MMPs And VEGF Were Increased By Irradiation Combined With Interventional Treatment In The Rabbit VX2 Liver Tumor

Background and Objection:Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in our country. Asians have high incidence and mortality rates compared with other racial/ethnic groups. However, recent 1-year survival rates remained less than 50% in advanced cases.Transarterial chemoembolization (TACE) using iodized oil as a carrier of chemotherapeutic agents has been routinely performed to control HCCs.TACE has become the important non-surgical method for treating HCC.The potent local antitumor effect of radiotherapy should be considered seriously as a part of the treatment strategy. Novel radiotherapy technologies have made it possible to deliver higher doses of radiation to the tumor while avoiding damage to critical normal tissues adjacent to the tumor. Radiotherapy might play a significant role in treating unresectable HCCs, alone or combined with other locoregional treatment such as TACE.The rabbit VX2 liver tumor is suitable for the experimental research of TACE and radiation therapy.Metastasis is an exceedingly complex process that includes cell adhesion to the ECM, protease secretion, ECM degradation, and tumor cell migration. Matrix metalloproteinases (MMPs) are the most important relevant extracellular proteolytic enzymes. MMP-2 and MMP-9 are two critical members that play important roles in HCC invasion and metastasis by degrading the basement membrane. MMP-2 (gelatinase A) and MMP-9 (gelatinase B) are cancer-associated, secreted, zinc-dependent endopeptidases. MMPs cleave many different targets (ECM, cytokines, growth factors, chemokines, and cytokine/growth factor receptors), which, in turn, regulate key signaling pathways in cell growth, migration, invasion, inflammation, and angiogenesis.Animal experiments and clinical studies have shown that the VEGF signaling pathway plays an important role in endothelial cell differentiation, vascular permeability, and promotion of new vessel growth. VEGF high expression are associated with recurrence rates and poor survival in patients with HCC. Expression of VEGF was found in the hepatocytes and HCC cells,The degree of VEGF expression was correlated with angiogenesis and cell proliferation activity.Recent evidences have shown that irradiation can promote the invasiveness of hepatocellular carcinoma cells with MMP-9,VEGF,MMP-2 expression. Recurrence and metastasis are frequently observed after radiotherapy for HCC. The upregulation of MMPs and VEGF that is induced by radiation has been claimed to be involved. Radiation may increase the invasion ability of HCC through activating the MMP proteolytic system; simultaneous administration of the MMP inhibitor during radiotherapy suppresses the radiation-enhanced invasion from latent type to active type.The increased expression of VEGF and MMP-9 in residual VX2 rabbit liver tumor cells and tumor angiogenesis postembolization are responsible for the increased metastatic potentiality and invasiveness of these cells. The residual surviving cancerous tissue in HCC after TACE has a rich vascularity. TACE increases VEGF expression in the residual surviving cancerous tissue.The purpose of the present study was to investigate whether the combination of TACE and radiation therapy will generate an effect on the expressions of MMPs and VEGF reflecting tumor invasion and metastasis in an experimentally induced rabbit VX2 liver tumor model.Methods and materials1. Animals and treatmentThe initial weight of the rabbits used in this study was median 4.6 kg (range 4.2-5.5 kg). For treatment and follow-up, the animals were anesthetized with 25 mg/kg sodium pentobarbital through an ear vein and immobilized on a surgical table. The VX2 cells were implanted into subcutaneous tissues of the limb of a carrier rabbit, finally developing VX2 tumors. Then, the donor tumor was excised and divided into 1 mm3 pieces. A midline abdominal incision was made to expose the liver in the recipient rabbits. The tumor tissue was inserted approximately 1.0 cm into the left lobe. A single dose of penicillin was intravenously injected via an auricular vein to prevent infection. Experiments were carried out 2-3 weeks later, which is a period required for the tumor cells to grow, when the tumors were anticipated to be 15 mm in diameter. Computed tomography (CT) scans of the liver were performed on all animals before treatment. A total of 40 tumor-bearing rabbits were randomly divided into four groups, with 10 rabbits in each group (n= 10/group). In group A (control), 0.5 mL of 0.9% saline was injected; in group B,1 mL of Lipiodol was injected into the hepatic artery; in group C, radiotherapy was given; and in group D, radiotherapy and TACE were given.2. TACE:General anesthesia was induced and the right femoral artery was exposed. A 3-F microcatheter (Cook, Bloomington, IN, USA) was inserted into the celiac artery. Celiac angiography was performed to identify the hepatic arterial anatomy and the feeder artery of the tumor, under a digital subtraction angiography unit (AlluraXper, Philips Healthcare, The Netherlands). A dose of 1 mL of Lipiodol (Guerbert Co., France) or 0.9% saline was injected carefully to avoid effluxion of the embolic materials out of the artery through a hyperselective microcatheter (Fig.1A). The catheter was then removed and the femoral artery was ligated to obtain hemostasis. CT (LightSpeed Ultra, GE Medical Systems, WI, USA) scans were obtained to evaluate the distribution of the iodized oil.3. Radiotherapy:The rabbits were fastened on the wooden base plate. CT scans for radiotherapy planning were done, and the rabbits were placed in a supine position (Fig. 1B). The data of the enhanced series were immediately sent to a treatment planning station, and the rabbits remained on the wooden base plate. The three-dimensional CT-based planning system (XIO-Release 4.80, Elekta, Ltd., Stockholm, Sweden) was used to cover adequately the tumor target with 0.8 cm margins, which covered the entire tumor and decreased the irradiated bowel, and the normal liver and kidneys (Fig.1C). The liver tumors were irradiated once with a 6MeV or 9MeV external electron beam (Fig. 1D) at a dose of 15 Gy using a linear accelerator (23EX, Varian Medical Systems, CA, USA, dose rate of 4 Gy/min).4. The animals were then sacrificed mercifully by an intravenous dose of sodium pentobarbital 1 week after the therapy. The tumor tissue was collected. The experimental protocols were approved by the Nanhai Affiliated Hospital of Southern Medical University Animal Care and Use Committee and were performed in accordance with the Southern Medical University Guidelines for the Care and Use of Laboratory Animals.5. ImmunohistochemistryThe EnVision two-step method was performed according to the manufacturer’s instructions. Consecutive sections were deparaffinized with xylene and dehydrated by a series of ethanol solutions (100%,95%,80%, and 70%). The sections were first washed with distilled water, and then washed three times with phosphate-buffered saline (PBS) for 5 min each. The endogenous peroxidase was inactivated using 3% hydrogen peroxide for 10 min, and then again for 15 min. The sections were rinsed in PBS three times for 10 min each and were blocked with 5% normal goat serum for 30 min. MMP-2 antibody, MMP-9 antibody, and VEGF antibody were employed for detecting the respective proteins, with anti-rabbit Envison-PO (Dako) secondary antibodies. The antibody binding sites were visualized using 3,3’-diaminobenzidine substrate, and the slides were examined after counterstaining with hematoxylin. The result was considered positive when a brown precipitate localized in the cytoplasm. Pictures were captured and pixels were counted for quantification using the Image-Pro Plus Version 6.0 software.6. Total RNA extraction and real-time PCR assaysQuantitative real-time polymerase chain reaction (PCR) was performed to examine the changes in the VEGF and MMP mRNA expression of different groups in the rabbit liver tumor. Total RNA was extracted from tissues using a TRIzol reagent, according to the manufacturer’s instructions. RNA (2 Mg) was converted to cDNA using a RevertAid First Strand cDNA Synthesis Kit. Total RNA was transcribed to cDNA with a primer and was amplified by the SYBR Green real-time PCR reaction system. The relative expression was calculated using the comparative 2 △△ Ct method; 2△△ Ct>3 or<1/3 was deemed statistically significant.7. Protein isolation and Western blot analysisProteins from tissues were resolved using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. Briefly,15 mg of total protein lysates were resolved using SDS-PAGE and transferred to polyvinylidene difluoride membranes by electroblotting. Antigen-antibody complexes were visualized using a chemiluminescence kit (BCA Protein Assay Kit).8. Statistical analysisData were shown as the mean ± standard deviation (SD). Differences between groups were analyzed using one-way analysis of variance and Bonferroni test. Statistical analysis was performed using the SPSS software for Windows version 16.0. P<0.05 was considered as statistically significant.Results:1.General observation and Histological analysis of residual CarcinomaThe untreated tumor consisted of viableVX2 cells with bright and large nucle.After transcatheter lipiodol embolization was given through the hepatic artery,we found that lipiodol was mainly penetrated in the periphery of tumors.But only a few lipiodol deposited in the central region where was poorly vascularized.A few viable VX2 cells,inflammatory cell infiltration and necrosis was seen in all the treatment groups.Necrosis area was different from the pretreatment showing as hard,pale and dull shape.After irradiation at a dose of 15 Gy,the number of viable tumor cells was markedly decreased.The VX2 cells were replaced by the fibrous tissue.2.The expression of VEGF,MMP-2,MMP-9 in tumor cellsThe location of VEGF,MMP-2,MMP-9 in tumor tissue was displayed in all groups by immunohistochemical staining.They were mainly expressed in the cytoplasm of tumor cells.Quantitation of the protein content of VEGF,MMP-2 and MMP-9 were assessed by computerized planimetry in the carcinoma cells in immunohistochemically stained slides by using Image-Pro Plus 6.0.In B,C and D group, the levels of VEGF,MMP-2 and MMP-9 were significantly increased compared to A group (P<0.05). However,The levels of VEGF and MMP-2,9 were showed no significant difference among B, C and D group (P>0.05).3.Western blot analysis was also performed to measure the expressions of VEGF,MMP-2,MMP-9.The expression of VEGF,MMP-2 and MMP-9 were also increased in B,C and D group compared to A group.We compared the the transcription levels of VEGF,MMP-2 and MMP-9 mRNA of residual tumor cells by real time PCR. According to Ct values from the results of quantitative real-time PCR,the difference between A group and treatment groups(B,C and D group) was statistically significant for VEGF,MMP-2 and MMP-9(P<0.05),but there were no significances between B,C and D group (P>0.05).ConclusionIn this study, we showed that VEGF,MMP-2,9 expression were significantly increased in the group TACE,irradiation and TACE plus irradiation compared with the control group.The expression VEGF,MMP-2,9 slightly were decreased in TACE plus radiation group compared to monotherapy.However there was no significant difference between the rabbits treated with TACE/irradiation alone and TACE plus radiation therapy.