Apoptotic and inhibitory effects on cell proliferation of hepatocellular carcinoma HepG2 cells by methanol leaf extract of Costus speciosus

Abstract

Costus speciosus is a medicinal plant commonly known as wild ginger distributed in South and Southeast Asian countries. Leaves of this plant are used for ayurvedic treatment regimes in malignancies and mental illness. Rhizome extract from the plant is used to treat malignancies, pneumonia, urinary disorders, jaundice, rheumatism, and diabetes. The goal of this study was to investigate the effects of methanol extract of leaves of C. speciosus on the growth of human hepatocellular carcinoma (HepG2) cells and understand possible mechanisms of its action. Viability of HepG2 cells were measured by MTS assay after 24 h and 48 h treatment with extracts of 1, 10, 50, 100, and 200 μg/mL concentrations. Cell cycle analysis and apoptosis were evaluated by flow cytometry and caspase-3 induction. HepG2 cells treated with 100 μg/mL methanol leaf extract for 24 h displayed a significant reduction in cell viability (P ≤ 0.05). The methanol extract perturbed cell cycle progression, modulated cell cycle and regulated, signal molecules were involved in induction of apoptosis in HepG2 cells. Our findings indicate that phytochemicals of leaves of C. speciosus shows potential for natural therapeutic product development for hepatocellular carcinoma. This is the first report to demonstrate in vitro anticancer activity of leaf extract of C. speciosus in relation to liver cancer.

Figure 1

Inverted phase contrast microscopy showing methanolic extract of Costus speciosus leaves or sorafenib (positive control) induced changes of HepG2 cells. The control cells were well adhered, displaying the normal morphology of HepG2 cells. In contrast, majority of HepG2 cells treated with methanol extract at the concentration of 100 μg/mL or more became round and shrunken and could not be affixed to the walls and floating in the medium (×400 magnification). (a) 50 μg/mL methanol extract; (b) 100 μg/mL methanol extract; (c) 200 μg/mL methanol extract; (d) 50 μg/mL sorafenib; (e) 100 μg/mL sorafenib; (f) 200 μg/mL sorafenib; (g) control.

Figure 2

FACS analyses of Annexin V and 7-AAD staining. HepG2 cells were treated with methanolic extract of Costus speciosus leaves or sorafenib (100 μg/mL) for 24 h. Lower right quadrant (D4) Annexin V positive/7-AAD negative cells denote early apoptosis cells and upper right quadrant (D2) Annexin V positive/7-AAD positive cells denote necrosis or late apoptotic cells. Images are the representative of three independent experiments.

Figure 3

Effects of methanolic extract of Costus speciosus leaves in HepG2 cells. Caspase-3 activity significantly increased on treatment with methanol extract or sorafenib at a concentration of 100 μg/mL for 24 h compared to control. Data are expressed as mean ± SD (n = 3). Columns not sharing the same superscript letter differ significantly (P < 0.05).

Figure 4

Effect of methanolic extract of Costus speciosus leaves on mitochondrial membrane potential (ΔΨm). (a) Mitochondrial membrane potential (ΔΨm) was measured by JC-1 fluorescence [fluorescence of JC-1 monomers (em 535 nm)/aggregates (em 590 nm)] in HepG2 cells treated with 100 μg/mL of methanol extract or sorafenib for 24 h. Methanol extract or sorafenib showed significant decrease in ΔΨm in HepG2 cells. Data are expressed as mean ± SD (n = 3). Columns not sharing the same superscript letter differ significantly (P < 0.05%). (b) Untreated control cells showed that cells have higher mitochondrial ΔΨm as they are abundant in J-aggregates which is indicated by intense red fluorescence; methanol extract or sorafenib treated cells showed lower mitochondrial ΔΨm which is represented by monomeric JC-1 with green fluorescence.

Figure 5

Effect of methanolic extract of Costus speciosus leaves on cell cycle distribution. Methanolic extract or sorafenib treatment significantly increased the proportion of S phase while significant decrease of G1/G0 phase. (a) Cell cycle analysis by flow cytometry. After exposure of HepG2 cells to 100 μg/mL of the methanol extract or sorafenib for 24 h, cells were harvested, stained with propidium iodide, and analyzed by flow cytometry. Flow cytometric histograms are representative of two separate experiments. (b) Percentage of cell populations. The data are from one of two independent experiments with similar results. Data are expressed as mean ± SD (n = 3). Columns not sharing the same superscript letter differ significantly (P < 0.05%).