Continuous and low-energy 125I seed irradiation changes DNA methyltransferases expression patterns and inhibits pancreatic cancer tumor growth
- Jian-xia Ma†1,
- Zhen-dong Jin†1,
- Pei-ren Si1,
- Yan Liu1,
- Zheng Lu1,
- Hong-yu Wu1,
- Xue Pan1,
- Luo-wei Wang1,
- Yan-fang Gong1,
- Jun Gao1 and
- Li Zhao-shen1Email author
© Ma et al; licensee BioMed Central Ltd. 2011
Received: 12 January 2011
Accepted: 2 April 2011
Published: 2 April 2011
Iodine 125 (125I) seed irradiation is an effective treatment for unresectable pancreatic cancers. However, the radiobiological mechanisms underlying brachytherapy remain unclear. Therefore, we investigated the influence of continuous and low-energy 125I irradiation on apoptosis, expression of DNA methyltransferases (DNMTs) and cell growth in pancreatic cancers.
Materials and methods
For in vitro125I seed irradiation, SW-1990 cells were divided into three groups: control (0 Gy), 2 Gy, and 4 Gy. To create an animal model of pancreatic cancer, the SW 1990 cells were surgically implanted into the mouse pancreas. At 10 d post-implantation, the 30 mice with pancreatic cancer underwent 125I seed implantation and were separated into three groups: 0 Gy, 2 Gy, and 4 Gy group. At 48 or 72 h after irradiation, apoptosis was detected by flow cytometry; changes in DNMTs mRNA and protein expression were assessed by real-time PCR and western blotting analysis, respectively. At 28 d after 125I seed implantation, in vivo apoptosis was evaluated with TUNEL staining, while DNMTs protein expression was detected with immunohistochemical staining. The tumor volume was measured 0 and 28 d after 125I seed implantation.
125I seed irradiation induced significant apoptosis, especially at 4 Gy. DNMT1 and DNMT3b mRNA and protein expression were substantially higher in the 2 Gy group than in the control group. Conversely, the 4 Gy cell group exhibited significantly decreased DNMT3b mRNA and protein expression relative to the control group. There were substantially more TUNEL positive in the 125I seed implantation treatment group than in the control group, especially at 4 Gy. The 4 Gy seed implantation group showed weaker staining for DNMT1 and DNMT3b protein relative to the control group. Consequently, 125I seed implantation inhibited cancer growth and reduced cancer volume.
125I seed implantation kills pancreatic cancer cells, especially at 4 Gy. 125I-induced apoptosis and changes in DNMT1 and DNMT3b expression suggest potential mechanisms underlying effective brachytherapy.
Pancreatic cancer is a devastating disease that is generally detected at a late stage. Surgical resection is the only potentially curative treatment; however, only 10 to 20% of patients are candidates for curative surgical resection due to advanced diagnosis, poor patient condition and tumor location. The remaining patients have to seek alternative therapies [1–3]. Even with resection, long term survival remains poor, with a median survival of 12 - 20 months. The survival rate of pancreatic cancer patients is so short, that treatment tends to be palliative. Recently, palliative surgery, endoscopic drainage, chemotherapy or brachytherapy alone or in combination have been used to elongate the survival and alleviate pain or jaundice symptoms [4–7].
Iodine-125 (125I) brachytherapy with either external beam radiation therapy (EBRT) or interstitial brachytherapy (IBT) improve local control and increase survival [8–10]. However, EBRT requires high doses of irradiation for efficacy . Moreover, the very radioresponsive organs surrounding the pancreas adversely affect the dose of radiation used to target the tumor on radiation treatment . Fractionated EBRT is only effective on cancer cells before metastasis occurs, and the efficiency of EBRT is usually impaired because, between irradiation treatments, tumor cells in the stationary phase enter the mitotic stage [8, 9]. As a result, IBT has been introduced as treatment for unresectable pancreatic cancers to maximize local dose and minimize irradiation of the surrounding normal tissue . Recently, 125I seed implantation, an efficient IBT technique, has attracted increasing attention because of its specific advantages: 1) effective irradiation dose applied in a single procedure; 2) reduced irradiation outside the target tumor; 3) elongating the tumor killing over several weeks or months; 4) percutaneous implantation under the guidance of ultrasound or CT [11, 12].
Cancer irradiation therapy may keep tumor cells in the sensitive resting period, resulting in tumor cell apoptosis, inducing epigenetic changes to reactivate silenced tumor suppressor genes, and damaging DNA to kill the cancer cells. However, the radiobiological effect of persistent and low-energy 125I irradiation, especially on epigenetic modifications and apoptosis are not fully understood. Cancer cell apoptosis is an indicator of response to cancer treatment. Aberrant DNA methylation in cancer cells is a critical epigenetic process involved in regulating gene expression. DNA hypermethylation is associated with tumor suppressor gene silencing and defects in cell cycle regulation, resulting in tumor development and progression [13, 14]. The DNA methyltransferases DNMT1, DNMT3a, and DNMT3b are the three main functional enzymes that are responsible for establishing and maintaining DNA methylation patterns in mammalian cells. The purpose of this study is to investigate the effect of persistent and low-energy 125I seed irradiation on apoptosis and the expression patterns of DNMTs in a mouse model of pancreatic cancer.
Materials and methods
Cell lines and cell culture
Human SW-1990 pancreatic cancer cell lines obtained from the American Type Culture Collection (Manassas, VA) were maintained in DMEM (pH 7.4; Sigma, St. Louis, MO) supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 10 ng/ml streptomycin in a humidified atmosphere of 95% air and 5% CO2 at 37°C.
In vitro125I seed irradiation model
125 I irradiation and Cell Group
The adherent SW-1990 cells were detached by 0.25% trypsin-EDTA until cells became a single cell suspension when observed under the microscope. The digestion was terminated by adding DMEM containing 10% fetal calf serum. The single cell suspension was diluted to a concentration of 1 × 105 cells/ml and was transferred to culture dishes with DMEM. Exponentially-growing SW1990 cells in a cell culture dish were irradiated using the in-house 125I seed irradiation model. The cell culture dishes were placed on the top of the in vitro125I seed irradiation model and placed in the incubator. The culture dishes were rotated clockwise at specific time intervals to guarantee even irradiation of the cells. The cultured cells were randomly divided into three groups: control group (0 Gy, without the embedded seed in the paraffin), 2 Gy, and 4 Gy.
Apoptosis analysis by flow cytometry
Adherent SW 1990 cells cells were trypsinized and centrifuged for 5 min at 220xg. Cells were then washed three times in ice cold PBS and suspended in binding buffer (0.01 M Hepes, pH 7.4; 0.14 M NaCl; 2.5 mM CaCl2) at 1 × 106 cells/ml. The cells were stained with annexin V-FITC (1 μl/ml) and propidium iodide (5 μg/ml) for 15 min in the dark as described previously . Cells were analyzed by fluorescence-activated cell sorting (FACS) using a Coulter EPICS and MOdFit SOFTWARE (Verity Software House, Topsham, MN). Each test was performed 3 times.
Real-time polymerase chain reaction (PCR)
Total RNA was retracted from SW 1990 cells using Trizol reagent (Invitrogen, Carlsbad, CA). The housekeeping gene glyceraldehyde-3-phosphate dehydrogenase GAPDH was used as an internal reference . Real-time PCR was performed by using the following primers: for DNMT1, upstream primer 5'-GTGGGGGACTGTGTCTCTGT-3' downstream primer 5'-TGAAAGCTGCATGTCCTCAC-3', and amplified fragment length of 204 bp; for DNMT3a, upstream primer 5'-ATCTCGCGATTTCTCGACTC- 3', downstream primer 5'-GCTGAACTTGGCTATCCTGC -3', and amplified fragment length of 180 bp; for DNMT3b, upstream primer 5'-TTGAATATGAAGCCCCCAAG- 3', downstream primer 5'-TGATATTCCCCTCGTGCTTC -3', amplified fragment length of 160 bp; for GAPDH, upstream primer 5'-GCACCGTCAAGGCTGAGAAC-3', downstream primer 5'-ATGGTGGTGAAGACGCCAGT-3', amplified fragment length of 142 bp. Cycling parameters: pre-denaturation 1 min, 95°C; denaturation 15 s, 95°C; annealing 15 s, 60°C; extension 45 s, 72°C, 40 cycles; final extension 5 min, 70°C. The PCR was repeated three times for each sample. The standard curve was generated with the ABI 7500 Real Time PCR system (Applied Biosystems, Carlsbad, CA, USA) to describe the linear relationship between threshold cycle (Ct) value and relative quantity (RQ). RQ values were obtained from measured Ct value with the following formula: 2(-ΔΔCt), where ΔΔCt = ΔCtT; ΔCtS = (ΔCtT - ΔCtTE ) - (ΔCtS - ΔCtSE), T is the target sample, S is the SW-1990 cell sample, and E is the reference. The RQ of mRNA in all groups were calculated relative to the RQ value in control group 1.
Western blotting was performed as described previously [17, 18]. Nuclear protein was prepared from SW-1990 pancreatic cancer cells with a Nuclear Protein extraction kit (Fermentas, Ontario, CA). The total protein concentration was determined by the Bradford assay using the Coomassie Protein Assay Reagent Kit (Pierce Biotechnology, Rockford, IL). Prepared protein samples (20 μg each) were boiled for 5 min and loaded onto a 12% SDS polyacrylamide gel. After separation by electrophoresis and electroblotting to nitrocellulose membranes, membranes were blocked by with 5% nonfat dry milk in 0.05% Tween 20 Tris-buffered saline (TTBS) at 4°C for 2 h. Membranes were incubated with rabbit anti-human anti-DNMT1 antibody (1:1000; Abcam, Cambridge, MA), DNMT3a (1:1000; Epitomics, Burlingame, CA) and DNMT3b (1:1000; Imagenex, Port Coquitlam, BC) at room temperature overnight. After three washes with TTBS, blots were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG antibody (1:5000) for 2 h at room temperature. The membranes were visualized with an enhanced chemiluminescence (ECL) detection system (Pierce) and images acquired using a Fluores-max instrument (Alpha Innotech, Santa Clara, CA). The gray scale value of the respective bands was quantified using Quantity One imaging software (Bio-Rad Laboratories, Hercules, CA).
Animal model of pancreatic cancer and animal group
The animals used in this study received humane care in compliance with the Guide to the Care and Use of Experimental Animals formulated by the Medical Ethical Committee on animal experiments of the Second Military Medical University. Twenty four 4 week old nude mice weighing 18 to 20 g were anesthetized by intraperitoneal injection of sodium pentobarbital (50 mg/kg). In a mini-laparotomy, the recipient rat pancreas was exposed and a small stab wound made in the pancreas parenchyma with a knife blade. The SW1990 cell suspension (1 × 105 cells/ml, 0.2 ml) was inoculated under the parenchyma of the pancreatic tail. Any leakage of the cell suspension into abdominal cavity was carefully removed with 75% ethanol to avoid peritoneal metastasis. Ten days later, the ultrasonic images demonstrated the formation of in situ pancreatic cancer with a tumor diameter of 1.52 ± 0.31 cm.
After the diagnosis of pancreatic cancer was established by ultrasound images during laparotomy, the 18-gauge needles were implanted into the visible mass at the tail of pancreas, and spaced in a parallel array at intervals of approximately 0.5 cm. After the needles were implanted, 125I seeds were implanted using a Mick-applicator with the spacing maintained at approximately 0.5 cm. The mice with pancreatic cancer were randomly divided into three groups. Groups I, II, and III underwent the implantation of 0 Gy, 2 Gy, and 4 Gy 125I seeds, respectively. The 2 Gy or 4Gy irradiation were achieved through implantation of 1 or 2 seeds, respectively, into the pancreatic tumor. The 125I seed have a average activity of 0.5 - 0.8 mCi. No seed implantation was performed in the 0 Gy irradiation group. After 125I seed implantation, two mice in the 0 Gy group died; however, no death was observed in the 2 Gy and 4 Gy groups.
Measurement of tumor volume by ultrasonic images
Ultrasonic inspection was performed through using a GF-UCT240-AL5 (Olympus Co Ltd, Tokyo, Japan) endoscopic ultrasound (EUS) 0 and 28 d post-implantation with a probe frequency of 12 MHz. After anesthetizing the animals by intraperitoneal injection of sodium pentobarbital (50 mg/kg), the mouse abdomen was soaked with sterile deionized water. The ultrasonic images were acquired using the EUS probe with a water bag and the direct contact method. The long (a) and short (b) diameters were measured from the ultrasonic images. The volume of tumor was calculated according to the following formula: a × b2/2.
TUNEL staining was described previously . Formalin-fixed tissues were dehydrated, embedded in paraffin, and sectioned. Tissue sections were deparaffinized with xylene and rehydrated with graded dilution of ethanol and fixed by 4% paraformaldehyde. The tissue sections were incubated in 0.1% Triton X-100 in 0.1% sodium citrate (SSC) for 15 min and 0.3% H2O2 for 3 - 5 min. The slides were washed three times in phosphate-buffered saline (PBS) and incubated with 50 μl of TUNEL reaction mixture (TdT and fluorescein-labeled dUTP) in a humid atmosphere for 60 min at 37°C. After three washes in PBS, the sections were incubated for 30 min with an antibody specific for fluorescein-conjugated horseradish peroxidase. The TUNEL stain was visualized with a DAB substrate system in which nuclei with DNA fragmentation stained brown. Slides were mounted in neutral gum medium and were observed with an IX71 light microscope (Olympus, Tokyo, Japan). A commercial fluorometric TUNEL system (DeadEnd; Promega, Madison, WI) was used for analysis of apoptosis. Tissue sections were examined microscopically using a 40× objective; apoptotic cells were counted in 200 fields. Alternatively, lenses were dissected from Formalin-fixed eyeballs and pictures were taken with an MZ FLIII stereomicroscope (Leica Microsystems, Deerfield, IL) with bright-field transmitted light. All pictures were processed in ImageJ to measure the surface area and height of each lens for comparison.
Immunohistochemical analysis was conducted as described previously . Tissues were obtained from pancreatic cancer approximately 5 mm distant from the center of the implanted 125I seed. Formalin-fixed tissues were dehydrated, embedded in paraffin, and sectioned. Tissue sections were deparaffinized, rehydrated, and incubated for 30 min in 0.3% hydrogen peroxide in methanol and then for 10 min with 1% goat serum in TBS. Then the sections were incubated with rabbit anti-human anti-DNMT1 antibody (Abcam), DNMT3a (Epitomics) and DNMT3b (Imagenex; all at 1:100) at room temperature overnight. After washing three times in TBS, the sections were incubated with biotinylated mouse anti-rabbit IgG (1:5000; Abcam) for 30 min and followed by three 5 min wash in TBS. The final incubation was for 30 min with HRP-avidin D at 37°C. The peroxidase was detected with 0.05% 3,3-diaminobenzidine tetrahydrochloride (DAB). The sections were counterstained with hematoxylin and mounted in neutral gum medium for light microscopy . Positive protein expression was visualized as nuclear localization of granular brown-yellow precipitate. The counts were performed in 3 high power fields of vision under a high magnification (400×) for each section. The percentage of positive cells was calculated as the ratio of positive cells to the total number of cells. The scoring scale for the percentage of positive cells was: 0, less than 1%; 1, 1 - 24%; 2, 25 - 50%; 3, 51 - 75%; 4, more than 75%. The scoring scale for staining intensity was: 0, no color; 1, bright yellow; 2, yellow; 3, brown yellow; 4, brown. The final score was obtained by multiplying the percentage of positive cells by the staining intensity score.
All data were plotted as mean ± standard deviation. Statistical analysis was performed with SPSS 13.0 software. (SPSS Inc., Chicago, IL). Student's t test was used for comparisons. Differences were considered significant when the P was less than 0.05.
The continuous and low-energy 125I seed irradiation-induced cell apoptosis
Expression changes of DNMTs in SW-1990 cells after 125I seed irradiation
The number of apoptotic cells in pancreatic cancer after 125I seed implantation
Immunohistochemistrical stains for DNMTs in pancreatic cancer after 125I seed implantation
The positive expression scoring of DNMTs protein in 125I pancreatic cancers
Control Group (0Gy)
6.72 ± 2.63
4.72 ± 2.16
2.61 ± 1.24
9.11 ± 3.64*
7.27 ± 3.76*
3.22 ± 1.30Δ
6.50 ± 2.85#Δ
3.11 ± 2.42*#
3.06 ± 2.13Δ
Histopathology of in pancreatic cancer after 125I seed implantation
Tumor volume of pancreatic cancer at 0 and 28 days after 125I seed implantation
Epigenetic changes in cells are closely linked to tumor occurrence, progression and metastases. DNA methylation is a crucially important epigenetic alteration by which the tumor suppressor gene expression and cell cycle regulation may be substantially altered. Three different DNMTs, specifically DNMT1, DNMT3a and DNMT3b, have critical roles in establishing and maintaining DNA methylation. Many chemotherapeutic agents exert their antitumor effects by inducing apoptosis in cancer cells. The purpose of this study is to investigate whether 125I seed irradiation significantly influences the expression of DNA methyltransferases, promote the cell apoptosis and inhibit the pancreatic cancer growth.
SW-1990 pancreatic cancer cells were cultured ex vivo and implanted into the pancreas to create the animal model. The 125I seed irradiation induced apoptosis in SW-1990 cells. Likewise, large numbers of apoptotic cells were present in pancreatic cancer receiving 125I seeds implantation. Irradiation-induced apoptosis became more obvious when the radiation dose increased from 2 Gy to 4 Gy. DNMT1 and DNMT3b mRNA and protein expression was increased substantially in 2 Gy 125I irradiated SW-1990 cells, whereas 125I irradiation with 4 Gy inhibited DNMT3b mRNA and protein expression. The expression of DNMT3a mRNA did not change regardless of the 125I irradiation dose. The similar DNMT expression patterns were confirmed by immunohistochemical staining in 125I seed implanted pancreatic cancer. Most importantly, the 2 Gy 125I seed implantation limited the growth of the pancreatic tumor, while 4 Gy 125I seed implantation substantially decreased pancreatic tumor volume.
Our results demonstrated that apoptosis may have an important role in the therapeutic effects when pancreatic cancer is exposed to continuous low-energy 125I irradiation. The apoptosis in the 4 Gy group was more obvious than in the 2 Gy group, which is in agreement with the fact that cancer treatment is more effective at 4 Gy than at 2 Gy. Similar irradiation-induced apoptosis patterns were also observed in the other cancer cell lines . The 125I irradiation induced apoptosis was the primary mechanism of CL187 colonic cancer cell-killing under low dose treatment . Ionizing radiation can generate the reactive oxygen species (ROS), which induce apoptosis . The ROS damages critical cellular components such as DNA, proteins, and lipids, eventually causing cellular apoptosis . Therefore, the 125I irradiation-induced apoptosis is a key mechanism underlying the therapeutic effect of 125I seed implantation in pancreatic cancer.
Our results demonstrated that altered DNA methylation patterns might have a pivotal role in tumor inhibition resulting from consecutive low-energy irradiation. The 2 Gy irradiation caused a significant increase in DNMTs expression, whereas 4 Gy irradiation was associated with decreased DNMTs expression. However, a substantial reduction in tumor volume was only observed in 4 Gy irradiation group rather than in 2 Gy group at 28 d after 125I seed implantation. There are a strong and positive correlation between DNA methylation and expression of DNMTs, because DNMTs maintain DNA methylation patterns . Therefore, it is reasonable to speculate that DNA hypomethylation significantly inhibits cancer cell proliferation or impairs cell survival potentially to an even greater extent than DNA hypermethylation. X- and γ-radiation induce DNA hypomethylation paralleled by decreased DNMTs expression in somatic cells [25–28]. Actually, low-dose irradiation (2Gy) predominantly resulted in reversible DNA damage, which was associated with DNA repair. The DNMTs are the key enzyme for DNA repair. As a result, the increase in reactive DNMTs expression reflects active DNA repair. Thus, 125I irradiation-induced DNA hypomethylation could be the key mechanism by which 125I seed implantation lead to tumor growth inhibition.
Aberrant de novo DNA methylation is commonly associated with cancer, and DNA methylation in mammalian cells largely occurs on cytosine residues at CpG dinucleotides in genomic DNA. The de novo methylation of CpG islands in promoter regions of tumor suppressor genes can result in silencing . Thus, DNA hypermethylation might lead to cancer generation and progression . The irradiation-induced DNA demethylation, as the result of decreased DNMTs expression, can reactivate the tumor suppressor gene and inhibit tumor growth. The inhibitory effect of DNA demethylation on cancer was also demonstrated by the demethylating agent 5-aza-cytidine (AZA) and zebularine. Incorporation of a demethylating agent (like a cytidine analog) into DNA during replication inhibited DNMTs enzyme activity and demethylated the tumor suppressor genes, eventually leading to tumor growth inhibition [30, 31]. AZA demethylates the P16 and pMLHI gene promoters and reactivates these previously silenced tumor suppressor genes [30, 32]. Zebularine administration depleted DNMT1 and the demethylation of the P16 and RASSFIA gene promoters [33, 34]. Activation of the tumor suppressor genes RASSF1A and P16 inhibited cell proliferation by inhibiting accumulation of cyclin D, which arrests cell cycle progression at the G1/S phase transition . G1 includes a restriction point beyond which the cell is committed to undergo division independent of growth regulatory signals. As a result, the mechanisms underlying the inhibitory effect of DNA hypomethylation on tumors could be related to reactivating tumor suppressor genes and negative regulation of cell cycle progression.
In conclusion, our study provides important insight into the mechanism by which 125I seed irradiation affects pancreatic cancer. 125I seed implantation effectively inhibited tumor growth and reduced tumor volume, especially at 4 Gy. 125I irradiation-induced apoptosis and DNA hypomethylation are two key mechanisms underlying the therapeutic effect of low-energy 125I seed implantation.
This work is supported by National Natural Science Foundation of China (2008, C171006)
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