Kaempferia parviflora Extract Exhibits Anti-cancer Activity against HeLa Cervical Cancer Cells

Abstract

Kaempferia parviflora (KP) has been traditionally used as a folk remedy to treat several diseases including cancer, and several studies have reported cytotoxic activities of extracts of KP against a number of different cancer cell lines. However, many aspects of the molecular mechanism of action of KP remain unclear. In particular, the ability of KP to regulate cancer cell growth and survival signaling is still largely unexplored. The current study aimed to investigate the effects of KP on cell viability, cell migration, cell invasion, cell apoptosis, and on signaling pathways related to growth and survival of cervical cancer cells, HeLa. We discovered that KP reduced HeLa cell viability in a concentration-dependent manner. The potent cytotoxicity of KP against HeLa cells was associated with a dose-dependent induction of apoptotic cell death as determined by flow cytometry and observation of nuclear fragmentation. Moreover, KP-induced cell apoptosis was likely to be mediated through the intrinsic apoptosis pathway since caspase 9 and caspase 7, but not BID, were shown to be activated after KP exposure. Based on the observation that KP induced apoptosis in HeLa cell, we further investigated the effects of KP at non-cytotoxic concentrations on suppressing signal transduction pathways relevant to cell growth and survival. We found that KP suppressed the MAPK and PI3K/AKT signaling pathways in cells activated with EGF, as observed by a significant decrease in phosphorylation of ERK1/2, Elk1, PI3K, and AKT. The data suggest that KP interferes with the growth and survival of HeLa cells. Consistent with the inhibitory effect on EGF-stimulated signaling, KP potently suppressed the migration of HeLa cells. Concomitantly, KP was demonstrated to markedly inhibit HeLa cell invasion. The ability of KP in suppressing the migration and invasion of HeLa cells was associated with the suppression of matrix metalloproteinase-2 production. These data strongly suggest that KP may slow tumor progression and metastasis in patients with cervical cancer. Taken together, the present report provides accumulated evidence revealing the potent anti-cancer activities of Kaempferia parviflora against cervical cancer HeLa cells, and suggests its potential use as an alternative way for cervical cancer prevention and therapy.

Keywords: Kaempferia parviflora; MAPK pathway; PI3K/AKT pathway; anti-cancer; apoptosis; cervical cancer; invasion; migration.

FIGURE 1

The effects of KP on HeLa cell viability. The bars indicate percent cell viability of HeLa cells treated with different concentrations of KP extract (0–1 mg/mL) for 24 h with cell viability measured by the MTT assay. Data represent mean ± SD of three independent experiments. ^∗p < 0.05.

FIGURE 2

Morphological changes and nuclear fragmentation of HeLa cells exposed to KP extract. (A) Phase-contrast images of HeLa cells treated with 0.5 mg/mL of KP extract taken at different time points (0, 3, 6, and 24 h). (B) The nuclei of HeLa cells treated with KP extract at different concentrations (0.1–0.5 mg/mL) or with DMSO (0.01–0.05%), stained with Hoechst33342, and visualized using a fluorescent microscope. Arrows indicate cells with nuclear fragmentation or nuclear deformity, and magnified views of cells indicated with arrows are shown at the bottom right corners. Data are representative of three replicates.

FIGURE 3

The effects of KP on inducing HeLa cell apoptosis. (A) Representative figures from flow cytometry showing HeLa cells undergoing apoptotic cell death upon incubation with different concentrations (0–0.5 mg/mL) of KP extract for 6 h. (B) Quantitative analysis of percentage cell apoptosis from flow cytometry. Data are representative of three replicates and are expressed as mean ± SD. (C) A representative western blot of caspase 9, caspase 7, and BID from HeLa cells treated with different concentrations (0–0.5 mg/mL) of KP extract for 6 h. Two immunoreactive bands of cleaved-caspase 9 indicate a p35 subunit and a p37 subunit of active caspase 9. Beta-actin was used as a loading control.

FIGURE 4

The effects of KP on HeLa cell migration and invasion. (A) Scratch wounds of monolayers HeLa cells treated with KP extract (0.01, 0.05, and 0.1 mg/mL) for 40 h. Cell migration was monitored with 4x magnification, and phase-contrast images of cell migration were taken at the time of the scratch and at 24 and 40 h post-scratch. (B) Quantitative analysis of cell migration into the scratch wound at 40 h post-scratch. Data are expressed as mean ± SD. Asterisks indicate significantly different from the control groups (untreated groups) (^∗p < 0.05). (C) Representative images of HeLa cell invasion treated with KP extract (0.01, 0.05, and 0.1 mg/mL) and examined by the Transwell invasion assay. Vehicle is the control group where cells were treated with the highest concentration of DMSO (0.01%) which corresponded to the concentration present in 0.1 mg/mL of KP. (D) Quantitative analysis of percent of cell invasion in KP-treated cells compared to the vehicle control. Data are representative of three replicates and are expressed as mean ± SD. ^∗p < 0.05 compared with the vehicle control.

FIGURE 5

Effects of KP extract on suppressing MMP-2 activity. (A) Zymographic analysis for MMP-2 activity in HeLa cells treated with KP extract at various concentrations (0–0.1 mg/mL) with or without the presence of 100 ng/mL of EGF. Western blot for beta actin was used as a loading control. (B) Quantitative analysis of MMP-2 level using ImageJ software. Beta actin from the western blot was used as an internal control for normalization. Data are expressed as mean ± SD. ^∗p < 0.05 compared with untreated cells.

FIGURE 6

The effects of KP extract on suppressing growth and survival signal transduction pathways. (A) Immunofluorescence of pERK1/2 in HeLa cells treated with KP extract. (B) Western blot showing immunoreactive bands of pPI3K, pAKT, pERK1/2, pElk1, and β-actin of HeLa cells stimulated with 100 ng/mL EGF and treated with different concentrations of KP extract. (C) Quantitative analysis of phosphorylation status of PI3K, AKT, ERK1/2, and Elk1 of HeLa cells treated with 100 ng/mL EGF and different concentrations of KP extract. Beta actin was used as an internal control and for normalization. Data are representative of three independent replicates and are expressed as mean ± SD. ^∗p < 0.05 compared with untreated cells.