thiourea and 3-methyladenine

Topics: Adenine; Autophagosomes; Autophagy; Cell Line, Tumor; Hepatitis B virus; Heterocyclic Compounds, 2-Ring; Humans; Lysosomes; Membrane Fusion; rab GTP-Binding Proteins; rab7 GTP-Binding Proteins; Thiourea

The aim of the present study was to investigate whether endoplasmic reticulum (ER) stress is involved in MG‑132‑induced autophagy, and to determine the effects of the inhibition of autophagy and ER stress on cell viability following MG‑132 treatment. The proteasome inhibitor, MG‑132, was used to induce autophagy in MCF‑7 cells, and 3‑methyladenine (3‑MA) and salubrinal were used to inhibit autophagy and ER stress, respectively. An MTT assay was used to analyze cell viability. Apoptosis and the cell cycle were analyzed using flow cytometry. The expression levels of apoptosis‑ and ER stress‑associated genes were investigated using western blot and reverse transcription‑quantitative polymerase chain reaction analyses. MG‑132 inhibited cell proliferation, and induced apoptosis and cell cycle arrest at the G2 phase of the cell cycle. Notably, MG‑132 increased the autophagy‑associated conversion of microtubule‑associated protein 1 light chain 3 (LC3)‑I to LC3‑II, which was partially attenuated by the ER stress inhibitor, salubrinal. In addition, MG‑132 inhibited the protein expression of the anti‑apoptotic protein, B‑cell lymphoma (Bcl)‑2, whereas the expression levels of Bcl‑2‑associated X protein and caspase‑3 were upregulated. These effects were enhanced by co‑treatment with either 3‑MA or salubrinal. Furthermore, the mRNA and protein levels of the ER stress‑associated genes, glucose‑regulated protein 78, growth arrest and DNA damage induced gene‑153, and caspase‑12, were upregulated by MG132, and these levels were significantly inhibited by co‑treatment of the cells with salubrinal. Taken together, the results of the present study indicated that the induction of autophagy by the proteasome inhibitor was associated with ER stress in the MCF‑7 cells, and that the inhibition of autophagy or ER stress enhanced MG‑132‑induced apoptosis. These findings suggest the potential application of inhibitors of ER stress and autophagy, in combination with proteasomal inhibitors, for the development of combinatorial targeted cancer therapy.

Topics: Adenine; Apoptosis; Autophagy; Cinnamates; Drug Synergism; Endoplasmic Reticulum Stress; Humans; Leupeptins; MCF-7 Cells; Proteasome Inhibitors; Thiourea

Recent studies have suggested that autophagy plays a prosurvival role in ischemic preconditioning (IPC). This study was taken to assess the linkage between autophagy and endoplasmic reticulum (ER) stress during the process of IPC. The effects of IPC on ER stress and neuronal injury were determined by exposure of primary cultured murine cortical neurons to 30 min of OGD 24 h prior to a subsequent lethal OGD. The effects of IPC on ER stress and ischemic brain damage were evaluated in rats by a brief ischemic insult followed by permanent focal ischemia (PFI) 24 h later using the suture occlusion technique. The results showed that both IPC and lethal OGD increased the LC3-II expression and decreased p62 protein levels, but the extent of autophagy activation was varied. IPC treatment ameliorated OGD-induced cell damage in cultured cortical neurons, whereas 3-MA (5-20 mM) and bafilomycin A 1 (75-150 nM) suppressed the neuroprotection induced by IPC. 3-MA, at the dose blocking autophagy, significantly inhibited IPC-induced HSP70, HSP60 and GRP78 upregulation; meanwhile, it also aggregated the ER stress and increased activated caspase-12, caspase-3 and CHOP protein levels both in vitro and in vivo models. The ER stress inhibitor Sal (75 pmol) recovered IPC-induced neuroprotection in the presence of 3-MA. Rapamycin 50-200 nM in vitro and 35 pmol in vivo 24 h before the onset of lethal ischemia reduced ER stress and ischemia-induced neuronal damage. These results demonstrated that pre-activation of autophagy by ischemic preconditioning can boost endogenous defense mechanisms to upregulate molecular chaperones, and hence reduce excessive ER stress during fatal ischemia.

Topics: Adenine; Animals; Apoptosis; Autophagy; Brain Ischemia; Caspase 12; Caspase 3; Cells, Cultured; Cerebral Cortex; Cinnamates; Cytoprotection; Disease Models, Animal; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Glucose; Heat-Shock Proteins; HSP70 Heat-Shock Proteins; Ischemic Preconditioning; Male; Mice; Neurons; Oxygen; Rats; Rats, Sprague-Dawley; Sirolimus; Thiourea; Transcription Factor CHOP

Adaptation to hypoxia through activation of the hypoxia inducible factor-1 (HIF-1) is crucial for tumor cells survival. Here we describe the antitumoral effects of the new molecule CR 3294 on tumor cells in the presence of hypoxia. Treatment of the breast carcinoma cell line MDA-MB-231 with CR 3294 in 1% O(2) resulted in an in vivo and in vitro inhibition of tumor growth. CR 3294 induced accumulation of autophagosomes in hypoxic MDA-MB-231 cells as assessed by both transmission electron microscopy (TEM) and the autophagic marker LC3-II. TEM analysis revealed the presence of invaginations of the cytoplasm into the nucleus. Autophagosomes were present in such invaginations. Moreover, CR 3294 inhibited both the DNA binding of HIF-1alpha and VEGF mRNA synthesis. Immunoprecipitation and immunofluorescence studies showed an interaction between LC3 and HIF-1alpha. We next detailed the effect of inhibitors and activators of autophagy on both HIF-1alpha and LC3. In particular, 3 methyladenine (3MA) and wortmannin, two macroautophagic inhibitors, prevented both the decrease of HIF-1alpha protein levels and LC3 processing in cells treated with CR 3294. Bafilomycin and leupeptin, inhibitors of lysosomes, prevented HIF-1alpha decrease without affecting LC3 processing. By contrast, treating hypoxic MDA-MB-231 cells with trifluoperazine (TFP) or serum withdrawal (SW), two activators of autophagy, diminished HIF-1alpha levels and stimulated LC3 processing. These results indicate that activation of the autophagic pathway in hypoxic cells by the new molecule CR 3294, as well as by TFP or SW, can have potentially important implications for cancer treatment.

Topics: Adenine; Amidines; Androstadienes; Animals; Anti-Inflammatory Agents, Non-Steroidal; Antineoplastic Agents; Autophagy; Cell Hypoxia; Cell Line, Tumor; Cell Proliferation; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Mice; Mice, Transgenic; Microtubule-Associated Proteins; Neoplasms; Phagosomes; Thiourea; Trifluoperazine; Vascular Endothelial Growth Factor A; Wortmannin