Saussurea lappa Exhibits Anti-Oncogenic Effect in Hepatocellular Carcinoma, HepG2 Cancer Cell Line by Bcl-2 Mediated Apoptotic Pathway and Mitochondrial Cytochrome C Release

Abstract

Background and Objectives: Saussurea lappa (S. lappa) is an important species of the Asteraceae family with several purposes in traditional medicine. This study intended to explore the cytotoxic effect of S. lappa on HepG2 cancer cell proliferation. Materials and Methods: The effects of an S. lappa n-butanol extract on the induction of apoptosis were investigated by flow cytometry and mitochondrial cytochrome C-releasing apoptosis assay. Additionally, real-time PCR was employed to confirm apoptosis initiation. Further, qualitative estimation of the active constituent of S. lappa was done by gas chromatography-mass spectroscopy (GC-MS). Results: The cell viability study revealed that the n-butanol extract of S. lappa demonstrated potent cytotoxicity against HepG2 cancer cells, with an IC50 value of 56.76 μg/mL. Cell morphology with dual staining of acridine orange (AO)-ethidium bromide (EB) showed an increase in orange/red nuclei due to cell death by S. lappa n-butanol extract compared to control cells. Apoptosis, as the mode of cell death, was also confirmed by the higher release of cytochrome C from mitochondria, the increased expression of caspase-3 and bax, along with down regulation of Bcl-2. Conclusion: These findings conclude that S. lappa is a cause of hepatic cancer cell death through apoptosis and a potential natural source suggesting furthermore investigation of its active compounds that are responsible for these observed activities.

Keywords: Bcl-2; HepG2; Saussurea lappa; anticancer; apoptosis; cytochrome C release.

Figure 1

Images of (A) Saussurea lappa root and (B) coarse powder.

Figure 2

Major constituents found in n-butanol S. lappa root extract.

Figure 2

Major constituents found in n-butanol S. lappa root extract.

Figure 3

Chromatogram of n-butanol root extract of Saussurea lappa by GC–MS.

Figure 4

(A) Free radical scavenging activity of the n-butanol extracts from roots of S. lappa and positive controls measured in DPPH assay. (B) Absorbance of FRAC assay of root extract at different concentrations.

Figure 4

(A) Free radical scavenging activity of the n-butanol extracts from roots of S. lappa and positive controls measured in DPPH assay. (B) Absorbance of FRAC assay of root extract at different concentrations.

Figure 5

In vitro anticancer activity of root extract of Saussurea lappa ((A)—untreated, (B)—positive control, (C)—12.5 µg/mL, (D)—25 µg/mL, (E)—50 µg/mL, (F)—100 µg/mL, (G)—200 µg/mL). (the images were captured at 10× magnification by inverted biological microscope).

Figure 6

Graph showing the percentage cell viability of root extract against HepG2 cell line after 24 h.

Figure 7

S. lappa n-butanol root extract-induced apoptosis. Dual staining study of HepG2 cells by acridine orange (AO) and ethidium bromide (EB) (AO represents viable cells and EB represents dead cells). Untreated and treated with n-butanol extract (56.76 µg/mL) representing the changes in nuclear morphology of cells (the images were captured at 40× magnification).

Figure 8

Histograms representing cytochrome C expression study with (A)—control, (B)—positive control-treated, and (C)—S. lappa n-butanol root extract treated with IC50 concentration, viz., 56.76 ug/mL against the HepG-2 cells using BD FACS Calibur, Cell Quest Pro Software (Version: 6.0).

Figure 9

Relative mRNA expression of caspase-3, Bcl-2, Bax, and GAPDH in Untreated, S. lappa extract, and positive control-treated HepG2 cells by RTqPCR. The significant differences from control are indicated by * p < 0.05.