bafilomycin-a1 and Hypoxia

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

Longitudinal bone growth takes place in epiphyseal growth plates located in the ends of long bones. The growth plate consists of chondrocytes traversing from the undifferentiated (resting zone) to the terminally differentiated (hypertrophic zone) stage. Autophagy is an intracellular catabolic process of lysosome-dependent recycling of intracellular organelles and protein complexes. Autophagy is activated during nutritionally depleted or hypoxic conditions in order to facilitate cell survival. Chondrocytes in the middle of the growth plate are hypoxic and nutritionally depleted owing to the avascular nature of the growth plate. Accordingly, autophagy may facilitate their survival. To explore the role of autophagy in chondrocyte survival and constitutional bone growth, we generated mice with cartilage-specific ablation of either Atg5 (Atg5cKO) or Atg7 (Atg7cKO) by crossing Atg5 or Atg7 floxed mice with cartilage-specific collagen type 2 promoter-driven Cre. Both Atg5cKO and Atg7cKO mice showed growth retardation associated with enhanced chondrocyte cell death and decreased cell proliferation. Similarly, inhibition of autophagy by Bafilomycin A1 (Baf) or 3-methyladenine (3MA) promoted cell death in cultured slices of human growth plate tissue. To delineate the underlying mechanisms we employed ex vivo cultures of mouse metatarsal bones and RCJ3.IC5.18 rat chondrogenic cell line. Baf or 3MA impaired metatarsal bone growth associated with processing of caspase-3 and massive cell death. Similarly, treatment of RCJ3.IC5.18 chondrogenic cells by Baf also showed massive cell death and caspase-3 cleavage. This was associated with activation of caspase-9 and cytochrome C release. Altogether, our data suggest that autophagy is important for chondrocyte survival, and inhibition of this process leads to stunted growth and caspase-dependent death of chondrocytes.

Topics: Adenine; Animals; Apoptosis; Autophagy; Autophagy-Related Protein 5; Autophagy-Related Protein 7; Caspases; Cell Death; Cell Line; Cell Proliferation; Cell Survival; Chondrocytes; Collagen Type II; Cytochromes c; Gene Deletion; Growth Plate; Humans; Hypoxia; Immunohistochemistry; In Situ Hybridization; Macrolides; Metatarsal Bones; Mice; Mice, Knockout; Microtubule-Associated Proteins; Proteins; Rats; Ubiquitin-Activating Enzymes

The neuropilins-1 and -2 (NRP1 and NRP2) function as receptors for both the semaphorins and vascular endothelial growth factor. In addition to their contribution to the development of the nervous system, NRP1 and NRP2 have been implicated in angiogenesis and tumor progression. Given their importance to cancer and endothelial biology and their potential as therapeutic targets, an important issue that has not been addressed is the impact of metabolic stress conditions characteristic of the tumor microenvironment on their expression and function. Here, we demonstrate that hypoxia and nutrient deprivation stimulate the rapid loss of NRP1 expression in both endothelial and carcinoma cells. NRP2 expression, in contrast, is maintained under these conditions. The lysosomal inhibitors chloroquine and bafilomycin A1 prevented the loss of NRP1 expression, but proteasomal inhibitors had no effect. The hypothesis that NRP1 is degraded by autophagy is supported by the findings that its expression is lost rapidly in response to metabolic stress, prevented with 3-methyladenine and induced by rapamycin. Targeted depletion of NRP2 using small hairpin RNA revealed that NRP2 can function in the absence of NRP1 to mediate endothelial tube formation in hypoxia. Studies aimed at assessing NRP function and targeted therapy in cancer and angiogenesis should consider the impact of metabolic stress.

Topics: Adenine; Autophagy; Cell Line, Tumor; Cell Membrane; Chloroquine; Culture Media; Endothelium, Vascular; Enzyme Inhibitors; Gene Expression Regulation; Humans; Hypoxia; Lysosomes; Macrolides; Neuropilin-1; Neuropilin-2; Sirolimus

Under anoxia, Artemia franciscana embryos downregulate metabolic processes and approach an ametabolic state. Entrance into this quiescent state is accompanied by a profound acidification of the intracellular space, and more than two decades of research now clearly demonstrates that this acidification is critical to metabolic downregulation in anoxic embryos. However, the proximal mechanisms responsible for the pH shift remain largely unidentified. Here, we report evidence demonstrating expression of the V-ATPase in encysted embryos and present an argument for its involvement in the intracellular acidification induced by anoxia. We identified a single B-subunit cDNA sharing the greatest degree of sequence similarity with ;generalist-type' homologues from mammals (brain-type) and invertebrates. Quantitative analysis of B-subunit mRNA demonstrates differential expression throughout early development, and western blot analyses confirm the expression of at least six V-ATPase subunits in both heavy membranes and microsomal vesicles. The critical need for proton pumping during the anoxia-tolerant stage of development is demonstrated by incubation with the V-ATPase inhibitor bafilomycin A1, which halts embryonic development. Importantly, net proton flux from V-ATPase-acidified compartments to the surrounding cytoplasm is likely under anoxia and may significantly contribute to the enigmatic acidification critical to quiescence.

Topics: Acid-Base Equilibrium; Amino Acid Sequence; Analysis of Variance; Animals; Artemia; Base Sequence; Blotting, Northern; Blotting, Western; Cloning, Molecular; DNA Primers; Embryo, Nonmammalian; Energy Metabolism; Gene Expression Regulation, Developmental; Gene Expression Regulation, Enzymologic; Hydrogen-Ion Concentration; Hypoxia; Macrolides; Molecular Sequence Data; Protons; Sequence Alignment; Sequence Analysis, DNA; Utah; Vacuolar Proton-Translocating ATPases

The metabolic downregulation critical for long-term survival of Artemia franciscana embryos under anoxia is mediated, in part, by a progressive intracellular acidification. However, very little is known about the mechanisms responsible for the pH transitions associated with exposure to, and recovery from, oxygen deprivation. In the present study, we demonstrate with 31P-NMR that incubation of intact embryos with the V-ATPase inhibitor bafilomycin A1 severely limits intracellular alkalinization during recovery from anoxia without affecting the restoration of cellular nucleotide triphosphate levels. Based on these data, it appears that oxidative phosphorylation and ATP resynthesis can only account for the first 0.3 pH unit alkalinization observed during aerobic recovery from the 1 pH unit acidification produced during 1 h of anoxia. The additional 0.7 pH unit increase requires proton pumping by the V-ATPase. Aerobic incubation with bafilomycin also suggests that V-ATPase inhibition alone is not enough to induce an acute dissipation of proton gradients under anoxia. In intact embryos, the dissipation of proton gradients and uncoupling of oxidative phosphorylation with carbonyl cyanide 3-chlorophenylhydrazone (CCCP) leads to an intracellular acidification similar to that seen after 1 h of anoxia. Subsequent exposure to anoxia, in the continued presence of CCCP, yields little additional acidification, suggesting that proton gradients are normally dissipated under anoxia. When combined with protons generated from net ATP hydrolysis, these data show that the dissipation of proton chemical gradients is sufficient to account for the reversible acidification associated with quiescence in these embryos.

Topics: Acid-Base Equilibrium; Animals; Artemia; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Embryo, Nonmammalian; Hydrogen-Ion Concentration; Hypoxia; Macrolides; Magnetic Resonance Spectroscopy; Oxygen Consumption; Phosphorus Isotopes; Protons; Signal Transduction; Utah; Vacuolar Proton-Translocating ATPases