mk-2206 and Liver-Neoplasms

Hepatocellular carcinoma (HCC) is one of the most common causes of cancer-related deaths worldwide, and its incidence is rising. HCC develops almost exclusively on the background of chronic liver inflammation, which can be caused by chronic alcohol consumption, viral hepatitis, or an unhealthy diet. The key role of chronic inflammation in the process of hepatocarcinogenesis, including in the deregulation of innate and adaptive immune responses, has been demonstrated. The inhibition of Akt (also known as Protein Kinase B) directly affects cancer cells, but this therapeutic strategy also exhibits indirect anti-tumor activity mediated by the modulation of the tumor microenvironment, as demonstrated by using Akt inhibitors AZD5363, MK-2206, or ARQ 092. Moreover, the isoforms of Akt converge and diverge in their designated roles, but the currently available Akt inhibitors fail to display an isoform specificity. Thus, selective Akt inhibition needs to be better explored in the context of HCC and its possible combination with immunotherapy. This review presents a compact overview of the current knowledge concerning the role of Akt in HCC and the effect of Akt inhibition on the HCC and liver tumor microenvironment.

Topics: Aminopyridines; Carcinoma, Hepatocellular; Drug Delivery Systems; Heterocyclic Compounds, 3-Ring; Humans; Imidazoles; Liver Neoplasms; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Pyrimidines; Pyrroles; Tumor Microenvironment

Hepatitis B virus X (HBx), a viral onco-protein encoded by HBV, can promote oncogenesis of HCC. However, the mechanism of HBx in hepatocarcinogenesis is still unclear. In this study, we establish a new mouse model with normal immune system to investigate the role of HBx and its functional mechanisms under normal immune function. The animal model was established by injecting HBx-EGFP-14-19 cells into the hepatic portal vein of KM mice. To verify the mouse model, the expression of HBx in the liver tissue of mice was detected by qRT-PCR, western blotting and immunohistochemistry. The apoptosis index was calculated using the terminal deoxynucleotidyl transferase-dUTP nick-end labeling (TUNEL) assay, and the expression levels of apoptosis-related and cell cycle-related factors were measured. Moreover, expression of proteins in the protein kinase B/mammalian target of rapamycin (Akt/mTOR) signaling pathway was detected in HBx-EGFP-14-19 mice with and without use of an Akt inhibitor. The results showed the HBx was successfully overexpressed in liver of KM mice. After overexpressing HBx, the apoptosis index was downregulated in HBx-EGFP-14-19 liver tissue, and the expression levels of caspase-9 and Bad were reduced, but Bcl-xl was increased in HBx-EGFP-14-19 liver tissue. Overexpression of HBx increased the expression of the cyclin-dependent kinase 2 (CDK2), cyclinD1 and cyclinE. Moreover, compared with the low-level HBx group, p-Akt and p-mTOR were increased in the livers of mice with high levels of HBx. However, inactivation of apoptosis by overexpression of HBx was abolished by the treatment with an Akt inhibitor. These results indicate that HBx can induce anti-apoptosis mechanisms in hepatocytes in vivo, which is mediated by the Akt/mTOR signaling pathway.

Topics: Animals; Apoptosis; Cell Cycle; Cell Line; Cell Proliferation; Dependovirus; Disease Models, Animal; Hepatitis B virus; Hepatocytes; Heterocyclic Compounds, 3-Ring; Humans; Liver; Liver Neoplasms; Mice; Neoplasms, Experimental; Phosphorylation; Proto-Oncogene Proteins c-akt; Signal Transduction; TOR Serine-Threonine Kinases; Trans-Activators; Up-Regulation; Viral Regulatory and Accessory Proteins

Sorafenib, an orally available kinase inhibitor, is the standard first-line systemic drug for advanced hepatocellular carcinoma (HCC), and it exerts potent inhibitory activity against epithelial-mesenchymal transition (EMT) and multidrug resistance (MDR) by inhibiting mitogen-activated protein kinase (MAPK) signaling in HCC. However, after long-term exposure to sorafenib, HCC cells exhibit EMT and resistance to sorafenib. The activation of AKT by sorafenib is thought to be responsible for the development of these characteristics. The present study aims to examine the underlying mechanism and seek potential strategies to reverse this resistance and the progression to EMT. Sorafenib-resistant cells showed increased metastatic and invasive ability, with a higher expression of P-glycoprotein (P-gp), compared with the parental cells. This phenomenon was at least partially due to EMT and the appearance of MDR in sorafenib-resistant HCC cells. Moreover, MDR was a downstream molecular event of EMT. Silencing Snail with siRNA blocked EMT and partially reversed the MDR, thereby markedly abolishing invasion and metastasis in sorafenib-resistant HCC cells, but silencing of MDR1 had no effect on the EMT phenotype. Additionally, HCC parental cells that were stably transfected with pCDNA3.1-Snail exhibited EMT and MDR. Two sorafenib-resistant HCC cell lines, established from human HCC HepG2 and Huh7 cells, were refractory to sorafenib-induced growth inhibition but were sensitive to MK-2206, a novel allosteric AKT inhibitor. Thus, the combination of sorafenib and MK-2206 led to significant reversion of the EMT phenotype and P-gp-mediated MDR by downregulating phosphorylated AKT. These findings underscore the significance of EMT, MDR and enhanced PI3K/AKT signaling in sorafenib-resistant HCC cells.

Topics: Animals; Antineoplastic Agents; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Cell Survival; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Epithelial-Mesenchymal Transition; Heterocyclic Compounds, 3-Ring; Humans; Liver Neoplasms; Male; Mice, Inbred BALB C; Mice, Nude; Neoplasm Invasiveness; Neoplasm Transplantation; Niacinamide; Phenylurea Compounds; Phosphatidylinositol 3-Kinases; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Signal Transduction; Sorafenib

Sorafenib is a multikinase inhibitor for the treatment of hepatocellular carcinoma. However, most patients who initially respond to sorafenib become refractory. In a previous study, we demonstrated that sphere-forming cells derived from liver cancer cell lines possess the properties of liver cancer stem cells (LCSCs). In the present study, we found that successive passages of LCSCs were more resistant to sorafenib, and LCSCs treated with sorafenib showed an increase in spheroid formation with a lower inhibition rate. MK2206, but not various other inhibitors of cell signaling pathways, enhanced their sensitivity to sorafenib, increased the apoptotic rate, and suppressed the growth of LCSC xenografts in vivo (P < 0.01); sorafenib treatment decreased the level of active phosphorylated (p)Akt (Thr308) and reduced the levels of active pAkt (Ser473) and extracellular signal-regulated kinase (ERK) in LCSCs, whereas MK2206 reduced pAkt expression and increased pERK expression. Cotreatment with sorafenib and MK2206 reduced pAkt and pERK expression in LCSCs and xenografted tumors (P < 0.01). Treatment with either sorafenib or MK2206 decreased the expression of EpCAM and CD133 in LCSCs, which was more evident after combined treatment. Based on these results, we conclude that resistance to sorafenib is associated with weak ERK signaling and strong Akt signaling in LCSCs. By inhibition of Akt and upregulation of ERK, MK2206 overcomes the resistance of LCSCs to sorafenib.

Topics: Animals; Antineoplastic Agents; Apoptosis; Biomarkers, Tumor; Blotting, Western; Carcinoma, Hepatocellular; Cell Proliferation; Drug Resistance, Neoplasm; Extracellular Signal-Regulated MAP Kinases; Gene Expression Regulation, Neoplastic; Heterocyclic Compounds, 3-Ring; Humans; Immunoenzyme Techniques; Liver Neoplasms; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplastic Stem Cells; Niacinamide; Phenylurea Compounds; Phosphorylation; Proto-Oncogene Proteins c-akt; Signal Transduction; Sorafenib; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

We have shown that an Akt inhibitor, MK2206, reduces hepatocellular carcinoma (HCC) proliferation. To further delineate MK2206, we sought to investigate the Notch1 pathway and hypothesize that MK2206 treatment will result in Notch1 inhibition with either subsequent or parallel Akt suppression.. HCC cell lines were treated with various concentrations of MK2206. Cell proliferation was determined via real-time live cell imaging. Knockdown of Notch1 was used to observe interaction between Notch1 and pAkt. Cell lysates were analyzed via Western blotting for Notch and Akt pathway targets.. After treatment with MK2206 (up to 2 μM), there was a 60% reduction in cell viability at 48 hours with a concomitant reduction in Notch1 expression. Knockdown of Notch1 in HCC cell lines correlated with reduction in Akt phosphorylation.. MK2206 inhibits both the PI3-K/Akt and Notch1 pathways. Therefore, further characterization of MK2206 comparing the 2 pathways is warranted and the effect of dual targeting in HCC.

Topics: Blotting, Western; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Cell Survival; Heterocyclic Compounds, 3-Ring; Humans; Liver Neoplasms; Phosphorylation; Proto-Oncogene Proteins c-akt; Receptor, Notch1; Signal Transduction

To provide support for combined usage of phosphoinositide 3-kinase (PI3K) inhibitors or mitogen-activated protein kinase pathway inhibitors together with sorafenib in treatment of sorafenib-resistant hepatocellular carcinoma.. The sorafenib-resistant cell lines were established to evaluate the effects of MK-2206 2HCL, a dual PI3K/mammalian target of rapamycin (mTOR) inhibitor, and PD0325901, an rat sarcoma (RAS) and/or extracellular signal-regulated kinase (ERK) inhibitor, on cell proliferation and apoptosis, as both single and combined treatments with sorafenib. In addition, multidrug resistance 1 gene expression, mutation status of key members in PI3K/mTOR, and RAS/ERK pathways and pathway activation were analyzed to identify predictors of drug response.. Molecular studies reveal that combining MK-2206 2HCL or PD0325901 with sorafenib not only has a synergistic effect, in suppressing PI3K/protein kinase B/mTOR and RAS/MEK/ERK signaling more effectively than either treatment alone, but also prevents the cross activation of the other pathway that occurs with single treatments in both sorafenib sensitive and resistant lines. PD0325901 exhibited a stronger synergic effect with sorafenib than MK-2206 2HCL. Sorafenib-resistant cell lines were characterized by activation of both of the two pathways, as indicated by multidrug resistance 1 gene expression profiles and pathway activity analysis.. Our studies have showed that both inhibitors of PI3K/mTOR and RAS/ERK signaling are potentially effective antihepatocellular carcinoma drugs especially in treating sorafenib-resistant hepatocellular carcinoma.

Topics: Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Benzamides; Biomarkers, Tumor; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Cell Survival; Diphenylamine; Drug Resistance, Neoplasm; Heterocyclic Compounds, 3-Ring; Humans; Liver Neoplasms; Niacinamide; Phenylurea Compounds; Sorafenib

To explore whether combining inhibitors that target the insulin-like growth factor receptor (IGFR)/PI3K/Akt/mTOR signaling pathway (vertical blockade) can improve treatment efficacy for hepatocellular carcinoma (HCC).. HCC cell lines (including Hep3B, Huh7, and PLC5) and HUVECs (human umbilical venous endothelial cells) were tested. The molecular targeting therapy agents tested included NVP-AEW541 (IGFR kinase inhibitor), MK2206 (Akt inhibitor), BEZ235 (PI3K/mTOR inhibitor), and RAD001 (mTOR inhibitor). Potential synergistic antitumor effects were tested by median dose-effect analysis in vitro and by xenograft HCC models. Apoptosis was analyzed by flow cytometry (sub-G1 fraction analysis) and Western blotting. The activities of pertinent signaling pathways and expression of apoptosis-related proteins were measured by Western blotting.. Vertical blockade induced a more sustained inhibition of PI3K/Akt/mTOR signaling activities in all the HCC cells and HUVEC tested. Synergistic apoptosis-inducing effects, however, varied among different cell lines and drug combinations and were most prominent when NVP-AEW541 was combined with MK2206. Using an apoptosis array, we identified survivin as a potential downstream mediator. Over-expression of survivin in HCC cells abolished the anti-tumor synergy between NVP-AEW541 and MK2206, whereas knockdown of survivin improved the anti-tumor effects of all drug combinations tested. In vivo by xenograft studies confirmed the anti-tumor synergy between NVP-AEW541 and MK2206 and exhibited acceptable toxicity profiles.. Vertical blockade of the IGFR/PI3K/Akt/mTOR pathway has promising anti-tumor activity for HCC. Survivin expression may serve as a biomarker to predict treatment efficacy.

Topics: Animals; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Biomarkers, Tumor; Blotting, Western; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Survival; Drug Synergism; Enzyme Inhibitors; Everolimus; Flow Cytometry; Heterocyclic Compounds, 3-Ring; Humans; Imidazoles; Inhibitor of Apoptosis Proteins; Liver Neoplasms; Male; Mice, Inbred BALB C; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Pyrimidines; Pyrroles; Quinolines; Receptors, Somatomedin; Signal Transduction; Sirolimus; Survivin; TOR Serine-Threonine Kinases; Xenograft Model Antitumor Assays

Hepatocellular carcinoma (HCC) is commonly diagnosed at an advanced stage and has limited effective treatment options. The aberrant regulation of the phosphoinositide 3-kinase/Akt pathway in HCC makes it an attractive therapeutic target. The effect of MK2206, a novel, allosteric Akt inhibitor, on HCC cells is not yet fully understood. We hypothesized that inhibition of Akt by MK2206 would impact cellular viability.. Human Huh7, Hep3B, and HepG2 cell lines were treated with 0-2 μM of MK2206 for 96 h. Cell viability was determined by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Western blot analysis was used to examine the expression level of various protein markers to assess the mechanism of drug action and proliferation inhibition.. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay showed a reduction in cellular viability by ≥55% for all cell lines (control versus 2 μM MK2206; P <0.001). Western blot analysis revealed reduction in the level of phosphorylated AKT-Ser473 with no change in AKT-thr308 expression confirming the specificity of MK2206. There was an observed reduction in caspase-9 and survivin. Importantly, there were increases in p21 and p27 along with decreased cyclinD1 expression after treatment.. This study demonstrates the anti-tumor activity of MK2206 in HCC cells. The observed reduction in survivin and pro-caspase 9 suggests that MK2206 induces apoptosis. However, HCC proliferation is also halted via induction of cell cycle arrest as indicated by the increase in p21 and p27 expression and decrease in cyclinD1. Importantly, the concentration needed to achieve growth inhibition in HCC is lower than that needed for other cancer types.

Topics: Apoptosis; Carcinoma, Hepatocellular; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Heterocyclic Compounds, 3-Ring; Humans; Liver Neoplasms; Proto-Oncogene Proteins c-akt

It has become evident that AKT inhibitors have great potential in cancer treatment. In this study, we investigate the anticancer activity of MK-2206, a novel AKT inhibitor, on HepG2 hepatocellular carcinoma cell, and to show whether MK-2206 enhances the apoptosis-inducing potential of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). The cell growth inhibition was evaluated by MTT assay and colony formation assay. Cell cycle distribution was assessed by propidium iodide flow cytometry. Apoptosis was determined by AnnexinV-FITC/PI double staining assay and caspase-9, casapse-7, caspase-3, and PARP cleavage. The results of present study showed that MK-2206-induced G1-phase arrest was associated with a marked decrease in the protein expression of cyclin D1 with concomitant induction of p21 and p27. MK-2206-induced apoptosis was characterized by cleavage of a pro-caspase in a concentration-dependent manner. Moreover, the MAP family kinases p38 kinase and JNK were activated by exposure to MK-2206. SB203580, an p38-specific inhibitor, partially blocked MK-2206-induced death of HepG2 cells and caspase activation. A combination of MK-2206 with TRAIL significantly inhibited growth of TRAIL resistant HepG2 cells. Taken together, our findings provide a new insight to better understand anticancer mechanisms of MK-2206, at least in HepG2 cell. Using of MK-2206 as a potent sensitizer to TRAIL-induced apoptotic cell death offers a promising means of enhancing the efficacy of TRAIL-based HCC treatments.

Topics: Apoptosis; Carcinoma, Hepatocellular; Caspases; Cell Cycle Checkpoints; Cell Proliferation; Cell Survival; Down-Regulation; Drug Screening Assays, Antitumor; Enzyme Activation; Hep G2 Cells; Heterocyclic Compounds, 3-Ring; Humans; JNK Mitogen-Activated Protein Kinases; Liver Neoplasms; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Proto-Oncogene Proteins c-akt; TNF-Related Apoptosis-Inducing Ligand

Hepatocellular carcinoma (HCC) is one of the most common potentially lethal human malignancies worldwide. Advanced or recurrent HCC is frequently resistant to conventional chemotherapeutic agents and radiation. Therefore, targeted agents with tolerable toxicity are mandatory to improve HCC therapy and prognosis. In this neoplasia, the PI3K/Akt signaling network has been frequently shown to be aberrantly up-regulated. To evaluate whether Akt could represent a target for treatment of HCC, we studied the effects of the allosteric Akt inhibitor, MK-2206, on a panel of HCC cell lines characterized by different levels of Akt-1 activation. The inhibitor decreased cell viability and induced cell cycle arrest in the G0/G1 phase of the cell cycle, with a higher efficacy in cells with hyperphosphorylated Akt-1. Moreover, MK-2206 induced apoptosis, as documented by Annexin V labeling, and also caused autophagy, as evidenced by increased levels of the autophagy marker LC3A/B. Autophagy was shown to be a protective mechanism against MK-2206 cytotoxicity. MK-2206 down-regulated, in a concentration-dependent manner, the phosphorylation levels of Akt-1 and its downstream targets, GSK3 α/β and FOXO3A. MK-2206 synergized with doxorubicin, a chemotherapeutic drug widely used for HCC treatment. Our findings suggest that the use of Akt inhibitors, either alone or in combination with doxorubicin, may be considered as an attractive therapeutic regimen for the treatment of HCC.

Topics: Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Autophagy; Carcinoma, Hepatocellular; Cell Growth Processes; Doxorubicin; Drug Synergism; Heterocyclic Compounds, 3-Ring; Humans; Liver Neoplasms; Phosphorylation; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Signal Transduction

Due to the frequent dysregulation of the PI3K/AKT/mTOR signaling pathway, mTOR represents a suitable therapeutic target in hepatocellular carcinoma (HCC). However, emerging data from clinical trials of HCC patients indicate that mTOR inhibition by RAD001 (Everolimus) alone has only moderate antitumor efficacy which may be due to the feedback activation of AKT after mTOR inhibition. In this study, we analyzed the effects of dual inhibition of mTOR and AKT on the proliferation of HCC cell lines. In addition, we measured the feedback activation of each of the AKT isoforms after mTOR inhibition in HCC cell lines and their enzymatic activity in primary samples from HCC patients.. The activation status of specific AKT isoforms in human HCC samples and corresponding healthy liver tissue was analyzed using an AKT isoform specific in vitro kinase assay. AKT isoform activation after mTOR inhibition was analyzed in three HCC cell lines (Hep3B, HepG2 and Huh7), and the impact of AKT signaling on proliferation after mTOR inhibition was investigated using the novel AKT inhibitor MK-2206 and AKT isoform specific knockdown cells.. AKT isoforms become differentially activated during feedback activation following RAD001 treatment. The combination of mTOR inhibition and AKT isoform knockdown showed only a weak synergistic effect on proliferation of HCC cell lines. However, the combinatorial treatment with RAD001 and the pan AKT inhibitor MK-2206 resulted in a strong synergism, both in vitro and in vivo. Moreover, by analyzing primary HCC tissue samples we were able to demonstrate that a hotspot mutation (H1047R) of PI3KCA, the gene encoding the catalytic subunit of PI3K, was associated with increased in vitro kinase activity of all AKT isoforms in comparison to healthy liver tissue of the patient.. Our results demonstrate that dual targeting of mTOR and AKT by use of RAD001 and the pan AKT inhibitor MK-2206 does effectively inhibit proliferation of HCC cell lines. These data suggest that combined treatment with RAD001 and MK-2206 may be a promising therapy approach in the treatment of hepatocellular carcinoma.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Drug Delivery Systems; Drug Synergism; Everolimus; Feedback, Physiological; Female; Gene Knockdown Techniques; Hep G2 Cells; Heterocyclic Compounds, 3-Ring; Humans; Isoenzymes; Liver Neoplasms; Mice; Mice, SCID; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases