Study Of Effect On Rabbit Hepatic Implantation Tumor After Transcatheter Arterial Chemoembolization With Arsenic Trioxide And Lipiodol

Hepatocellular carcinoma (HCC) is one of the common malignant tumors in Asia, half of the death caused by HCC happened in china. Surgical resection is still the first choice for treating patients with HCC nowadays. However, because of tumor development, poor hepatic functional reserve due to underlying liver cirrhosis, or both, only a small portion of the patients are suitable candidates for surgery. Life span of untreatment advanced patients with HCC is from one to four months naturally. Transcatheter arterial chemoembolization (TACE) is considered to be an effective treatment for patients with HCC. TACE has anticancer effect through selectively blocking blood supply of tumor, the rate of tumor growth suppressed is 30% to 7o% in the near future. However, a prospective randomized controlled trial showed that the metastasis rate in lungs increased and the time of metastasis was ahead after TACE. Other studies showed that TACE before operation for patients who were candidates for surgical resection could promote extrahepatic metastasis and decrease life span. Arsenic trioxide (As₂O₃) has different effects on biological body. On one hand, it, as a toxicity material, could lead to damage on human being, on the other hand, could treat some disease, including tumor as a kind of drug. Many results of researchs, including cell culture, animal and clinical experiments, have proved that As₂O₃ has the effect on inhibiting the growth of HCC, the mechanisms of that are mainly in following aspects:â‘ cytoplast toxicity; â‘¡inducing tumor cell apoptosis; â‘¢inhibiting the activity of telomerase of tumor cell; â‘£inhibiting the expression level of vascular endothelial growth factor (VEGF) of tumor cell, at the same time inducing vascular endothelial cell apoptosis and shuting down the tumor blood supply selectively. The effect of As₂O₃ on HCC cells depends on dosage and time. TACE is the major therapeutic method for patients with HCC who are not candidates for surgical resection. However, complete tumor necrosis is rarely seen after TACE. Some studies showed that embolization leaded to tumor anoxia, which on one hand could reduce the size of tumor, on the other hand promote the expression of VEGF in the residual tumor tissue. VEGF is associated with metastases and poor prognosis in various cancers. This would be the main reason for poor prognosis in patients with HCC after TACE chronically. Because Arsenic trioxide (As₂O₃) could selectively induce apoptosis of hepatocellular carcinoma cell, at the same time it could inhibit neovascularization, and decrease the expression level of VEGF. Theoretically, embolization via hepatic artery with lipiodol combined As2O3 could inhibit tumor growth by blocking blood supply of tumor, leading to part or a large part of tumor necrosis, decrease the expression level of VEGF which may enhance the effect against HCC, reduce metastasis and relapse of tumors after TACE, without further deteriorating liver function. The objective of the study is to testify the hypothesis above. It is necessary to establish a reliable animal model bearing a single liver tumor for exactly assessing the effects of biological aspects, including treatment, prognosis of liver tumor and so on. Rabbits are the largest animal models bearing tumor and suitable for interventional therapy via hepatic artery nowadays. There are several methods to inoculate tumor, but the common problems are low successful rates and high ectopic rates of implantation. For raising the successful rate of implantation, we improved the method during the experiment. The animal experiment was divided into two parts: short-term and long-term observation. value of microvessel density(MVD), expression of VEGF and PCNA, apoptosis index of the tumors were evaluated, blood flow of the tumors was detected by contrast-enhanced power Doppler, and the partof liver function was determined in the short-term experiment (one week after treatment). The growth and metastasis of the tumors were observed in the long-term experiment (five weeks after treatment). For guiding the application of As₂O₃, we have investigated the slowly releasing properties of As₂O₃ in lipiodol in vivo and in vitro. Part one:Establishment of rabbit model bearing VX2 liver tumor and monitoring tumor angiogenesis with contrast-enhanced power Doppler Objective: To compare the features of establishing rabbit model bearing VX2 liver tumor in different methods, to observe the efficacy of agarose in preventing VX2 carcinoma cell leakage, the application of ultrasound in monitoring tumor growth and contrast-enhanced power Doppler for evaluating tumor angiogenesis Methods: Forty-five New Zealand white rabbits were divided into 3 groups (15 rabbits in each group): Group 1, The puncture site was gently compressed using an absorbent cotton for three minutes, after injecting VX2 tumor fragment suspension 0.3mL (10⁷-10⁸ cells/ mL) into left lobe of the liver percutaneously under the ultrasonographic(US) guidance; Group 2, VX2 tumor fragment suspension was injected in the same way as in Group 1, except 0.2ml of liquefied agarose was injected to seal the aperture site; Group 3, 3 pieces of 1mm³ fragment of VX2 carcinoma were inoculated into the sub-capsule of the left hepatic central lobe via laparotomy. Tumor growth was monitored by ultrasound and biological features of tumors were examined by routine histology. Three weeks after implanting VX2 tumor into liver, microbubble contrast agent intravenous bolus injection, blood flow signals were detected by power Doppler and mean color vessel density(MCVD) was calculated quantificationally with computer image analysis system. The rabbits were sacrificed after sonography, and MVD in the tumor tissues was examined by immunohistochemical method. The correlation of the MCVD and MVD was analysed Results: â‘ Tumors were successfully implanted in 3/15 (20.0%), 12/15(80.0%), and 11/15 (73.3%) of animals in Group 1, Group 2, and Group 3, respectively, with the respective ectopic implantation rates of 12/15 (80.0%), 3/15 (20.0%), and 2/13 (15.4%). There was significant difference between Group 1 and Group 2, Group 1 and Group 3. â‘¡Under the ultrasound, most tumors were isoechoic, some tumors were hypoechoic or slightly hyperechoic with surrounding echo halo. In all tumors, blood flow was detected by color Doppler or power Doppler. â‘¢Tumors were poorly demarcated from the surrounding liver parenchyma. Multiple mitotic figures were detected on histology. â‘£The maximum tumor diameters measured by ultrasound and macroscopic pathology were closely correlated (r = 0.946,P =0.000). ⑤There was positive correlation between MCVD and MVD of the tumors( r=0.746,P<0.05). Conclusions: VX2 liver tumor models were successfully established with percutaneous US-guided injecting tumor fragment suspension into liver parenchyma. Blockade of the tumor inoculation site with agarose plug is an useful method of preventing tumor cell leakage and ectopic implantation. Ultrasonography is useful in evaluating liver tumor growth. Contrast-enhanced power Doppler can be used for evaluating tumor angiogenesis in vivo. Part two: Study on controlled release of As₂O₃ -Lipiodol suspension and As₂O₃-Lipiodol emulsion Objective: To investigate the physical properties and controlled release of Arsenic trioxide(As₂O₃) in 40% Lipiodol(LIP) and ultra -fluid lipiodol(UFL) emulsions and suspensions for clinical application. Methods: Several types of emulsions and suspensions were prepared using LIP or UFL and As₂O₃ by"pumping technique". Physical stability of each preparation was examined macroscopically and microscopically. The drug release test was made through dialysis band from the phosphate buffer solution in 37±0.5℃, the As₂O₃ releasing rates were calculated by detecting the concentration of As₂O₃ in dialysis solution (releasing rate = concentration of As₂O₃ measured×100/As₂O₃ quantity in all×100 %) Results: Separation and sedimentation in LIP or UFL As₂O₃suspensions were presented after putting them some time. As₂O₃ was separated easier in LIP suspension than in UFL suspension. Separation between oil phase and water phase was presented immediately in both of emulsions without emulsifier, the emulsions with emulsifier were more stable. As₂O₃ can be released slowly in LIP or UFL emulsions, the release of As₂O₃ was slower in their emulsions with emulsifier than in those without emulsifier. The release of As₂O₃ in their suspensions was faster. Conclusion: Both LIP and UFL can serve as embolization materials and carrier of As₂O₃. Using their emulsions can increase the dose of As₂O₃ in order to improve therpeutic effect without concurrent increase of adverse reactions . Part three: Study on growth and angiogenesis in rabbit hepatic implantation tumor after TACE with As₂O₃ and Lipiodol Objective: To investigate the effect of TACE with As₂O₃ and Lipiodol on growth, angiogenesis, and VEGF expression of hepatic tumor. Methods: Forty New Zealand White rabbits were randomly divided into five groups and VX2 carcinoma was implanted in the left lobes of the livers. Two weeks later, a catheter was inserted into the hepatic artery of rabbit with VX2 hepatic tumor and infusion was performed via the hepatic artery using physiological saline (group A), As₂O₃(group B), Lipiodol (group C) , adriamycin(ADM) plus Lipiodol (group D) and As₂O₃ plus Lipiodol (group E). Tumor′s size was observed by sonography, volume and growth rates of tumors were calculated, blood flow of the tumors was studied by power Doppler sonography before and after intravenous administration of a microbubble contrast agent one week after treatment. MCVD was calculated, necrotic rate of tumors was assessed by routine histology; MVD and expression of VEGF of tumors were examined by immunohistochemistry. Results: The growth of tumors was suppressed in embolization groups. Necrotic rate of tumors was higher in group E than that in other groups. Blood flow of tumors revealed by unenhanced or contrast-enhanced power Dopplersonography significantly decreased after TACE treatment. There was no statistically significant difference between group C and group D , group C and group E before using microbubble contrast agent, but blood flow of the tumors was lower in group E than that in groups C and D after using microbubble contrast agent. MVD was slightly higher in groups C and D versus group A, the value of MVD was 23.4±4.7,22.3±5.3 and 21.8±6.3 respectively in these groups, but there was no statistically significant difference ( P>0.05). Expression of VEGF enhanced after embolization (groups C and D). The staining intensity of VEGF in groups C and D was 0.163±0.019 and 0.160±0.016 respectively, higher than that in group A (0.140±0.02, P<0.05). MVD and expression level of VEGF were significantly lower in group E(15.1±3.2 and 0.102±0.02 respectively) compared with that in other groups (P<0.05). There was positive correlation between the value of MVD and VEGF expression level. Conclusions: Embolization with As₂O₃ and Lipiodol can suppress the growth of tumor, increase the tumor necrotic rates, inhibit the development of neovascularization of residual tumors, reduce the expression of VEGF. Contrast-enhanced power Doppler sonography is an useful technique for assessing blood flow of the residual tumors after TACE treatment . Part four:Observation of apoptosis and proliferation of tumor cells, liver function of rabbits after treatment and the releasing effect of As₂O₃ in Lipiodol Objective: To investigate the effect on apoptotic index, expression of proliferating cell nuclear antigen (PCNA) in hepatic tumor tissue, the influence on liver function, and to observe the As₂O₃ release in Lipiodol after treatment of TACE with As₂O₃ and Lipiodol. Methods: Forty New Zealand White rabbits were randomly divided into five groups and VX2 carcinoma was implanted in the left lobes of the liver. Two weeks later, a catheter was inserted into the hepatic artery of rabbit with VX2 hepatic tumor and infusion was performed via the hepatic artery with physiological saline (group A), As₂O₃(group B), Lipiodol (group C) ,ADM plus Lipiodol (group D, ) and As₂O₃ plus Lipiodol (group E). Blood samples were collected from ear margin vein of rabbits, 3days before, 4 days and 7 days after treatment, respectively. The level of AST and ALT in serum was determined. Blood samples were collected from hepatic vein and a portion of tumor and normal hepatic tissue were excised in groups C and E, the quantities of arsenic in the biological tissue samples were determined. The apoptosis index and proliferation index in tumor tissue were examined one week after treatment by using TUNEL (in situ nick end-labeling) and anti-PCNA monoclonal antibody,respectively. Results: Serum AST and ALT level markedly increased 4 days after embolization treatment, there was no significant difference in group E versus group C (P>0.05), but there was statistically significant difference in group D versus other groups (P<0.05). 7 days after treatment, the AST and ALT level tended to reverse, in group D the AST and ALT level was still higher than in other groups. The quantity of arsenic in the liver tumor tissue still keeped in high level in group E 7 days after treatment, there was statistically significant difference compared with liver tissue of tumor –free hepatic right lobe or liver tumor tissue in group C (P<0.05). Moreover, the quantity of arsenic in the serum of hepatic vein in group E was higher than that in peripheral vein, or than that in serum collecting from other groups (P<0.05). Proliferation index (PI) of tumors was 1.53±0.42, 1.82±0.41, 2.66±0.54, 2.91±0.32 and 3.44±0.65, apoptosis index (AI) was 60.8±15.5, 55.9±14.8, 42.4±11.2, 40.6±8.8 and 28.5±5.7 in groups A, B, C, D and E, respectively, there was negative correlation between PI and AI . Conclusions: Embolization treatment via hepatic artery may lead to temporal rise of AST and ALT level, which reflect the damage of liver cell, the AST and ALT tend to reverse after one week treatment. The toxicity of As₂O₃ to normal liver tissue is lower than ADM. The apoptosis of residual tumor cell increases and the proliferation decreases in group E. This shows that inducing apoptosis of tumor cells would be one of main mechanisms for As₂O₃ fighting against tumor. The arsenic may slowly release in the Lipiodolretained in tumor tissue and operate against tumor continuously. Part five:Study on growth and metastasis of hepatic tumor in rabbit after TACE with As₂O₃ and Lipiodol Objective: To investigate the effect of TACE with As₂O₃ and Lipiodol on growth, metastasis of the implanted hepatic tumor in rabbits. Methods: Forty New Zealand White rabbits were randomly divided into five groups and VX2 carcinoma was implanted in the left lobes of the livers. Two weeks later, a catheter was inserted into the hepatic artery of rabbit with VX2 hepatic tumor and infusion was performed via the hepatic artery with physiological saline (group A), As₂O₃ (group B), Lipiodol (group C), ADM plus Lipiodol (group D) and As₂O₃ plus Lipiodol (group E). The dosage of As₂O₃ was 2mg/kg. Five weeks after tumor inoculation, all rabbits were sacrificed, the body weight and liver weight of animals were assessed, the hepatic Index(HI) was calculated, volume and necrotic area of the implanted tumor were measured. The presence of metastases in the liver, lungs and other organs was recorded . Results: Five weeks after inoculation, the body weight of all animals decreased, the liver weight increased, HI was 9.8±2.2, 9.7±2.1, 8.5±2.0, 8.4±2.1 and 6.4±1.2 in groups A, B, C, D and E, respectively. The mean volume and necrosis area of the implanted tumor were 35.5±7.6cm3 and 66.0±6.4% in group A, 32.2±9.7 cm 3 and 64.3±8.3% in group B, 21.2±8.5 cm 3 and 64.8±7.0% in group C, 20.9±11.3 cm 3 and 69.8±8.6% in group D, 11.8±4.0 cm 3 and 70.8±7.6% in group E, respectively. The difference of tumor volume between embolization treatment groups versus unembolization treatment groups, group E versus other groups were statistically significant, the difference of necrosis area of the tumors was not statistically significant. The numbers of metastasis in the lungs were 52.4±32.2, 51.8±26.3, 54.8±29.2, 53.5±30.7 and 19.6±17.0 , and the diameter of metastasis in the lungs was 3.8±1.2 mm, 3.6±1.1 mm, 3.9±1.3 mm, 3.5±1.6 mm and 2.2±0.7 mm in groups A, B, C, D and E, respectively, the difference between group E versus other groups (P<0.05) was statistically significant. The differences of...