Introduction

In addition to the classical genetic mechanisms of deletion or inactivating point mutations, growth regulatory gene can be functionally inactivated by promoter CpG island methylation without alterations of the primary sequence.[1-7] CpG island comprises stretches of DNA approximately 1 kb long that are rich in dinucleotides. The CpG site in these gene-associated regions is rarely methylated in normal cells with the exception of CpG island of genes on the inactivated X chromosome and CpG associated with imprinted genes.[8-11] It is now accepted that abnormal methylation is not restricted to cultured cells but also occurs during aging and tumor development.[12] The mechanism by which hypermethylation of selected CpG island occurs in cells remains to be elucidated. However, it has been shown that unmethylated CpG islands associated with a variety of genes become partially or fully methylated in tumors, leading to the abnormal expression of mRNA and protein.[13-18] This methylation status can be reactivated by 5-aza-2-deoxycytidine (ZdCyd).[19-23] ZdCyd, a methylation inhibitor, can eliminate the methylation status of tumor suppressor gene promoter, resulting in the re-expression of these genes and tumor inhibition.[24-26] Although the tumor suppressor gene is methylated, the DNA sequence is unchanged and this kind of change is reversible. This makes it possible that a methylated tumor suppressor gene can be demethylated and reactivated by ZdCyd for the purpose of cancer therapy.

ZdCyd has been used in the chemotherapy for a varity of cancers such as leukaemia.[27-29] Some studies discovered recently that many tumor suppressor genes were methylated and inactivated, suggesting that the promoter methylation of tumor suppressor gene may play an important role in the occurrence and progress of bile duct cancer. Since the surgical removal rate is low for bile duct cancer and the drugs used for chemotherapy are less effective,[30,31] new chemotherapeutic drugs are in pressing need. It is a possible way for bile duct cancer therapy via elimination of the tumor suppressor gene methylation status with ZdCyd. By now, there is still no systemic study of ZdCyd in the treatment of bile duct cancer. We studied the inhibitory effect of ZdCyd on bile duct cell line in vivo and in vitro and its possibility in chemotherapy of bile duct cancer.

Methods

Cell line and reagents

Bile duct cell line QBC939 was kindly provided by Professor ShuGuang Wang from the Third Military Medical University, Chongqing, China, and was kept in RPMI1640 medium supplemented with 10% fetal calf serum (FCS) at 37 ℃ in a 5% CO₂ incubator. ZdCyd was bought from Sigma, USA, and dissolved with dimethylsulfoxide (DMSO).

Inhibitory effect of ZdCyd on QBC939

QBC939 cells were placed in RPMI1640 supplemented with 10% FCS at 37 ℃ in a 5% CO₂ incubator, and digested with 0.25% trypsin in the log phase to make a single cell suspension with a concentration of 2?/SPAN>10⁵/ml counted under a microscope. The cells were planted into a 96-well plate with 200 μl per well. The blank well was only added with RPMI1640 medium without cells. The cells were incubated for 18 hours under the above conditions, then the cell culture medium was renewed, and added with ZdCyd with the final concentrations of 0.1 μmol/L, 0.5 μmol/L, 1.0 μmol/L, 5.0 μmol/L, 25 μmol/L, and 125 μmol/L. The cells of each concentration were added into 5 wells, and the control group was only given renewed culture medium without ZdCyd.

The plate was incubated in an incubator for additional 4, 12, 24, 36, 48 hours separately and then added with 20 μl methyl thiazoy tetrazolium (MTT) per well with the concentration of 5 mg/ml. The medium was discarded after incubation for additional 4 hours, and added with 150 μl DMSO per well. The optical density (OD) level under 490 nm was measured and the cell survival rate was calculated with the following formula: cell survival rate (%)=(OD level of experimental group-OD level of blank group)/(OD level of control group-OD level of blank group)?/SPAN>100%.

Cooperative effect of ZdCyd and other chemotherapeutic drugs

QBC939 cells was cultured and single cell suspension was made similarly. The cells were divided into two groups A and B. In group A, cells were treated with different concentrations of 5-FU, adriamycin (ADM), cisplatin; and in group B, 1.0 μmol/L ZdCyd was added into each subgroup. MTT was used, and the cell survival rate was calculated with the same formula.

Evaluation of cell apoptosis rate and cell cycle change by flow cytometry after use of ZdCyd

QBC939 cell suspension was made according to the process of MTT, and then planted into a 6well plate. ZdCyd was added after the cells were incubated for 18 hours. The groups of different ZdCyd dosage and different time were set up as those of the process of MTT. Cells were harvested using trypsin, washed twice with PBS, and then added with 70% cold ethanol which was pre-cooled at -20 ℃ drop by drop. The cells were subsequently stained with propidium iodide and the cell cycle and apoptosis rate were detected by flow cytometry.

Inhibitory effect of ZdCyd on QBC939 in nude mice

Twenty-five healthy nude mice of 4 weeks old were chosen and divided randomly into 3 groups A, B and C, and the number of mice of each group was 10, 10, 5 respectively. In group A, QBC939 was treated with 5 μmol/L ZdCyd for 24 hours, and single cell suspension was made with a concentration of 1?/SPAN>10⁷/ml; 0.2 ml of the suspension was injected subcutaneously into the flanks of the nude mice. In group B, all were the same except that the cells were not treated with ZdCyd before injection into the nude mice. In group C, 0.2 ml PBS solution was injected instead of the cells. After 6 weeks, when the tumor occurred, group B was subdivided into 2 groups equally and randomly. One group was injected with ZdCyd 2 μg/g (weight)?/SPAN>day, and the other was injected with saline instead. The mice were kept in sterile conditions and the size of tumors was measured every week with the following formula: tumor volume=transverse diameter²?/SPAN>largest diameter/2.

Statistical analysis

Statistical software SPSS was used, and a P value less than 0.05 was considered significant.

Results

Effect of ZdCyd on QBC939 proliferation

The cells were observed under an inverted microscope after different dose of ZdCyd was added. The cells treated by ZdCyd were found shrank, dead and had a lower density (Figs. 1-3). The effect of ZdCyd on cell proliferation was dose and time-dependent. When the dose was 0.5 μmol/L, the inhibitory effect appeared, and the IC₅₀ for 24 hours was 5.0 μmol/L. The effect was markedly increased with increased dose and prolonged time.

Cooperative effect of ZdCyd and other chemotherapeutic drugs

The IC₅₀ of 5-FU, ADM was 0.25 mg/ml and 12.5 μg/ml respectively when 1.0 μmol/L ZdCyd was added. The cell survival rate decreased to 31% and 20% respectively for 5-FU (0.25 mg/ml) and ADM (12.5 μg/ml). As to cisplatin, it had no effect on cell proliferation (Fig. 4).

Fig. 1. Effect of ZdCyd on QBC939 proliferation.

Fig. 2. The cells not treated with ZdCyd under an inverted microscope (original magnification?/SPAN>200).

Fig. 3. Treated with 5 μmol/L ZdCyd for 24 hours, the cells were shrunk, dead and decreased in density (original magnification?/SPAN>200).

Fig. 4. The cooperative effect of ZdCyd with other chemotherapeutic drugs.

Fig. 5. Flow cytometry results of the cells untreated with ZdCyd.

Fig. 6. Flow cytometry results of the cells treated with 5 μmol/L ZdCyd for 24 hours.

Effect of ZdCyd on QBC939 cell cycle and apoptosis rate

ZdCyd blocked the cell cycle at G1 phase and increased the apoptosis rate. The effect characterized by dose dependence is most significant at 5 μmol/L. However, when the concentration reached 125 μmol/L, most of the cells showed necrosis instead of apoptosis (Figs. 5 and 6, Table).

Effect of ZdCyd on QBC939 in vitro

The tumor occurrence rate was 20% (2/10), 100% (10/10), and 0% (0/5) for groups A, B, C respectively in the flank of the nude mice in 6 weeks after QBC939 cell injection, and the tumor size varied from 5 mm³ to 2700 mm³. In group B, tumor growth of the subgroup treated with ZdCyd was slow down, and some of the tumors became smaller, even disappeared (one tumor). In the subgroup treated with saline, tumor growth was not stopped and 2 mice died of cachexia in additional 6 weeks.

Discussion

A recent examination of more than 600 primary tumor samples of 15 tumor types showed that CpG island promoters of three or more genes were hypermethylated in 5%-10% of the samples.[32] At least one CpG island was methylated in 80% or more of samples for each tumor type. Using methods that allow genome wide screening of CpG islands has estimated that, on average, 1% of CpG islands in DNA from tumor tissues are abnormally methylated.

Caca et al[33] studied 9 bile duct cell lines and 21 cases of primary extrahepatic bile duct cancer and found that P16 expression inactivated in all the cell lines and in 11 of the 21 cases of primary extrahepatic bile duct cancer. In the 9 cell lines, 8 were inactivated by loss of heterozygosity (LOH) in chromosome 9P21, and the other was inactivated by promoter region hypermethylation. In 11 cases of bile duct cancer, however, 9 cases were inactivated by promoter methylation and they were P16 inactivated. The reported rates of P14, P16 promoter methylation in 51 cases of cholangicarcinoma were 25%, 76% respectively, shown by the method of methylation specific PCR (MSP-PCR).[34] Moreover, mRNA expression was lost in all the cases in which methylation occurred.

In our study, ZdCyd inhibited the proliferation of bile duct cancer cell line QBC939 at the concentration of 0.5 μmol/L, and this effect increased with increased drug doses and prolonged time in vitro. When the dosage reached 125 μmol/L, almost all the cells died in 24 hours, suggesting that ZdCyd could inhibit the proliferation of bile duct cancer cells. We also found that ZdCyd could have the cell cycle blocked at G1 phase and the number of apoptosic cells increased. The apoptosis rate was 49.01% when the drug dosage was 25 μmol/L. The survival rate decreased as long as the dosage increased, and the apoptosis rate decreased too. Broken cells and lots of cell pieces were found microscopically as the characteristics of cell necrosis. These findings were similar to cell apoptosis in low dosage and necrosis in high dosage of other drugs.

The curative effect of traditional drugs for the treatment of bile duct cancer is low when they were used singly.[35] We tried to combine ZdCyd with traditional drugs in dealing with bile duct cell proliferation and found that the cell proliferation inhibitory effect of the combination therapy is more powerful than that of single drug, suggesting that there is a cooperative killing effect on bile duct cancer cells. Possibly, ZdCyd may reactivate the inactivated tumor suppressor gene, while eliminating the methylation status of the gene promoter region and changing the cell cycle. ZdCyd could also inhibit the proliferation of bile duct cancer cell line QBC939 in vitro. In this study, the tumor occurrence rate of the pre-treated cells in nude mice was much lower than that of the untreated cells (20% vs 100%), although the number of cells injected was identical (2?/SPAN>10⁶ cells/per mouse). ZdCyd also prevented tumor growth in nude mice, and reduced tumor size after treatment with 2 μg/g per day in contrast to the control mice.

We conclude that ZdCyd can inhibit the proliferation of bile duct cancer cell line QBC939, block cell cycle at G1 phase, and induce cell apoptosis in vivo. Also it can prevent tumor recurrence and growth in nude mice, and its clinical implications await study.

Competing interest

No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

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Received September 2, 2003

Accepted after revision November 23, 2003