Effect of Asparagus saponins on HepG2 Apoptosis and mitochondrial membrane potential and ROS Level

JI Chen feng, JI Yu bin, YUE Lei

Abstract—To study the effect of Saponins of asparagus on HepG2 apoptosis, reactive oxygen species (ROS) and mitochondrial membrane potential (Δψm) levels. Saponins of asparagus with different concentration was treated with HepG2 at different time, MTT assay was used to detect inhibitory rate, fluorescence staining was used to observe apoptosis morphology, flow cytometry was used to detect apoptosis rate and cell cycle, also ROS and Δψm were measured by flow cytometry. Results showed Saponins of asparagus inhibited cell proliferation; the IC50 on HepG2 was 172.3 μg/mL. Apoptosis morphology was observed by fluorescence microscope, some of the cells had changed from irregular shapes into round cells, the nuclei had decreased in size, the number of nucleoli had decreased; the chromatin had condensed, with the cytoplasm becoming more concentrated. The cell cycle of HepG2 was arrested at S phase, G2/M phase percent decreased. After 72h the treated group appeared apoptosis peak, and apoptosis rate with high dose group 30.94±1.74%；After 48h the ROS in treated group increased with high dose group 77.7±4.5%; and Δψm decreased with high dose group 77.8±1.9% From the above it can be seen that Asparagus saponins have a relatively strong inhibitory effect on HepG2 cells, and the mechanism involved has to do with their role in increasing the production of intracellular ROS, which leads to the lowering of mitochondrial membrane potential, thus initiating the apoptosis mechanism that induces apoptosis in HepG2 cells.

I. INTRODUCTION Asparagus officinalis Linn is an herbaceous perennial

plant of the Liliaceae family. Modern scientific research has shown that Asparagus officinalis can act to prevent the proliferation of cancer cells, and is effective for treating many cancers, including lymphoma, carcinoma of the bladder, pulmonary carcinoma, hepatocarcinoma, gastric carcinoma, and skin carcinoma[1,2], with saponin as one of its major anti-tumor agents. In the present experiment, modern biotechnology was used to explore the effect of the

Manuscript received January 22, 2009. This work was supported in part by National Natural Science Foundation of China (No. 30400592) and Foundation of Harbin Bureau of Science and Technology.

JI Chen feng is with Engineering Research Center of Natural Anticancer Drugs, Ministry of Education, Harbin, China. He is also with the Institute of Materia Medica and Postdoctoral Program, Harbin University of Commerce, Harbin, China (phone: +86-451-8486-6922; fax: +86-451-8484-4417;e-mail: jichenfeng@ hrbcu. edu.cn).

JI Yu bin is with the Center of Research and Development on Life Sciences and Environmental Sciences, Harbin University of Commerce, Harbin, China (e-mail: smilejcf001@sina.com).

YUE Lei was with the Institute of Materia Medica and Postdoctoral Program, Harbin University of Commerce, Harbin, China. She is now with Engineering Research Center in Medicine and Biology, Harbin Institute of Technology, Harbin, China (e-mail: smileyl001@163.com).

total saponins of Asparagus officinalis on the apoptosis of HepG2 hepatocarcinoma and their cell cycle as well as their effect on the mitochondrial membrane potential and active oxygen species, thus exploring its anti-tumor mechanism from different perspectives.

II. MATERIALS AND METHODS

A. Materials 1) Materials and reagents: Asparagus saponins; FBS;

RPMI1640; dimethyl sulfoxide (DMSO); methyl thiazolyl tetrazolium (MTT); Hochest 33258; propidium iodide (PI); RNaseA; Triton-X-100;2'-7'-dichlorofluorescein-diacetate; rhodamine 123; 5-fluorouracil; adriamycin (ADM)

2) Cell line: Human hepatocarcinoma HepG2

3) Aparatuses: CO2 incubator (NBS); fluorescence invert microscope (Olympus); microplate reader (Bio-Rad); EPICS XL flow cytometer (Beckman).

B. Methods 1) Cell culture: Human hepatocarcinomatic cell line

HepG2 was cultured in RPMI1640 containing fetal bovine serum with a volume fraction of 10%. The culture was then incubated at 37°C and 5% CO2, and cells in the phase of logarithmic growth were used for the experiment.

2) MTT test: Cells were taken during the logarithmic growth phase, and whole-culture solution was used to adjust the cell concentration to 5×107/L. The culture was added to 96-well culture plate, 100 μL per well, and then incubated for 24 h in CO2 incubator at 37°C and 5% relative humidity. Asparagus saponins was added to a 96-well culture plate, 100 μL per well, and made into solutions with final concentrations of 1, 10, 100, and 1000 mg/L, respectively; for the blank, 100 μL of RPMI1640 culture was added to each well; for the positive control, 100 μL of adriamycin was added to each well, and made into solutions with final concentrations of 0.01, 0.1, 1, 10 mg/L, respectively, with 6 parallel wells used for each dose. The culture plates were incubated for 72 h more in CO2 incubator at 37°C and 5% relative humidity. The supernatant was discarded by quickly turning the culture plates over, and 100 μL of MTT test solution with a concentration of 0.5 g/L was added to each well, after which the culture plates were incubated for 4 h more before the supernatant was discarded. Then 200 μL of DMSO was added to each well, and the microplate reader was used

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to measure the absorbance with a detecting wavelength of 570 nm. The rate of inhibition on cell growth and IC50 of the drugs were calculated.

3) Apoptotic forms of cells observed with fluorescence microscope: Cells were taken during the logarithmic growth phase, and whole-culture solution was used to adjust the cell concentration to 3×108/L. The culture was added, 1 mL per well, to a 24-well culture plate with cover glass, and then incubated for 24 h in CO2 incubator at 37°C and 5% relative humidity. Asparagus saponins was added to the 24-well culture plate, 1 mL per well and 4 parallel wells for each group, with final concentrations of 50, 100, 200 mg/L, respectively. For the blank, 1 mL of RPMI1640 culture medium was added. For the positive control, 1 mL of adriamycin was added to each well, with a final concentration of 10 mg/L. The culture was incubated for 48 h more in CO2 incubator at 37°C and 5% relative humidity, after which the liquid in the culture plate was discarded, and the plate was rinsed once with PBS. 0.5 mL of fixing solution was added to fix the sample for 10 min at 4°C, after which 0.5 mL of Hoechest 33258 stain (5 mg/L) was added to each well to stain the sample for 15 min. Then the sample was observed under the fluorescence invert microscope.

4) Changes in cell cycle and apoptosis rate measured using flow cytometry: Cells were incubated for 24, 48, and 72 h, digested with pancreatin, suspended in PBS, and then rinsed twice with PBS (1500 rpm, 10 min). Cold ethanol (70%) was added to fix the sample for 12 h or more at 4°C. PI stain (PI 50 mg/L, RNaseA 10 mg/L, Triton-X-100 5 mL/L) was added to stain the sample for 30 min, after which the sample was filtered with a 200-mesh nylon net and then mounted onto the flow cytometer for measurement, with an excitation wavelength of 488 nm.

5) Effect of Asparagus saponins on the mitochondrial membrane potential (Δψm) of the cells: Cells were incubated for 48 h, digested with pancreatin, suspended in PBS, and then rinsed twice with PBS (1500 rpm, 10 min). 0.5 mL of rhodamine 123 (5 mg/L) was added (the operation was carried out away from light) to the sample, which was then placed in the incubator for the staining to proceed for 30 min. Then the sample was rinsed once with PBS (1500 rpm, 10 min), filtered with a 200-mesh nylon net, and then mounted onto the flow cytometer for measurement, with an excitation wavelength of 488 nm.

6) Effect of Asparagus saponins on reactive oxygen species (ROS) measured with flow cytometry: Cells were incubated for 48 h, digested with pancreatin, suspended in PBS, and then rinsed twice with PBS (1500 rpm, 10 min). 0.2 mL of DCFH-DA (5 μmol/L) was added to stain the sample for 20 min at room temperature. The sample was then rinsed once with RPMI1640 (1500 rpm, 10 min),

filtered with a 200-mesh nylon net, and then mounted onto the flow cytometer for measurement, with an excitation wavelength of 488 nm.

7) Statistical analysis: Data are presented in the form x ±s. Variance test is adopted for the analysis, with Dunnett test used to compare the different groups.

III. RESULTS

A. Effect of Asparagus saponins on the proliferation of tumor cells

The results show that Asparagus saponins all have an inhibitory effect on HepG2 cells, and that the effect is dose-dependent. The OD values for the high-dose groups are all significantly different from the control group (P < 0.01), as shown in TableⅠ.

TABLE Ⅰ INHIBITORY EFFECT OF ASPARAGUS SAPONINS ON HEPG2 CELLS（X±S，N=6）

Dose OD value Inhibition rate IC50

Control 1

10 100

1000

0.440±0.018 0.391±0.011** 0.376±0.009** 0.309±0.004** 0.118±0.003**

- 11.1% 14.6% 29.9% 73.1%

172.3 mg/L

*P<0.05 compared with control , **P<0.01 compared with control

B. Effect of Asparagus saponins on cell morphology After Asparagus saponins had acted on the samples for

48 h, the treated groups were found to have changed significantly in cell morphology compared with the untreated control. The changes include the following: the number of cells had declined slightly; the tendency of the cells to grow in clusters had weakened, with the cells more scattered; the number of cell division phases had clearly decreased; some of the cells had changed from irregular shapes into round cells, with the cell silhouette becoming quite clear; the nuclei had decreased in size, the number of nucleoli had decreased; the chromatin had condensed, with the cytoplasm becoming more concentrated; the nucleo-cytoplasmic ratio had been lowered; and the fluorescence intensity had increased.

A B

Figure1. M orphology of HepG2 cells(A. HepG2 cell morphology of the control group B. HepG2 cell morphology of the treated group)

C. Changes in cell cycle measured with flow cytometry After Asparagus saponins had acted on samples of

different concentrations for 24 h, 48 h, and 72 h, the cell cycle of HepG2 cells had clearly changed. Compared with the negative control at the same duration of treatment, the proportion of cells in the S phase in all three dose groups had seen clear increase, while that for cells in the G2/M phase had seen clear decrease. For the medium-dose group, after it had been treated for 48 h and 72 h, the number of cells in the G2/M phase was 0. In both the medium- and high-dose groups, the percentages of cells in the S phase were all above 50% after 72 h of treatment. These results are all presented in TableⅡ.

TABLEⅡ EFFECT OF ASPARAGUS SAPONINS ON THE CELL CYCLE OF HEPG2（X±S，N=3）

Group Time Cell cycle（%）

G0/G1 S G2/M

Control

24 64.17±0.48 26.51±0.79 9.32±1.02

48 65.28±0.53 25.95±1.12 8.77±0.89

72 65.16±0.67 22.71±1.17 12.14±0.64

5-Fu

24 73.84±0.65** 21.02±0.53** 5.14±0.39**

48 77.82±0.49** 22.18±0.84* 0±0**

72 90.40±0.59** 9.60±0.73** 0±0**

low

24 60.64±0.88** 38.12±0.74** 1.24±0.97**

48 47.80±1.11** 47.62±1.38** 4.57±0.71**

72 59.31±0.66** 35.37±0.59** 5.32±0.47**

medium

24 59.27±0.42** 40.41±0.38** 0.32±0.84**

48 48.13±1.23** 51.87±0.97** 0±0**

72 44.83±0.51** 55.17±0.43** 0±0**

high

24 63.95±0.69 34.59±0.52** 1.46±0.58**

48 50.60±0.52** 48.00±0.97** 1.40±0.71**

72 39.62±0.85** 58.75±0.94** 1.63±1.17** * P<0.05 compared with control , ** P<0.01 compared with control

D. Apoptosis rate measured with flow cytometry After the drug administered for 72 h, a clear apoptosis

peak appeared in HepG2 cells; as the concentration of Asparagus saponins increased, the number of apoptotic cells increased. See Table Ⅲ and Figure 2.

TABLE Ⅲ EFFECT OF ASPARAGUS SAPONINS ON THE APOPTOSIS RATE OF HEPG2（X±S，N=3）

Group Dose (mg/L) Apoptosis rate (%)

Control — —

ADM 25 74.23±1.97

Saponins of asparaus

50 9.93±0.81

100 12.77±1.84

200 30.94±1.74

E. Changes in the amount of ROS in HepG2 cells It can be seen from Figure 3 that, 48 h after Asparagus

saponins of different concentrations had acted on HepG2 cells, the level of intracellular ROS had increased significantly, and that, with increase in the concentration, the level of ROS also increased, with that for the high-dose group reaching 77.7%. This result suggests that Asparagus saponins can raise the level of ROS in HepG2 cells, with the ROS concentration being positively correlated to the duration of treatment.

A B

C D

Figure 3. Effect of Asparagus saponins on ROS in HepG2 cells at 48 h

F. Changes in the mitochondrial membrane potential (Δψm) in HepG2 cells

From Figure 4, it can be seen that after Asparagus saponins acted on HepG2 cells for 48 h, Δψm decreased slightly compared with the control, with increase in the concentration of Asparagus saponins, Δψm decreased more, or Δψm was shown to be inversely proportional to the concentration of Asparagus saponins administered.

A B

C D

Figure 4. Effect of saponins of asparagus on Δψm of HepG2 cells at 48 h

IV. DISCUSSION

At certain concentrations, Asparagus saponins was shown to have a proliferation-inhibiting and killing effect on HepG2 cells that is concentration-dependent [3-5]. After a high dose of Asparagus saponins had acted on the cells for 72 h, the rate of proliferation-inhibition for the HepG2 cells reached 73.1%. When observed under a fluorescence microscope, the cells were found to emit uneven masses or granules of rather strong and dense blue fluorescence. The cells had decreased in volume, forming apoptotic bodies. In contrast, the untreated cells emitted an even blue fluorescence. With increase in drug concentration, the number of apoptotic cells also increased

[6,7].

In this experiment, flow cytometry was used to analyze the cell cycle of HepG2 cells. The results show that after Asparagus saponins of different concentrations had acted on the cells for different durations, two phases in the cell cycle of the tumor cells had become abnormal. After Asparagus saponins had acted on the cells for 24 h, 48 h, and 72 h, the cell cycle of HepG2 cells shows a blockage at the S phase. Compared with the control group, the number of cells in the S phase for the treated groups have increased significantly (P < 0.01), while that of the cells in the G2/M phase decreased significantly (P < 0.01)[8]. With increase in the duration of treatment or in the concentration, the number of cells in the G2/M phase decreased gradually, while the number of cells in the G0/G1 phase is correlated neither with the duration of treatment nor with the concentration. The G2/M phase is the stage of mitosis and an important stage for cell proliferation [9-10]. Results from this experiment show that Asparagus saponins can affect the transition of HepG2 cells from the S phase to the G2/M phase, which leads to a large number of cells being accumulated at the S phase, thus decreasing the number of cells that enter the G2/M phase, decreasing the number of cells that undergo mitosis and go on to enter the next cycle of cell proliferation, and eventually leading to the rapid apoptosis of the tumor cells. In the DNA histogram on the flow cytometer, we observed the characteristic hypodiploid peak (the sub-G1 peak), the

appearance of which is one of the signs for apoptosis [11]. This further shows that the induction of apoptosis in HepG2 cells is the main anti-tumor mechanism of Asparagus saponins. Furthermore, after Asparagus saponins had acted on the cells for 72 h, the rate of apoptosis gradually increased the concentration increased. At 24 h and 48 h after Asparagus saponins was administered, however, the apoptosis peak on the DNA histogram of the flow cytometer was not very clear, while changes in the cell cycle were quite clear, which means that the drug induces apoptosis by first affecting the cell cycle, and that abnormal changes in the cell cycle is one of the mechanisms for apoptosis. This result also suggests a temporal continuity of these two processes [12].

In normal circumstances, ROS are required for cell metabolism, and oxidation and antioxidation are in equilibrium, thus low concentration of ROS can promote cell proliferation. When the equilibrium is broken due to certain factors and ROS production is increased, the inflow of Ca2+ would be increased, the expression of bax is adjusted upward, MPTP is opened, and Caspase is activated, leading to the apoptosis of cells. Research has shown that changes in the functions of mitochondria are closely associated with apoptosis. The transmembrane potential of mitochondria directly reflects changes in their functions. Under the effect of apoptosis-inducing factors, the mitochondrial membrane potential begins to lower before the nuclei show changes characteristic of apoptosis. In this experiment, FCM was used to measure the level of intracellular ROS and Δψm, and it was found that 48 h after Asparagus saponins was administered, the ROS level in HepG2 cells gradually increased, while at the same time Δψm was found to have decreased, and the amount of decrease went up with the increase in the dosage of the drug applied.

From the above it can be seen that Asparagus saponins have a relatively strong inhibitory effect on HepG2 cells, and the mechanism involved has to do with their role in increasing the production of intracellular ROS, which leads to the lowering of mitochondrial membrane potential, thus initiating the apoptosis mechanism that induces apoptosis in HepG2 cells.

REFERENCES [1] Wu X P,Guo Y F. Summatize of studying Asporagus officinalis

linn]. TCM Res,1996,9(4):54-56. [2] Sun C Y,Zhao B T,Yu Z F,. The development of chemical

components in asparagus officinalis and their medical effects. Chinese wild plant resource,2004,23(5):1-5.

[3] Habib M R,Joel A H,Qing F. Modulation of apoptosis by nitric oxide: I mplications in myocardial ischemia and heart failure. Pharmacology & Therapeutics,2005,106:147-162.

[4] Masahiko K,Takashi N,Masatoshi M. Capases cleave the aminoterminal calpain inhibitory unit of calpastain during apoptosis in human jurkat T cell.J Biochem,2005,127:297-305.

[5] Vries J F, Falkenburg J H, Willemze R,et al.The mechanisms of Ara-C-induced apoptosis of resting B-chronic lymphocytic leukemia cells. Haematologica,2006,91(7):912-919.

[6] Yeh C T, Yen G C. Effect of sulforaphane on metallothionein expression and induction of apoptosis in human hepatoma HepG2 cells. Carcinogenesis. 2005,26(12):2138-2148.

[7] Castellon E, Clementi M, Hitschfeld C,et al.Effect of leuprolide and cetrorelix on cell growth, apoptosis, and GnRH receptor expression in primary cell cultures from human prostate carcinoma.Cancer Invest. 2006,24(3):261-268.

[8] Fan H T,Xiong X Y,Cao W,. Effects of proliferation inhibition and apoptosis of resveratrol trinicotinate on HepG2 cells and its mechanism. Chin J Clin Pharmacol Ther,2006,11(5):540-544.

[9] Zhan H X,Zhong C G,Xu S Q. Relationships of hexavalent chromium(�) -induced apoptosis,oxidative damage and expressions of p53 and caspase3 in L-02 hepatocytes. Chin J Pharmacol Toxicol,2006,20(2):144-151.

[10] Zhan J,Yang J,Tang H B. Inhibitory effect of isoliquiritigenin on proliferation of human cervical cancer cells in vitro and in vivo. Chin J Pharmacol Toxicol,2005,19(6):436-442.

[11] Jia X D, Han C, Chen J S. Tea pigments induce cell-cycle arrest and apoptosis in HepG2 cells.World J Gastroenterol,2005,11(34): 5273-5276.

[12] Ralstin MC, Gage EA, Yip-Schneider MT, et al. Parthenolide cooperates with NS398 to inhibit growth of human hepatocellular carcinoma cells through effects on apoptosis and G0-G1 cell cycle arrest.Mol Cancer Res,2006,4(6):387-399.