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Arsenic trioxide (ATO) is of limited therapeutic benefit for the treatment of solid tumors. Genistein exhibits anticancer and pro-oxidant activities, making it a potential candidate to enhance the efficacy of ATO whose cytotoxicity is oxidation-sensitive. This study sought to determine whether genistein synergizes with ATO to combat hepatocellular carcinoma (HCC). Three human HCC cell lines, namely HepG2, Hep3B, and SK-Hep-1, were incubated with ATO, genistein, or ATO + genistein. The cells were also pretreated with antioxidant agents N-acetyl-L-cysteine (NAC) or butylated hydroxyanisole (BHA). Cell viability, apoptosis, intracellular reactive oxygen species (ROS), mitochondrial membrane potential (DeltaPsim), expression of Bcl-2, Bax, caspase-9, and -3, and release of cytochrome c into the cytosol were examined. The synergistic effect of ATO and genistein was also assessed using HepG2 xenografts subcutaneously established in BALB/c nude mice. The results show that genistein synergized with ATO to reduce viability, induce apoptosis, and diminish the DeltaPsim of cells. The combination therapy down-regulated Bcl-2 expression, up-regulated Bax expression, enhanced the activation of caspase-9 and -3, and increased the release of cytochrome c. The synergistic effect of ATO and genistein was diminished by pretreatment with NAC or BHA. Genistein increased the production of intracellular ROS, while ATO had little effect. Genistein synergized with a low dose of ATO (2.5 mg/kg) to significantly inhibit the growth of HepG2 tumors, and suppress cell proliferation and induce apoptosis in situ. There were no obvious side effects, as seen with a high dose of ATO (5 mg/kg). Combining genistein with ATO warrants investigation as a therapeutic strategy to combat HCC.

Arsenic trioxide (As2O3) is a potent antitumor agent used to treat acute promyelocytic leukemia (APL) and, more recently, solid tumors. However, the dose of As2O3 required to suppress human xenographs in mice is markedly higher than that used to treat APL in humans. Paradoxically, low doses of As2O3 stimulate angiogenesis, which might be expected to promote tumor growth. Clearly, appropriate dosages of As2O3 are required to treat human patients to avoid toxicity and undesirable side effects. In the present study, we investigated As2O3 with respect to its toxicity and effects on tumor growth, angiogenesis and cell apoptosis using H22 hepatocellular carcinoma (HCC) cells in a mouse model of HCC. As2O3 inhibited tumor growth and angiogenesis, and enhanced tumor cell apoptosis at doses greater than 1 mg/kg, but mice lost weight and failed to thrive at doses of 4 mg/kg and greater. In contrast, low doses (

The hypoxic microenvironment inside solid tumors, including hepatocellular carcinoma (HCC), is a major cause of tumor resistance to chemotherapy. The recently identified hypoxia‐inducible factor (HIF)‐2 executes the hypoxia response. Its expression feature and transcriptional targets indicate a possible dominance of HIF‐2 in regulating genes in HCC. The aim of the present study was to determine whether transfection of siRNA targeting HIF‐2α could enhance the efficacy of doxorubicin, the most commonly used drug in the treatment of HCC. Transfection of HIF‐2 siRNA into human HCC cells downregulated the expression of HIF‐2α, vascular endothelial growth factor (VEGF), transforming growth factor (TGF)‐α, and cyclin D1, but had little effect on the expression of HIF‐1α, fms‐related tyrosine kinase‐1 (Flt‐1), the glucose transporter (GLUT)‐1, and lactate dehydrogenase A (LDHA). Doxorubicin itself only downregulated VEGF expression. Furthermore, HIF‐2 siRNA inhibited proliferation, induced cell cycle arrest at the G(0)/G(1) phase, and acted synergistically with doxorubicin to inhibit the growth of human HCC cells in vitro. Transfection of HIF‐2 siRNA also downregulated tumoral expression of HIF‐2α, VEGF, TGF‐α, and cyclin D1 in vivo, and acted synergistically with doxorubicin to suppress the growth of HepG2 tumors established in immunodeficient mice by inhibiting cell proliferation, tumor angiogenesis and microvessel perfusion. The results of the present study suggest that targeting HIF‐2α with siRNA warrants investigation as a potential strategy to enhance the efficacy of doxorubicin in the treatment of HCC. (Cancer Sci 2012; 103: 528–534)

Hepatocellular carcinoma (HCC) is one of the most common cancer-related causes of death, and conventional treatments offer unsatisfactory response. We have previously reported that kallistatin gene therapy suppressed the growth of HCC tumors by its anti-angiogenic activity, and meloxicam, a selective COX-2 inhibitor, inhibited proliferation and induced apoptosis of human HCC cells in vitro. The aim of this study was to determine whether combining kallistatin gene therapy and meloxicam could offer a better therapeutic effect to combat HCC in mice. A kallistatin expression plasmid was constructed and its expression was detected after intratumoral gene transfer. Both kallistatin gene therapy and meloxicam suppressed the growth of subcutaneous human HepG2 tumors established in BALB/c nude mice, and the combinational therapy showed a stronger effect in suppressing tumor growth, tumor angiogenesis and cell proliferation, and increasing cell apoptosis, than the respective monotherapies. Gene transfer of kallistatin inhibited tumor angiogenesis, and slightly inhibited cell proliferation and increased cell apoptosis in situ, but had no effect on expression of vascular endothelial growth factor, basic fibroblast growth factor, proliferating cell nuclear antigen, Bcl-2, Bax, or activation of caspase-3. Meloxicam therapy inhibited cell proliferation, induced cell apoptosis, reduced expression of proliferating cell nuclear antigen, increased activation of caspase-3, and upregulated Bax. Meloxicam also slightly inhibited tumor angiogenesis with no effect on the expression of vascular endothelial growth factor or basic fibroblast growth factor. Combining two novel anticancer agents, kallistatin targeting tumoral vascularization and meloxicam targeting cell proliferation and apoptosis, warrants investigation as a therapeutic strategy to combat HCC.

Hepatocellular carcinoma (HCC), one of the most common cancers worldwide, is resistant to anticancer drugs. Hypoxia is a major cause of tumor resistance to chemotherapy, and hypoxia-inducible factor (HIF)-1 is a key transcription factor in hypoxic responses. We have previously demonstrated that gene transfer of an antisense HIF-1alpha expression vector downregulates expression of HIF-1alpha and vascular endothelial growth factor (VEGF), and synergizes with immunotherapy to eradicate lymphomas. The aim of the present study was to determine whether gene transfer of antisense HIF-1alpha could enhance the therapeutic efficacy of doxorubicin to combat HCC. Both antisense HIF-1alpha therapy and doxorubicin suppressed the growth of subcutaneous human HepG2 tumors established in BALB/c nude mice, tumor angiogenesis, and cell proliferation, and induced tumor cell apoptosis. The combination therapy with antisense HIF-1alpha and doxorubicin was more effective in suppressing tumor growth, angiogenesis, and cell proliferation, and inducing cell apoptosis than the respective monotherapies. Gene transfer of antisense HIF-1alpha downregulated the expression of both HIF-1alpha and VEGF, whereas doxorubicin only downregulated VEGF expression. Antisense HIF-1alpha and doxorubicin synergized to downregulate VEGF expression. Both antisense HIF-1alpha and doxorubicin inhibited expression of proliferating cell nuclear antigen, and combined to exert even stronger inhibition of proliferating cell nuclear antigen expression. Antisense HIF-1alpha therapy warrants investigation as a therapeutic strategy to enhance the efficacy of doxorubicin for treating HCC.