Cationic amphiphilic drugs induce elevation in lysoglycerophospholipid levels and cell death in leukemia cells.

INTRODUCTION: Repurposing of cationic amphiphilic drugs (CADs) emerges as an attractive therapeutic solution against various cancers, including leukemia. CADs target lysosomal lipid metabolism and preferentially kill cancer cells via induction of lysosomal membrane permeabilization, but the exact effects of CADs on the lysosomal lipid metabolism remain poorly illuminated. OBJECTIVES: We aimed to systematically monitor CAD-induced alterations in the quantitative lipid profiles of leukemia cell lines in order to chart effects of CADs on the metabolism of various lipid classes present in these cells. METHODS: We conducted this study on eight cultured cell lines representing two leukemia types, acute lymphoblastic leukemia and acute myeloid leukemia. Mass spectrometry-based quantitative shotgun lipidomics was employed to quantify the levels of around 400 lipid species of 26 lipid classes in the leukemia cell lines treated or untreated with a CAD, siramesine. RESULTS: The two leukemia types displayed high, but variable sensitivities to CADs and distinct profiles of cellular lipids. Treatment with siramesine rapidly altered the levels of diverse lipid classes in both leukemia types. These included sphingolipid classes previously reported to play key roles in CAD-induced cell death, but also lipids of other categories. We demonstrated that the treatment with siramesine additionally elevated the levels of numerous cytolytic lysoglycerophospholipids in positive correlation with the sensitivity of individual leukemia cell lines to siramesine. CONCLUSIONS: Our study shows that CAD treatment alters balance in the metabolism of glycerophospholipids, and proposes elevation in the levels of lysoglycerophospholipids as part of the mechanism leading to CAD-induced cell death of leukemia cells.

Comprehensive Evaluation of a Quantitative Shotgun Lipidomics Platform for Mammalian Sample Analysis on a High-Resolution Mass Spectrometer.

Shotgun lipidomics is a powerful tool that enables simultaneous and fast quantification of diverse lipid classes through mass spectrometry based analyses of directly infused crude lipid extracts. We present here a shotgun lipidomics platform established to quantify 38 lipid classes belonging to four lipid categories present in mammalian samples and show the fine-tuning and comprehensive evaluation of its experimental parameters and performance. We first determined for all the targeted lipid classes the collision energy levels optimal for the recording of their lipid class- and species-specific fragment ions and fine-tuned the energy levels applied in the platform. We then performed a series of titrations to define the boundaries of linear signal response for the targeted lipid classes, and demonstrated that the dynamic quantification range spanned more than 3 orders of magnitude and reached sub picomole levels for 35 lipid classes. The platform identified 273, 261, and 287 lipid species in brain, plasma, and cultured fibroblast samples, respectively, at the respective optimal working sample amounts. The platform properly quantified the majority of these identified lipid species, while lipid species measured to be below the limit of quantification were efficiently removed from the data sets by the use of statistical analyses of data reproducibility or a cutoff threshold. Finally, we demonstrated that a series of parameters of cell culture conditions influence lipidomics outcomes, including confluency, medium supplements, and use of transfection reagents. The present study provides a guideline for setting up and using a simple and efficient platform for quantitatively exploring the mammalian lipidome.

Cell Death Induced by Cationic Amphiphilic Drugs Depends on Lysosomal Ca2+ Release and Cyclic AMP.

Repurposing cationic amphiphilic drugs (CAD) for cancer treatment is emerging as an attractive means to enhance the efficacy of chemotherapy. Many commonly used CADs, including several cation amphiphilic antihistamines and antidepressants, induce cancer-specific, lysosome-dependent cell death and sensitize cancer cells to chemotherapy. CAD-induced inhibition of lysosomal acid sphingomyelinase is necessary, but not sufficient, for the subsequent lysosomal membrane permeabilization and cell death, while other pathways regulating this cell death pathway are largely unknown. Prompted by significant changes in the expression of genes involved in Ca2+ and cyclic AMP (cAMP) signaling pathways in CAD-resistant MCF7 breast cancer cells, we identified here an early lysosomal Ca2+ release through P2X purinergic receptor 4 (P2RX4) and subsequent Ca2+- and adenylyl cyclase 1 (ADCY1)-dependent synthesis of cAMP as a signaling route mediating CAD-induced lysosomal membrane permeabilization and cell death. Importantly, pharmacologic and genetic means to increase cellular cAMP levels either by activating cAMP-inducing G-protein-coupled receptors (GPR3 or ß2 adrenergic receptor) or ADCY1, or by inhibiting cAMP-reducing guanine nucleotide-binding protein G(i) subunit α2, C-X-C motif chemokine receptor type 4, or cAMP phosphodiesterases, sensitized cancer cells to CADs. These data reveal a previously unrecognized lysosomal P2RX4- and ADCY1-dependent signaling cascade as a pathway essential for CAD-induced lysosome-dependent cell death and encourage further investigations to find the most potent combinations of CADs and cAMP-inducing drugs for cancer therapy.