Synergistic anti-tumor efficacy of doxorubicin and flavopiridol in hepatocellular carcinoma

Abstract

Introduction: Hypoxic hepatocellular carcinoma (HCC) cells have been reported to be less sensitive than normoxic HCC cells to doxorubicin due to N-myc downstream-regulated gene-1 (NDRG1) expression. Additionally, a previous in vitro study showed that flavopiridol, a cyclin-dependent kinase inhibitor, suppresses NDRG1 expression and sensitizes hypoxic HCC cells to doxorubicin cytotoxicity by increasing apoptosis. Based on this, we investigated whether the combination treatment of doxorubicin and flavopiridol has a synergistic anti-tumor efficacy against an in vivo model of HCC. Methods: An in vivo HCC mouse model was established by implanting C3H/He mouse with MH134 HCC cells. The mice were divided into 4 groups, and each group was administered DMSO (control group), doxorubicin, flavopiridol, or the doxorubicin + flavopiridol combination. We evaluated necrotic area and hexokinase II expression to evaluate whether hypoxic condition was achieved in HCC. The anti-tumor efficacy was evaluated by measuring tumor volumes, and the anti-tumor mechanism was investigated by quantifying apoptotic cells, microvessel densities, and NDRG1 expression through immunohistochemical staining of tumor tissue. In the in vitro portion of the study, HCC cell viability was assessed using the MTS assay. The apoptotic signaling pathway and expression of NDRG1 were evaluated with immunoblotting. Results: In all mouse HCC tumor tissue, hypoxic condition was achieved. Tumor growth was significantly suppressed in the doxorubicin+flavopiridol combination group compared to the control, doxorubicin-only, or flavopiridol-only group. On immunohistochemical staining of tumor tissue, the percentage of apoptotic cells was significantly high in the doxorubicin+flavopiridol combination group

however, microvessel densities were not statistically different across groups. Additionally, flavopiridol suppressed the expression of NDRG1 in mouse tumor tissue. In the in vitro study, the doxorubicin+flavopiridol combination treatment significantly suppressed cell viability by increasing apoptosis through the caspase 7-dependent pathway. Flavopiridol also suppressed NDRG1 expression in MH134 cells in vitro. Conclusions: These results show that combination treatment of doxorubicin and flavopiridol has a synergistic anti-tumor effect in an HCC model, both in vivo and in vitro. This can be interpreted that flavopiridol killed doxorubicin-resistant cells under hypoxic condition, which continued to exist after doxorubicin treatment. Synergistic effect of doxorubicin and flavopiridol combination therapy was attributed to increased apoptosis by suppressing NDRG1 expression rather than decreased angiogenesis.