bafilomycin-a1 and Carcinoma--Hepatocellular

Despite pharmacological advances such as lenvatinib approval, therapeutic failure of hepatocellular carcinoma (HCC) remains a big challenge due to the complexity of its underlying molecular mechanisms. Neuropilin-1 (NRP1) is a co-receptor involved in several cellular processes associated to chemoresistance development. Since both the double-edged process of autophagy and hypoxia-derived response play crucial roles in the loss of therapeutic effectiveness, herein we investigated the interplay among NRP1, autophagy and hypoxia in development of lenvatinib resistance in HCC cell lines. We first analyzed NRP1 expression levels in human HCC samples from public databases, found significantly increased NRP1 expression in human HCC samples as well as its correlation with advanced tumor and metastasis stages. Among 3 HCC cell lines (HepG2, Huh-7 and Hep3B), Hep3B and Huh-7 cells showed significantly increased NRP1 expression levels and cell migration ability together with higher susceptibility to lenvatinib. We demonstrated that NRP1 gene silencing significantly enhanced the anticancer effects of lenvatinib on Hep3B and Huh-7 cells. Furthermore, lenvatinib suppressed NRP1 expression through promoting autophagy in Hep3B and Huh-7 cells; co-treatment with bafilomycin A1 attenuated the antitumor effects of lenvatinib, and NRP1 silencing prevented this loss of in vitro effectiveness of lenvatinib even in the presence of bafilomycin A1. In addition, exposure to a hypoxic microenvironment significantly decreased NRP1 expression through autophagy in Hep3B and Huh-7 cells. Under hypoxia, HIF-1α directly modulated NRP1 expression; HIF-1α silencing not only enhanced the anticancer effects of combined lenvatinib and hypoxia, but also prevented the loss of effectiveness caused by bafilomycin A1, highlighting the potential role of HIF-1α-derived hypoxia response in the adaptive cellular response to lenvatinib and promoting resistance acquisition by autophagy modulation. Overall, NRP1 may constitute a potential therapeutic target to prevent lenvatinib failure derived from a hypoxia-associated modulation of autophagy in advanced HCC.

Topics: Autophagy; Carcinoma, Hepatocellular; Cell Line, Tumor; Drug Resistance, Neoplasm; Gene Expression Regulation, Neoplastic; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Liver Neoplasms; Neuropilin-1

Aberrant autophagic processes have been found to have fundamental roles in the pathogenesis of different kinds of tumors, including hepatocellular carcinoma (HCC). P300/CBP-associated factor (PCAF), a histone acetyltransferase (HAT), performs its function by acetylating both histone and non-histone proteins. Our previous studies showed that PCAF was downregulated in HCC tissues and its high expression was significantly associated with patient survival after surgery, serving as a prognostic marker. In this study we found that overexpression of PCAF induced autophagy of HCC cells and its knockdown depressed autophagy. As type II programmed cell death, autophagy induced by PCAF-elicited cell death in HCC cells. In vivo experiments confirmed that PCAF-induced autophagy inhibited tumor growth. Subsequent in vitro experiments showed that PCAF promoted autophagy by inhibiting Akt/mTOR signaling pathway. Our findings show that PCAF is a novel modulator of autophagy in HCC, and can serve as an attractive therapeutic strategy of HCC treatment.

Topics: Adenine; Animals; Apoptosis; Autophagy; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Female; Gene Knockdown Techniques; Humans; Liver Neoplasms; Macrolides; Mice, Inbred BALB C; Mice, Nude; Oligopeptides; p300-CBP Transcription Factors; Proto-Oncogene Proteins c-akt; Signal Transduction; TOR Serine-Threonine Kinases; Xenograft Model Antitumor Assays

Hepatocellular carcinoma (HCC) is refractory to chemotherapies, necessitating novel effective agents. The lysosome inhibitor Bafilomycin A1 (BafA1) at high concentrations displays cytotoxicity in a variety of cancers. Here we show that BafA1 at nanomolar concentrations suppresses HCC cell growth in both 2 dimensional (2D) and 3D cultures. BafA1 induced cell cycle arrest in the G1 phase and triggered Cyclin D1 turnover in HCC cells in a dual-specificity tyrosine phosphorylation-regulated kinase 1B (DYRK1B) dependent manner. Notably, BafA1 induced caspase-independent cell death in HCC cells by impairing autophagy flux as demonstrated by elevated LC3 conversion and p62/SQSTM1 levels. Moreover, genetic ablation of LC3 significantly attenuated BafA1-induced cytotoxicity of HCC cells. We further demonstrate that pharmacological down-regulation or genetic depletion of p38 MAPK decreased BafA1-induced cell death via abolishment of BafA1-induced upregulation of Puma. Notably, knockdown of Puma impaired BafA1-induced HCC cell death, and overexpression of Puma enhanced BafA1-mediated HCC cell death, suggesting a role for Puma in BafA1-mediated cytotoxicity. Interestingly, pharmacological inhibition of JNK with SP600125 enhanced BafA1-mediated cytotoxicity both in vitro and in xenografts derived from HCC cells. Taken together, our data suggest that BafA1 may offer potential as an effective therapy for HCC.

Topics: Animals; Autophagy; Carcinoma, Hepatocellular; Caspases; Female; Hep G2 Cells; Humans; Liver Neoplasms; Macrolides; MAP Kinase Signaling System; Mice; Mice, Nude; Neoplasm Proteins; Xenograft Model Antitumor Assays

Platycodin D (PD), isolated from the Chinese medicinal herb named Platycodonis Radix, is a triterpenoid saponin with well-known anti-tumor effects. In this study, we provided reliable evidence that PD triggered autophagy in a number of cell lines in vitro. PD-triggered autophagy was identified by observation of cytoplasmic vacuole, up-regulation of microtubule-associated protein 1 light chain 3 II (LC3-II), and accumulation of autophagosomes. The Akt/mammalian target of rapamycin (mTOR) pathway may be not involved in PD-triggered autophagy, as evidenced by the increased phosphorylation of Akt (Thr308), mTOR (Ser2448), ribosomal protein S6 kinase (Ser371), and ULK1 (Ser757). However, the extracellular signal-regulated kinase (ERK) was activated after PD treatment. The decreased ERK phosphorylation caused by pretreatment with U0126, an inhibitor of MEK, suppressed the expression of LC3-II compared with PD treatment alone, suggesting that ERK pathway may have a critical function in PD-triggered autophagy. In addition, the PD-induced proliferative inhibition and apoptosis were enhanced when pretreatment with autophagy inhibitor chloroquine (CQ) or bafilomycin A1 (BAF), indicating that PD may trigger a protective autophagy in HepG2 cells. To the best of our knowledge, this paper is the first to report that PD triggers autophagy in a series of cell lines and ERK activation is important for PD-triggered autophagy in hepatocellular carcinoma HepG2 cells. The combined treatment with PD and CQ or BAF may be a promising regimen for hepatocellular carcinoma treatment.

Topics: Autophagy; Carcinoma, Hepatocellular; Cell Line, Tumor; Chloroquine; Extracellular Signal-Regulated MAP Kinases; Humans; Liver Neoplasms; Macrolides; Saponins; Triterpenes

Liver cancer is the second-most frequent cause of cancer death in the world and is highly treatment resistant. We reported previously that inhibition of neddylation pathway with specific NAE inhibitor MLN4924, suppressed the malignant phenotypes of liver cancer. However, during the process, MLN4924 induces pro-survival autophagy as a mechanism of drug resistance. Here, we report that blockage of autophagy with clinically-available autophagy inhibitors (e.g. chloroquine) significantly enhanced the efficacy of MLN4924 on liver cancer cells by triggering apoptosis. Mechanistically, chloroquine enhanced MLN4924-induced up-regulation of pro-apoptotic proteins (e.g. NOXA) and down-regulation of anti-apoptotic proteins. Importantly, the down-regulation of NOXA expression via siRNA silencing substantially attenuated apoptosis of liver cancer cells. Further mechanistic studies revealed that blockage of autophagy augmented MLN4924-induced DNA damage and reactive oxygen species (ROS) generation. The elimination of DNA damage or blockage of ROS production significantly reduced the expression of NOXA, and thereby attenuated apoptosis and reduced growth inhibition of liver cancer cells. Moreover, blockage of autophagy enhanced the efficacy of MLN4924 in an orthotopic model of human liver cancer, with induction of NOXA and apoptosis in tumor tissues. These findings provide important preclinical evidence for clinical investigation of synergistic inhibition of neddylation and autophagy in liver cancer.

Topics: Antineoplastic Agents; Apoptosis Regulatory Proteins; Autophagy; Carcinoma, Hepatocellular; Cell Line, Tumor; Chloroquine; Cyclopentanes; DNA Damage; Drug Synergism; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; Macrolides; Molecular Targeted Therapy; NEDD8 Protein; Neoplasm Proteins; Protein Processing, Post-Translational; Proto-Oncogene Proteins c-bcl-2; Pyrimidines; Random Allocation; RNA, Small Interfering; Transfection; Ubiquitins; Xenograft Model Antitumor Assays

Autophagy is a lysosomal pathway for cellular homeostasis control. Both non-selective bulk autophagy and selective autophagy of specific proteins or organelles have been found. Selective autophagy prevents cells from pathogen invasion and stress damage, but its role in regulating transcriptional factors is not clear. Using a macrophage cell differentiation model, the role of autophagy in nuclear factor-κB (NF-κB) regulation is investigated. The bone marrow-derived macrophages (BMDMs) will differentiate into a M2-like phenotype in the presence of hepatoma tumor cell condition medium (CM). The TLR2 signaling drives this M2 polarization and causes NF-κB p65 degradation via lysosome-dependent pathway. The CM-induced ubiquitinated- NF-κB p65 forms aggresome-like structures (ALS) in the cytoplasm of cultured and hepatoma-associated M2 macrophages. This NF-κB p65-contained ALS is recognized by p62/SQSTM1 and degraded by selective autophagy. Treatment with the lysosomal inhibitor bafilomycin A1 or the knockdown of Atg5 can prevent CM-induced NK-κB p65 degradation and induce M2 macrophages to produce a high level of pro-inflammatory cytokines. Furthermore, TLR2 signal induces sustained phosphorylation of extracellular signal-regulated kinase 1/2 to facilitate this autophagy-dependent NF-κB regulation. Our finding provides a novel pathway of NF-κB regulation by p62/SQSTM1-mediated selective autophagy.

Topics: Adaptor Proteins, Signal Transducing; Animals; Autophagy; Autophagy-Related Protein 5; Bone Marrow Cells; Carcinoma, Hepatocellular; Cell Differentiation; Cell Line, Tumor; Culture Media, Conditioned; Cytokines; Female; Lysosomes; Macrolides; Macrophages; Mice; Mice, Inbred BALB C; Mice, Knockout; Microtubule-Associated Proteins; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Phosphorylation; RNA Interference; RNA, Small Interfering; Sequestosome-1 Protein; Toll-Like Receptor 2; Transcription Factor RelA

Pigment epithelium-derived factor (PEDF) has several biological actions on tumor cells, but its effects are cell-type dependent. The aim of this study was to examine the pathophysiological role of PEDF in hepatocellular carcinoma (HCC). PEDF expression was examined in various hepatoma cell lines and human HCC tissues, and was seen in various hepatoma cell lines including HepG2 cells. In human HCC tissues, PEDF expression was higher than in adjacent non-HCC tissues. In addition, serum PEDF levels were higher in HCC patients than in non-HCC patients, and curative treatment of HCC caused significant reductions in serum PEDF levels compared with pretreatment levels. In vitro experiments, camptothecin (CPT) was used to induce apoptosis and the effect of PEDF was investigated by knockdown of the PEDF gene in CPT-treated HepG2 cells. Knockdown of the PEDF gene enhanced CPT-induced apoptosis, simultaneously down-regulating Bcl-xL expression in HepG2 cells. Expression of apoptosis-related molecules and effects of bafilomycin A1 on CPT-induced apoptosis were also examined in PEDF gene knockdown HepG2 cells. Treatment with bafilomycin A1 suppressed CPT-induced decreases in Bcl-xL expression and increases in apoptosis in PEDF gene knockdown HepG2 cells. PEDF may, therefore, exert anti-apoptotic effects through inhibition of lysosomal degradation of Bcl-xL in CPT-treated HepG2 cells.

Topics: Apoptosis; bcl-X Protein; Camptothecin; Carcinoma, Hepatocellular; Cell Cycle; Cyclophilins; Densitometry; Eye Proteins; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Hep G2 Cells; Humans; Leupeptins; Liver; Liver Neoplasms; Lysosomes; Macrolides; Nerve Growth Factors; Protein Processing, Post-Translational; Recombinant Proteins; RNA, Messenger; Serpins

Extracellular ATP represents an important autocrine/paracrine signaling molecule within the liver. The mechanisms responsible for ATP release are unknown, and alternative pathways have been proposed, including either conductive ATP movement through channels or exocytosis of ATP-enriched vesicles, although direct evidence from liver cells has been lacking. Utilizing dynamic imaging modalities (confocal and total internal reflection fluorescence microscopy and luminescence detection utilizing a high sensitivity CCD camera) at different scales, including confluent cell populations, single cells, and the intracellular submembrane space, we have demonstrated in a model liver cell line that (i) ATP release is not uniform but reflects point source release by a defined subset of cells; (ii) ATP within cells is localized to discrete zones of high intensity that are approximately 1 mum in diameter, suggesting a vesicular localization; (iii) these vesicles originate from a bafilomycin A(1)-sensitive pool, are depleted by hypotonic exposure, and are not rapidly replenished from recycling of endocytic vesicles; and (iv) exocytosis of vesicles in response to cell volume changes depends upon a complex series of signaling events that requires intact microtubules as well as phosphoinositide 3-kinase and protein kinase C. Collectively, these findings are most consistent with an essential role for exocytosis in regulated release of ATP and initiation of purinergic signaling in liver cells.

Topics: Adenosine Triphosphate; Animals; Autocrine Communication; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Size; Enzyme Inhibitors; Epithelial Cells; Exocytosis; Hepatocytes; Liver Neoplasms; Macrolides; Microscopy, Confocal; Paracrine Communication; Protein Kinase C; Rats; Receptors, Purinergic; Signal Transduction; Transport Vesicles; Vacuolar Proton-Translocating ATPases

Hepatocellular carcinoma (HCC) is the fifth most common cancer and the third leading cause of cancer death worldwide. Drug treatments for HCC have been largely unsuccessful. Histone deacetylase inhibitors can reactivate tumor suppressor genes in cancer cells and serve as potential anti-cancer drugs. Two potent HDAC inhibitors OSU-HDAC42 and SAHA induced autophagy in HCC cells as revealed by transmission electron microscopy, immunofluorescence and LC3-II accumulation. We found that SAHA and OSU-HDAC42 induced autophagy through downregulation of Akt/mTOR signaling and induction of ER stress response. Inhibition of autophagy by 3-MA or Atg5 knockout reduced SAHA-induced cytotoxicity, indicating that SAHA-induced autophagy led to cell death. Our results show that the combination of autophagy inducers with SAHA might be attractive for the treatment of HCC and pharmacological targeting of autophagy provides promise for the management of cancer therapy.

Topics: Adenine; Animals; Antineoplastic Agents; Apoptosis; Autophagy; Carcinoma, Hepatocellular; Cell Line, Tumor; Drug Screening Assays, Antitumor; Drug Synergism; Endoplasmic Reticulum; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Liver Neoplasms; Macrolides; Mice; Microtubule-Associated Proteins; Phagosomes; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Signal Transduction; Stress, Physiological; TOR Serine-Threonine Kinases; Vorinostat