lithium-chloride and Liver-Neoplasms

The current study was designed and performed to investigate the effect of mefloquine on the proliferation and tumor formation potential of liver cancer stem cells. CD133 + HepG2 cells were identified using MACS and showed markedly higher tumor formation potential compared to the parental cells. The secondary tumors formed by CD133 + cells were markedly large in size and more in number compared to the parental cells. Mefloquine treatment of CD133 + HepG2 cells inhibited the proliferation selectively in concentration based manner. The rate of proliferation was inhibited to 82 and 12% in parental and CD133 + sphere forming cells, respectively on treatment with 10 μM concentration of mefloquine. The number of secondary tumors formed by primary tumors was decreased significantly on treatment with 10 μM mefloquine concentration. Treatment of the liver cancer stem cells with mefloquine markedly decreased the potential to undergo self-renewal at 10 μM concentration after 48 h. The results from western blot analysis showed significantly higher expression of cancer stem cell molecules β-catenin and cyclin D1 in LCSCs. Treatment of the LCSCs with various concentrations of mefloquine reduced the expression levels of β-catenin and cyclin D1. Administration of the CD133 + cell tumor xenografts in the mice led to the formation of large sized tumors in the control group. However, the tumor growth was inhibited significantly in the mice on treatment with 10 mg/kg doses of mefloquine after day 21. The tumor weight was significantly lower in the animals of mefloquine treatment group compared to the control group. Thus, mefloquine treatment inhibits self-renewal and proliferation potential of cells through targeting β-catenin pathway.

Topics: AC133 Antigen; Animals; beta Catenin; Carcinoma, Hepatocellular; Cell Proliferation; Cell Survival; Cyclin D1; Disease Models, Animal; Drug Combinations; Hep G2 Cells; Humans; Lithium Chloride; Liver Neoplasms; Male; Mefloquine; Mice; Mice, Inbred BALB C; Neoplastic Stem Cells; Transplantation, Heterologous

The present study was designed to investigate the effect of cantharidin on cell proliferation, ability of selfrenewal, cell cycle arrest and induction of apoptosis in HepG2 hepatocellular carcinoma stem cells (HCSCs). It was observed that cantharidin treatment exhibited dose- and time-dependent inhibitory effect on the viability of HCSCs. The inhibition of cell viability by cantharidin in HepG2 CD133+ and parental cells was significant at the concentration 5 and 15 µM, respectively after 48 h. Cantharidin treatment inhibited the self-renewal ability of the HCSCs and the expression of β-catenin and cyclin D1. Flow cytometry revealed that cantharidin treatment at 5 µM concentration significantly increased the cell population in G2/M phase and decreased the population in the G1 phase. Cantharidin treatment in the HCSCs for 48 h increased expression of histone H2AX, Myt1, cyclin A2, cyclin B1, p53 and cdc2 (Tyr15) phosphorylation significantly compared to the parental cells. Exposure of the HCSCs to cantharidin for 48 h at a concentration of 5 µM caused a significant increase in the proportion of apoptotic cells. Therefore, cantharidin is a promising agent for the hepatocellular carcinoma treatment.

Topics: Animals; Antineoplastic Agents; Apoptosis; beta Catenin; Cantharidin; Carcinoma, Hepatocellular; Cell Proliferation; Cell Self Renewal; Cyclin D1; Drug Screening Assays, Antitumor; G2 Phase Cell Cycle Checkpoints; Hep G2 Cells; Humans; Lithium Chloride; Liver Neoplasms; Male; Mice, Inbred BALB C; Mice, Nude; Neoplasm Transplantation; Neoplastic Stem Cells

The Wnt signaling pathway is involved in many differentiation events during embryonic development and can lead to tumor formation after aberrant activation of its components. beta-catenin, a cytoplasmic component, plays a major role in the transduction of canonical Wnt signaling. The aim of this study was to identify novel genes that are regulated by active beta-catenin/TCF signaling in hepatocellular carcinoma-derived Huh7 cells with high (transfected) and low beta-catenin/TCF activities. High TCF activity Huh7 cells led to earlier and larger tumor formation when xenografted into nude mice. SAGE (Serial Analysis of Gene Expression), genome-wide microarray and in silico promoter analysis were performed in parallel, to compare gene expression between low and high beta-catenin/TCF activity clones, and also those that had been rescued from the xenograft tumors. SAGE and genome-wide microarray data were compared and contrasted. BRI3 and HSF2 were identified as novel targets of Wnt/beta-catenin signaling after combined analysis and confirming experiments including qRT-PCR, ChIP, luciferase assay and lithium treatment.

Topics: Animals; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; beta Catenin; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Genome; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Heat-Shock Proteins; Humans; Lithium Chloride; Liver Neoplasms; Membrane Proteins; Mice; Mice, Nude; Nerve Tissue Proteins; Oligonucleotide Array Sequence Analysis; Promoter Regions, Genetic; Signal Transduction; Transcription Factor 4; Transcription Factors; Wnt Proteins

Hepatocellular carcinoma (HCC) is a common human cancer with high mortality, and currently, there is no effective chemoprevention or systematic treatment. Recent evidence suggests that cyclooxygenase-2 (COX-2)-derived PGE(2) and Wnt/beta-catenin signaling pathways are implicated in hepatocarcinogenesis. Here, we report that omega-3 polyunsaturated fatty acids (PUFA), docosahexaenoic acid (DHA), and eicosapentaenoic acid (EPA) inhibit HCC growth through simultaneously inhibition of COX-2 and beta-catenin. DHA and EPA treatment resulted in a dose-dependent reduction of cell viability with cleavage of poly ADP ribose polymerase, caspase-3, and caspase-9 in three human HCC cell lines (Hep3B, Huh-7, HepG2). In contrast, AA, a omega-6 PUFA, exhibited no significant effect. DHA and EPA treatment caused dephosphorylation and thus activation of GSK-3beta, leading to beta-catenin degradation in Hep3B cells. The GSK-3beta inhibitor, LiCl, partially prevented DHA-induced beta-catenin protein degradation and apoptosis. Additionally, DHA induced the formation of beta-catenin/Axin/GSK-3beta binding complex, which serves as a parallel mechanism for beta-catenin degradation. Furthermore, DHA inhibited PGE(2) signaling through downregulation of COX-2 and upregulation of the COX-2 antagonist, 15-hydroxyprostaglandin dehydrogenase. Finally, the growth of HCC in vivo was significantly reduced when mouse HCCs (Hepa1-6) were inoculated into the Fat-1 transgenic mice, which express a Caenorhabditis elegans desaturase converting omega-6 to omega-3 PUFAs endogenously. These findings provide important preclinical evidence and molecular insight for utilization of omega-3 PUFAs for the chemoprevention and treatment of human HCC.

Topics: Animals; Apoptosis; Axin Protein; beta Catenin; Carcinoma, Hepatocellular; Cell Growth Processes; Cell Line, Tumor; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Dinoprostone; Docosahexaenoic Acids; Eicosapentaenoic Acid; Fatty Acids, Unsaturated; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Hydroxyprostaglandin Dehydrogenases; Lithium Chloride; Liver Neoplasms; Male; Mice; Mice, Transgenic; Repressor Proteins; Signal Transduction; TCF Transcription Factors; Transcriptional Activation; Transfection; Wnt Proteins; Wnt3 Protein; Xenograft Model Antitumor Assays

Hypoxia initiates an intracellular signaling pathway leading to the activation of the transcription factor hypoxia-inducible factor-1 (HIF-1). HIF-1 activity is regulated through different mechanisms involving stabilization of HIF-1alpha, phosphorylations, modifications of redox conditions, and interactions with coactivators. However, it appears that some of these steps can be cell type-specific. Among them, the involvement of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway in the regulation of HIF-1 by hypoxia remains controversial. Here, we investigated the activation state of PI3K/Akt/glycogen synthase kinase 3beta (GSK3beta) in HepG2 cells. Increasing incubation times in hypoxia dramatically decreased both the phosphorylation of Akt and the inhibiting phosphorylation of GSK3beta. The PI3K/Akt pathway was necessary for HIF-1alpha stabilization early during hypoxia. Indeed, its inhibition was sufficient to decrease HIF-1alpha protein level after 5-h incubation in hypoxia. However, longer exposure (16 h) in hypoxia resulted in a decreased HIF-1alpha protein level compared with early exposure (5 h). At that time, Akt was no longer present or active, which resulted in a decrease in the inhibiting phosphorylation of GSK3beta on Ser-9 and hence in an increased GSK3beta activity. GSK3 inhibition reverted the effect of prolonged hypoxia on HIF-1alpha protein level; more stabilized HIF-1alpha was observed as well as increased HIF-1 transcriptional activity. Thus, a prolonged hypoxia activates GSK3beta, which results in decreased HIF-1alpha accumulation. In conclusion, hypoxia induced a biphasic effect on HIF-1alpha stabilization with accumulation in early hypoxia, which depends on an active PI3K/Akt pathway and an inactive GSK3beta, whereas prolonged hypoxia results in the inactivation of Akt and activation of GSK3beta, which then down-regulates the HIF-1 activity through down-regulation of HIF-1alpha accumulation.

Topics: Adjuvants, Immunologic; Carcinoma, Hepatocellular; Chromones; DNA-Binding Proteins; Enzyme Inhibitors; Gene Expression; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Hypoxia; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; Lithium Chloride; Liver Neoplasms; Morpholines; Nuclear Proteins; Phosphatidylinositol 3-Kinases; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Transcription Factors; Tumor Cells, Cultured