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.
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.
Nutritional stress caused by amino acid starvation involves a coordinated cellular response that includes the global decrease of protein synthesis and the increased production of cell defense proteins. Part of this response is the induction of transport system A for neutral amino acids that leads to the recovery of cell volume and amino acid levels once extracellular amino acid availability is restored. Hypertonic stress also increases system A activity as a mechanism to promote a rapid recovery of cell volume. Both a starvation-dependent and a hypertonic increase of system A transport activity are due to the induction of SNAT2, the ubiquitous member of SLC38 family. The molecular mechanisms underlying SNAT2 induction were investigated in tissue culture cells. We show that the increase in system A transport activity and SNAT2 mRNA levels upon amino acid starvation were blunted in cells with a mutant eIF2␣ that cannot be phosphorylated. In contrast, the induction of system A activity and SNAT2 mRNA levels by hypertonic stress were independent of eIF2␣ phosphorylation. The translational control of the SNAT2 mRNA during amino acid starvation was also investigated. It is shown that the 5-untranslated region contains an internal ribosome entry site that is constitutively active in amino acid-fed and -deficient cells and in a cell-free system. We also show that amino acid starvation caused a 2.5-fold increase in mRNA and protein expression from a reporter construct containing both the SNAT2 intronic amino acid response element and the SNAT2-untranslated region. We conclude that the adaptive response of system A activity to amino acid starvation requires eukaryotic initiation factor 2␣ phosphorylation, increased gene transcription, and internal ribosome entry site-mediated translation. In contrast, the response to hypertonic stress does not involve eukaryotic initiation factor 2␣ phosphorylation, suggesting that SNAT2 expression can be modulated by specific signaling pathways in response to different stresses.The severe nutritional stress caused by amino acid starvation triggers several adaptive changes. One of these is the stimulation of transport system A for neutral amino acids. Adaptive regulation of system A has been described in most amino acid-starved animal cells (1-3). Work from several laboratories (4 -6) has demonstrated that this regulatory mechanism is associated with an increase in the mRNA for SNAT2, the ubiquitously expressed member of the sodium-coupled neutral amino acid transporter family (7). The induction of SNAT2 transcription leads to the increased synthesis of SNAT2 carriers, which are found in greater amounts on the membranes of starved cells (5,8). Recently, an amino acid-responsive element has been identified in the first intron of the mouse and human SNAT2 genes (9), supporting the hypothesis that transcriptional activation contributes to the adaptive regulation of system A.Unlike transcriptional activation, the translation of SNAT2 during amino acid starvation has received little attention ...
The copper(II) complex A0 induces a type of non-apoptotic cell death also known as paraptosis. Paraptosis involves extensive endoplasmic reticulum vacuolization in the absence of caspase activation. A wide panel of human cancer cell lines was used to demonstrate differences in cytotoxicity by the paraptosis-inducing drug A0 and the metal-based pro-apoptotic drug cisplatin. Gene expression profiling of the human fibrosarcoma HT1080 cells showed that, while cisplatin induced p53 targets, A0 up-regulated genes involved in the unfolded protein response (UPR) and response to heavy metals. The cytotoxic effects of A0 were associated with inhibition of the ubiquitinproteasome system and accumulation of ubiquitinylated proteins, in a manner dependent on protein synthesis. Cycloheximide inhibited the accumulation of ubiquitinylated proteins and hampered A0-induced cell death process. The occurrence of the UPR during A0-induced death process was shown by the increased abundance of spliced XBP1 mRNA, transient eIF2␣ phosphorylation, and a series of downstream events, including attenuation of global protein synthesis and increased expression of ATF4, CHOP, BIP, and GADD34. Mouse embryonic fibroblasts expressing a mutant eIF2␣, which could not be phosphorylated, were more resistant to A0 than wild type cells, pointing to a pro-death role of eIF2␣ phosphorylation. A0 may thus represent the prototypical member of a new class of compounds that cause paraptotic cell death via mechanisms involving eIF2␣ phosphorylation and the UPR.
The use of an inexpensive and simple modification of Costar 24-well cluster trays is described in a rapid and reproducible method for measuring substrate fluxes in adherent cultured eukaryotic cells.
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