Study on arsenic trioxide inducing in vitro apoptosis of gastric cancer cell lines

INTRODUCTION

Cell apoptosis, which involves the biologic regulation of the numbers and vital activity of cells, is an important metabolic process in both normal cells and tumor cells. Delayed process of cell apoptosis will probably lead to a disturbance of metablism, and occurrence and development of neoplasms. Song et al[1] have proved the relationship between apoptosis delay and tumor development through inhibition of cell apoptosis induced by tumor promotor. Thus, induction of cell apoptosis could be a new strategic measure against tumor. In this paper, we studied whether arsenic oxide will induce apoptosis of gastric cancer cell lines (GCCL) to explore the use of such chemical agent against gastric cancer in clinic.

MATERIALS AND METHODS

Target cells

Human gastric cancer cell lines, MKN45 and SGC7901 (provided by Chinese Academy of Sciences) were used as target cells. Human leukemia cell line K562 and human peripheral blood lymphocytes (PBL) were used as controls. All of cells were cultured in RMPI 1640 medium (GIBICO-BRL), supplemented with 10% heat-inactivated fetal calf serum, 100 U/mL penicillin and 100 mg/mL streptomycin, in a humidified atmosphere of 95% air/5% CO₂ at 37 °C. The numbers of four kinds of cells were maintained at 1 × 10⁶/mL by daily adjusting cell concentration.

Product of arsenic trioxide (As₂O₃)

Ai-Lin No. 1 containing As₂O₃ was prepared by pharmacy of our hospital. Stock solution was made at the concentration of 1 mmol/L with phosphate-buffered saline (PBS) and diluted with RMPI 1640 to working concentration before use.

Cytocide test

MTT [3-(4,5-dimethythiazoyl-2-yl)2,5-diphenylte-trozoliumbromide] colori metric analysis was used to measure the cytocide rate of As₂O₃. Well grown cells were collected and were put into 96 well culture plate at 1 × 10⁴/well. As₂O₃ was added to culture plate at the concentration of 5 μmol/well, while cells stuck on wall, 6 duplicate wells were set up in each sample in both experimental and control groups. Cells were cultrured for 24 h, 48 h and 72 h separately, MTT(5 mg/mL) was add ed at 10 μL/well. Acidulated isopropanol was finally added at 100 μL/well-after 2 h culture; values of OD570 were obtained on autocolorimeter. Cytocide rate was calculated by the following formula:

Cytocide rate (%) = [(Control OD - Experimental OD)/Control OD] × 100%

Apoptosis detection

Cell treatment MKN45 and SGC7901 cells of 10⁶ were treated with As₂O₃ of the concentration of 5 μmol and 10 μmol, or with 5-Fu (0.5 g/L), or without any treatment as control. Cells were harvested after 24 h, 48 h and 72 h culture for apoptosis detection.

TDT label Consulting Gregory modified method[2]: PBS containing 1% formalin was added to cells at 4 °C. Thirty min later, it was washed twice with PBS, then reacted with 0.5 μg terminal deoxynucleotidyl transferase (TDT) and 0.5 μmol biotinylted dUTP at 37 °C for 30 min. Afterwards it was washed again with PBS, finally labelled by affinitin-fluorescin isothiocyanate (FITC) at room temperature for 30 min. DNA strand will be broken off during apoptosis, which can be labelled by dUTP and the positive observable fluorescences were seen under fluorescent microscope.

Flow cytometry (FCM) assays and fluorescent photographing The stimulating wave length of FCM (Becton Dickson FAC Scan)is 488 nm, FITC detection spectrum is between 80 and 630 nm. A total of 2000 cells were counted. Results were recorded and analyzed automatically.

Preparation of specimen for electronic microscopy MKN45 cells treated with As₂O₃ of concentration of 10 μmol for 72 h were washed with PBS and fetal calf serum; 25% glutaraldehyde was added to fix the cell specimens for 12 h. Then it was washed twice with 0.1 mol/L phosphoric buffer solution; the specimens were fixed with 1% osmi c acid, and were dehydrated step by step with alcohol and acetone; then covered up with Epon 812 epoxy vesin. Sections were made by ultramicrotome and dyed with uranium acetate and citric acid, and finally observed under transmission electronic microscope.

RESULTS

Cytocide rate of As₂O₃ on GCCL

Our results showed that As₂O₃ had higher cytotoxicity on GCCL than on K562 (P < 0.05). The effect of cytocide was observed at 24 h after reaction, and it increased with time (Table 1).

Table 1.

Cytocide effects of As₂O₃ (5 μmol) on various cell categories (%,^-x ± s)

Group	MKN45	7901	K562	PBL
24 h	69.45 ± 11.12	68.27 ± 8.27	51.36 ± 10.25a	30.31 ± 5.12b
48 h	71.40 ± 10.15	69.04 ± 11.31	57.11 ± 7.45a	35.67 ± 4.37b
72 h	80.53 ± 10.18	82.74 ± 9.14	65.33 ± 8.24a	39.74 ± 8.27b

Comparison of cytocide rate between GCCL and controls by As₂O₃,

^aP < 0.05 (t > 2.2262),

^bP < 0.01 (t > 3.250)

GCCL apoptosis rate induced by As₂O₃

As shown in Table 2, apoptosis rates of 2 kinds of GCCL induced by 5-Fu were also obviously higher than natural cell apoptosis rate, of controls, which suggested that antitumor drug can kill tumor cells by inducing cell apoptosis. Nevertheless, a more significant cytocide effect of As₂O₃ on GCCL was demonstrate d in our study compared with that of 5-Fu. We found that apoptosis rate of GCCL induced by As₂O₃ is correlated with the concentration and reaction time of As₂O₃ (Table 3).

Table 2.

Apoptosis rate (%) of MKN45 by different treatment methods (^-x ± s)

Group	Controls	5-Fu	As2O3 1	As2O3 2
24 h	4.71 ± 0.36	18.04 ± 1.50	32.33 ± 3.75a	60.02 ± 7.41b
48 h	14.90 ± 0.94	24.39 ± 3.45	90.59 ± 10.35b	97.05 ± 8.24b
72 h	28.98 ± 3.12	41.47 ± 2.24	98.10 ± 13.24b	98.75 ± 11.53b

Comparison of apoptosis rate between As₂O₃ and 5-Fu group,

^aP < 0.05 (t > 2.2262),

^bP < 0.01 (t > 3.250).

Table 3.

Apoptosis rate (%) of SGC7901 by different treatment methods (^-x ± s)

Group	Controls	5-Fu	As2O3 1	As2O3 2
48 h	7.44 ± 0.60	34.28 ± 4.15	51.33 ± 5.16a	78.31 ± 9.14b
72 h	30.90 ± 2.54	56.32 ± 4.56	78.15 ± 6.78a	89.28 ± 10.26b

Comparison of apoptosis rate between As₂O₃ and 5-Fu group,

^aP < 0.05 (t > 2.2262),

^bP < 0.01 (t > 3.250)

The morphology of GCCL apoptosis under fluorescent microscope:

Under fluorescent microscope, the apoptosis cell can be seen after being terminally labelled (positive), but nonapoptotic cells were not labelled by fluorescin isothiocyanate (negative). The positive staining showed bright green fluorescence. Fluorescent spots appeared in early stage, and these fluorescent bodies gathered like a bunch of grapes in late stage. The cellular volume can be seen shrunken under the microscope.

The morphology of GCCL apoptosis under electronic microscope:

Under the transmission electronic microscope, typical morphologic changes of apoptotic GCCL (mainly cell nucleus) took place after treatment of As₂O₃. These changes included cell nucleus fixation and shrinkage of GCCL, chromatin condensation, and fragmentation of apoptotic bodies. These changes coexisted.

DISCUSSION

Some researchers have proposed that the uncontrolled growth of neoplasms would b e due to the loss of the nature of autoapoptosis rather than over proliferation. Previous studies have proved that there existed autoapoptosis blockage in tumor cells. Lauwers et al[3] examined bcl-2 gene in 46 cases of gastric adenocarcinoma by immunochemical method, revealling 75% positivity. Of bcl-2 gene in tumor tissues, which indicated that apoptosis was blocked in gastric cancer. Bcl-2 has been considered as one of survival genes which plays an important role in the specific-inhibition of tumor cell apoptosis[4]. Based on these findings, a new proposal of inducing apoptosis to inhibit tumor growth was introduced[5]. Many factors such as high temperature, cytokine, radiations and all kinds of anti-tumor chemotherapy drugs have a certain effect on inducing tumor cells apoptosis. But some of these are not satisfactory. Our aim is to find a specific-agent which can induce apoptosis of tumor cells. Vollmers et al[6] reported the suppressive effects of monoclonal antibody (SC-1) on both in vitro proliferation of gastric cancer cell line and growth of a tumor inoculated on nude mice. The inhibition of proliferation of tumor cells was produced through the induction of autoapoptosis, which has been proved by the observation of ultrastructure.

Arsenic is a major composition of traditional Chinese medicine, white arsenic. White arsenic has been considered as a carcinogen. It can inactivate some important enzymes in cells, change the metabolic process and induce chromosome aberration[7]. Zhang et al[8] reported a satisfactory result by using As₂O₃ for the treatment of early acute promyelocytic leukemia (APL). Complete remission was 73.3% in patients after the first therapeutic course, and 52.83% in recurrent patients. The longest remission period of APL patient was over 10 years. No obvious toxic reactions were found when As₂O₃ was given by iv drip, which is appropriate. Similar result has also been obtained by researchers at Shanghai Institute of Hematology of Shanghai Ruijin Hospital[9,10]. Recently, Zhang et al[11] demonstrated that Arsenic Oxide can inhibit growth of lymphosarcoma cells and induce apoptosis to these cells.

Based on the above studies, we applied As₂O₃ for the treatment of GI solid tumor. The results from in vitro study are impressive. Proliferation of MKN45 and SGC7901 was inhibited by As₂O₃ through apoptosis induction. Results also showed that As₂O₃ has a stronger effect of apoptosis induction than 5-Fu. Induction of apoptosis was enhanced with increase of concentration and time of As₂O₃. The question is what the optimal dosage is for clinical use so as to produce maximal effect with no toxicity. Further comprehensive researches are needed to clarify the significance of As₂O₃ for the treatment of GI solid tumors.