Abstract
OBJECTIVE To explore the inhibitory effect of arsenic trioxide on growth and telomerase activity of BEL-7402 and SMMC-7721 hepatocarcinoma cells, and to measure their GSH level.
METHODS Cell culture and trypan blue exclusion were used to examine the inhibitory effect of arsenic trioxide on BEL-7402 and SMMC-7721 hepatocarcinoma lines. A GSH kit and telomerase kit were used to measure the GSH content in cells and telomerase activity.
RESULTS The growth of BEL-7402 cells was significantly inhibited at a level of 0.50 μmol/L of arsenic trioxide by 24 h. The inhibitory effect increased with time and concetration of arsenic trioxide. The telomerase activity of BEL-7402 cells was also significantly inhibited at a level of 0.50 MITIOI/L of arsenic trioxide by 24 h, after which the inhibitory effect increased with time. On the other hand, at 24 h of incubation a level of 2.00 μmol/L of arsenic trioxide was required to significantly inhibit growth of SMMC-7721 cells, and only after 48 h with 2.00 μmol/L of arsenic trioxide did telomerase activity significantly decline. The GSH content of the BEL-7402 and SMMC-7721 cells was 18.7±1.4 and 50.8±5.2 nmol/mg protein respectively, a significant difference.
CONCLUSION Different concentrations of arsenic trioxide are required to inhibit growth and telomerase activity of SMMC-7721 and BEL-7402 cells. Perhaps BEL-7402 cells are more sensitive to arsenic trioxide because of their low level of GSH content, which results in a low capacity for oxidation reduction and poorer detoxification mechanisms in BEL-7402 cells.
keywords
Arsenic trioxide, a poisonous material for living things, is the main component of white arsenic of Chinese Traditional Medicine. As reported by Zhang [1] in 1971, arsenic trioxide was used to treat patients with acute promyelocytic leukemia (APL), resulting in successful therapy. The pilot study showed that the main mechanism for arsenic trioxide to cure APL was to induce apoptosis and differentiation of the APL cells.[2][3] In recent years, it was found that growth of some hepatocarcinoma cells could be inhibited by an apoptotic mechanism.[4] Our study was undertaken to determine the sensitivity of hepatocarcinoma cells to the killing effects of arsenic trioxide.
The sensitivity of the cells to arsenic trioxide, as well as possible mechanism of its action on the BEL-7402 and SMMC-7721 hepatocarcinoma cell lines were explored by measuring cellular telomerase activity and GSH levels.
Materials And Methods
Arsenic Irioxide
Arsenic trioxide was purchased from the Harbin Yida Limited Co., and diluted to a concentration of 0.5 mol/L when used.
Cell culture
The hepatocarcinoma cell lines, BEL-7402 and SMMC-7721, were obtained from the Tumor Hospital of Zhongshan University. The cells were incubated in RPMI 1640 supplemented with 5% new bom bovine serum, 100 U/ml penicillin and 100 mg/L streptomycin at 37 °C, under 5% C02, and saturation humidity. The medium was changed every 2-3 days and when the cell density reached 80% confluency, the cells were sub-cultured. Log-phase cells were used in the study.
Treatment of cells with arsenic trioxide
The cells were treated with arsenic trioxide concentrations of 0.0, 0.25, 0.50, 1.00 and 2.00 p,mol/L. When the cells doubled, 1/2 of the corresponding concentration of arsenic trioxide was added into the culture. The experiments were performed in 3 wells for each concentration on 24-well culture plates. The effect of arsenic trioxide on growth and telomerase activity of the hepatocarcinoma cells were determined in triplicate at different time points (0, 24, 48, 72, 96 h).
Cell count and inhibitory effect
The adherent layer of cells from the control (no arsenic trioxide) and treated groups were digested with 0.25% trypsin, centrifuged and resuspended in RPMI 1640 medium. The cells were stained with 0.4% trypan blue and the viable cells counted microscopically. Each group consisted of 3 replicate wells. The inhibitory rates for each group were calculated as follows: Inhibitory rate(%)= [cell no. of control group—cell no. of group treated with arsenic trioxide] cell no. of control group.
Telomerase activity assays
Telomerase activity was measured using a telomerase PCR ELISA assay kit (Boehringer Mannheim). The assay was conducted following instructions in the kit. Briefly,[5] cell extracts were prepared at different times of PS-ODN treatment. Positive and negative control groups were established for each experiment; cell extracts were heated to 65 °C for 10 min for a negative control, and an extract of the 293 cell line having telomerase activity as a positive control (from kit). The telomeric repeat amplification protocol (TRAP reaction) was conducted follows: (1) primer elongation : transfer 25 xl reaction mixture into a tube suitable for PCR amplification, then add 2 p,l of cell extract and sterile water to a final volume of 50 xl; transfer the tubes for thermal cycling and perform one cycle at 25 °C for 30 min; (2) telomerase inactivation: perform one cycle at 94 °C for 5 min; (3) amplification: 50 p.1 reaction mixture contains dNTP, Taq polymerase, the biotin-labeled Pl-TS primer and P2 primer. Perform 30 cycles, at 94 °C for 30 s for dénaturation, at 50 °C for 30 s for annealing, at 72 °C for 90 s for polymerization, then at 72 °C for 10 min for balance. Hybridization and ELASA procedure: transfer 20 |xl dénaturation reagent into a suitable tube, then add 5 pi of amplification product, incubate it at 20 °C for 10 min, add 225 pi hybridization buffer (containing DIG-POD) and mix thoroughly by briefly vortexing. Transfer 100 pi of the mixture on a nuclease-free microtiter plate and shake it at 37 °C for 2 h. Add 100 pi anti-DIG-POD at room temperature for 30 min. Then, add 100 pi TMB substrate solution and incubate it for 10 min at room temperature for color development, followed by the adding of 100 pi stop reagent to prevent further reaction. The absorbance was determined at 450-655 nm wave-length in order to calculate A=A450-A655.
Assay of GSH content in hepatocarcinoma cells
The assay of GSH content in the hepatocarcinoma cells was performed with a Cay GSH assay kit (Promega Co.). In brief, 5×l06 cells were collected by centrifugation and resuspened in PBS (1 mmol/L EDTA), and following recentrifugation, the supernatant discarded. Metaphosphoric acid was added to the cells, which were swirled gently to prepare a uniform cell suspension. Five min later, the cells were centrifuged at 4000 xg at 4°Cfor 10 min and the supernatant collected. The absorbancy of the supernatant was determined at A405nm, and the GSH content calculated as nmol/mg protein.
Statistical analysis
An independent-sample t test using SPSS software was used to analyze our results.
Results
Effect of different concentrations of arsenic trioxide on growth of BEL-7402 and SMMC-7721 heptocarcino-ma cells.
As shown in Table 1, there was a significant decrease in growth of BEL-7402 heptocarcinoma cells treated with arsenic trioxide at concentrations of 0.5-2.0 xmol/L for 24 h, and at 0.25 imol/L for 96 h. However, there was no significant effect on growth of SMMC-7721 cells treated with 0.25-0.50 xmol/L of arsenic trioxide up to 96 h. When the level of arsenic trioxide was increased to 2.00 (xmol/L for 24 h, there was a significant inhibitory effect on growth of SMMC-7721 cells (P<0.05), which showed an increase in effect with time. It can be seen in Table 1, in comparing the 2 cell types treated with different levels (0.25, 0.50, 1.00, 2.00 p,mol/L) of arsenic trioxide for 72 h, that the inhibitory effect on the BEL-7402 cells was much greater than the effect on the SMMC-7721 cells (P<0.01).
Effect of different concentrations of arsenic trioxide with time on cell growth of BEL-7402 and SMMC-7721 hepatocarcinoma cells (x±s) xl05/mL
Effect of different concentrations of arsenic trioxide on telomerase activity of BEL-7402 and SMMC-7721 heptocarcinoma cells
Table 2 shows there is no effect of arsenic trioxide on BEL-7402 cells at a concentration of 0.25 μmol/L over a 48 h period. At the longer times of 72-96 h, telomerase activity started to decline (P>0.05). However, at a level of 0.50 μmol/L, there was a significant inhibition of telomerase activity at 24 h (P>0.05), with the inhibition increasing with time up to 96 h. At a level of 1.00 μmol/L or above, telomerase activity of the BEL-7402 cells showed a time and concentration inhibitory effect of arsenic trioxide.
For concentrations of 0.25~0.50 μmol/L, there was no inhibitory effect of arsenic trioxide on telomerase activity of SMMC-7721 cells during the 24-96 h period (F>0.05). At a level of 1.00 pumol/L, it required 96 h for telomerase activity to decline significantly (P> 0.05). At a level of 2.00 p,mol/L, 48 h were needed for telomerase activity to decrease significantly (E>0.05), as shown in Table 2. Comparaison of the 2 cell types at an intermediate arsenic trioxide level of 0.50 μmol/L, showed there was an inhibitory effect of arsenic trioxide on telomerase of the BEL-7402 cells at 24 h, which increased with time. However, there was no inhibitory effect on the telomerase activity of the SMMC-7721 cells at any time at this concentration of arsenic trioxideTable 2.
Effect of different concentrations of arsenic trioxide on telomerase activity of BEL-7402 and SMMC-7721 heptocarcinoma cells (x±.s)
GSH contents in BEL-7402 and SMMC-7721 cells
As shown in Table 3, there was a big difference in the amount of GSH in BEL-7402 verus SMMC-7721 hepatocarcinomar cells. SMMC-7721 cells contained 50.8 ±5.2 nmol/mg protein whereas BEL-7402 cells had only 18.7 ±1.4 nmol/mg protein. The amount of GSH in SMMC-7721 cells was 2,7 times that of BEL-7402 cells.
Comparison of GSH content in BEL-7402 verus SMMC-7721 cells (x±s, n=3)
Discussion
In the present study, we found that arsenic trioxide at a level of 0.25 jiinol/L and incubation for 24 h significantly inhibited the growth of BEL-7402 hepatocarcinoma cells and depressed telomerase activity. However, for the SMMC-7721 hepatocarcinoma cells, a concentration of 2.00 μmol/L and incubation for 48 h was needed to inhibit growth, ie, a 8 fold concentration and longer time. Although the BEL-7402 and SMMC-7721 hepatocarcinoma cells are both liver carcinomas, there is a difference in sensitivity to arsenic trioxide. This finding would suggest that it is necessary to determine if a carcinoma is sensitive to arsenic trioxide prior to its use for therapy.
As reported Chen et al.,[6] the concentration needed in blood for APL therapy is 0.50~3.0 μmol/L, a level which is considered safe. In our study, the effective concentration for arsenic trioxide to inhibit growth of SMMC-7721 cells was 0.5~2.0 μmol/L, which is believed to be safe for arsenic trioxide and is used in clinical applications.
There is no telomerase activity in normal human liver cells, however, there exists high telomerase activity in 85% of hepatocarcinoma cells, and the degree of malignancy is in proportion to telomerase activity. As reported in recent years, inhibition of telomerase activity increases the sensitivity to chemotherapy-drugs.[7][12] Our study showed that arsenic trioxide inhibited the telomerase activity, and demonstrated the difference between BEL-7402 and SMMC-7721 cells. This phenomenon is parallel to the effect of arsenic trioxide on the growth of hepatocarcinoma cells.
Transformation of hepatocytes into carcinoma cells involves changes in gene expression. If the oxidation-reduction system in cells is strengthened, especially the GSH system, detoxification capacity is strengthened. Our results show that there is big difference between the GSH level in SMMC-7721 (GSH: 50.8±5.2) and BEL-7402 (GSH: 18.7+1.4) cells, which influences their sensitivity to arsenic trioxide.[13]
Arsenic trioxide has very strong affinity for sulfhydryl groups,[14][15][16] which serve as an important chemical receptors for arsenic trioxide resulting in a lowing mitochondrial transmembrane potential and inducing apoptosis.[17] Arsenic trioxide causes oxidation of sulfhydryl groups, which inhibits mitochondrial function and enhances apoptosis.1'81 We believe the difference in GSH content in cells is the important reason for the difference in sensitivity of the BEL-7402 and SMMC-7721 cells to arsenic trioxide. However, further studies are needed to verity this possibility.
Footnotes
This work was supported by the grant form the Natural Foundation of Guang-dong Province(No.021195)and Guangzhou City(No.2001-Z-037-01).
- Received November 23, 2005.
- Accepted April 21, 2006.
- Copyright © 2006 by Tianjin Medical University Cancer Institute & Hospital and Springer
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