. Blots were normalized for total protein loading. Representative data from three independent experiments are shown. (b) Apoptosis indicated by poly(ADP-ribose) polymerase (PARP) cleavage. Western blot analysis for poly(ADP-ribose) polymerase (upper panel) and Bax protein expression (lower panel). Cells were treated with or without 100–500 μg/mL range of Fenugreek seed crude extract, FCE, for 48 h. Blots were normalized for total protein loading.

Figure 6

GC-MS chromatogram of methanol extract of Trigonella foenum-graecum, Fenugreek, FCE.