Experimental Study on Apoptosis Induced by Elemene in Glloma Cells

 

Zhou Hong-Yu1, Shen Jian-Kang1, Hou Ju-Sheng2, Qiu Yong-Ming3, Luo Qi-Zhong3

 

[Abstract]

BACKGROUND & OBJECTIVE: elemene, isolated from Chinese medical herb Rhizoma Zedoariae, was shown to exhibit antitumor activity. Our previous studies showed that elemene had a markedly antineoplastic activity on glioma. This study was designed to investigate the proliferation inhibitory effect and the apoptosis-inducing activity of elemene on glioma cells.  METHODS: The effects of elemene on cell proliferation were confirmed by Hoechst 33258/PI staining. The apoptosis was evaluated by flow cytometry analysis and agarose gel electrophoresis.  RESULTS: Elemene exhibited a marked antiproliferative effect on rat glioma cell C6 and human glioma cell SHG-44. The fifty percent inhibition concentration (IC50) of elemene against glioma cell lines at different time points (D1-D4) by 3H-TdR incorporation was C6 7.33-11.02 mg/L, SHG-44 13.29-27.16mg/L. At the same concentration, human glioma cell line SHG-44 was found to be less sensitive to elemene compared to rodent cell line C6. The characteristic nucleolus alternations under fluorescent microscope included condensation of chromatin arranged under the nuclear membrane and apoptotic bodies. With a low nuclear/cytoplasmic ratio, in flow cytometry analysis, a typical subdiploid peak before Phase G0/G1 (apoptotic peak) was detected in DNA frequency distribution histograms. Also the apoptosis in glioma cells was confirmed by DNA ladder formation on gel elextrophoresis.  CONCLUSION: Elemene exhibited a marked antiproliferative effect on glioma cells, and it could induce apoptosis in vitro.

Keywords: Glioma; Apoptosis; Elemene

 

Elemene, isolated from Chinese medical herb Rhizoma Zedoariae, is a chemical withβ-elemene as the major component, which is a new second-line non-cytotoxic anti-tumor drug. Elemene has been used for the treatment of glioma with significant effect [1]; it is also observed that the proliferation of glioma cells can be slowed down after treatment of elemene in vitro and characteristic morphologic changes occur [2]. But the mechanism of anti-tumor hasn’t been explored extensively. The research is aimed to probe into the anti-proliferation of elemene on glioma cells, and related mechanism from the perspective of inducement of apoptosis, so as to provide theoretical evidence and experiment base for the treatment of glioma with elemene.

1 Material and Method

1.1 Cell Culture

Classical glioma cell lines C6 (rate derived) are purchased from Shanghai Institute Chinese academy of science, SHG-44 (human derived) are generously given by Professor Huang Qiang of Suzhou Medical School affiliated Second Hospital. Two kinds of glioma are respectively cultured and multiplied in DMEM and RPMI-1640 culture medium (produced by Gibco Company) containing 10% newborn calf serum (produced by Hangzhou Si Ji Qing Company).

1.2 Major Agent and Devices

The 10 ml 2% elemene solution injection is produced by Dalian Medical Science Institute. methyl - 3 HThymidine is purchased from Shanghai Nuclear Institute, Hoechst 33258 is purchased from Shanghai Huashun Company, and propidium iodide (PI) is product of Sigma Company, the rest are analytical reagents domestically produced. Lambda DNA/Hind III makers are used. The major devices used are as the following: liquid scintillation counter (Wallac 2405 Mictobeta), fluorescent microscope (Olympus), flow cytometer (Becton Dickinson) and horizontal gel system (DY III-3).

 

1.3 Proliferation Iinhibitory Effect of Elemene on Glioma Cells Examined with 3H-TdR Incorporation  

C6/SHG-44 glioma cells are incubated on the 96 well cell culture plate, 100μl per well, at a density of 5×104/ml. There are two groups, i.e. elemene group (C6 glioma cell of 5, 10, 15, 20, 25, 30 mg/l; SHG-44 cells of 10, 20, 30, 40, 50 mg/L) and blank control group, with five duplicate wells. After one day, conditioned culture medium with various concentrations of elemene were changed according to different groups, the blank control group was changed for equal amount of culture medium without elemene, 37 kBq (1μCi)3H-TdR was added into the medium after constant treatment of 1, 2, 3, 4 days, then after10h coculture, the cells were collected and measured cpm value with liquid scintillation counter. Finally, IC50 was analyzed with medical statistical software ED 50 plus v1.0.

1.4 Observations on Morphologic Changes of Cellular Apoptosis Using Hoechst 33258/PI Fluorescent Staining Analysis [3]

C6/SHG-44 glioma cells were cultured in a routine manner and divided into elemene treated group (C6 glioma cells of 10, 20, 30, and 40mg/L; SHG-44 glioma cells of 20, 30, 40, 50, 60mg/L) and blank control group, which should change solutions every two to three days. Two groups should be collected respectively in 1, 2, 3, and 4 days for cell counting. We sampled 1ml of cell suspension, with 10μl 10% Hoechst 33258 dye solution or 250mg/LPI dye solution, which was stored without light for 15 min at the temperature of 37.The cells were suspended after 10 min centrifugation at 1000r/min, then made into cell smears, and fixed for observation under fluorescence microscope.

 

1.5 Measurements of Cellular Apoptosis with Flow Cytometry [4]

A certain amount of logarithmic phase C6/SHG-44 gioma cells should be isolated and inculcated in 25 cm2 culture bottles at a density of 5×104/ml, with 5ml in each bottle, and the grouping method is as mentioned above in section 1.4. After 24 hours, conditioned culture medium with different concentration of elemene should be exchanged for corresponding groups. The cells of each group should be collected in different stages after treatment, with PBS washing twice, and centrifugation of 1000r/min for 5min. Then the cells should be suspended with 4PBS and counted, made into 1×106/ml single cell suspension. Two times volume of -20℃ precooled 95% ethanol solution should be slowly added and fully mixed on the oscillator and fixed later. Finanlly it is stored in refrigerator at 4℃ before measurement in two weeks. The fixed cells should be PBS washed twice before measurement, centrifuged 1000r/min for 5 min, added with RNase A 100U/ml and put in water bath of 4 on shaking table for 30min. After that, the sample should be stained with 10μg/ml PI dye solution containing Triton X-100, and stored without light for 30min at the temperature of 4℃. Filtrated with nylon net, and measured with flow cytometry. According to the apoptotic peak in DNA histogram, and analysis result of CellQuest software, the apoptosis rate can be calculated.  

 

1.6 Gel Electrophoresis [5]

C6 glioma cells were treated with the methods mentioned in section 1.5 and collected for gel electrophoresis. (1) Isolation of DNA with Phenol-chloroform method. The cells were collected and washed by 1000r/min PBS for twice, added into 500μl lysate [100μg/ml protein kinase K, 10mmol/L Tris-HCl (pH 8.0)15mmol/L Nacl, 10mmol/L EDTA, 500μl 0.4% Sodium Dodecyl Sulfate (SDS)], and the cells are suspended again, stored at 37 for 12h. Then 500μl phenol is added and mixed, centrifuged for 1000r/min 10min. Upper water phase is transferred, added into 450μl chloroform: mixed with isoamyl alcohol (24:1), centrifuged for 1000r/min 10minUpper water phase is transferred, added into 1/10 volume of 3 mmol/L sodium acetate and 2.5 times volume of anhydrous ethanol, stored at -20 for 1h. With 1000r/min centrifugation for 15min DNA gets precipitatedwashed with 70% alcohol once and naturally dried. Then they are dissolute in 100μl TE buffer containing RNase A.2Agarose Gel Electrophoresis. With 1.5% agarose gel-loaded, voltage 70V, 1h  electrophoresis, and observing the images in ultraviolet lamp.

 

1.7 Data processing

The data are expressed with mean±standard deviation, and the comparison between groups should go through t-test. 

 

 

2 Results

2.1 Proliferation Inhibitory Effect of Elemene on Gioma Cells

Elemene can inhibit the malignant proliferation of C6 and SHG-44 lines of glioma cells. Most C6 cells are observed to be tiny spindle shaped in appearance by inverted microscope, with rapid rate of proliferation. And the cells of control group has been densely lined and overlapped in three days; while SHG-44 cells shows long fusiform with cell processes vanished and accelerated growth rate. After constant treatment with elemene, the amount of C6 cells significantly decreases, and some cells die from detachment, so the percentage rises with the increase of elemene concentration and treatment time. In addition, some adherent cells are observed to have larger volume, more cytoplasm, and smaller nucleus while their processes become more and longer, some sort of cell contact inhibition also exists.   

With the same concentration of elemene, the rate C6 glioma cells are more sensitive than human SHG-44 glioma cells. We used 3H-TdR incorporation method to measure the IC50 value of C6/SHG-44 glioma cells treated with elemene at different time, finding that the IC50 value of C6 cells is among 7.33-11.02mg/L, while that of SHG-44 cells is 13.29-27.16mg/L (tab.1).

 

Tab. 1 The fifty Percent Inhibition Concentration (IC50) of Elemene against C6/SHG-44 Glioma Cell Lines at Different Time Points (±s,n=5)

Time (days)

IC50 value (mg/L)

C6

SHG-44

1

7.33±1.10

27.16±7.61

2

11.02±1.23

16.5±2.76

3

7.98±0.25

18.53±2.00

4

7.61±0.85

13.29±1.62

 

 

2.2 Observations on Morphology of Nucleolus Using Fluorescence Staining

Most glioma cells treated with elemene are observed with early morphological changes of apoptosis, such as karyopyknosis, DNA concentration and closer with nuclear membrane, decreased percentage of karyoplasm and cellular shrinkage. Because of DNA fracture, the nuclears of some cells appear massive or dotty, with apoptotic bodies.  They are easy to distinguish from normal cells under fluorescence microscope, considering the morphologic feature of normal cells is loose and uniform, and fluorescence staining of cellular nuclear is lighter and uniform. C6 cells are taken as an example shown in fig. 1.    

Fig. 1 Fluorescence micrographs of C6 glioma cells after treatment with elemene

A. C6 glioma cells treated with 40mg/L elemene for 3d, stained with Hoechst 33258, the characteristic nucleolus alternation, apoptotic bodies, were observed. Original magnification×200; B. C6 glioma cells treated with 20gm/L elemene for 3d, stained with PI; The nuclei of most cells were condensed and fragmented. Original magnification×100; C.C6 control D3, stained with PI. Original magnification×100.

 

2.3 Flow Cytometry Analysis

The results of flow cytometry analysis showed that the natural apoptosis rate of C6/SHG-44 cells is quite low without treatment of elemene. Apoptosis can be induced if treated with elemene in a certain period: an obvious apoptotic peak can be observed before G0/G1 peak on the histogram of DNA content, which is characteristic compared with that of cells in controlled group. The percentage of apoptotic cells is shown in tab. 2, taking C6 glioma cell as an example.

 

Tab.2  The Rates of Apoptosis in C6 Glioma cell at Different Time Points Following Elemene Treatment (±s,n=5)

Group

Apoptosis rate(%)

D0

D1

D3

D5

Control

1.58±1.92

1.54±4.30

0.83±0.69

1.45±1.81

Elemene 10 mg/L

1.58±1.92

5.83±7.60

4.74±3.81

5.40±5.20

Elemene 20 mg/L

1.58±1.92

2.24±2.71

3.65±2.33

12.31±8.25*

Elemene 30 mg/L

1.58±1.92

1.91±1.07

13.69±2.72

28.75±4.05*

Elemene 40 mg/L

1.58±1.92

17.66±15.53*

21.57±6.35*

41.81±11.08*

*P<0.05, compared with control group

 

With the same concentration, as the prolongation of drug treating time, the apoptosis rate of tumor cells tends to increase. Taking C6 glioma cell as an example, after treatment of 30mg/L elemene solution for 0 (control), 1, 3 and 5 days, the apoptosis rate was respectively 1.58%, 1.91%, 13.68% and 28.75%similarly with C6 glioma cellafter treatment with 40mg/L elemene solution for 0 (control), 1, 3, 5, 7 and 9 days,the apoptosis rate of SHG-44 cells was respectively 1.18%, 2.84%, 2.29%, 9.59% and 39.18% (Fig. 2).

 

 

Fig.2 Flow cytometry analysis of C6/SHG-44 glioma cells apoptosis after treatment with elemene

C6 control (A) and SHG-44 control (D), no apoptotic peak; C6 cells treated with 30mg/L elemene for 3d (B), 5d (C) and SHG-44 cells treated with 40 mg/L elemene for 5d (E), 9d (F), typical subdiploid peak before Phase G0/G1 was detected, and the rates of apoptosis were in a time-dependent manner.

2.4 DNA Gel Electrophoresis

The DNA of C6 glioma cell treated with elemene is isolated and examined by electrophoresis in agarose gel containing EB. While the DNA of control group is a single trap, that of cells treated with elemene disaplays characteristic ladder (Fig. 3).

 

Fig. 3 DNA electrophoresis in C6 glioma cells treated with elemene

1. Marker; 2. C6 control D3; 3. 30mg/L elemene D3; 4. 30mg/L elemene D5; 5. 20mg/L elemene D5; 6. C6 control D5

 

3 Discussions

Elemene is a new non-cytotoxic anti-tumor drug which is independently developed by China; it has unique effect on the improvement of immune function, characterized by specific target, zero inhibition against bone marrow, and low drug tolerance [6]. The drug is small molecular chemical, which is able to beach through blood brain barrier, so it proper for treatment of brain cancer. It is found that elemene has significant effect on inhibition of glioma through clinical trail and experiments in vitro [1,2], but there is no further reports on related basic researches recently. The study probed into the proliferation inhibitory effect of elemene on glioma, and discussed the mechanism in terms of apoptosis inducement.

 

It is proven that apoptotic cell density tends to decrease in human astrocytoma with the rise of malignant grade, which implicates that the inhibition of cellular apoptosis might play an important role during the growth and development of glioma [7,8]. Selective inducement of tumor cell apoptosis has become a new target of tumor biotherapy.

Elemene can effectively inhibit the proliferation of glioma cells at a relatively low concentration. The proliferation inhibitory effect on glioma is dependent on dosage, that is, with the increase of drug concentration, the inhibition of elemene gets stronger. With the assumption of the same concentration, the inhibitory effect relies on period. As the prolongation of action period, the effect is enhanced, which is revealed by the reduction of RA53 value. The result can be demonstrated through analysis of flow cytometer.

Hoechst 33258 and PI are fluorescent dyes combined with DNA, which can generate yellowish green and red fluorescence if excited. The structure of nuclear can be clearly illustrated with fluorescent microscope. The morphologic changes of apoptotic cells are observed with fluorescent staining analysis in the experiment, which proves that staining of glioma cellular nuclear is significantly enhanced, nuclear chromatin condensed and closer to nuclear membrane, and DNA fractured to pieces and formation of apoptotic bodies after treatment of elemene. While the Nucleoplasm DNA of normal cells is loose and homogeneous, and the staining of nuclear is thin and even after fluorescent staining. Flow cytometry analysis displays that the natural apoptosis rate of C6/SHG-44 cells is lower without treatment, cellular apoptosis can be significantly induced after a period of treatment with elemene of various concentrations. An obvious peak of apoptosis can be seen before G0/G1 peak from the histogram of DNA concentration, with characteristic difference compared with that of controlled group. An obvious ladder can be observed from DNA gel electrophoresis of C6 glioma cell after treatment with elemene, while there is no DNA ladder seen from that of controlled group. 

As mentioned above, the study demonstrated the mechanism of apoptosis inducement participates in the proliferation inhibitory effect of elemene from different aspects, such as morphologic observation, flow cytometry analysis and DNA biochemical alteration. It should be noted that the inducement of cellular apoptosis largely occurs in three days, and becomes more significant in five days, which implies that the concentration and action period of drug should be increased if possible during clinical practice.

 

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