Sage Journals: Discover world-class research

Abstract

Gypenosides (Gyp), found in Gynostemma pentaphyllum Makino, has attracted more attention owing to its wide bioactivities. However, the effects of Gyp on esophageal cancer cells and colon cancer cells are still unknown. The present study was to investigate the possible anti-proliferative and anti-migration activity of Gyp on human colon cancer cells SW620 and esophageal cancer cells Eca-109. Cell viability was evaluated using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell membrane integrity was evaluated using flow cytometry following propidium iodide staining. Apoptotic cell death was determined by nuclear 4′-6-diamidino-2-phenylindole staining. Generation of intracellular reactive oxygen species (ROS) and changes in mitochondrial membrane potential (Δψ _m) was analyzed by flow cytometry using 2′,7′-dichlorofluorescein–diacetate and rhodamine 123 staining, respectively. Wound healing assay was carried out to investigate Gyp-inhibited migration of SW620 and Eca-109 cells. The results indicated that Gyp inhibited cell proliferation and migration in SW620 and Eca-109 cells in dose- and time-dependent manner. Gyp elevated intracellular ROS level, decreased the Δψ _m, and induced apoptotic morphology such as cell shrinkage and chromatin condensation, suggesting oxidative stress and mitochondria-dependent cell apoptosis that might be involved in Gyp-induced cell viability loss in SW620 and Eca-109 cells. The findings indicate Gyp may have valuable application in clinical colon cancer and esophageal cancer treatments.

Keywords

Gypenosides SW620 cells Eca-109 cells antitumor anti-migration

Introduction

Colon cancer is one of the most common malignancies diagnosed all over the world. In recent years, the morbidity and mortality of colon cancer in Asian countries tend to increase.¹ This disease is ranked as the fifth frequent malignancy following pulmonary, hepatic, gastric, and esophageal cancers.² It has been listed in one of the eight important cancers needed to be intensively treated and prevented in China from 2004 to 2010.³ Esophageal carcinoma (EC) is prevalent in some regions of the world and occurs at a very high frequency in certain parts of China, and the mortality rate is ranked the fourth among cancer-related death.^4,5

The traditional cancer therapies, such as surgery, chemotherapy, and radiotherapy, show some limitations because of serious side effects and poor prognosis. Now, natural medicine in cancer therapy has aroused wide concern at home and abroad, because of its safety, efficiency, and minimal side effects.

A number of compounds extracted from plants and their derivatives have been demonstrated to show antitumor activity, such as paclitaxel, which is derived from Taxus brevifolia. ^6,7 “Jiao-Gu-Lan” in Chinese, Gynostemma pentaphyllum Makino, is a well-known herb tea in Asia. The gypenosides (Gyps) are the major components purified from G. pentaphyllum Makino, a popular folk medicine in China. It is a saponin-rich plant and contains about 90 dammarane-type glycosides, which are closely related to the component saponins in ginseng, and hence, cheap G. pentaphyllum is regarded as “second ginseng.”^8

–14 In recent years, G. pentaphyllum has attracted more attention owing to its wide bioactivities for the anti-inflammatory, antioxidative,¹⁵ and antithrombotic¹⁶ effects and treatments of hepatitis, chronic bronchitis, hypertension, gastritis, cancer, and some other diseases.^8,14 Gyp have antifibrotic effects in rats,¹⁷ and it has antiproliferative effects in rat hepatic stellate cells.¹⁸ Gyp induced apoptosis in human hepatoma cells,¹⁹ and human tongue cancer SCC4 cells through endoplasmic reticulum stress and mitochondria-dependent pathways.²⁰ For this reason, drinking herb tea of G. pentaphyllum could promote health and alleviate the severity of many disorders.

In order to investigate whether Gyp could induce some antitumor efficacy on colon cancers and esophageal carcinoma, in the present study, we selected human colon cancer SW620 cells and esophageal cancer Eca-109 cells for examining cell apoptosis induction and migration inhibition after Gyp treatment. We specially focused on cell viability loss, membrane integrity damage, cellular apoptotic response, reactive oxygen species (ROS) production, mitochondrial membrane potential (Δψ _m) loss, and migration inhibition of Eca-109 cells and SW620 cells treated by Gyp. The obtained findings may provide valuable data for Gyp clinical application.

Materials and methods

Chemicals and reagents

Gyp was obtained from Ankang Pharmaceutical Institute of the Beijing University, People’s Republic of China. The Gyp powder was dissolved in 80% ethanol (EtOH) to get a stock solution of 100 mg/ml, which was passed through 0.22 μm filter for use in subsequent experiments. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), rhodamine 123 (Rh123), 4′-6-diamidino-2-phenylindole (DAPI), and propidium iodide (PI) were purchased from the Sigma Chemical Company (St Louis, Missouri, USA). 2′,7′-Dichlorodihydrofluorescein–diacetate (DCFH-DA) was supplied by Molecular Probes Inc. (Invitrogen, Carlsbad, California, USA).

Cell culture

The human colon cancer SW620 cells were obtained from the cell bank of the Chinese Academy of Science (Shanghai, China.) The cell line was cultured in L-15 medium containing 10% fetal bovine serum (FBS), 1% penicillin–streptomycin (100 U/ml penicillin and 100 μg/ml streptomycin), and 1% glutamine in 100 cm² tissue culture flasks under a humidified 5% carbon dioxide (CO₂) and 95% air atmosphere at 37°C. The human esophageal cancer Eca-109 cells, obtained from the cell bank of the Chinese Academy of Science, were cultured in RPMI-1640 medium containing 10% FBS, 1% penicillin–streptomycin (100 U/ml penicillin and 100 μg/ml streptomycin), and 1% glutamine in 100 cm² tissue culture flasks under a humidified 5% CO₂ and 95% air atmosphere at 37°C.

Isolation of normal mouse PBMC

The peripheral blood mononuclear cells (PBMCs) isolated from healthy mouse (Kunming, female, weighing 18–22 g) were used as normal cells compared with SW620 and Eca-109 tumor cells. PBMCs were isolated with the lymphocyte separation kit (Applygen Technologies Inc., Beijing, China) and the procedure was performed according to the instructions provided by the company. The viability of the obtained PBMCs was assessed using trypan blue exclusion test and the viability was above 95% in the experiment.

Assessment of cell viability after Gyp treatment

To investigate the effect of Gyp on SW620 and Eca-109 cell proliferation, cells were seeded in 96-well plates. Various concentrations (0, 50, 100, 150, and 200 μg/ml; 80% EtOH was used as the solvent control) of Gyp were added, and the cells were incubated for various periods of time, at a density of 1 × 10⁵ and 5 × 10⁴ cells/ml, respectively. The cell viability was determined using MTT assay.²¹ The absorbance at 570 nm was recorded using a microplate reader (ELX800, Bio-Tek Laboratories, Seattle, Washington, USA). The cell viability of Gyp-treated samples was then obtained by comparing with the control.

By comparison, PBMCs were used as normal cells to evaluate the cytotoxicity of Gyp. PBMCs (100 μl) were seeded at a density of 1 × 10⁵ cells/ml in 96-well culture plates and incubated with Gyp at doses of 0, 50, 100, 150, and 200 μg/ml for 24 h (80% EtOH was used as the solvent control). Cells were then harvested and stained with 0.4% trypan blue exclusion test. The stained and unstained cells were counted under an optical microscope using a hemocytometer. The cell survival rate was determined using the following equation

c e l l s u r v i v a l r a t e (%) = \frac{n u m b e r o f u n s t a i n e d c e l l s}{n u m b e r o f c e l l s i n t h e c o n t r o l g r o u p} \times 100 .

Each experiment was repeated three times to ensure reliable results.

Flow cytometry analysis for cell membrane integrity

To monitor membrane permeability in parallel, PI was added. PI is a fluorescent membrane-impermeant dye that stains the nuclei by intercalating between the stacked bases of nucleic acid. Since PI enters the cell only if the cell membrane becomes permeable, it is widely used in cell death research to measure the integrity of the plasma membrane. When bound to nucleic acids, the absorption maximum for PI is 535 nm and the fluorescence emission maximum is 617 nm.²² Cells in 12-well plates were treated with the indicated concentration of Gyp for 6, 12, 24, and 48 h. The cells were harvested and washed two times with phosphate-buffered saline (PBS), stained with 5 μg/ml PI for 5 min in the dark, and analyzed by flow cytometry (Guava easyCyte 8HT, Millipore, Billerica, Massachusetts, USA). Histograms were analyzed using Flow Cytometry Analysis (FCS) Express V3.

DAPI staining

In order to detect changes in nuclei morphology of tumor cells after Gyp treatment, DAPI staining was performed. After treatment with the indicated concentration of Gyp for 24 and 48 h, cells were stained with 4 μg/ml DAPI for 30 min at room temperature. Then, the stained cells were washed three times with PBS and observed using a fluorescence microscopy with standard excitation filters (Nikon, Japan). Excitation wavelength was 364 nm and emission wavelength was 454 nm.

Detection of intracellular ROS generation

In order to determine changes in the ROS level, we measured the oxidative conversion of the sensitive fluorescent probe DCFH-DA to fluorescent 2′,7′-dichlorofluorescein (DCF). DCFH-DA readily diffuses through the cell membrane and is enzymatically hydrolyzed by intracellular esterases to form nonfluorescent DCFH, which is then rapidly oxidized to form highly fluorescent DCF in the presence of ROS, and the fluorescence intensity is proportional to ROS production. Cells in 12-well plates were treated with the indicated concentration of Gyp for 4 h. The cells were harvested and washed two times with PBS, resuspended in 500 μl of 10 μM DCFH-DA, and incubated at 37°C for 30 min in the dark. The samples were then immediately detected by flow cytometry. Histograms were analyzed using FCS Express V3.

Examination of Δψ _m

To study the Δψ _m changes, cells were stained with Rh123, which selectively enters mitochondria with an intact membrane potential and is retained in the mitochondrial.²³ Once Δψ _m is lost, Rh123 is subsequently washed out of the cells. Cells in 12-well plates were treated with the indicated concentration of Gyp for 4 and 8 h. The cells were harvested and washed two times with PBS, resuspended in 500 μl of 1 μg/ml Rh123, and incubated at 37°C for 30 min in the dark. The samples were then immediately detected by flow cytometry. Histograms were analyzed using FCS Express V3.

Wound healing assay

Cells were plated in 24-well plates for 24 h, then the cells in individual wells were wounded by scratching with a pipette tip, and the cells were incubated with individual medium and treated with the indicated concentration of Gyp for 24 h. The cells were photographed under phase contrast microscopy (200× magnification).

Statistical analysis

Data are expressed as mean ± SD of at least three independent experiments. Statistical analysis was performed by one-way analysis of variance. Statistical significance was established at a value of p < 0.05.

Results

Effects of Gyp on cell viability

Data in Figure 1(a) and (b) showed that Gyp inhibited SW620 and Eca-109 cell proliferation in a dose- and time-dependent manner. As shown in Figure 1(a), in SW620 cells, the Gyp dose at 50 μg/ml showed no cell damage compared with the control (p > 0.05), while there was significant cell damage when Gyp dose was 100 μg/ml or above (p < 0.01), and the prolonged incubation enhanced the viability loss. The half maximal inhibitory concentration (IC₅₀) values were about 108.81, 89.29, and 61.68 μg/ml when cells were incubated with Gyp for 24, 48, and 72 h, respectively.

Figure 1.

Effects of Gyp on cell viability. (a) SW620 cells and (b) Eca-109 cells were seeded in 96-well plates and treated with 0, 50, 100, 150, and 200 μg/ml of Gyp for 24, 48, and 72 h. Cell viability was measured by MTT assay. (c) Comparison of PBMC, SW620, and Eca-109 cells to Gyp at the same experimental conditions for 24 h. Each value is expressed as a mean ± SD of at least three independent determinations. One-way ANOVA was used for comparisons of multiple group means followed by Dunnett’s t test. *p < 0.05 versus the control; **p < 0.01 versus the control (error bars = SD, n = 3). Gyp: gypenoside; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PBMC: peripheral blood mononuclear cell; ANOVA: analysis of variance.

Figure 1(b) showed similar Gyp dose-dependent cell damage on Eca-109 cells. However, the difference is that Gyp has a two-phase effect: low doses promote the proliferation of Eca-109 cells, while high doses inhibit the proliferation. When Gyp was 50 μg/ml, the cell viability of Eca-109 cells was increased by 17.71% and 23.68% after 24 and 48 h incubation (p < 0.01), respectively, but further incubations did not produce much more cell viabilities increase. The viability of Eca-109 cells treated with 100 μg/ml Gyp was 120.55%, increased by 20.55% after 24 h incubation, but the prolonged incubation did not enhance the viability increase; after 72 h incubation, the cell viability was 71.56%, which decreased by 28.44% (p < 0.01). The viability was significantly decreased when Gyp dose was 150 μg/ml or above (p < 0.01). The IC₅₀ values were about 149.39, 139.21, and 112.39 μg/ml when cells were incubated with Gyp for 24, 48, and 72 h, respectively.

As shown in Figure 1(c), SW620 and Eca-109 cells showed much more sensitivity to Gyp than PBMCs when Gyp dose was above 100 and 150 μg/ml, respectively. PBMCs exhibited significant cell viability loss when Gyp was above 150 μg/ml. After 24 h incubation, when Gyp was 200 μg/ml, the cell viability of PBMC, SW620, and Eca-109 cells were 86.23%, 6.29%, and 11.66%, respectively.

Gyp-induced plasma membrane damage of SW620 and Eca-109 cells

PI staining combined with flow cytometry was used to evaluate Gyp-induced cell membrane damage. In SW620 cells (Figure 2), Gyp-induced cell membrane damage was an early event. At 6 h after incubation, the percentage of cells with higher PI fluorescence gradually increased from 15.3 to 39.83% when cells were exposed to Gyp dose range from 70 to 130 μg/ml. Cells exposed to 70 μg/ml Gyp did not show much increased cell membrane damage with the prolonged incubation time. After 100 and 130 μg/ml Gyp treatment, the cell membrane damage was greatly increased by the prolonged incubation time until 24 h, but further increasing the incubation time to 48 h did not produce significant difference compared with the incubation at 24 h.

Figure 2.

Effects of Gyp on the cell membrane integrity of SW620 cells. Cells were treated with Gyp for 6, 12, 24, and 48 h, then stained with PI, and finally analyzed using flow cytometry. Histograms show number of cell channels (vertical axis) versus PI fluorescence (horizontal axis). Gyp: gypenoside; PI: propidium iodide.

As shown in Figure 3, in Eca-109 cells, compared with control, cells in 50 and 100 μg/ml Gyp-treated groups did not show much increased PI fluorescence after different incubation times. Cells in 150 μg/ml Gyp-treated group showed time-dependent cell membrane damage, about 93.2% of cells displayed high PI fluorescence at 48 h after treatment. While in 200 μg/ml Gyp-treated group, the percentage of cells with high PI fluorescence increased to 93.8% after 24 h of incubation.

Figure 3.

Effects of Gyp on the cell membrane integrity of Eca-109 cells. Cells were treated with Gyp for 6, 12, 24, and 48 h, then stained with PI, and finally analyzed using flow cytometry. Histograms show number of cell channels (vertical axis) versus PI fluorescence (horizontal axis). Gyp: gypenoside; PI: propidium iodide.

Gyp-induced morphological changes of SW620 and Eca-109 cells

As shown in Figure 4(a), in SW620 cells, DAPI staining in control cells were slightly blue and homogeneous, and the contrast phase microscopic results indicated normal spindle cell morphology. Cells in Gyp-treated groups showed enhanced DAPI staining and changed cell morphology in a Gyp-dose and incubation time-dependent manner. When Gyp dose was above 100 μg/ml, SW620 cells were seriously damaged with bright blue nuclear staining and the phase images indicated cells were shrunken to abnormal round type, and the cell number was significantly decreased.

Figure 4.

Gyp effects on the cell morphology and nucleus in (a) SW620 and (b) Eca-109 cells. Cells were treated with Gyp at the indicated concentrations for 24 and 48 h and then stained with DAPI for 15 min in the dark at 37°C. Morphological analysis of nuclear chromatin in cells was observed by fluorescence and morphological changes were observed by the homologous phase contrast images (×200 magnification), as described in “Materials and Methods” section. Gyp: gypenoside; DAPI: 4′-6-diamidino-2-phenylindole.

In Eca-109 cells, as shown in Figure 4(b), the control cells displayed uniform DAPI blue staining, and the contrast phase showed normal cell morphology with intact membrane in the form of polygon. Similarly, as shown in SW620 cells, Gyp-induced Eca-109 cell damage also presented Gyp-dose and incubation time-dependent manner. At 24 h after treatment, when Gyp was 100 μg/ml or above, obvious condensed chromatin with bright blue dots was observed in the cellular nuclear, and many spaces also appeared in the nucleus (pointed out by arrow), indicating that nuclear damage and DNA fragmentation might have occurred after Gyp treatment. The corresponding phase contrast images suggested that the cell morphology has seriously changed from polygonal type to round type, and some cell lysis with broken cell membrane was also observed. After 48 h of incubation, at the same Gyp concentration, the cell damage phenomena became more serious.

Gyp-induced cellular ROS generation in SW620 and Eca-109 cells

The intracellular ROS production was analyzed by flow cytometry with DCFH-DA staining. The data shown in Figure 5 suggested that the intracellular ROS levels were greatly increased after Gyp treatment, and this phenomenon in SW620 cells was more obvious than that in Eca-109 cells. In SW620 cells, at 4 h after treatment, there were about 27.33%, 69.04%, and 47% of cells in 70, 100, and 140 μg/ml Gyp-treated groups showing bright DCF fluorescence, while only 1.43% of cells in control group showed bright DCF fluorescence. In Eca-109 cells, compared with 2.37% of cells in the control, there were 22.5%, 28.07%, and 20.8% of cells in 100, 140, and 180 μg/ml Gyp-treated groups showing bright DCF fluorescence, respectively. In these two cell lines, we found that the ROS generation was first increased with increasing Gyp concentration and then slightly decreased at much higher Gyp dose, which may be due to more cell lysis caused by Gyp treatment at much higher drug concentration.

Figure 5.

Gyp-induced production of ROS in (a) SW620 and (b) Eca-109 cells. Cells were treated with Gyp for 4 h and labeled with DCFH-DA and the fluorescence intensity of the oxidized product DCF in individual cells was detected by flow cytometry. The percentage of fluorescent cells in each group was shown. ROS: reactive oxygen species; Gyp: gypenoside; DCFH-DA: 2′,7′-dichlorodihydrofluorescein–diacetate; DCF: 2′,7′-dichlorofluorescein.

Gyp-induced Δψ _m loss in SW620 and Eca-109 cells

Rh123 staining combined with flow cytometry was used to evaluate Gyp-induced Δψ _m changes. The results in Figure 6 showed the same trend for the two cell lines after Gyp treatment. Gyp induced the loss of Δψ _m quite early and caused the decrease in Δψ _m in a dose- and time-dependent manner. After 4 h of incubation, the percentage of cells with Δψ _m loss in two cell types increased from 35.25 to 46.35% and from 8.35 to 43.85% when Gyp increased from 70 to 130 μg/ml and from 100 to 180 μg/ml, respectively. When the incubation time increased from 4 to 8 h, in SW620 cells, the percentage of cells with Δψ _m loss increased from 35.85 to 77.1% when Gyp was 100 μg/ml; in Eca-109 cells, it increased from 22.85 to 63.35% when Gyp was 140 μg/ml.

Figure 6.

Effects of Gyp on Δψ _m of (a) SW620 and (b) Eca-109 cells. Cells were treated with Gyp for 4 and 8 h and labeled with Rho123 and analyzed using flow cytometry. Histograms show number of cell channel (vertical axis) versus Rho123 fluorescence (horizontal axis). Gyp: gypenoside; Δψ _m: mitochondrial membrane potential; Rho123: rhodamine 123.

Gyp inhibited migration of SW620 and Eca-109 cells in vitro

To determine whether Gyp affects the migration ability of SW620 and Eca-109 cells, a wound healing assay was conducted (Figure 7). The wound healing ability of cells reflects their movement and migration on the surface on which they are anchored to for growth.

Figure 7.

Effects of Gyp on the migration of (a) SW620 and (b) Eca-109 cells in vitro. Cells in 24-well plates were wounded by scratching with a pipette tip and the cells were incubated with Gyp for 24 h. The cells were photographed under phase contrast microscopy (×200 magnification). Gyp: gypenoside.

In SW620 cells, compared with 0 h after wounding, after 24 h of incubation, very dense cells in control gradually grew to the interspace of wound, cells in 70 μg/ml Gyp-treated group showed no significant difference with control, while cells in 100 and 130 μg/ml Gyp-treated groups rarely grew to the interspace of wound, and the cell density was seriously decreased. Similarly, higher Gyp concentrations leading to greater inhibition of cell migration was also observed in Eca-109 cells.

Discussion

G. pentaphyllum is a well-known medicinal plant in China. Pharmacological studies of G. pentaphyllum extracts have demonstrated a variety of health benefits, and the dammarane saponins (Gyps or gynosaponins) are believed to be the most active ingredients responsible for the biological activities of G. pentaphyllum. ^24
–26 There is, however, no available information on Gyp affecting human colon cancer SW620 cells and esophageal cancer Eca-109 cells. We showed in our studies that Gyp decreased the percentage of viable cells in two cell types. Meanwhile, the cytotoxic effect of Gyp on normal PBMCs was much less than on SW620 and Eca-109 cells. These results suggest that Gyp is capable of exerting different alternative cytotoxicity in cancer cells and normal cells, which might be potentially useful as a cancer preventive or treatment agent.

Staining with PI and analyzing using flow cytometry demonstrated that Gyp significantly damaged the plasma membrane of SW620 and Eca-109 cells. Subsequent DAPI staining assay displayed some apoptosis characteristics^27
–29 after Gyp treatment such as cell shrinkage, chromatin condensation with margination of chromatin to the nuclear membrane, and fragmented punctate blue nuclear fluorescence in SW620 and Eca-109 cells.

It is well known that apoptosis is a complex mechanism, which can be induced and regulated by many signal stimulus pathways. Apoptosis induction is arguably the most potent defense against cancer.³⁰ Dysregulation of apoptosis is usually considered as a major cancer hallmark.^31,32 Many studies have shown that apoptosis is an important mechanism by which various anticancer agents exert anticancer effects.^33,34 Recent investigations have revealed that a number of compounds extracted from plants and their derivatives could induce apoptosis in many cancer cells. Therefore, many researchers nowadays have performed screening for proapoptotic compounds from herbal extracts as well as natural compounds.^35

–41 In the present study, we found that Gyp of a well-known Chinese herb G. pentaphyllum could inhibit cell proliferation and exert apoptosis induction in SW620 and Eca-109 cells, suggesting Gyp has great potential in clinical cancer treatment.

At present, the proposed mechanisms of compounds extracted from plants that induced antitumor characteristics mainly focus on the mitochondrial-dependent pathway. Enhancement of ROS production has been associated with the apoptotic response induced by several proapoptotic compounds.^42,43 Investigations also show that the ROS generation, stimulated by the damaged mitochondria, could activate the signaling pathways that regulate cell apoptosis.^44,45 To confirm this, we next examined intracellular ROS formation and Δψ _m changes after different concentrations of Gyp treatment in SW620 and Eca-109 cells. Our results showed that, at early 4 h after Gyp treatment, intracellular ROS levels were greatly increased by Gyp in a dose-dependent manner in SW620 and Eca-109 cells, suggesting that an oxidative stress mechanism might be involved in response to Gyp treatment in SW620 and Eca-109 cells. The decrease in Δψ _m as a result of mitochondria depolarization in association with oxidative stress-induced apoptosis appears to be a common event.²¹ To study whether Δψ _m loss occurred during Gyp treatment, we monitored the Δψ _m changes of SW620 and Eca-109 cells using Rh123 staining. An initial drop in Δψ _m was detected as shown in Figure 6, indicating that the mitochondria were seriously damaged after Gyp treatment, and the Gyp-induced cell apoptosis in SW620 and Eca-109 cells might be mitochondria dependent.

Additionally, metastasis is one of the major challenges for a successful cancer treatment.⁴⁶ Therefore, the prevention of cancer metastasis is an important target for improving a patient’s prognosis. However, until now, there is no available information to address the effects of Gyp on the invasion and migration of cancer cells. In this study, we investigated whether Gyp could inhibit the migration of SW620 and Eca-109 cells. Results in Figure 7 suggested Gyp inhibited the migration of the two cell types in a Gyp in dose-dependent manner.

In conclusion, the present study evaluated the cytotoxicity of Gyp in SW620 and Eca-109 cells and demonstrated that Gyp could cause cell membrane integrity damage, promote ROS production, decrease Δψ _m level, and initiate apoptotic response in SW620 and Eca-109 cells. In addition, our study also showed that Gyp could exert an inhibitory effect on cell migration in vitro in the two cell types, suggesting Gyp may have great value in human colon cancer and esophageal cancer treatments. Further investigations are needed to explain the molecular mechanisms of Gyp in cancer therapy.

Footnotes

Conflict of interest

The authors declared no conflicts of interest.

Funding

This work was supported by National “Twelfth Five-Year” Plan for Science and Technology Support (2011BAI06B05).

References

Yako-Suketomo

Marugame

. Comparison of time trends in colon, rectum and anus cancer incidence (1973–2002) in Asia, from “cancer incidence in five continents. Vols IV-IX”. Jpn J Clin Oncol 2009; 39: 196–198.

Ministry of Public Health of People’s Republic of China. China Heath Statistics Yearbook 2009, http://www.moh.gov.cn/zwgkzt/ptjnj/200908/42635.shtml (2009).

Ministry of Public Health of People’s Republic of China. Program for National Cancer Prevention and Control in 2004–2010. Chin J Oncol (Chinese) 2004; 13: 65–68.

Zou

Taylor

Mark

Seasonal variation of food consumption and selected nutrient intake in Linxian, a high risk area for esophageal cancer in China. Int J Vitam Nutr Res 2002; 72: 375–382.

Jemal

Bray

Center

Global cancer statistics. CA Cancer J Clin 2011; 61: 69–90.

Pezzuto

. Plant-derivated anticancer agents. Biochem Pharmacol 1997; 53: 121–133.

Lilenbaum

Green

. Novel chemotherapeutic agents in the treatment of non-small-cell lung cancer. J Clin Onco 1993; 11: 1391–1402.

Aktan

Henness

Roufogalis

Gypenosides derived from Gynostemma pentaphyllum suppress NO synthesis in murine macrophages by inhibiting iNOS enzymatic activity and attenuating NF-κB-mediated iNOS protein expression. Nitric Oxide 2003; 8: 235–242.

Circosta

De Pasquale

Occhiuto

. Cardiovascular effects of the aqueous extract of Gynostemma pentaphyllum Makino. Phytomedicine 2005; 12: 638–643.

10.

Ia Cour

Molgaard

. Traditional Chinese medicine in treatment of hyperlipidaemia. J Ethnopharmacol 1995; 46: 125–129.

11.

Cui

Eneroth

Bruhn

. Gynostemma pentaphyllum: identification of major sapogenins and differentiation from Panax species. Eur J Pharm Sci 1999; 8: 187–191.

12.

Huang

Razmovski-Naumovski

Salam

A novel LXR-alpha activator identified from the natural product Gynostemma pentaphyllum . Biochem Pharmacol 2005; 70: 1298–1308.

13.

Lin

Huang

Lin

. Antioxidant and hepatoprotective effects of Anoectochillus formosanus and Gynostemma pentaphyllum . Am J Chin Med 2000; 28: 87–96.

14.

Attawish

Chivapat

Phadungpat

Chronic toxicity of Gynostemma pentaphyllum . Fitoterapia 2004; 75: 539–551.

15.

Jiao

Lau

. Protective effect of gypenosides against oxidative stress in phagocytes, vascular endothelial cells and liver microsomes. Cancer Biother 1993; 8: 263–272.

16.

Tan

Liu

. Antithrombotic effect of Gynostemma pentaphyllum . Zhongguo Zhong Xi Yi Jie He Za Zhi. 1993; 13: 278–280, 261.

17.

Chen

Tsai

Chen

Therapeutic effect of gypenoside on chronic liver injury and fibrosis induced by CCl4 in rats. Am J Chin Med 2000; 28: 175–185.

18.

Chen

Wang

The molecular mechanism of gypenosides-induced G1 growth arrest of rat hepatic stellate cells. J Ethnopharmacol 2008; 117: 309–317.

19.

Wang

Chiang

Gypenosides induce apoptosis in human hepatoma Huh-7 cells through a calcium/reactive oxygen species-dependent mitochondrial pathway. Planta Med 2007; 73: 535–544.

20.

Chen

Tsai

Gypenosides induced G0/G1 arrest via CHk2 and apoptosis through endoplasmic reticulum stress and mitochondria-dependent pathways in human tongue cancer SCC-4 cells. Oral Oncol 45: 273–283.

21.

Cheng

Xiao

Wang

Anti-tumor and pro-apoptotic activity of ethanolic extract and its various fractions from Polytrichum commune L.ex Hedw in L1210 cells. J Ethnopharmacol 2012; 143: 49–56.

22.

Krysko

Berghe

D’Herde

Apoptosis and necrosis: detection, discrimination and phagocytosis. Methods 2008; 44, 205–221.

23.

Wang

Liu

Zhang

Reactive oxygen species, but not mitochondrial membrane potential, is associated with radiation-induced apoptosis of AHH-1 human lymphoblastoid cells. Cell Biol Int 2007; 31: 1353–1358.

24.

Hsu

Yang

An experimental study on the antileukemia effects of gypenosides in vitro and in vivo. Integr Canc Ther 2011; 10: 101–112.

25.

Qian

Wang

Tang

. Effects of Gynostemma pentaphllum Makino polysaccharide (PGP) on immunefunction. J China Pharm Uni 1998; 30: 51–53.

26.

Tanner

Steimle

The direct release of nitric oxide by gypenosides derived from the herb Gynostemma pentaphyllum . Nitric Oxide 1999; 3: 359–365.

27.

Rello

Stockert

Moreno

Morphological criteria to distinguish cell death induced by apoptotic and necrotic treatments. Apoptosis 2005; 10: 201–208.

28.

Ndozangue-Touriguine

Hamelin

Bréard

. Cytoskeleton and apoptosis. Biochem Pharmacol 2008; 76: 11–18.

29.

Kerr

Wyllie

Currie

. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Canc 1972; 26: 239–257.

30.

Lipponen

. Apoptosis in breast cancer: relationship with other pathological parameters. Endocr Relat Canc 1999; 6: 13–16.

31.

Hanahan

Weinberg

. The hallmarks of cancer. Cell 2000; 100: 57–70.

32.

Abbott

Forrest

Pienta

. Simulating the hallmarks of cancer. Artif Life 2006; 12: 617–634.

33.

Wang

M(2)-A induces apoptosis and G(2)-M arrest via inhibiting PI3K/Akt pathway in HL60 cells. Canc Lett 2009; 283: 193–202.

34.

Sun

Geng

Huang

Coleusin factor exerts cytotoxic activity by inducing G0/G1 cell cycle arrest and apoptosis in human gastric cancer BGC-823 cells. Canc Lett 2011; 301: 95–105.

35.

Lee

Tse

Paeoniae Radix, a Chinese herbal extract, inhibit hepatoma cells growth by inducing apoptosis in a p53 independent pathway. Life Sci 2002; 71: 2267–2277.

36.

Cheng

Lee

Lin

Anti-proliferative activity of Bupleurum scrozonerifolium in A549 human lung cancer cells in vitro and in vivo . Canc Lett 2005; 222: 183–193.

37.

Kwon

Hong

Kim

Methanolic extract of Pterocarpus santalinus induces apoptosis in HeLa cells. J Ethnopharmacol 2006; 105: 229–234.

38.

Park

Jin

Kim

Induction of apoptosis by esculetin in human leukemia U937 cells through activation of JNK and ERK. Toxicol Appl Pharmacol 2008; 227: 219–228.

39.

Liu

Tang

Liang

Growth inhibitory and apoptosis inducing by effects of total flavonoids from Lysimachia clethroides Duby in human chronic myeloid leukemia K562 cells. J Ethnopharmacol 2010; 131: 1–9.

40.

Engel

Oppermann

Falodunb

Proliferative effects of five traditional Nigerian medicinal plant extracts on human breast and bone cancer cell lines. J Ethnopharmacol 2011; 137: 1003–1010.

41.

Wang

Liu

Cell cycle arrest and cell apoptosis induced by Equisetum hyemale extract in murine leukemia L1210 cells. J Ethnopharmacol 2012; 144: 322–327.

42.

Antosiewicz

Herman-Antosiewicz

, Marynowski SW, et al. c-Jun NH(2)-terminal kinase signaling axis regulates diallyl trisulfide-induced generation of reactive oxygen species and cell cycle arrest in human prostate cancer cells. Canc Res 2006; 66: 5379–5386.

43.

Chang

Shyue

Emodin induces apoptosis in human lung adenocarcinoma cells through a reactive oxygen species-dependent mitochondrial signaling pathway. Biochem Pharmacol 2005; 70: 229–241.

44.

Choi

Chun

Shin

Curcumin attenuates cytochrome P450 induction in response to 2,3,7,8-tetrachlorodibenzo-p-dioxin by ROS-dependently degrading AhR and ARNT. Canc Sci 2008; 99: 2518–2524.

45.

Ozben

. Oxidative stress and apoptosis: impact on cancer therapy. J Pharm Sci 2007; 96: 2181–2196.

46.

Chen

Lai

Gypenosides inhibits migration and invasion of human oral cancer SAS cells through the inhibition of matrix metalloproteinase-2-9 and urokinase-plasminogen by ERK1/2 and NF-kappa B signaling pathways. Hum Exp Toxicol 2011; 30: 406–415.

Antiproliferation and anti-migration induced by gypenosides in human colon cancer SW620 and esophageal cancer Eca-109 cells

Abstract

Keywords

Introduction

Materials and methods

Chemicals and reagents

Cell culture

Isolation of normal mouse PBMC

Assessment of cell viability after Gyp treatment

Flow cytometry analysis for cell membrane integrity

DAPI staining

Detection of intracellular ROS generation

Examination of Δψ m

Wound healing assay

Statistical analysis

Results

Effects of Gyp on cell viability

Gyp-induced plasma membrane damage of SW620 and Eca-109 cells

Gyp-induced morphological changes of SW620 and Eca-109 cells

Gyp-induced cellular ROS generation in SW620 and Eca-109 cells

Gyp-induced Δψ m loss in SW620 and Eca-109 cells

Gyp inhibited migration of SW620 and Eca-109 cells in vitro

Discussion

Footnotes

Conflict of interest

Funding

References

Examination of Δψ _m

Gyp-induced Δψ _m loss in SW620 and Eca-109 cells