nutlin-3a and Liver-Neoplasms

Tumor necrosis factor-associated apoptosis-inducing ligand (TRAIL) is a potent anticancer agent, which could specifically target cancerous cells. Nutlin-3, a small-molecular inhibitor of murine double minute 2 (MDM2), shows oncogenic potential in a variety of human cancers. It has also been found to promote the TRAIL-induced apoptosis of cancer cells in esophageal squamous cancer, but its potential role and underlying mechanisms in the TRAIL-treated hepatocellular carcinoma (HCC) remains to be elucidated.. The combination of Nutlin-3 and TRAIL facilitated the apoptosis and increased the levels of mitochondrial cleaved-caspase3/7 and 9 in HCC cells compared with TRAIL treatment alone, both in a concentration-dependent way. Nutlin-3 also upregulated the expression of p53 and DR5, while knockdown of p53 significantly hindered the pro-apoptotic effect of Nutlin-3. Further studies revealed that Nutlin-3 downregulated the expression of survivin and bcl-2, both of which could be reversed by p53 knockdown. Moreover, survivin suppressant YM155 and bcl-2 inhibitor YM155 further enhanced the apoptosis of HCC cells in the presence of Nutlin-3 and TRAIL.. These results suggested that Nutlin-3 facilitated TRAIL-induced apoptosis of HCC cells by activating the p53-survivin/bcl-2 pathway, which provided novel insights into the mechanism of Nutlin-3 and confirmed the potential of combination of Nutlin-3 and TRAIL as an adjuvant in HCC therapy.

Topics: Animals; Antineoplastic Agents; Apoptosis; Carcinoma, Hepatocellular; Caspase 3; Cell Line, Tumor; Humans; Imidazoles; Ligands; Liver Neoplasms; Mice; Piperazines; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-mdm2; RNA, Messenger; Survivin; TNF-Related Apoptosis-Inducing Ligand; Tumor Suppressor Protein p53

Aspirin as an antitumor drug has been studied in various malignancies with regards to its effects on apoptosis, proliferation, metastasis and senescence of tumor cells. However, the clinical application is limited by its side effects. Nutlin‑3 is a novel antitumor compound, which has not been clinically approved. The present study investigated the value of combining aspirin and nutlin‑3 on hepatocellular carcinoma (HCC) cells. MTT was performed to detect the proliferation of HepG2 cells treated with aspirin or/and nutlin‑3. Transwell invasion assays were performed to estimate the invasion ability of HepG2 cells treated with aspirin or/and nutlin‑3. Then the apoptotic analysis of HepG2 cells evaluated the synergistic effect of aspirin and nutlin‑3. Apoptosis markers, including B‑cell lymphoma 2 (Bcl‑2), Bcl‑2‑associated X protein (Bax), caspase‑3, caspase‑8 and caspase‑9 were estimated by western blot analysis at various time points. In addition, a Xenograft mouse model was established by infection with HepG2 cells, and aspirin and/or nutlin‑3 was administrated to verify the anti‑apoptotic effect of the two drugs in vivo. A high dose of aspirin and nutlin‑3 inhibit the proliferation and apoptosis of HepG2 cells. The antitumor effect was enhanced with the combined treatment of the two drugs, particularly in the group with a low concentration of aspirin and nutlin‑3. Nutlin‑3 was able to increase the level of Bax in HepG2 cells treated with aspirin significantly after treatment for 8 h. When treated with a low concentration of aspirin and nutlin‑3, the level of Bax in HepG2 cells was enhanced for 2 h. In the animal model, tumor volume and tumor angiogenesis were significantly decreased in combination group compared with other groups (P<0.01). Although there were side effects in the group treated with aspirin alone, no side effects were observed in the combination group. Nutlin‑3 enhanced the apoptotic effect of a low dose of aspirin by upregulating Bax expression in the HepG2 cell line and in vivo. The synergistic effect of nutlin‑3 in aspirin antitumor therapy contributed to diminishing the dose of aspirin required and decreased the occurrence of adverse drug events in HCC through targeting the Bcl‑2/Bax signaling pathway.

Topics: Animals; Antineoplastic Agents; Apoptosis; Aspirin; bcl-2-Associated X Protein; Carcinoma, Hepatocellular; Caspases; Cell Movement; Drug Synergism; Hep G2 Cells; Humans; Imidazoles; Liver Neoplasms; Mice; Mice, Inbred BALB C; Mice, Nude; Piperazines; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; Transplantation, Heterologous; Vascular Endothelial Growth Factor A

Nutlin-3, a small molecule regulator of the tumor suppressor p53, targets the interaction between p53 and murine double minute 2 (MDM2) thereby promoting stabilization of p53 and subsequent p53‑dependent induction of apoptosis and cell cycle arrest. Recent studies have demonstrated that Nutlin‑3 plays a critical role in regulating tumor cell migration, invasion, metastasis, and drug resistance. Although these studies identified various biological functions of Nutlin‑3, our understanding of the exact molecular mechanisms of Nutlin‑3‑mediated antitumor activity remains incomplete. In this study, we elucidated a role of Nutlin‑3 in reversing the epithelial‑mesenchymal transition (EMT) in gemcitabine-resistant (GR) hepatocellular carcinoma (HCC) cells. We assessed the effect of Nutlin‑3 treatment on cell growth, migration, and invasion in both parental HCC cells and GR HCC cells. Moreover, we detected the expression of EMT markers in GR HCC cells treated with Nutlin‑3 by real‑time RT‑PCR and western blot analysis, respectively. We found that Nutlin-3 inhibited cell migration and invasion in the GR HCC cells. Additionally, Nutlin‑3 treatment increased E-cadherin protein levels, but decreased the protein levels of vimentin, Snail and Slug in the GR HCC cells. Furthermore, we found that Smad2 was highly expressed in the GR HCC cells compared with their parental HCC cells, and Nutlin-3 treatment downregulated Smad2 expression in the GR HCC cells. Depletion of Smad2 retarded cell migration and regulated the expression of EMT markers in GR HCC cells similarly to Nutlin‑3 treatment. Our findings highlight an important role of Nutlin‑3 in reversing EMT in GR cells through regulation of Smad2 expression, suggesting that Nutlin-3 could be a potential agent for the treatment of HCC patients with gemcitabine resistance.

Topics: Apoptosis; Cadherins; Carcinoma, Hepatocellular; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Movement; Deoxycytidine; Down-Regulation; Drug Resistance, Neoplasm; Epithelial-Mesenchymal Transition; Gemcitabine; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Humans; Imidazoles; Liver Neoplasms; Neoplasm Invasiveness; Piperazines; Smad2 Protein; Snail Family Transcription Factors; Tumor Suppressor Protein p53; Vimentin

Interferon-γ inducible protein 16 (IFI16), a DNA sensor for DNA double-strand break (DSB), is expressed in most human hepatocellular carcinoma cell (HCC) lines. In this study we investigated the re-localization of chromatin-bound IFI16 by Nutlin-3, a DNA damage agent, in HCC cells in vitro, and the potential mechanisms.. Human HCC SMMC-7721 (wild-type TP53), Huh-7 (mutant TP53), Hep3B (null TP53) and normal fetal liver L02 cell lines were examined. DSB damage in HCC cells was detected via γH2AX expression and foci formation assay. The expression of IFI16 and IFNB mRNA was measured using RT-PCR, and subcellular localization and expression of the IFI16 protein were detected using chromatin fractionation, Western blot analysis, and fluorescence microscopy.. Treatment of SMMC-7721 cells with Nutlin-3 (10 μmol/L) or etoposide (40 μmol/L) induced significant DSB damage. In SMMC-7721 cells, Nutlin-3 significantly increased the expression levels of IFI16 and IFNB mRNA, and partially redistributed chromatin-bound IFI16 protein to the cytoplasm. These effects were blocked by pretreatment with pifithrin-α, a p53 inhibitor. Furthermore, Nutlin-3 did not induce ectopic expression of IFI16 protein in Huh-7 and Hep3B cells. Moreover, the association of IFI16 with chromatin and Nutlin-3-induced changes in localization were not detected in L02 cells.. Nutlin-3 regulates the subcellular localization of IFI16 in HCC cells in vitro in a p53-dependent manner.

Topics: Carcinoma, Hepatocellular; Cell Line; Cell Line, Tumor; Chromatin; Humans; Imidazoles; Liver Neoplasms; Nuclear Proteins; Phosphoproteins; Piperazines; Tumor Suppressor Protein p53

Drug-resistance and imbalance of apoptotic regulation limit chemotherapy clinical application for the human hepatocellular carcinoma (HCC) treatment. The reactivation of p53 is an attractive therapeutic strategy in cancer with disrupted-p53 function. Nutlin-3, a MDM2 antagonist, has antitumor activity in various cancers. The post-translational modifications of p53 are a hot topic, but there are some controversy ideas about the function of phospho-Ser392-p53 protein in cancer cell lines in response to Nutlin-3. Therefore, we investigated the relationship between Nutlin-3 and phospho-Ser392-p53 protein expression levels in SMMC-7721 (wild-type TP53) and HuH-7 cells (mutant TP53). We demonstrated that Nutlin-3 induced apoptosis through down-regulation phospho-Ser392-p53 in two HCC cells. The result suggests that inhibition of p53 phosphorylation on Ser392 presents an alternative for HCC chemotherapy.

Topics: Antineoplastic Agents; Apoptosis; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Cell Survival; Humans; Imidazoles; Liver Neoplasms; Phosphorylation; Phosphoserine; Piperazines; Tumor Suppressor Protein p53

Arsenic trioxide has been demonstrated as an effective anti-cancer drug against leukemia and solid tumors both in vitro and in vivo. However, recent phase II trials demonstrated that single agent arsenic trioxide was poorly effective against hepatocellular carcinoma (HCC), which might be due to drug resistance.. Mutation detection of p53 gene in arsenic trioxide resistant HCC cell lines was performed. The therapeutic effects of arsenic trioxide and Nutlin-3 on HCC were evaluated both in vitro and in vivo. A series of experiments including MTT, apoptosis assays, co-Immunoprecipitation, siRNA transfection, lentiviral infection, cell migration, invasion, and epithelial-mesenchy-mal transition (EMT) assays were performed to investigate the underlying mechanisms.. The acquisition of p53 mutation contributed to arsenic trioxide resistance and enhanced metastatic potential of HCC cells. Mutant p53 (Mutp53) silence could re-sensitize HCC resistant cells to arsenic trioxide and inhibit the metastatic activities, while mutp53 overexpression showed the opposite effects. Neither arsenic trioxide nor Nutlin-3 could exhibit obvious effects against arsenic trioxide resistant HCC cells, while combination of them showed significant effects. Nutlin-3 can not only increase the intracellular arsenicals through inhibition of p-gp but also promote the p73 activation and mutp53 degradation mediated by arsenic trioxide. In vivo experiments indicated that Nutlin-3 can potentiate the antitumor activities of arsenic trioxide in an orthotopic hepatic tumor model and inhibit the metastasis to lung.. Acquisitions of p53 mutations contributed to the resistance of HCC to arsenic trioxide. Nutlin-3 could overcome arsenic trioxide resistance and inhibit tumor metastasis through p73 activation and promoting mutant p53 degradation mediated by arsenic trioxide.

Topics: Animals; Antineoplastic Agents; Arsenic Trioxide; Arsenicals; Carcinoma, Hepatocellular; Cell Line, Tumor; DNA-Binding Proteins; Drug Resistance, Neoplasm; Drug Synergism; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Humans; Imidazoles; Liver Neoplasms; Lung Neoplasms; Mice; Mice, Nude; Mutation; Neoplasm Transplantation; Nuclear Proteins; Oxides; Piperazines; Signal Transduction; Tumor Protein p73; Tumor Suppressor Protein p53; Tumor Suppressor Proteins

Topics: Animals; Antineoplastic Agents; Apoptosis; Carcinoma, Hepatocellular; Cell Cycle; Cell Proliferation; Cell Survival; DNA-Binding Proteins; Enzyme Inhibitors; Humans; Imidazoles; Liver Neoplasms; Mutation; Nuclear Proteins; Piperazines; Proto-Oncogene Proteins c-mdm2; Tumor Protein p73; Tumor Suppressor Protein p53; Tumor Suppressor Proteins

Nutlin-3, a selective small-molecule inhibitor of the p53-MDM2 interaction, has been shown to have antitumor activities in various tumors with wild-type p53. However, its effect on hepatocellular carcinoma (HCC) with different types of p53 remains unclear. This study is designed to determine nutlin-3's antitumor efficacy and underlying mechanisms of action in human HCC cells.. Cell viability assay, cell cycle analysis, apoptosis assay, western blot, co- immunoprecipitation and siRNA experiments were analyzed in three human HCC cells. Anti-tumoral effects of nutlin-3 targeting the p53 and p73 pathways were evaluated in HCC cell lines.. Nutlin-3 exerted the greatest anti-tumoral effect to three human HCC cells with wild-type p53, mutant p53 and p53-null. Nutlin-3 not only upregulated p53 in HepG2 cells, but also p73 in Huh7 and Hep3B cells, and disrupted p53-MDM2 and p73-MDM2 complexes in HCC cells. The compound inhibited cell proliferation, induced G0/G1 phase arrest, decreased the levels of CyclinD1, CyclinE, CDK2, CDK4, PCNA and E2F-1, and increased the levels of p21 and p27. It also induced apoptosis, increased the Bax/Bcl-2 ratio, then activated caspase-9 and caspase-3.. Nutlin-3 has significant anticancer effects against human HCC cells, regardless of p53 status, indicating that it is a promising therapy for human hepatocellular carcinoma.

Topics: Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Blotting, Western; Carcinoma, Hepatocellular; Cell Cycle; Cell Cycle Proteins; Cell Proliferation; Cell Survival; DNA-Binding Proteins; Dose-Response Relationship, Drug; Enzyme Inhibitors; Flow Cytometry; Hep G2 Cells; Humans; Imidazoles; Immunoprecipitation; Liver Neoplasms; Mutation; Nuclear Proteins; Piperazines; Proto-Oncogene Proteins c-mdm2; RNA Interference; Time Factors; Tumor Protein p73; Tumor Suppressor Protein p53; Tumor Suppressor Proteins

Despite recent advances in chemotherapeutic agents for Hepatocellular carcinoma (HCC) treatment, the results of chemotherapy remain unsatisfactory. Doxorubicin (DOX) still represents the cornerstone in HCC chemotherapy, but resistance and toxicity to normal cells are major obstacles to successful chemotherapy. Therefore, new active agents in HCC chemotherapy and agents that increase the chemosensitivity of HCC cells to DOX are still urgently required. Nutlin-3 is a small-molecule inhibitor that acts to inhibit murine double minute-2 (MDM2) binding to p53 or p73, and subsequently activates p53- or p73-dependent apoptosis signaling pathway. This study was designed to investigate whether Nutlin-3 alters cell toxicity to HCC cells following DNA damage and to assess the suitability of DOX/Nutlin-3 as a chemotherapeutic combination in HCC chemotherapy.. Four human HCC cells were analyzed using cell proliferation assay, apoptosis assay, western blotting, co-immunoprecipitation and siRNA experiments. Anti-tumoral effects of Nutlin-3/DOX targeting the p53/MDM2 and p73/MDM2 pathways were evaluated in HCC cell lines.. Nutlin-3 enhances the growth inhibition by DOX and potentates the apoptotic effect in all HCC cell lines with different p53 types. Nutlin-3 acts through the disruption of p53-MDM2 binding in HepG2, and the disruption of p73-MDM2 in Huh-7 and Hep3B cell lines with subsequent activation of the apoptotic pathway, which leads to the increase in chemosensitivity to DOX in HCC cells.. Taken together, our findings suggest that Nutlin-3 will be active in the treatment of HCC and offers new prospects for overcoming DOX resistance.

Topics: Antibiotics, Antineoplastic; Apoptosis; Carcinoma, Hepatocellular; Cell Line, Tumor; DNA-Binding Proteins; Doxorubicin; Drug Synergism; Humans; Imidazoles; Liver Neoplasms; Nuclear Proteins; Piperazines; Proto-Oncogene Proteins c-mdm2; RNA, Small Interfering; Signal Transduction; Tumor Protein p73; Tumor Suppressor Protein p53; Tumor Suppressor Proteins

The interplay among hypoxia-inducible factor 1-alpha (HIF-1alpha), p53 and human orthologue of murine double minute 2 (Hdm2) has been introduced as a key event in tumor promotion and angiogenesis. Recently, nutlin-3, a small-molecule antagonist of Hdm2, was demonstrated to inhibit the HIF-1-mediated vascular endothelial growth factor production and tumor angiogenesis. Yet, the mechanism by which nutlin-3 inhibits HIF-1 is an open question. We here addressed the mode-of-action of nutlin-3 with respect to the HIF-1alpha-p53-Hdm2 interplay. The effect of nutlin-3 on HIF-1alpha function was examined by reporter analyses, immunoprecipitation and immunoblotting. Nutlin-3 downregulated HIF-1alpha, which occurred p53-dependently but von Hippel-Lindau-independently. On the contrary, nutlin-3 blunted the hypoxic induction of vascular endothelial growth factor by inactivating HIF-1 even in p53-null cells. The C-terminal transactivation domain (CAD) of HIF-1alpha was inactivated by nutlin-3, and furthermore, the factor-inhibiting hypoxia-inducible factor (FIH) hydroxylation of Asn803 was required for the nutlin-3 action. In terms of protein interactions, Hdm2 competed with FIH in CAD binding and inhibited the Asn803 hydroxylation both in vivo and in vitro, which facilitated p300 recruitment. Moreover, nutlin-3 reinforced the FIH binding and Ans803 hydroxylation by inhibiting Hdm2. In conclusion, Hdm2 functionally activates HIF-1 by inhibiting the FIH interaction with CAD, and the Hdm2 inhibition by nutlin-3 results in HIF-1 inactivation and vascular endothelial growth factor suppression. The interplays among HIF-1alpha, Hdm2, FIH and p300 could be potential targets for treating tumors overexpressing HIF-1alpha.

Topics: Aryl Hydrocarbon Receptor Nuclear Translocator; Carcinoma, Hepatocellular; Cell Line, Tumor; Colonic Neoplasms; Gene Expression Regulation, Neoplastic; Genes, Reporter; HCT116 Cells; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Imidazoles; Kidney Neoplasms; Liver Neoplasms; Mixed Function Oxygenases; Piperazines; Plasmids; Proto-Oncogene Proteins c-mdm2; Repressor Proteins; Reverse Transcriptase Polymerase Chain Reaction; Transfection; Vascular Endothelial Growth Factor A