Depletion of intracellular Ca ²⁺ stores, phosphorylation of eIF2α, and sustained inhibition of translation initiation mediate the anticancer effects of clotrimazole

Abstract

Regulation of translation initiation plays a critical role in the control of cell growth and division in eukaryotic cells. Translation of many growth regulatory proteins including cyclins depends critically on translation initiation factors because their mRNAs are translated inefficiently. We report that clotrimazole, a potent antiproliferative agent both in vitro and in vivo, inhibits cell growth by interfering with translation initiation. In particular, clotrimazole causes a sustained depletion of intracellular Ca 2؉ stores, which results in activation of PKR, phosphorylation of eIF2␣, and thereby in inhibition of protein synthesis at the level of translation initiation. Consequently, clotrimazole preferentially decreases the expression of the growth promoting proteins cyclin A, E and D1, resulting in inhibition of cyclindependent kinase activity and blockage of cell cycle in G 1 .

PERK mediates cell-cycle exit during the mammalian unfolded protein response

“…However, as cyclin D1 loss is not completely attenuated in PERK⌬C-3T3 cells, we cannot rule out the possibility that other eIF2␣ protein kinases such as PKR also participate in the regulation of protein translation after activation of the UPR. The capacity of the UPR to activate PKR (7) and the ability of PKR to regulate cyclin D1 translation in response to certain stimuli (35) provide support for this supposition. However, our data are entirely consistent with a model wherein ER stress promotes the activation of PERK via stress-dependent oligomerization (27), resulting in a net increase in eIF2␣ phosphorylation.…”

“…Although two members of this family are activated by the UPR, PERK and PKR (7) and both are capable of inhibiting cyclin D1 translation (ref. 35 and data herein), PERK is likely to be the primary cellular mediator of UPR-dependent signals. Previous work indicated that PKR is dispensable for UPRdependent inhibition of protein synthesis (11), whereas our data demonstrate that PERK is critically required.…”

Clotrimazole Binds to Heme and Enhances Heme-dependent Hemolysis

“…Depletion of intracellular Ca 2ϩ induces the activation of protein kinase R and phosphorylation of eukaryotic translation initiation factor 2␣, thereby inhibiting protein synthesis in the parasite (7). However, the actual mechanism of CLT antimalarial action at the molecular level remains equivocal.…”

The antifungal antibiotic clotrimazole alters calcium homeostasis of leukemic lymphoblasts and induces apoptosis

“…7,[10][11][12][13][14][15] To determine whether clotrimazole affected Ca 2+ homeostasis in ALL cells, we measured changes in cytosolic Ca 2+ concentration in the leukemic cell line 380. Exposure to 10 m clotrimazole induced a detectable increase in cytosolic Ca 2+ concentration, as shown by an increased Indo-1 fluorescence signal ratio; this increase became clearly evident after exposure to 50 m of the drug (Figure 7).…”

“…[3][4][5][6][7][8][9] Clotrimazole disrupts cellular Ca 2+ homeostasis by releasing Ca 2+ from intracellular stores while inhibiting Ca 2+ influx and blocking IK channels. 7,[10][11][12][13][14][15] It is being evaluated clinically for the treatment of IK channel-driven erythrocyte dehydration in sickle cell dis- ease 16,17 and the treatment of secretory diarrheas that involve chloride secretion through IK channels. 18 Interest in clotrimazole as a potential antileukemic compound derives from the following observations.…”