Combined inhibitors of angiogenesis and histone deacetylase: efficacy in rat hepatoma

Aim: To evaluate the antitumoral effect of combined inhibitors of angiogenesis and histone deacetylases in an experimental rat hepatoma model.

Methods: MH7777A hepatoma cells were injected into the liver of male Buffalo rats. After 7 d treatment with the vascular endothelial growth factor receptor antagonist PTK787/ZK222584 (PTK/ZK), the histone deacetylase inhibitor MS-275, tamoxifen (TAM) and/or retinoic acid was initiated (n ≥ 8 animals/group). Natural tumor development was shown in untreated control groups (control 1 with n = 12, control 2 with n = 8). The control groups were initiated at different time points to demonstrate the stability of the hepatoma model. For documentation of possible side effects, we documented any change in body weight, loss of fur and diarrhea. After 21 d treatment, the rats were euthanized. Main target parameters were tumor size and metastasis rate. Additionally, immunohistochemistry for the proliferating cell nuclear antigen (PCNA) and TdT-mediated dUTP-biotin nick end labeling (TUNEL) assay were performed.

Results: The control groups developed large tumor nodules with extrahepatic tumor burden in the lung and abdominal organs (control 1: 6.18 cm(3) ± 4.14 cm(3) and control 2: 8.0 cm(3) ± 4.44 cm(3) 28 d after tumor cell injection). The tumor volume did not differ significantly in the control groups (P = 0.13). As single agents MS-275 and PTK/ZK reduced tumor volume by 58.6% ± 2.6% and 48.7% ± 3.2% vs control group 1, which was significant only for MS-275 (P = 0.025). The combination of MS-275 and PTK/ZK induced a nearly complete and highly significant tumor shrinkage by 90.3% ± 1% (P = 0.005). Addition of TAM showed no further efficacy, while quadruple therapy with retinoic acid increased antitumoral efficacy (tumor reduction by 93 ± 1%) and side effects. PCNA positive cells were not significantly reduced by the single agents, while dual therapy (MS-275 and PTK/ZK) and quadruple therapy reduced the PCNA-positive cell fraction significantly by 9.1 and 20.6% vs control 1 (P < 0.05). The number of TUNEL-positive cells, markers for ongoing apoptosis, was increased significantly by the single agents (control 1: 6.9%, PTK/ZK: 11.4%, MS-275: 12.2% with P < 0.05 vs control 1). The fraction of TUNEL-positive cells was upregulated highly significantly by dual therapy (18.4%) and quadruple therapy (24.8%, P < 0.01 vs control 1). For the proliferating (PCNA positive) and apoptotic cell fraction, quadruple therapy was significantly superior to dual therapy (P = 0.01).

Conclusion: Combined PTK/ZK and MS-275 were highly effective in this hepatoma model. Quadruple therapy enhanced the effects microscopically, but not macroscopically. These results should be investigated further.