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Clinical Cancer Research Vol. 12, 2706-2715, May 1, 2006
© 2006 American Association for Cancer Research

Human Cancer Biology

Sublethal Irradiation Induces Vascular Endothelial Growth Factor and Promotes Growth of Hepatoma Cells: Implications for Radiotherapy of Hepatocellular Carcinoma

Yih-Lin Chung1,5, James Jer-Min Jian1, Skye Hongiun Cheng1, Stella Y.C. Tsai1, Vincent P. Chuang2, Thomas Soong2, Yu-Mong Lin3 and Cheng-Fang Horng4

Authors' Affiliations: Departments of 1 Radiation Oncology, 2 Radiology, 3 Internal Medicine, and 4 Clinical Protocol Office, Koo Foundation Sun Yat-Sen Cancer Center; and 5 School of Medicine, National Yang-Ming University, Taipei, Taiwan

Requests for reprints: Yih-Lin Chung, Department of Radiation Oncology, Koo Foundation Sun Yat-Sen Cancer Center, No. 125 Lih-Der Road, Taipei, Taiwan. Fax: 886-2-27020372; E-mail: ylchung{at}mail.kfcc.org.tw.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: To investigate the clinical benefit of additional radiotherapy to patients with unresectable hepatocellular carcinoma treated with transcatheter arterial chemoembolization (TACE) and the molecular effects of radiation on gene expression in hepatoma cells.

Experimental Design: Between August 1996 and August 2003, 276 and 64 patients with American Joint Committee on Cancer stage T3N0M0 hepatocellular carcinoma receiving TACE alone and TACE followed by three-dimensional conformal radiotherapy, respectively, at our institution were studied. Clinical outcome and pattern of failure were analyzed for the association of survival benefit with radiotherapy. The molecular effects of radiotherapy were studied in vitro and in vivo using human hepatoma cells with different p53 mutation and hepatitis B virus infection status.

Results: Median follow-up and survival time in the TACE alone and TACE + radiotherapy groups were 39 and 19 months, and 51 and 17 months, respectively. Additional radiotherapy to TACE did not improve overall survival (P = 0.65). However, different failure patterns were noted after TACE and after radiotherapy. Although all irradiated tumors regressed substantially, radiotherapy rapidly enhanced both intrahepatic and extrahepatic tumor progression outside the radiotherapy treatment field in a significant portion of patients, which offset the benefit of radiotherapy on overall survival. In molecular analysis of the radiation effects on human hepatoma cells, radiotherapy rapidly induced p53-independent transcriptional up-regulation of vascular endothelial growth factor (VEGF), increased VEGF secretion in a dose-, time-, and cell type–dependent manner, and promoted hepatoma cell growth in vivo with enhanced intratumor angiogenesis, which correlated well with elevated levels of serum VEGF.

Conclusions: Radiotherapy to eradicate a primary hepatocellular carcinoma might result in the outgrowth of previously dormant microtumors not included in the radiotherapy treatment field. Radiotherapy-induced VEGF could be a paracrine proliferative stimulus. Therapeutic implications of the study justify the combination of three-dimensional conformal radiotherapy with anti-VEGF angiogenic modalities for the treatment of unresectable hepatocellular carcinoma to reduce relapses.

Hepatocellular carcinoma is a major health problem worldwide (1). It frequently occurs in the setting of chronic liver disease and cirrhosis, most commonly associated with hepatitis B virus (HBV) or C infection (2). The development of hepatocellular carcinoma is generally the terminal event of long-standing, typically asymptomatic ever-increasing hepatic fibrosis over decades after the beginning of liver insult; thus, hepatocellular carcinoma is typically diagnosed late, with the median survival following diagnosis of ~6 to 20 months (3).

Resection for patients with early tumors and excellent liver functional reserve, and liver transplantation for patients with one hepatocellular carcinoma <5 cm, up to three nodules <3 cm, or advanced liver dysfunction may achieve the best outcomes (5-year survival of 60-70%; refs. 4–6). However, >80% of patients are not surgical candidates at the time of diagnosis because of vascular invasion, multifocality, and large tumor size (7–9). Therefore, nonresectional therapies [i.e., percutaneous ethanol injection, radiofrequency ablation, transcatheter arterial chemoembolization (TACE), and three-dimensional conformal radiotherapy] have been introduced to try to improve survival in patients with advanced hepatocellular carcinoma. Although percutaneous ethanol injection, radiofrequency ablation, and TACE are generally effective for patients with one to three lesions <4 cm in diameter, they are not considered as a curative treatment and have achieved very limited success in eradicating larger hepatocellular carcinomas, and repeated treatments are often necessary until there is tumor progression to extensive disease (9–12). With the development of three-dimensional conformal radiotherapy techniques, radiotherapy can be more safely given to patients with larger tumor-bearing parts of liver for whom percutaneous ethanol injection, radiofrequency ablation, and TACE are no longer possible (13–16).

Among the nonresection therapies, only TACE has been shown to improve survival in patients with intermediate hepatocellular carcinoma (17, 18), but complete necrosis is seldom observed when the tumor size is >5 cm (19). The residual viable tumor cells are noted around the necrotic center of the tumor following TACE (20). Thus, TACE combined with radiotherapy has been suggested for treating large hepatocellular carcinoma (21–24). Although radiotherapy seems to increase the tumor response rate, it has not been definitively shown to prolong the survival in unresectable hepatocellular carcinoma (22–25). The goal of this study was to analyze the clinical and molecular effects of radiotherapy in hepatocellular carcinomas and hepatoma cells to find out if radiotherapy alters the pattern of failure in a way that might balance out any prolongation of survival resulting from enhanced tumor control, and if radiotherapy modulates gene expression that could affect tumor cell growth.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Patient character, evaluation, treatment, and follow-up. Between August 1996 and August 2003, 340 patients with unresectable hepatocellular carcinoma, American Joint Committee on Cancer stage T3N0M0 receiving treatment at our hospital were reviewed. Among them, 276 patients received TACE alone and 64 patients had TACE followed by three-dimensional conformal radiotherapy. Patients were referred for radiotherapy after TACE based on physician assessment of inadequate tumor treatment with TACE alone, including poor uptake/retention of lipiodol in the tumor bed or decreasing liver function. The inclusion criteria for the study were as follows: (a) histologically proven hepatocellular carcinoma; (b) no obstruction of the main portal trunk; (c) Child's class A; (d) Karnofsky performance status ≥90%; (e) no liver resection; (e) no prior radiotherapy to the liver or systemic chemotherapy; and (f) no radiotherapy-induced liver disease manifested by the development of abnormal liver function in the absence of documented tumor progression within 3 months of completion of radiotherapy. The pretreatment work-up consisted of a complete history and physical examination, chest radiograph, abdominal helical computed tomography (CT) scan with contrast enhancement, angiography, complete hematology, renal, liver, electrolyte, {alpha}-fetoprotein, HBV surface antigen, anti-HCV, and prothrombin time studies, an indocyanine green test, and a core biopsy or aspiration cytology for liver tumor. For follow-up evaluation, tumor response was based on serial CT scans obtained before treatment, 3 to 6 weeks after TACE or radiotherapy, and then every 3 to 6 months. Radiotherapy-associated intrahepatic tumor progression was defined as evidence of multiple newly developed tumors elsewhere within the liver outside the field of radiotherapy (not seen in the previous angiography and CT scans, and not extending from the target volume of radiotherapy), seen in the first follow-up CT scans taken 3 to 6 weeks after completion of radiotherapy.

TACE. TACE was done with the infusion of a mixture of iodized oil contrast medium (Lipiodol 6-20 mL), Ivalon particles (47 µm, 50-100 mg, polyvinyl alcohol foam), and chemotherapeutic agents, including doxorubicin (25-50 mg), cisplatin (50-100 mg), and mitomycin C (5-10 mg). An interval of 3 months between each TACE, and at least 30 days between TACE and radiotherapy, was necessary if viable tumors or tumor progression was noted by the CT scan images.

Radiotherapy. CT scans for radiotherapy planning were done with patients placed in a supine position with both arms raised above the head. The three-dimensional CT-based planning system (FOCUS, CMA, Ltd., St. Louis, MO) was used to decrease the irradiated bowel, spare the normal liver and kidneys, and cover adequately the tumor target with 1.5 to 3 cm margins. The radiotherapy dose determination was based on the following guideline for dose-volume criteria: (a) if nonirradiated liver volume was ≤1/3, then radiotherapy dose was ≤40 Gy; (b) if nonirradiated liver volume was between 1/3 and 1/2, then radiotherapy dose was prescribed to 50 Gy; and (c) if nonirradiated liver volume was ≥1/2, then radiotherapy dose was given to 60 to 66 Gy. No patient received radiation to the whole liver. Radiotherapy was delivered with a 6 MV or 18 MV linear accelerator in a daily fraction of 1.8 to 2 Gy. Weekly verification films were routinely obtained for review. Patients were monitored weekly with physical examination, complete blood counts, and liver function tests.

Statistical methods. For clinical data analysis, overall survival was calculated from the day of commencement of TACE or radiotherapy as indicated until the day of death or loss of follow-up. The probability of survival was calculated according to the methods of Kaplan-Meier. The log rank or Wilcoxon test was used in the analyses of survival outcome. For experimental data analysis, the paired Student's t test was used. Two or more separate experiments were done for each case. P < 0.05 was considered to show a significant difference between the experimental and control groups.

Cell lines and treatment. Human hepatoma (HepG2, Hep3B, PLC/PRF/5, and HuH7) cells were obtained from American Type Culture Collection (Manassas, VA). HuH7 cells were maintained in RPMI 1640 (Life Technologies, Grand Island, NY) with 1% fetal bovine serum (Hyclone, Logan, UT). Other cell lines were maintained in RPMI 1640 supplemented with 10% fetal bovine serum. Cells were incubated at 37°C with 5% CO2. Tumor cells in log-growth phase were irradiated with a dose of 0, 2, 4, or 6 Gy at room temperature using a 6 MV X-ray from a linear accelerator (Primus, Siemens, Erlangen, Germany) with a delivering rate of 2 Gy/min. After 24 to 72 hours, medium was collected and cells were harvested.

Clonogenic assay. Cells (5 x 104) plated in 25 cm2 flasks were irradiated with doses of 0 to 8 Gy at room temperature using 6 MV X rays from a linear accelerator (Primus, Siemens) at a dose rate of 2 Gy/min. Cultures were returned to the incubator for 14 days, after which they were stained with crystal violet, colonies were counted, and the survival percentage was determined for clonogenic survival after correcting for plating efficiency.

Experimental animal study. For in vivo study, human hepatoma cells were irradiated before inoculation on mice to avoid the radiotherapy effects on the mouse endothelial and mesenchymal cells. HepG2 cells were suspended in 250 µL Matrigel (Collaborative Biomedical Products, Bedford, MA) and inoculated s.c. on the hind back of severe combined immunodeficient/nonobese diabetic male mice. The tumor size was calculated weekly with a caliper according to the formula ab2/2, where a and b are the larger and smaller diameters in millimeters, respectively. All experimental and surgical procedures done on mice were in accordance with the NIH guidelines outlined in the Guide for Care and Use of Laboratory Animals (NIH publication 85-23).

Protein array. To analyze the protein profile, the conditioned medium and cell lysates of the cultured cells with or without radiotherapy were collected, respectively, and used immediately for simultaneous detection of multiple cytokine expression levels, using human protein arrays (RayBio Human Cytokine Array V and 5.1, RayBiotech, Inc., Norcross, GA) coated with antibodies recognizing cytokines related to angiogenesis and cell growth, following the procedure recommended by the manufacturer. The signals on array chips were quantified using a chemiluminescene imaging device.

ELISA. Tumor cells (2 x 105) were plated in six-well plates. When the cultures reached 80% confluence, fresh medium was applied and collected after radiotherapy as indicated, and then clarified of cells and cell debris by centrifugation. The cells were harvested with trypsin-EDTA and counted. The conditioned medium samples from cell culture and the serum samples from experimental mice were used for measurement of vascular endothelial growth factor (VEGF)-A, using quantitative immunometric sandwich ELISA, following the procedure recommended by the manufacturer (Assay Designs, Inc., Ann Arbor, MI).

Reverse transcription-PCR analysis. Total RNA was extracted from hepatoma cells using Trigent (Molecular Research Center, Inc., Cincinnati, OH). cDNA was synthesized with Superscript II reverse transcriptase (Life Technologies, Gaithersburg, MD) and subjected to 30 cycles of PCR to amplify human VEGF and ß-actin cDNA. The PCR cycle was 94°C for 1 minute, 58°C for 1 minute, and 72°C for 2 minutes. The products were fractionated by 1% agarose gel electrophoresis and stained with ethidium bromide. The nucleic acid sequences of the specific PCR primers were as follows: VEGF (377 bp), 5'-CTGCTGTCTTGGGTGCATTCT-3'(sense), 5'-TTCACATTTGTTGTGCTGTAG-3' (antisense); and ß-actin (661 bp), 5'-TGACGGGGTCACCCACACTGTGCCCATCTA-3' (sense), 5'-CTGAAGCATTTGCGGTGGACGATGGAGGG-3' (antisense).

Immunohistochemistry. The tumor tissues were fixed in 10% formalin-buffered solution and embedded in paraffin wax. Serial 3 µm sections were cut, dewaxed, and deparaffinized by xylene and rehydrated by sequential concentrated alcohols. The slides were subjected to microwave antigen retrieval (800 W, twice for 5 minutes each) in 0.01 mol/L sodium citrate buffer (pH 6.0), then incubated with 3% H2O2 for 10 minutes, then a protein-blocking agent (DAKO, Glostrup, Denmark) for 20 minutes, and then treated with the goat polyclonal anti-CD31 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 hour at room temperature, followed by the secondary anti-goat antibody incubation for 30 minutes and then washed with PBS thrice. The peroxidase reaction was visualized using 3-amino-9-ethylcarbazole (3% in N,N-dimethylformamide) as chromagen.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Effects of radiotherapy in patients with unresectable hepatocellular carcinoma after TACE: different failure patterns after TACE and after radiotherapy. Since the first course of TACE was done, the median follow-up was 39 months in 276 patients of the TACE-alone group with 97 patients alive and 179 dead, and 51 months in 64 patients of the TACE + radiotherapy group with 10 patients alive and 54 dead. The survival rates in the TACE-alone and TACE + radiotherapy groups were comparable at 1 year (58% versus 65%), 2 years (44% versus 35%), and 3 years (35% versus 26%; Fig. 1 ). The median survival time in the TACE-alone and TACE + radiotherapy groups was 19 and 17 months, respectively, after TACE was started. Additional radiotherapy to TACE did not result in further improvement of the overall survival (P = 0.65).


Figure 1
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  		Fig. 1. Overall survival. Patients with unresectable hepatocellular carcinoma received TACE alone or TACE followed by radiotherapy (TACE alone vs. TACE + RT, P = 0.65).

 
However, three-dimensional conformal radiotherapy sparing the normal-appearing liver without adversely affecting liver function did substantially induce regression in the irradiated tumors (Fig. 2 ). We then analyzed whether radiotherapy altered the pattern of failure that might balance out any prolongation of survival resulting from enhanced local tumor control. The cause of death or the natural course in the vast majority of patients with advanced hepatocellular carcinoma was progression of intrahepatic multifocal tumors in 1 to 3 years. Different failure patterns were noted after treatment with TACE or radiotherapy. The failure pattern in almost all patients after several courses of TACE showed that tumor progression developed gradually at the periphery of the tumor that had been embolized well in the central area (Fig. 2A). In contrast, the failure pattern of radiotherapy seemed to enhance intrahepatic and extrahepatic tumor progression soon (Fig. 2B). Multiple newly developed intrahepatic tumors outside the treatment field of radiotherapy, not seen in the previous angiography and CT scans, arose immediately in 24 (37.5%) of the 64 patients receiving radiotherapy at the first follow-up CT scans taken 3 to 6 weeks after completion of radiotherapy. Ten (42%) of the 24 patients had synchronous extrahepatic metastasis (seven in lung, two in bone, and one in lung and bone). The extrahepatic progression-free survival was significantly shorter for patients in the TACE + radiotherapy group than in the TACE group (2-year rates: TACE + radiotherapy 35%, TACE 100%, P = 0.002). If the radiotherapy-associated tumor progression occurred, additional radiotherapy to TACE shortened the median overall survival to 9 months (P = 0.027) when compared with TACE alone; otherwise, radiotherapy prolonged the median overall survival to 26 months after start of TACE (P = 0.026; Fig. 3A ). These results indicate that focal radiotherapy or three-dimensional conformal radiotherapy can produce objective tumor response and may prolong survival, but the association of radiotherapy with rapid development of intrahepatic and extrahepatic recurrence outside the radiotherapy treatment field in some patients offsets the benefit of radiotherapy in the overall survival.


Figure 2
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  		Fig. 2. Radiotherapy-associated tumor progression. Comparison of preradiotherapy and postradiotherapy CT scan images to show that radiotherapy (RT) rapidly enhances both intrahepatic and extrahepatic tumor progression. A, case 1. Although the liver tumor shrank in the irradiation field, rapid intrahepatic tumor progression out of the radiotherapy field developed in the lateral segment of liver that did not show any tumor growth in the preradiotherapy images and angiography. Arrowheads, lipiodol retention due to previous courses of TACE. B, case 2. Both intrahepatic tumor progression out of the radiotherapy field in the right lobe of liver and extrahepatic multiple lung metastases developed soon after radiotherapy, whereas the irradiated tumor shrank significantly.

 

Figure 3
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  		Fig. 3. Survival with or without radiotherapy-associated rapid intrahepatic progression. Radiotherapy-associated intrahepatic tumor progression is defined as evidence of multiple newly developed tumors elsewhere within the liver outside the field of radiotherapy (not extending from the target volume of radiation), seen in the first follow-up CT scans taken 3 to 6 weeks after completion of radiotherapy. A, survival curve after start of TACE in the TACE + radiotherapy group. Additional radiotherapy to TACE caused intrahepatic tumor progression out of the radiotherapy field in a substantial portion of patients, which decreased the benefit of radiotherapy on overall survival rate. B, survival curve after start of radiotherapy. The patients receiving TACE followed by radiotherapy had a similar interval between TACE and radiotherapy, but the survival rate after the start of radiotherapy was significantly different between patients with and without radiotherapy-associated rapid intrahepatic progression out of the radiotherapy field.

 
The study includes a clinical experience, not a randomized controlled trial. Thus, prognostic factors might not be balanced at baseline so it is possible that the worse survival in some patients receiving radiotherapy was due to more advanced disease. However, the characteristics of the patient profile and treatment variables in patients with or without radiotherapy-associated tumor progression did not show significant differences (Table 1 ). The mean and median time intervals between TACE and radiotherapy for patients with or without radiotherapy-associated progression were almost the same at ~9.4 and 5 months, and 8.9 and 4 months, respectively, but the median survival time was 4 and 17 months, respectively, after start of radiotherapy. Therefore, the difference in survival rate was not associated with TACE treatment but appeared immediately after start of radiotherapy (Fig. 3B).


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  		Table 1. Characteristics of patients

 
Up-regulation of VEGF mRNA and protein by radiotherapy. The rapid development of intrahepatic and extrahepatic recurrence outside the radiotherapy treatment field prompted us to hypothesize that radiotherapy might increase production and secretion of certain growth factors that result in paracrine growth stimulation. Because progression of hepatocellular carcinoma is closely linked to neoangiogenic activity (26), we then used a protein array system to analyze the angiogenic growth factor and cytokine profile in hepatoma cells receiving radiation. As shown in Fig. 4 , among 79 important angiogenic factors, a single radiation dose of 2 and 4 Gy to the HepG2 human hepatoma cells caused a significant increase of VEGF protein in the cell as well as secretion into the medium in 24 to 48 hours. Because p53 and viral infection might influence cell growth and gene modulation in response to radiation, in addition to HepG2 cells, other human hepatoma cell lines with different p53 and HBV infection status were also chosen in further studies to compare the VEGF expression and secretion after different doses of radiation. HepG2 without HBV infection contains the wild-type p53, Hep3B with HBV infection completely lacks p53 expression, and PLC/PRF/5 with HBV infection and HuH7 without HBV infection carry different mutant forms of p53. Each cell line was irradiated with single 0, 2, 4, and 6 Gy radiation and further incubated for 24, 48, and 72 hours. The amount of VEGF protein in the medium measured by ELISA increased with time after radiation, but higher radiation dose did not result in more VEGF secretion in the medium (Fig. 5A ). The increase in VEGF protein levels in the medium in response to radiation was transcriptionally regulated (Fig. 5B). The up-regulation of VEGF mRNA in the cell correlated with the increase of VEGF protein in the medium after radiotherapy. Although the up-regulation of VEGF in hepatoma cells in response to radiation is independent of p53 presence, the sensitivity to radiation is still different between each cell line. That a single 6 Gy radiation did not elicit greater up-regulation of VEGF expression than 2 Gy radiation might be to the result of too much DNA damage and cell death from 6 Gy radiation (Fig. 6A ). When HepG2 cells were exposed to single 2, 4, 6, and 8 Gy radiation, we observed loss of reproduction or cell death in >90% of cells above the 4 Gy single dose.


Figure 4
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  		Fig. 4. Analysis of the production and secretion of angiogenic cytokine profile in response to radiation in human hepatoma cells. HepG2 cells were exposed to single 2 Gy radiation using 6 MeV X-rays from a linear accelerator. After 24 to 48 hours, irradiated and control cells were harvested. The cell lysate and cultured medium were incubated, respectively, with a human cytokine antibody array covering 79 important cytokines related to apoptosis, angiogenesis, cell growth, and differentiation. A, images of protein chips and the corresponding array map. Only the production and secretion of VEGF was up-regulated in the 24 to 48 hours after radiation. Ang, angiogenin; OSM, oncostatin M; TPO, thrombopoietin; Pos, positive control; Neg, negative control. All others are standard abbreviations. B, relative signal intensity on chips is expressed as percentage of positive control for comparison and shows the increase of VEGF production in the cell and secretion in the medium with time after radiation.

 

Figure 5
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  		Fig. 5. Effect of radiation on VEGF expression in hepatoma cells with different p53 and HBV infection status. Four human hepatoma cells, HepG2 (HBV-negative) with wild-type p53, Hep3B (HBV-positive) with depleted p53, and PLC/PRF/5 (HBV-positive) and HuH7 (HBV-negative) with different mutated forms of p53, were treated with single doses of 0 to 6 Gy, and incubated for 24 hours. A, the secretion of VEGF into cultured medium was measured by ELISA. Radiation caused significant up-regulation in the secretion of VEGF with time, but the higher radiation dose did not result in more VEGF secretion. Representative experiment of three done in triplicate (control: Hep G2 = 8.0 ± 1.6 pg/mL; Hep 3B = 13.1 ± 2.9 pg/mL; PLC/PRF/5 = 40.7 ± 8.2 pg/mL; HuH7 = 16.3 ± 3.1 pg/mL). B, the expression level of VEGF mRNA was detected by reverse transcription-PCR. ß-actin was used as an internal control. The radiation-induced VEGF was transcriptionally up-regulated, independent of p53 status.

 

Figure 6
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  		Fig. 6. Radiotherapy enhances hepatoma cell growth in vivo. After 2 Gy single radiotherapy, human HepG2 hepatoma cells were inoculated into the backs of severe combined immunodeficient mice. Tumor formation incidence, tumor growth volume, serum VEGF concentration, and microvessel density were measured. A, the survival percentage of HepG2 cells exposed to different single radiotherapy doses was determined by clonogenic assay after correction for plating efficiency. Points, mean of two to three separate experiments, each plated in triplicate; bars, SD. Above single 4 Gy radiotherapy, >90% cells were killed. B, tumor formation incidence represents the ratio of the number of mice with tumor formation to the number of injected mice. Injection of unirradiated cell numbers fewer than 107 did not form any palpable tumors in mice. In contrast, only injection with 105 irradiated cells was enough to see tumor formation. C, the size of tumor growing from the irradiated cells exceeded the size from the control cells by 3 months. *, P < 0.05 in comparison with the mean tumor size of six mice in each group. The in vivo experiments were repeated twice. D, the serum VEGF concentration was higher in the mice with tumor formation from the irradiated cells than from the unirradiated cells. *, P < 0.05 (control: 48 ± 3.8 pg/mL, n = 6). E, immunohistochemical staining for the expression of CD31, a vessel endothelial marker, shows the increased microvessel density or angiogenesis in the tumor growing from the irradiated cells.

 
Exposure of hepatoma cells to radiation enhances tumor growth and angiogenesis in vivo. We next analyzed whether increased VEGF expression and secretion induced by sublethal radiation can lead to increased tumorigenicity in vivo. In addition to rapid induction of VEGF, radiotherapy has also effects in rapidly and persistently altering the tissue microenvironment, which affects the physical interactions and signaling between cells and causes a microenvironment-mediated damage response program that includes production of reactive oxygen species, chronic inflammation, and remodeling of the extracellular matrix. Therefore, to avoid the interference from the radiotherapy effects on the tissue microenvironment and to focus on the radiation effects on the tumor cells, HepG2 cells were irradiated before inoculation on mice.

Because increasing the intensity of radiation above 4 Gy did not result in a further increase in the level of VEGF gene expression, to ensure cell viability, we used 2 Gy radiation in in vivo experiments (Fig. 6B). Each mouse was injected s.c. with unirradiated or irradiated cell numbers from 104 to 108. Tumor formation incidence, tumor growth rate, and serum VEGF were monitored weekly up to 3 months. Unirradiated cells did not form palpable tumors 3 months after injection with cell numbers fewer than 107, which can induce tumor formation in 50% of mice. In contrast, injection of as few as 105 irradiated cells was enough to produce a tumor, and injection of 106 irradiated cells resulted in tumor formation in 50% of mice. Tumor growth to 0.5 cm in diameter with injection of 107 irradiated and unirradiated cells needed 1.5 to 2 months and 1 month, respectively, but by 3 months after injection the tumor size of 3 to 3.5 cm in the irradiated group was larger than the tumor size of 2 to 2.5 cm in the unirradiated group (Fig. 6C). The serum VEGF concentration in the mice with tumor formation increased after injection (Fig. 6D). The increased rate of VEGF level was higher in the irradiated group than in the unirradiated cells and correlated with the growth rate of tumor size.

At 3 months, the tumors were harvested to analyze the microvessel density (Fig. 6E). After being stained with CD31, a marker of vessel endothelium, the tumor growing from irradiated cells showed a higher density of intratumor angiogenesis than the tumor growing from unirradiated cells. These results suggest the rapidly induced VEGF expression by radiation in HepG2 cells could be an initial factor for tumor growth. Crosstalk between tumor and endothelial cells by the secreted VEGF should then promote neovascularization and tumor growth in vivo.


   Discussion
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 Abstract
 Materials and Methods
 Results
 Discussion
References
 
The role of radiotherapy for advanced hepatocellular carcinoma remains unclear. The use of radiotherapy had been limited in hepatocellular carcinoma before the development of the three-dimensional conformal radiotherapy technique because of the sensitivity of the liver to radiation. Limited experience with local radiotherapy using three-dimensional conformal radiotherapy for unresectable hepatocellular carcinoma suggests that it is tolerable and induces a substantial tumor response (27, 28). However, in our analysis, one problem of focal radiotherapy or three-dimensional conformal radiotherapy is that outgrowth of intrahepatic and extrahepatic tumors was accelerated outside the radiotherapy treatment field in a significant portion of patients, which appeared immediately after radiotherapy and offset the overall survival benefit.

Virtually every modality used in the treatment of cancer has shown an adverse effect on tumor progression or metastasis under some conditions.

Rapid growth of tumors or metastases could be induced by surgical removal of a primary tumor (29). Exposure of melanoma cells to chemotherapy agents, such as dacarbazine, could result in enhanced tumor growth and metastasis in vivo (30). Enhancement of metastases following local tumor irradiation has also been noted as an untoward effect of ionizing radiation (31). The first major work used a transplantable mouse mammary carcinoma to show that mice receiving radiotherapy to the transplantable tumor developed pulmonary metastases more frequently than unirradiated control mice (32). Further studies showed that radiotherapy to the implanted lung tumors in the thighs of mice accelerated distant pulmonary metastatic growth (33); irradiation to primary cutaneous melanoma promoted tumorigenic and metastatic properties (34–36); radiotherapy enhanced migration and invasiveness of brain glioma cells (37); and radiotherapy induced an increase in invasive potential of pancreatic cancer cells (38). Several possible mechanisms for the radiotherapy-associated tumor progression have been proposed, including radiotherapy-induced DNA changes that increase the metastatic propensity of the tumor cells (39); radiotherapy-induced vascular damage that facilitates tumor cell intravasation (40); radiotherapy-induced increases in fraction of tumor hypoxia that up-regulates urokinase-type plasminogen activator receptor (36, 41); and radiotherapy-induced decreased production of an angiogenesis inhibitor from primary tumors (33). However, the pathomechanisms of rapidly developed intrahepatic and extrahepatic recurrence out of the radiotherapy treatment field after radiotherapy to primary hepatocellular carcinoma are unclear and comparatively little research has been devoted to their elucidation.

Evidence from CT scans, liver transplants, recurrence rates after resection, and animal hepatocarcinogenesis models have shown that hepatocellular carcinoma tends to be a whole-organ disease, is usually multifocal, and has characteristic vascular abnormalities in the portal vein and hepatic artery. Hepatocellular carcinoma has the propensity to invade the normal portal veins. In view of the anatomic structure of the portal system, it is possible that radiotherapy to unresectable hepatocellular carcinoma with microinvasion to the branches of portal vein causes tumor spread via the efferent route that leads to intrahepatic metastasis out of the radiotherapy field. However, because the rapid development of intrahepatic and extrahepatic recurrence outside the radiotherapy field is noted almost just after radiotherapy, the time might be too short for circulating malignant cells to dwell in the otherwise normal liver parts and other organs and grow into visible tumors. On the other hand, neovasculature, in which small branches of the hepatic artery grow, is required for the growing tumor to develop beyond a certain size (42). Neovascularization both precedes and is necessary for tumor progression and metastasis; thus, the rapid development of intrahepatic and extrahepatic tumor growth outside the radiotherapy field is more likely to result from the outgrowth of preexisting micrometastases or microtumors through certain mechanisms. First, the outgrowth of remote micrometastases or microtumors may be promoted by inhibition of the primary tumor because the production of antiangiogenic factors by the primary tumor suppresses distant tumor growth (29, 33). Second, dormant tumor cells may be stimulated by paracrine proliferative stimuli or proangiogenic factors that are up-regulated and released during cell response to radiotherapy-induced cell damage, tissue injury, and/or hypoxia (30, 36, 41).

Our molecular study suggests that sublethal dose of irradiation promoted hepatoma cell growth in vivo concomitant with elevated serum VEGF levels and enhanced intratumor angiogenesis. The biological effect of irradiation in human hepatoma cells was p53 independent and involved transcriptional up-regulation of VEGF. Overexpression of VEGF, an important growth factor controlling angiogenesis, has been associated with tumor progression, metastasis, and reduced survival in hepatocellular carcinoma and other tumors (43, 44). In some tumors, in contrast to normal tissues, VEGF is not produced by the endothelial cells but by tumor cells, which is consistent with a paracrine mode of action (45). Because in clinical radiotherapy, the total radiation dose for hepatocellular carcinoma treatment is delivered in a series of equal fractions separated by time intervals sufficiently long for the repair of sublethal DNA damage and induction of gene expression (46), the irradiated hepatocellular carcinoma cells receiving a sublethal dose in each fraction might induce VEGF expression and increase VEGF secretion between fractions until the accumulated radiotherapy doses reach the tumoricidal level. The radiotherapy-induced VEGF could be a paracrine proliferative stimulus to accelerate the growth of microtumors not included in the radiotherapy field. Due to field cancerization, the multiclonal displacement of normal epithelium by a genetically altered but microscopically undistinguishable homologue may explain the origin of recurrences that arise in the residual liver not involved in the radiotherapy. On the other hand, VEGF secretion can enhance distant metastasis, but metastasis formation also depends on the stroma reaction and cell type. That we did not see metastasis in the animal model might be because the microenvironment is not damaged by radiotherapy and the HepG2 cell line is nonmetastatic in nude mice (47). However, we did note that radiotherapy promoted both intrahepatic and extrahepatic tumor progression in hepatocellular carcinoma patients.

A further study to see if increase of serum of VEGF levels in patients with hepatocellular carcinoma receiving radiotherapy correlates with radiotherapy-associated tumor progression is warranted. The therapeutic implication derived from the present study could be a significant clinical relevance, justifying the combination of three-dimensional conformal radiotherapy with anti-VEGF angiogenic modalities for the treatment of unresectable hepatocellular carcinoma to reduce relapses. A better understanding of hepatocellular carcinoma behavior in response to radiotherapy through identification of more prognostic markers shown by gene and/or protein microarrays would allow a more rational basis for patient selection for radiotherapy or combined treatment.


   Footnotes
 
Grant support: National Science Council, Taipei, Taiwan, grants NSC 92-2320-B-368-001 and NSC 93-2314-B-368-002 (Y-L. Chung).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 12/16/05; revised 2/ 1/06; accepted 2/24/06.


   References
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 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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