We report a quantitative structure-activity relationship study of a new class of pyrazole-pyridine copper complexes that establishes a clear correlation between the ability to promote copper accumulation and cytotoxicity. Intracellular metal accumulation is maximized when ligand lipophilicity allows the complex to rapidly cross the membrane. Copper and ligand follow different uptake kinetics and reach different intracellular equilibrium concentrations. These results support a model in which the ligand acts as an ionophore for the metal ion, cycling between intra- and extracellular compartments as dissociated or complexed entities. When treating cancer cells with structurally unrelated disulfiram and pyrazole-pyridine copper complexes, as well as with inorganic copper, the same morphological and molecular changes were reproduced, indicating that copper overload is responsible for the cytotoxic effects. Copper-based treatments drive sensitive cancer cells toward paraptotic cell death, a process hallmarked by endoplasmic reticulum stress and massive vacuolization in the absence of apoptotic features. A lack of caspase activation, as observed in copper-treated dying cells, is a consequence of metal-mediated inhibition of caspase-3. Thus, copper acts simultaneously as an endoplasmic reticulum (ER) stress inducer and a caspase-3 inhibitor, forcing the cell into caspase-independent paraptotic death. The establishment of a mechanism of action common to different copper binding agents provides a rationale for the exploitation of copper toxicity as an anticancer tool.
Key Points• Myeloma cells produce ammonium in the presence of glutamine, showing high glutaminase and low glutamine synthetase expression.• Myeloma cells show high expression of glutamine transporters and inhibition of ASCT2 transporter hinders myeloma growth.The importance of glutamine (Gln) metabolism in multiple myeloma (MM) cells and its potential role as a therapeutic target are still unknown, although it has been reported that human myeloma cell lines (HMCLs) are highly sensitive to Gln depletion. In this study, we found that both HMCLs and primary bone marrow (BM) CD138 1 cells produced large amounts of ammonium in the presence of Gln. MM patients have lower BM plasma Gln with higher ammonium and glutamate than patients with indolent monoclonal gammopathies. Interestingly, HMCLs expressed glutaminase (GLS1) and were sensitive to its inhibition, whereas they exhibited negligible expression of glutamine synthetase (GS). High GLS1 and low GS expression were also observed in primary CD138 1 cells. Gln-free incubation or treatment with the glutaminolytic enzyme L-asparaginase depleted the cell contents of Gln, glutamate, and the anaplerotic substrate 2-oxoglutarate, inhibiting MM cell growth. Consistent with the dependence of MM cells on extracellular Gln, a gene expression profile analysis, on both proprietary and published datasets, showed an increased expression of the Gln transporters SNAT1, ASCT2, and LAT1 by CD138 1 cells across the progression of monoclonal gammopathies. Among these transporters, only ASCT2 inhibition in HMCLs caused a marked decrease in Gln uptake and a significant fall in cell growth. Consistently, stable ASCT2 downregulation by a lentiviral approach inhibited HMCL growth in vitro and in a murine model. In conclusion, MM cells strictly depend on extracellular Gln and show features of Gln addiction. Therefore, the inhibition of Gln uptake is a new attractive therapeutic strategy for MM. (Blood. 2016;128(5):667-679)
Background: Protein synthesis control is important for -cell fate during ER stress. Results: Increased protein synthesis during chronic ER stress in -cells involves the transcriptional induction of an amino acid transporter network. Conclusion: Increased amino acid uptake in -cells during ER stress promotes apoptosis. Significance: Induced expression of a network of amino acid transporters in islets can contribute to chronic ER stress-induced diabetes.
This study reports the structure-activity relationship of a series of 8-hydroxoquinoline derivatives (8-HQs) and focuses on the cytotoxic activity of 5-Cl-7-I-8-HQ (clioquinol, CQ) copper complex (Cu(CQ)). 8-HQs alone cause a dose-dependent loss of viability of the human tumor HeLa and PC3 cells, but the coadministration of copper increases the ligands effects, with extensive cell death occurring in both cell lines. Cytotoxic doses of Cu(CQ) promote intracellular copper accumulation and massive endoplasmic reticulum vacuolization that precede a nonapoptotic (paraptotic) cell death. The cytotoxic effect of Cu(CQ) is reproduced in normal human endothelial cells (HUVEC) at concentrations double those effective in tumor cells, pointing to a potential therapeutic window for Cu(CQ). Finally, the results show that the paraptotic cell death induced by Cu(CQ) does not require nor involve caspases, giving an indication for the current clinical assessment of clioquinol as an antineoplastic agent.
Hypoxia-inducible factor (HIF) prolyl hydroxylases (PHDs) are α-ketoglutarate (αKG)-dependent dioxygenases that function as cellular oxygen sensors. However, PHD activity also depends on factors other than oxygen, especially αKG, a key metabolic compound closely linked to amino-acid metabolism. We examined the connection between amino-acid availability and PHD activity. We found that amino-acid starvation leads to αKG depletion and to PHD inactivation but not to HIF stabilization. Furthermore, pharmacologic or genetic inhibition of PHDs induced autophagy and prevented mammalian target of rapamycin complex 1 (mTORC1) activation by amino acids in a HIF-independent manner. Therefore, PHDs sense not only oxygen but also respond to amino acids, constituting a broad intracellular nutrient-sensing network.
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