HET was obtained from Tsumura Co. (Tokyo, Japan). HET is a mixture of spray-dried powder from hot water extracts obtained from the following ten medicinal plants: Ginseng radix (16.7%), Atractylodis rhizoma (16.7%), Astragali radix (16.7%), Angelicae radix (12.5%), Ziziphi fructus (8.3%), Bupleuri radix (8.3%), Glycyrrhizae radix (6.3%), Zingiberis rhizoma (2.0%), Cimicifuga rhizoma (4.2%) and Aurantii nobilis pericarpium (8.3%). HET powder was dissolved at 10 mg/ml in culture medium at 37°C for 10 min and thoroughly mixed. The solution was passed through a 0.22-µm filter to sterilize and remove any insoluble components. Cisplatin was purchased from Sigma-Aldrich (St. Louis, MO, USA).

The human cervical cancer cell line HeLa, obtained as previously described (16) was used in the present study. The cells were cultured in Eagle's Minimum Essential Medium (EMEM; Nissui, Tokyo, Japan) supplemented with 10% calf serum (Gibco-BRL, Invitrogen Life Technologies, Carlsbad, CA, USA). Cells were grown at 37°C in a humidified atmosphere containing 5% CO₂.

The colony survival assay was used to determine the dose of HET that did not affect cell viability (17). Briefly, HeLa cells were plated in 100-mm dishes (1×10³ cells/dish) in medium containing HET at various concentrations (0, 5, 25, 50, 250 and 500 µg/ml). After culturing for 14 days, the colonies were stained with 0.2% methylene blue (Wako Pure Chemical Industries, Ltd., Osaka, Japan) in 30% methanol (Wako Pure Chemical Industries, Ltd.), and visible colonies with a diameter greater than 3 mm (containing greater than 50 cells) were counted by eye.

The sensitivity of cells to cisplatin was measured by colony survival and crystal violet assays (18). For the colony survival assay, cells were plated in 100-mm dishes (1×10³ cells/dish) in medium with or without 50 µg/ml HET. After culturing for 24 h, the medium was changed to serum-free medium containing the indicated concentrations of cisplatin (0, 0.31, 0.63, 1.3, 2.5 or 5.0 µM). After incubation in the cisplatin-containing medium for 2 h, the HET-pre-treated cells (cells treated with cisplatin following HET pre-treatment) or HET-untreated cells (cells treated with cisplatin without HET pre-treatment) were cultured in fresh medium for 14 days followed by colony counting.

For the crystal violet assay, cells were plated in 60-mm dishes (5×10⁵ cells/dish) in medium with or without 50 µg/ml HET and incubated for 24 h. Cells were harvested, plated onto 96-well plates (4×10³ cells/well) containing cisplatin at the indicated concentrations (0, 0.39, 0.78, 1.6 or 3.1 µM), and cultured for 2 days. After culture, viable cells were stained with crystal violet (Wako Pure Chemical Industries, Ltd.) followed by measurement of absorbance at 595 nm. Cell viability was calculated from the absorbance using an Emax Precision Microplate reader (Molecular Devices, LLC, Sunnyvale, CA, USA) and expressed as the percentage of surviving cells with regard to the group treated without cisplatin.

Cells that were pre-treated with and without HET (50 µg/ml) for 24 h were then treated with cisplatin (0, 15 and 30 µM) at the indicated concentrations for 24 h. The HET-pre-treated or -untreated cells were harvested and the cell population in sub-G₁ phase (apoptotic fraction) was analyzed with Guava Cell Cycle Reagent (EMD Millipore, Billerica, CA, USA) and an Accuri C6 cytometer (Tomy Digital Biology, Encyclopedia Circle, Fremont, CA, USA). The data were analyzed with FlowJo software, version 7.6 (FlowJo, LLC, Ashland, OT, Canada) as described previously (19).

Apoptosis-associated molecules were analyzed by immunoblotting (19). Cells were cultured in medium in the presence or absence of 50 µg/ml HET for 24 h and then treated with cisplatin at indicated concentrations for 24 h. Next, whole-cell lysates in an SDS sampling buffer (62.5 mM Tris-Cl, pH 6.8, 2.3% SDS, 10% glycerol, 5% 2-mercaptoethanol, 0.01% bromophenol blue) were prepared from the HET-pre-treated or -untreated cells. Briefly, cells were harvested with phosphate-buffered saline, cell number was counted, then whole cells were dissolved directly with the SDS sampling buffer. The whole-cell lysates from approximately 2×10⁴ cells were applied to each lane of SDS-PAGE. Electrophoresis and blotting were performed with constant 15 mA and 250 mA current using a Tris-glycine buffer system, for 2 h and 3 h, respectively. All reagents for the western blotting were obtained from Wako Pure Chemical Industries, Ltd. The active form of caspase-3 protein was detected using rabbit anti-cleaved caspase-3 antibody (5A1E; Cell Signaling Technology, Inc., Beverly, MA, USA; 1:500 dilution). Bcl-2, Bax, p-Akt and p53 proteins were detected using mouse anti-Bcl-2 monoclonal antibody (sc-7832; Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1:1,000 dilution), mouse anti-Bax monoclonal antibody (sc-7480; Santa Cruz Biotechnology; 1:1,000 dilution), rabbit anti-p-Akt antibody (193H12; Cell Signaling Technology, Inc.; 1:1,000 dilution), and mouse anti-p53 monoclonal antibody (sc-126; Santa Cruz Biotechnology; 1:1,000 dilution), respectively. The secondary antibodies used were as follows: Horseradish peroxidase (HRP)-linked anti-rabbit antibody (NA934V; GE Healthcare Life Sciences, Chalfont, UK; 1:4,000 dilution) and HRP-linked anti-mouse antibody (NA931V; GE Healthcare Life Sciences; 1:4,000 dilution). The protein signals were visualized by the Enhanced Chemiluminescence system reaction (GE Healthcare Life Sciences). Protein levels of actin were also analyzed using mouse anti-actin antibody (C4; ICN Biomedicals, Costa Mesa, CA, USA; 1:10,000 dilution) as a loading control. The intensities of the protein signals were quantified using Multi Gauge 2.2 image analysis software (Fuji Foto Film, Tokyo, Japan) and expressed as a relative value to that of actin. Immunoblotting analysis was performed three times using cell samples prepared twice independently.

P-values were calculated to compare the HET-pre-treated cells and the HET-untreated cells by Student's t-test with Microsoft Excel 2010 software (Microsoft Corporation, Redmond, WA, USA). Values are expressed as the mean ± standard deviation. P<0.05 was considered to indicate a statistically significant difference between values.

The present study first examined the effect of HET alone on HeLa cell viability. No growth suppression by HET was observed at concentrations of up to 500 µg/ml (Fig. 1).

The present study therefore adopted 50 µg/ml HET to exclude any cytotoxic effects. Next, the sensitivity of HeLa cells to cisplatin after pre-treatment in medium containing 50 µg/ml of HET for 24 h was assessed. The colony survival assay indicated that the HET-pre-treated cells had a significantly higher sensitivity to cisplatin-induced cell death than the HET-untreated cells (Fig. 2A).

In addition, the crystal violet assay showed that HET-pre-treated HeLa cells were more sensitive to the inhibitory effect of cisplatin on cell viability than HET-untreated HeLa cells (Fig. 2B). These findings suggested that the anti-cancer effect of cisplatin was enhanced by pre-treatment with HET.

A previous study by our group reported that cisplatin induced apoptosis in HeLa cells and other cancer cell lines (20). Therefore, the present study examined whether the cell death induced by cisplatin and HET is induced via apoptotic pathways. Flow cytometric analysis indicated a distinct increase in the population of cells in the sub-G₁ phase (apoptotic fraction) after cisplatin treatment in the cells pre-treated with HET compared with that in the HET-untreated cells (Fig. 3). While the sub-G₁ population in the HET-untreated HeLa cells was 7.8±0.71, 10.3±3.0 and 72.2±6.7% after treatment with 0, 15 and 30 µM cisplatin, respectively, the sub-G₁ fractions were increased to 9.8±3.6, 36.8±6.4 (P<0.005) and 88.6±4.3% (P<0.05), respectively, in the HET-pre-cultured HeLa cells.

Caspase-3 is one of the effector caspases, which is cleaved and activated by initiator caspases, and executes apoptosis when activated (21). The levels of active caspase-3 increased by ~1.7-fold after incubation of HET-pre-treated HeLa cells with 20 µM cisplatin, compared with those in the HET-untreated cells (Fig. 4). These results supported that HET-pre-treatment enhanced cisplatin-induced apoptosis, which proceeded via a caspase-dependent pathway.

Bcl-2 is an anti-apoptotic protein, and Bax is a pro-apoptotic protein (22). The relative expression ratios of anti-apoptotic proteins to pro-apoptotic proteins have been reported to correlate with cellular sensitivity to the lethal effects of anti-cancer drugs (23). The Bcl-2-to-Bax ratio at 0 and 20 µM cisplatin in the HET-pre-cultured HeLa cells was decreased by 23 and 68%, respectively, compared with that in the HET-untreated cells (Fig. 5).

Next, the present study determined the cellular levels of p-Akt and p53 as candidates for upstream molecules to regulate the enhancement of the cisplatin-induced apoptosis by HET. Akt is negatively regulated by p53 (24) when apoptosis occurs. Fig. 6 shows the effect of HET on the protein levels of p-Akt and p53 in HeLa cells incubated with various concentrations of cisplatin. In the HET-untreated cells as well as in HET-treated cells, p-Akt expression was decreased at cisplatin concentra tions of >20 µM, compared with that in control cells without cisplatin treatment. In addition, at almost all of the tested concentrations of cisplatin (0, 5, 10 and 20 µM), the p-Akt levels in the HET-pre-treated cells were lower than those in the HET-untreated cells. The difference between the two groups was significant at cisplatin concentrations other than 10 and 30 µM (Fig. 6A and B). Conversely, the protein levels of p53 were increased at cisplatin concentrations of >10 µM compared with those in the cisplatin-untreated groups. Of note, p53 levels were markedly elevated in the HET-pre-treated cells as compared with those in the HET-untreated cells (Fig. 6A and C).