Treatment Of Hepatocellular Carcinoma(HCC) With The Combination Of Trans-catheter Arterial Embolization(TAE) And Sunitinib: An Experimental Study

Part 1 Expression of angiogenesis-related factors and receptors after the treatment of hepatocellular carcinoma with trans-catheter arterial embolization(TAE)ObjectiveTo study the expression of angiogenesis-related factors and receptors after the treatment of hepatocellular carcinoma(HCC) with trans-catheter arterial embolization (TAE).Materials and methods43 cases of second-stage surgery of HCC after TACE(second-stage group) and 20 of direct surgery(direct surgery group) were taken and made into paraffin-embedded slices.Vascular endothelial growth factor receptor(VEGFR-2),phosphorylated VEGFR-2(p VEGFR-2),platelet-derived growth factor receptor-β(PDGFR-β), hypoxia induced factor-1α(HIF-1α) and microvessel density(MVD)(labeled by CD31)were detected with the immunohistochemical method.Rat HCC models,which were established with rat HCC line McA-RH7777 and Buffalo rats,were divided into 2 groups:trans-catheter arterial embolization with lipiodol(TAE group) or with saline(control group).Tumor volume and initial slope(IS) percent were measured with MRI at different times:6 hours before,1 hour after,6 hours after,24 hours after,3 days after and 7 days after surgery.MVD(labeled by CD31),HIF-1α,VEGFR-2,p VEGFR-2,PDGFR-βand p PDGFR-βof tumor were also detected with IHC and/or western blotting(WB).All data were processed by SPSS 11.0.ResultsIn clinical samples,The necrosis of cancerous tissue was obvious in second-stage group with 6 cases of complete necrosis.The density means(DM) of phosphorylated VEGFR-2 and HIF-1αin the cancerous tissue of HCC were(0.03376±0.01648) and(0.04705±0.02128) in second-stage group,and(0.02352±0.00893) and (0.03532±0.01623) in direct-surgery group,which showed statistical difference.The DMs of VEGFR-2,phosphorylated VEGFR-2 and HIF-1αin the peri-cancerous tissue were(0.03962±0.01680),(0.03064±0.01138) and(0.03749±0.01517) respectively in direct-surgery group,and(0.03022±0.01481),(0.02010±0.00827) and(0.02383±0.01390) respectively in direct-surgery group,all of which showed statistical difference.MVD of peri-cancerous tissue was(58.3±15.2) in second-stage group and(44.4±10.5) in direct-surgery group,both of which were different statistically.In the rat samples,tumor volume of TAE group was slightly smaller than control group,but it showed no significant difference(P＞0.05).Tumors' signal of both groups were almost even in MRI,while some dot-like necrosis were found in TAE group.The difference of initial slope(IS) percent between two groups were statistically significant(F=430.2,P＜0.001),and IS percent of TAE group at 1hours, 6hours,24hours and 3 days were all lower than control group significantly.MVD of TAE group was not significantly different than control group(U=59.0,P＞0.05). Differences of HIF-1αand p VEGFR-2 between two groups were significant statistically(U=22.0,26.0,both P value less than 0.05).VEGFR-2 and PDGFR-βshowed no significant difference between two groups(U=46.0,48.0,both P value more than 0.05).All of the above were detected with IHC.Expression of p VEGFR-2 and p PDGFR-β,detected with WB,were higher at 6 hours,3 days after surgery than before surgery and control group.Expressions of them in control group were not significantly different between before and after surgery.VEGFR-2 and PDGFR-βin both groups showed no significant difference between every two times.Conclusions1.Human HCC after TACE has obvious necrosis,but the cancerous and peri-cancerous tissues are more hypoxic and both the expression and function of VEGFR-2 enhanced.2.Rat HCC after TAE shows less blood supply in short period,and the tumor is more hypoxic,and eventually its function of VEGFR-2 and PDGFR-βenhanced.Part 2 Sunitinib suppresses hepatocellular carcinoma(HCC) cells and vascular endothelial cell in vitro ObejectiveTo study the suppression of HCC cells and vascular endothelial cell(VEC) with sunitinib,a multi-targeted receptor tyrosine kinase(RTK) inhibitor.Materials and methodsSeveral cell lines were used in this part including human HCC lines(Hep G2, Hep 3B),rat HCC line(McA-RH7777),human umbilical vein endothelial cell(HUVEC) and human normal liver cell(L-02).WST-1 method was applied to detect the proliferation or toxicity of VEGF-165,PDGF-BB and sunitinib for these cells.The apoptosis of cells with sunitinib was also detected with PI staining method by flow cytometry.Cell scratch test was used to find the movement inhibition of sunitinib for HUVEC.Cell immunofluorescence(IF) and western blotting(WB) were applied to detect the inhibition of VEGFR-2,p VEGFR-2,PDGFR-βand p PDGFR-βwith sunitinib in different concentration.All data were processed by SPSS 11.0.ResultsVEGF and PDGF promoted the proliferation of HUVEC at 25ng/mL and above(F=8.953,9.731,P=0.001),while they didn't show such effect for HepG2,Hep 3B,McA-RH7777 and L-02.Sunitinib suppressed the proliferation of HUVEC signigicantly at 0.01μM and above(F=41.003,P＜0.001),while it also suppressed Hep G2 and McA-RH7777 significantly at 10μM(F=3.367,4.518,both P value less than 0.05).Scratch test showed HUVEC didn't cross the scratch after incubation with sunitinib.Flow cytometry revealed the apoptosis of HUVEC was 19.2%at 0.1μM of sunitinib,36.7%at 1μM,which of Hep G2,Hep 3B and McA-RH7777 were 9.8%,9.2%and 14.5%at 1μM,and 15.6%,16.3%and 19.5%at 10μM respectively.HUVEC,Hep G2 and McA-RH7777 demonstrated the expression of VEGFR-2,p VEGFR-2,PDGFR-βand p PDGFR-β.After incubation with sunibinib,the phosphorylation of both receptors were suppressed,and furthmore it's correlated with the dose.Conclusions1.Sunitinib suppresses the proliferation of HUVEC and promotes its apoptosis, which is the main mechanism of anti-angiogenesis.2.Sunitinib also shows some kind of inhibition for HCC cells at high concentration,which non-anti-angiogenesis way may play a role in. Part 3High performance liquid chromatography(HPLC) detects sunitinib pharmacokinetics(PK) difference between different administration methodsObejectiveTo set up a method of HPLC to detect the concentration of sunitinib in blood and liver samples,and study the pharmacokinetics(PK) difference between oral and arterial administration.Materials and methodsHPLC with ultra-violet(420nm and 268nm) detected the sunitinib concentration in rat blood and liver samples using chrysin as internal standard.Standard curve and its linear scope were set up and their accuracy,precision and minimum limit of quantitation were validated.Rat HCC models were randomly divided into sunitinib oral administration group(oral group,sunitinib 10mg/kg,10 days continually) and TAE with sunitinib group(TAE group,sunitinib 10mg/kg signally with lipiodol).Blood and liver samples were harvested at 1,6,24,30,48 hours and 3,5,7,9 and 14 days after first dose in oral group,while at 0.5,1,6,12,24,30,48 hours and 3,5,7,9 and 14 days after TAE in TAE group.All samples were detected by the validated methods.ResultsThe standard curves were set up for blood and liver samples,which regression equation was y=2197.3×C-2.2907 for blood and y=4723.5×C+33.246 for liver. With the detection of quality concentrations,accuracy at different concentrations was (-6.4～-4.4)%,in-day precision was(5.4～10.6)%,and between-day precision was (3.1～9.5)%in blood samples.Accuracy at different concentrations was(-8.0～3.1) %,in-day precision was(11.4～13.3)%,and between-day precision was(1.5～2.4) %in liver samples.All accuracy and precision were under 15%,which demonstrate the method was stable and reliable.The minimum limit of quantification was 20ng/ml for blood and 25ng/ml for liver.In oral group,the concentration of sunitinib in blood and liver was peaking within 6～10 hours.The concentration at 6 hours after first dose was about 140ng/ml in blood,and 200ng/ml in liver.Its half-life period(T_1/2) was about 6-12 hours in blood.Peak concentration was gotten at about 6 hours after repeated dose.Then detected at every 24 hours after administration,blood concentration was(30-70)ng/ml and liver concentration was(100-130)ng/g at 9 days after the first dose.At 14 days, blood concentration was under the minimum limit and liver concentration was about 45ng/g.In TAE group,the concentration was peaking at about 30 minutes after TAE in blood and liver.The concentration in blood was(30-40)ng/ml at 24-30 hours after TAE,and under minimum limit at 48 hours and after.The concentration at 6 hours and before in liver were all above the maximum limit of qualification(1500ng/g). Then it decreased slowly comparing to blood concentration,was about 100ng/g at 5 days and under the minimum limit at 9 days and after.Conclusions1.The methods of HPLC to detect the concentration of sunitinib in blood and liver samples are stable and reliable,which can satisfy this experiment.2.The PK difference of sunitinib between oral and arterial administration provides the pharmacological basis for the combination of sunitinib and TAE.Part 4 Treatment of hepatocellular carcinoma(HCC) with the combination of sunitinib and trans-catheter arterial embolization(TAE) in rat modelsObejectiveTo investigate the effect of the combination of TAE and sunitinib for HCC,and study the mechanism of angiogenesis and molecular level.Materials and methodsRat HCC models,established with McA-RH7777 and Buffalo strait rats,were divided randomly into five groups:saline group(group A,control group),TAE group(group B),TAE+sunitinib iA group(group C),TAE+sunitinib p.o.group(group D) and TAE+sunitinib iA&p.o,group(group E).Rat survival time and tumor volume were evaluated.MRI plain scan and dynamic enhancement were applied for five groups at 1 week,2 weeks,4 weeks and 8 weeks after surgery and at the same time, some rats were sacrificed for the detection of MVD(labeled by CD31),HIF-1α, VEGFR-2,p VEGFR-2,PDGFR-βand p PDGFR-βwith IHC and/or WB.All data were processed by SPSS 11.0.ResultsSurvival time among five groups were different statistically(X²=10.8,P＜0.05),in which E group was longest(verses A group,X²=5.9,P＜0.05).Tumor volume was also different statistically(F=4.149,P＜0.001).Among them,tumors in C,D group were retarded(verses A group,both P value less than 0.05),and ones in E group were found little growth(verse A～D group,all P value less than 0.05),some of which even decreased.MRI demonstrated that the scope of necrosis increased obviously.IS percent among groups was different significantly(F=3.311,P＜0.001),in which E group decreased mostly(verse A group,P＜0.001).MVD in C～E group was reduced at 1 week,2 weeks after surgery with comparison to before surgery and A group,which among groups was different(F=33.005,P＜0.001).HIF-1αin C～E group increased more than A group,which among groups showed significant difference(X²=22.5,P＜0.001).VEGFR-2 in C～E group at 1 week,2 weeks after surgery also increased at different grade,which difference in groups was significant(X²=23.2,P＜0.001).But p VEGFR-2 in C～E group at 1 week,2 weeks after surgery was inhibited significantly(X²=9.38,P＜0.05).PDGFR-βamong groups also had significant difference(X²=13.3,P＜0.05),among which it in E group expressed strongly and persistently.WB showed that the phosphorylation of VEGFR-2 and PDGFR-βwas inhibited significantly.Among them,the inhibition of phosphorylation in E group at 7 and 14 days was more obvious than C and D group.However,VEGFR-2 and PDGFR-βwere not different significantly.Conclusions:1.Sunitinib,with combination of TAE for treatment of HCC,inhibits several angiogenesis-related receptors,suppresses angiogenesis,and enhances the effect of TAE.2.Sunitinib,through arterial administration after emulsification with lipiodol,can suppress angiogenesis of HCC more effectively in short period.3.Sunitinib with the combination of arterial administration after emulsification with lipiodol and oral administration continually,can perform more strong and persistent effect of anti-angiogenesis,which is more effective with the combination with TAE for HCC.