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Induction of biliary cholangiocarcinoma cell apoptosis by 103Pd cholangial radioactive stent γ-rays

HE, Gui-jin; SUN, Dan-dan; JI, Da-wei; SUI, Dong-ming; YU, Fa-qiang; GAO, Qin-yi; DAI, Xian-wei; GAO, Hong; JIANG, Tao; DAI, Chao-liu

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Abstract

Extra-hepatic cholangiocarcinoma and ampullary carcinoma primarily infiltrate local tissues. Thus, the malignant obstructive jaundice caused by the carcinoma develops progressively. Dealing with this jaundice in clinical practice is difficult, and the surgical excision rate is very low (only 10%-20%).1 The main causes of death are local infiltration and obstructive jaundice; the key to improve the quality of life and to prolong the life span is to control the growth of tumor. Although radiotherapy with a high dosage (55 Gy) in vitro can prolong the life span of patients,2,3 it is difficult to accumulate the radioactive materials up to the required dosage as there are many radiation-sensitive organs (e.g., the liver, kidney, and gastrointestinal tract). In recent years, interventional therapy of tumors has involved the implantation of intra-cholangial metal stents through percutaneous trans-hepatic puncture. This has provided a new method for treating cholangiocarcinoma, as it allows delivery of higher radioactive dosages, and over a shorter distance.4103Pd cholangial radioactive stents can concentrate a high radioactive dosage in the malignant tumor, while simultaneously having only a small effect on the surrounding normal tissues. As such, a stent can effectively kill tumor cells, and prevent re-stenosis of the lumen caused by a relapsed tumor. Thus, without surgical drainage, we can both improve the tumor control rate and reduce the rate of invasive surgery. In addition, the quality of life and the survival rate of patients are dramatically improved. The aim of the present study was to investigate the efficacy of γ-rays released by cholangial radioactive stents in treating cholangiocarcinoma.

METHODS

103Pd cholangial radioactive stents

When an optimum titanium stent punches 103Pd protons into 103Rh through the accelerator, one neutron will be released, producing [103Rh (p,h)103Pd]. During this process the 103Pd can be chemically purified. 103Pd in the energy state of 21–23 keV can release γ-rays with low energy. The half-life of 103Pd is 17 days, and as such, 103Pd is able to become 103Rh, its stable state after decay. The 103Pd stent was provided by the Radioisotope Laboratory of the Atomic Energy Research Institute of China, where 103Pd was electroplated onto a stent using chemical methods. The quantity of 103Pd on the surface of the stent was controlled by regulating the concentrations of 103Pd in the electroplating solution, and in that time metal stent γ-rays were made to release lower energy rates.

Cultivation of the cholangiocarcinoma cell line

The cholangiocarcinoma cell line was provided by the Hepatobiliary Surgery Department of the Affiliated Southwest Hospital of the Third Military Medical University of the People's Liberation Army. These cancer cells were digested by 0.25% trypsin and then sub-cultured. We chose the cells ranging from the 5th to the 15th generations, in the exponential growth stage.

Effects of γ-rays on cholangiocarcinoma cell growth

Cholangiocarcinoma cells were digested in a culture flask into a cell suspension. The cells suspension was transferred into two plastic freeze-storage ducts separately at 105 cells/ml. Cells were then divided into an irradiation group and a control group. In the irradiation group, 103Pd stents with different radioactive dosages (37, 197.321, 245.865, 395.445, 345.062, and 393.569 MBq) were used, while non-radioactive stents were used in the control group. First, the sterile stents were sheared longitudinally, and laid on the bottom of a 25 ml culture flask. Secondly, 10 ml of culture solution were added to each culture flask containing 5×106 cholangiocarcinoma cells, and were placed into an incubator at 37°C and CO2 volume percentage of 5%. Thirdly, the cholangiocarcinoma cells were irradiated with 103Pd for 12, 24, 48, or 72 hours, and were then removed afterwards. After cultivation for 1–5 days, the cells were then implanted into a 96 well culture plate with the addition of a culture solution containing 10% fetal bovine serum. Finally, the culture plate was placed into an incubator, and the culture solution changed every two days.

Cell growth curves and growth inhibition rates

We examined the cell growth curve and the growth inhibition rate with a modified 3-(4,5-dimethy thiazol-2-yl)-2,5-diphenyl terazolium-bromide (MTT) technique. First, cells in the logarithmic growth phase were irradiated with γ-rays. Secondly, these cells were implanted into a 96 well culture plate at 5000 cells per well with 200 μl per well of culture solution containing 10% fetal bovine serum. Thirdly, these cells were divided into either irradiation or control groups. Each group of cells was implanted into four culture plates, and from each group five wells were chosen as duplicates. Cells were then cultivated for 12, 24, 48, or 72 hours. Finally, the absorbance rate of each well was measured using a Full Automatic Enzyme Standardized Instrument (Tokyo, Japan). The measuring wavelength was set to 570 nm, and the reference wavelength to 630 nm. The value shown on the enzyme standardized instrument was A570-A630. The growth curve of each group was calculated by averaging A. Cell growth inhibition rates were then calculated using the following formula: inhibition rate (%) = (A value of the control group-A value of the irradiation group)/A value of the control group×100%.

Observation of cell ultrastructure

The cell suspension was collected into an agar tube, and then centrifuged at 2000 r/min to conglobate the cells. The supernatant was discarded, and the cell conglobation was fixed with a 2% glutaraldehyde for 30 minutes at 4°C. The cell conglobation was stripped and then fixed with 1% of osmic aceticaldehyde for 1–2 hours. Ultra-thin sections were prepared. Finally, the changes in the ultrastructure of the cells were observed using a transmission electron microscope.

DNA agarose gel electrophoresis

The collected cells were washed twice with phosphate buffered saline (PBS), and then centrifuged for 5 minutes at 3000 r/min. A total of 500 μl of DNA extract was added to the cell sedimentation, and then digested overnight in a 55°C water bath. After extraction by phenol, chloroform, and isoamyl alcohol, the DNA was precipitated by anhydrous alcohol, doubling the volume. DNA was dissolved in a 37°C water bath with TE:Rnase (1 ml:1 μl) for 4 hours, and then the sample and the supernatant buffer were mixed together in a 1:2 ratio. The mixed solution was separated by 1.2% agarose gel electrophoresis, and then stained with ethidium bromide. Finally, DNA electrophoresis bands were observed under the UV-light lamp.

Flow cytometric analysis

The cholangiocarcinoma cell suspensions from the 103Pd stent group and the control group were implanted into 50 ml culture flasks. After 12–72 hours of incubation, the attached cells were digested with the enzyme. The single cell suspensions (106 cells/ml) of the control group and irradiation groups were then collected and irradiated by 103Pd radioactive stents with dosages of 37, 197.321, 245.865, 395.445, 345.062, and 393.569 MBq. Next, the cells were washed with PBS, centrifuged for 5 minutes at 800–1000 r/min, then fixed with 70% ethanol and stored at 4°C until staining by addition of 500 μl of propidium iodide (PI) solution (PI 100 μg/ml, ribonuclease 500 μg/ml). The cells were then incubated for 30 minutes in a 37°C water bath, and analyzed by computer with an excitation wavelength of 488 nm.

Statistical analysis

All data were expressed as mean ± standard deviation (SD). Qualitative results were compared with a χ2 test. Quantitative results were compared using t test. Data analyses were performed with SAS software (Cary, Notrh Carolina, USA). A P value less than 0.05 was considered statistically significant.

RESULTS

Cell growth curves

The cholangiocarcinoma cell growth rate decreased with increased irradiation dosage of the 103Pd stent. Although the growth rate of the cholangiocarcinoma cells irradiated by a 1.0 mCi γ-ray dosage was the same as that of the control group, the growth rate and the growth curve of the cells irradiated by γ-rays with a dosage larger than 197.321 MBq were flat (Figure 1).

Figure 1.
Figure 1.:
Growth curves of cholangiocarcinoma cells irradiated by different dosages of γ-rays.

Growth inhibition rates of cholangiocarcinoma cells

The growth inhibition rate of cholangiocarcinoma cells increased with irradiation dosage (P <0.01), and the inhibition rate of the cholangiocarcinoma cells irradiated for 48 hours was much higher than those irradiated for 24 hours. Furthermore, the growth inhibition rate of cholangiocarcinoma cells which were irradiated for 72 hours was much higher than those irradiated for 48 hours (Figure 2).

Figure 2.
Figure 2.:
The growth rate of cholangiocarcinoma cells after irradiation by different dosages of γ-rays.

Influence of γ-rays on the ultrastructure of cholangiocarcinoma cells

Using transmission electron microscopy, the cells of the control group were observed to have larger nuclei, uniform karyotin, and abundant amounts of rough endoplasmic reticulum and mitochondria. In the 197.321 MBq γ-ray irradiation group, characteristic morphological hallmarks of apoptosis were observed including smaller nucleus, agglutinated chromatin, and many spherical prominences that coated the surface of cells, as well as the cytoplasm, organelle, nuclear debris, or an integral condensed nucleus. Some prominences separated from the cells, forming apoptotic bodies, appearing on one side of the cells. The cells disaggregated into some spherical apoptotic bodies of which the membrane still intact with organelles inside. Some apoptotic bodies exhibited evidence of being phagocytized by the surrounding cells. In the group receiving an γ-ray irradiation dosage larger than 245.865 MBq, the following phenomena were observed: the cells were swollen; at an early stage, the membranes and nuclei of cells were intact morphologically; in the cytoplasm there were more vacuoles, less endoplasmic reticulum, swollen mitochondria, or lytic mitochondria; at a late stage, the cell membrane was destroyed; and the nucleus burst releasing the cellular contents (Figure 3).

Figure 3.
Figure 3.:
Following a γ-ray irradiation dosage larger than 245.865 MBq, the nuclei of the cells were shrunken, with chromatin agglutination.

DNA agarose gel electrophoresis

The group irradiated with a γ-ray dosage of 197.321 MBq for 48 hours showed the typical laddered band pattern, while the group irradiated with a γ-ray dosage larger than 245.865 MBq showed smeared bands. The control group showed genomic bands near the electrophoresis samples (Figure 4).

Figure 4.
Figure 4.:
The irradiation group showed the typical laddered band patterns on DNA electrophoresis. Lanes 1–4 and 8–11: γ-ray dosage of 245.865 MBq, showing smeared bands; Lanes 5–7: γ-ray dosage of 197.321 MBq, showing the typical laddered band patterns.

Flow cytometry

The cholangiocarcinoma cell apoptotic rates in the 103Pd stent group at 24, 48, and 72 hours were 24.3%, 56.8%, and 91.3%, respectively. There was also a typical apoptotic peak (G1 peak) in the DNA histogram.

DISCUSSION

Apoptosis was previously described as an active process that required gene expression, protein synthesis, and energy.5 However, high dosages of radioactivity can destroy gene transcription and membrane integrity, disrupting the cells ionic balance. As a result, the apoptotic pathway would not be able to be initiated. In the present study, we showed that as a passive process, high dosages of radiation induced cell necrosis, without the characteristic changes indicative of apoptosis. In the same cell lines, the dosages of radiation required for induction of apoptosis were much lower than those for necrosis. One of the differences we observed between apoptosis and necrosis of therapeutic significance was that necrosis caused cellular swelling or cell lysis. This induced further release of lysosomal enzymes, injuring the surrounding cells. During apoptosis, cells form apoptotic bodies which are phagocytized, lysed, and digested by the surrounding cells, without arousing local inflammatory reactions.6–8

In the present study, the γ-rays released by 103Pd stents inhibited the growth of cholangiocarcinoma cells in vitro, as well as possessing lethal effects. Using transmission electron microscopy, two stages of apoptosis were observed after the cells were irradiated with a 197.321 MBq γ-ray dosage: (1) the nucleus became smaller, chromatin agglutinated, there were many spherical prominences on the surface of cells, and apoptotic bodies were formed, and (2) the apoptotic bodies were eliminated by phagocytes, and even digested or degraded by the surrounding cells. In the cholangiocarcinoma cells irradiated by γ-rays with a dosage larger than 245.865 MBq, a necrotic change in cells was observed with the cells becoming swollen until the cell membrane ruptured and the nucleus dissolved, ultimately resulting in the intracellular contents being released. With regards to our results demonstrating that low dosage γ-rays could cause apoptosis of cholangiocarcinoma cells, the ladder-patterns spaced over bands (shown in the diagram of DNA agar gel electrophoresis and the characteristic G1 peak examined by flow cytometry) are important indices for differentiation of apoptotic processes.9–11 In the present study, the group irradiated with a 5.333 mCi dosage of γ-rays showed typical ladder-patterns spaced over bands under DNA electrophoresis, while the group irradiated with a larger than 245.865 MBq dosage of γ-rays showed smeared bands without spaces. In the group irradiated with a 197.321 MBq dosage of γ-rays, the characteristic G1 peak was observed with flow cytometry. Thus, these data suggest that low dosages of γ-rays can induce apoptosis of cholangiocarcinoma cells.

Recently, it has been suggested that the time of cellular apoptosis induced by irradiation may differ between different types of cells. In vivo, cellular apoptosis occurs rapidly, with an early appearance peak, while in vitro, cellular apoptosis occurs later.12–15 However, there have been few studies regarding the time of cholangiocarcinoma cell apoptosis induced by irradiation. The results from the present study demonstrated that the cholangiocarcinoma cell apoptotic rates at 24, 48, and 72 hours after γ-ray irradiation were obviously higher than at 12 hours.

In summary, we suggest that cholangiocarcinoma cell apoptosis, as induced by γ-rays, is an important mechanism for the treatment of cholangiocarcinoma, with a close relationship between the irradiation dosage and apoptosis. These results provide an experimental foundation, as well as a theoretical basis, for the treatment of cholangiocarcinoma with radioactive therapy using an intra-cavity 103Pd radioactive stent. Therefore, we believe that γ-rays at low dosages are the best choice of radioactive therapy in treating cholangiocarcinoma. We have labeled an intra-cavity metal stent with 103Pd nuclides in a low dosage, followed by implantation of the metal stent into the bile duct. This is a new radiological method for treating cholangiocarcinoma.

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Keywords:

γ-ray; cell apoptosis; cholangiocarcinoma cells; radiotherapy

© 2008 Chinese Medical Association