Experimental Study On Inhibitory Effect Of 10-hydroxycamptothecin On Hypoxia-inducible Factor-1α Expression And Angiogenesis In Liver Tumors After Transcatheter Arterial Embolization

PART I:Effect of 10-hydroxycamptothecin on expression of hypoxia-induced factor-1αand vascular endothelial growth factor induced by hypoxia in human HepG2 cellsPurpose:To determine the inhibitory effect of 10-hydroxycamptothecin (HCPT), a semisynthetic analog of camptothecin, on hypoxia-induced expression of hypoxia-induced factor-1α(HIF-1α) and it target gene vascular endothelial growth factor (VEGF) in human hepatoma cell line HepG2 in vitro.Materials and Methods:HepG2 cells were cultured under normoxic and hypoxic conditions respectively. The cells were treated with different concentrations of HCPT for 16 hours. Cell viability was determined by Cell Counting Kit-8 assay. Expression of HIF-1αand VEGF mNRA was examined by real-time reverse transcription-polymerase chain reaction (Real-time PCR). Expression of HIF-1αand VEGF protein was examined by western blot analysis.Results:No significant difference in cytotoxicity exerted by HCPT was noted between normoxic and hypoxic HepaG2 cells. Expression of HIF-1αprotein but not its mRNA was induced by hypoxia. HCPT down-regulated the hypoxia-induced HIF-1αprotein expression, but it did not affect HIF-1αmRNA expression. In contrast, hypoxia increased VEGF expression at both mRNA and protein levels, and HCPT showed potent inhibitory activity on the VEGF mRNA and protein overexpression. Although HCPT inhibited the expression of HIF-1αand VEGF in a dose-dependent manner, a low concentration of the drug not exerting significant cytotoxicity is sufficient to achieve potent inhibition. Conclusions:HCPT inhibits the hypoxia-induced expression of HIF-1αprotein and VEGF mRNA and protein in HepG2 cells, and the inhibitory effect is independent of its cytotoxicityPARTⅡ:Expression of hypoxia-inducible factor-1αin rabbit VX2 liver tumors after transcatheter arterial embolizationPurpose:To examine the effect of transcatheter arterial embolization (TAE) of hepatic cancers on hypoxia-inducible factor-1α(HIF-1α) expression in the residual viable tumor in an animal model.Materials and Methods:A total of 30 New Zealand White rabbits implanted with VX2 liver tumor were divided into 2 groups. TAE-treated group animals (n=15) were subjected to TAE with 150-250μm polyvinyl alcohol (PVA) particles. Control group animals (n=15) underwent sham embolization with distilled water. Six hours,3 days or 7 days after TAE, the animals were sacrificed, and samples of tumor and adjacent normal liver tissue were harvested. Expression of HIF-1αprotein was examined immunohistochemically. Real-time PCR was performed to examine the HIF-1αmRNA levels. Results:HIF-la protein was expressed in the VX2 tumors but not in the adjacent normal liver tissue. The HIF-la-positive tumor cells were located predominantly at the periphery of necrotic tumor regions. The levels of HIF-1αprotein were significantly higher in TAE-treated tumors than those in control tumors (P=0.002). Among the three sacrificing time points, significant increase in HIF-la protein was found between the two groups at the sacrificing time point of 6 h and 3 days after TAE (P=0.020, P= 0.031, respectively), whereas no significant increase in HIF-1αprotein was noted 7 days after TAE (P=0.502). In contrast, although HIF-la mRNA was expressed in TAE-treated and control VX2 tumors, there existed no significant difference in its level between the two groups (P=0.372).Conclusions:TAE of hepatic tumors increases the expression of HIF-1αat protein level in the residual viable tumor, which could be attributed to hypoxia generated by the procedure.PARTⅢ:Correlation of hypoxia-inducible factor-la with angiogenesis in rabbit VX2 liver tumors after transcatheter arterial embolizationPurpose:To determine the expression of HIF-1αand its relation to angiogenesis in liver tumors after transcatheter arterial embolization (TAE) in an animal model.Materials and Methods:A total of 20 New Zealand White rabbits were implanted with VX2 tumor in the liver. TAE-treated group animals (n=10) received TAE with polyvinyl alcohol (PVA) particles. Control group animals (n=10) received sham embolization with distilled water. Six hours or three days after TAE, animals were sacrificed, and tumor samples were harvested. Immunohistochemical staining was performed to evaluate HIF-1αand VEGF protein expression and microvessel density (MVD). Real-time PCR was performed to examine VEGF mRNA levels.Results:The levels of HIF-1αprotein were significantly higher in TAE-treated tumors than those in the control tumors (P=0.001). HIF-1αprotein was expressed in viable tumor cells that were located predominantly at the periphery of necrotic tumor regions. The levels of VEGF protein and mRNA, and mean MVD were significantly elevated in TAE-treated tumors compared with the control tumors (P=0.001,0.000 and 0.001, respectively). HIF-1αprotein level was significantly correlated with VEGF mRNA (r =0.612, P=0.004) and protein (r=0.554, P=0.011), and MVD (r=0.683, P= 0.001).Conclusion:HIF-la is overexpressed in VX2 tumors treated with TAE as a result of intratumoral hypoxia generated by the procedure and involved in activation of the TAE-associated tumor angiogenesis. HIF-1αmight represent a promising therapeutic target for anti-angiogenesis in combination with TAE against liver tumors.PARTⅣ:Inhibitory effect of 10-hydroxycamptothecin on hypoxia-inducible factor-1αexpression and angiogenesis in rabbit VX2 liver tumors after transcatheter arterial embolizationPurpose:To evaluate the effect of transcatheter administration of 10-hydroxycamptothecin (HCPT), a hypoxia-inducible factor-1α(HIF-1α) inhibitor, on HIF-1αexpression and angiogenesis in liver tumor after transcatheter arterial embolization (TAE) in an animal model.Materials and Methods:VX2 tumors were implanted in the livers of 30 rabbits. The animals were randomly divided into three groups of 10 animals each. Group 1 animals received hepatic intraarterial infusion of distilled water. Group 2 animals received iodized oil infusion followed by embolization with 150-250μm polyvinyl alcohol (PVA) particles. Group 3 animals received infusion of a mixture of HCPT (1 mg/kg body weight) with iodized oil followed by the particle embolization. Six hours or three days after transcatheter treatment, the animals were sacrificed, and the tumor samples were harvested. Immunohistochemical staining was performed to evaluate the levels of HIF-1αand vascular endothelial growth factor (VEGF) protein, and microvessel density (MVD).Results:The levels of HIF-1αand VEGF and MVD in tumors of group 2 were significantly higher than those of group 1 or 3 (P<0.05). However, no significant differences were noted in tumors between group 1 and 3 (P>0.05). HIF-1αlevels were significantly correlated with VEGF levels (r=0.587, P=0.001) and MVD (r= 0.527, P=0.003).Conclusion:Transcatheter infusion of HCPT has inhibitory effect on HIF-1αexpression and angiogenesis in liver tumors after TAE.