akt-i-1-2-compound and Liver-Neoplasms

Molecule-targeted therapy has become the research focus for hepatocellular carcinoma (HCC). Persistent PI3K-AKT activation is often detected in HCC, representing a valuable oncotarget for treatment. Here, we tested the anti-HCC activity by a potent AKT inhibitor: AKT inhibitor 1/2 (AKTi-1/2). In both established (HepG2 and Huh-7) and primary human HCC cells, treatment with AKTi-1/2 inhibited cell survival and proliferation, but induced cell apoptosis. AKTi-1/2 blocked AKT-mTOR activation, yet simultaneously provoked cytoprotective autophagy in HCC cells. The latter was evidenced by ATG-5 and Beclin-1 upregulation, p62 downregulation as well as LC3B-GFP puncta formation. Autophagy inhibition, via pharmacological inhibitors (3-methyladenine, ammonium chloride, and bafilomycin A1) or Beclin-1 siRNA knockdown, significantly potentiated AKTi-1/2-induced HepG2 cell death and apoptosis. In nude mice, AKTi-1/2 intraperitoneal injection inhibited HepG2 tumor growth. Significantly, its anti-tumor activity in vivo was further sensitized when combined with Beclin-1 shRNA knockdown in HepG2 tumors. Together, these results demonstrate that autophagy activation serves as a main resistance factor of AKTi-1/2 in HCC cells. Autophagy prevention therefore sensitizes AKTi-1/2-induced anti-HCC activity in vitro and in vivo.

Topics: Animals; Antineoplastic Agents; Autophagy; Benzylamines; Carcinoma, Hepatocellular; Cell Line, Tumor; Drug Synergism; Female; Hep G2 Cells; Humans; Liver Neoplasms; Mice; Mice, Inbred C57BL; Mice, Nude; Molecular Targeted Therapy; Quinoxalines; Treatment Outcome

Oct4 protein encoded by POU5F1 plays a pivotal role in maintaining the self‑renewal of pluripotent stem cells; however, its presence in cancer cells remains controversial. In the present study, we provided evidence that the transcripts of authentic OCT4 gene (OCT4A) and its multiple pseudogenes were detected in a variety of cancer cell lines. A few major bands were also detected by western blotting using an anti‑Oct4A monoclonal antibody. Moreover, an anti‑Oct4‑pT235 antibody was used to identify a band in the majority of the tested cancer cell lines that coincided with one of the anti‑Oct4A bands which was decreasable by a specific shRNA. The Oct4‑pT235 signals were also detected in human glioblastoma and liver cancer specimens by immunofluorescence microscopy and immunohistochemistry. U87 glioblastoma cells were cultured in a neural stem cell medium to induce the formation of neurospheres rich in stem‑like cancer cells. The levels of Oct4‑pT235 in the sphere cells were markedly increased compared to their monolayer parental cells, a result that was accompanied by upregulation of the PI3K‑Akt pathway. Akti‑1/2, a specific inhibitor of Akt, effectively reduced the level of Oct4‑pT235 and attenuated the proliferation of U87 sphere cells. ITE, an agonist of the aryl hydrocarbon receptor, also significantly attenuated the Akt‑mediated phosphorylation of Oct4 in glioblastoma and liver cancer cells, and reduced their tumorigenic potential in a xenograft tumor model. Taken together, we concluded that the Akt‑mediated phosphorylation of Oct4A or its homolog protein was associated with the proliferation of stem‑like cancer cells that may serve as a novel biomarker and drug target for certain types of cancer.

Topics: Animals; Benzylamines; Brain Neoplasms; Carcinoma, Hepatocellular; Cell Division; Cell Line, Tumor; Cell Nucleus; Culture Media; Glioblastoma; Heterografts; Humans; Indoles; Liver Neoplasms; Mice; Mice, Nude; Neoplasm Proteins; Neoplastic Stem Cells; Octamer Transcription Factor-3; Phosphorylation; Protein Processing, Post-Translational; Proto-Oncogene Proteins c-akt; Quinoxalines; Receptors, Aryl Hydrocarbon; RNA Interference; RNA, Small Interfering; Signal Transduction; Spheroids, Cellular; Thiazoles

The serine/threonine kinase Akt, a downstream effector of phosphatidylinositol 3-kinase (PI3K), is involved in cell survival and anti-apoptotic signaling. Akt has been shown to be constitutively expressed in a variety of human tumors including hepatocellular carcinoma (HCC). In this report we analyzed the status of Akt pathway in three HCC cell lines, and tested cytotoxic effects of Akt pathway inhibitors LY294002, Wortmannin and Inhibitor VIII. In Mahlavu human hepatoma cells Akt was constitutively activated, as demonstrated by its Ser473 phosphorylation, downstream hyperphosphorylation of BAD on Ser136, and by a specific cell-free kinase assay. In contrast, Huh7 and HepG2 did not show hyperactivation when tested by the same criteria. Akt enzyme hyperactivation in Mahlavu was associated with a loss of PTEN protein expression. Akt signaling was inhibited by the upstream kinase inhibitors, LY294002, Wortmannin, as well as by the specific Akt Inhibitor VIII in all three hepatoma cell lines. Cytotoxicity assays with Akt inhibitors in the same cell lines indicated that they were all sensitive, but with different IC50 values as assayed by RT-CES. We also demonstrated that the cytotoxic effect was through apoptotic cell death. Our findings provide evidence for its constitutive activation in one HCC cell line, and that HCC cell lines, independent of their Akt activation status respond to Akt inhibitors by apoptotic cell death. Thus, Akt inhibition may be considered as an attractive therapeutic intervention in liver cancer.

Topics: Androstadienes; Antineoplastic Agents; Apoptosis; Benzimidazoles; Carcinoma, Hepatocellular; Cell Line, Tumor; Chromones; Humans; Inhibitory Concentration 50; Liver Neoplasms; Morpholines; Proto-Oncogene Proteins c-akt; Quinoxalines; Signal Transduction; Wortmannin

Abnormal expression of the hepatic gluconeogenic genes (glucose-6-phosphatase [G6Pase] and PEPCK) contributes to hyperglycemia. These genes are repressed by insulin, but this process is defective in diabetic subjects. Protein kinase B (PKB) is implicated in this action of insulin. An inhibitor of PKB, Akt inhibitor (Akti)-1/2, was recently reported; however, the specificity and efficacy against insulin-induced PKB was not reported. Our aim was to characterize the specificity and efficacy of Akti-1/2 in cells exposed to insulin and then establish whether inhibition of PKB is sufficient to prevent regulation of hepatic gene expression by insulin.. Akti-1/2 was assayed against 70 kinases in vitro and its ability to block PKB activation in cells exposed to insulin fully characterized.. Akti-1/2 exhibits high selectivity toward PKBalpha and PKBbeta. Complete inhibition of PKB activity is achieved in liver cells incubated with 1-10 mumol/l Akti-1/2, and this blocks insulin regulation of PEPCK and G6Pase expression. Our data demonstrate that only 5-10% of maximal insulin-induced PKB is required to fully repress PEPCK and G6Pase expression. Finally, we demonstrate reduced insulin sensitivity of these gene promoters in cells exposed to submaximal concentrations of Akti-1/2; however, full repression of the genes can still be achieved by high concentrations of insulin.. This work establishes the requirement for PKB activity in the insulin regulation of PEPCK, G6Pase, and a third insulin-regulated gene, IGF-binding protein-1 (IGFBP1); suggests a high degree of functional reserve; and identifies Akti-1/2 as a useful tool to delineate PKB function in the liver.

Topics: Animals; Benzylamines; Carcinoma, Hepatocellular; Cell Line, Tumor; Enzyme Inhibitors; Genes, Reporter; Gluconeogenesis; Insulin; Liver; Liver Neoplasms; Proto-Oncogene Proteins c-akt; Quinoxalines; Rats; Reverse Transcriptase Polymerase Chain Reaction