dihydroceramide and Glioblastoma

Autophagy consists on the delivery of cytoplasmic material and organelles to lysosomes for degradation. Research on autophagy is a growing field because deciphering the basic mechanisms of autophagy is key to understanding its role in health and disease, and to paving the way to discovering novel therapeutic strategies. Studies with chemotherapeutic drugs and pharmacological tools support a role for dihydroceramides as mediators of autophagy. However, their effect on the autophagy outcome (cell survival or death) is more controversial.. We have examined the capacity of structurally varied Des1 inhibitors to stimulate autophagy (LC3-II analysis), to increase dihydroceramides (mass spectrometry) and to reduce cell viability (SRB) in T98G and U87MG glioblastoma cells under different experimental conditions.. The compounds activity on autophagy induction took place concomitantly with accumulation of dihydroceramides, which occurred by both stimulation of ceramide synthesis de novo and reduction of Des1 activity. However, autophagy was also induced by the test compounds after preincubation with myriocin and in cells with a reduced capacity to produce dihydroceramides (U87DND). Autophagy inhibition with 3-methyladenine in the de novo dihydroceramide synthesis competent U87MG cells increased cytotoxicity, while genetic inhibition of autophagy in U87DND cells, poorly efficient at synthesizing dihydroceramides, augmented resistance to the test compounds.. Dihydroceramide desaturase 1 inhibitors activate autophagy via both dihydroceramide-dependent and independent pathways and the balance between the two pathways influences the final cell fate.. The cells capacity to biosynthesize dihydroceramides must be taken into account in proautophagic Des1 inhibitors-including therapies.

Topics: Adenine; Autophagy; Cell Line, Tumor; Cell Survival; Ceramides; Enzyme Inhibitors; Fatty Acids, Monounsaturated; Glioblastoma; Humans; Oxidoreductases

Glioblastomas (GBMs) are very aggressive tumors with low chemosensitivity. The DNA-alkylating agent temozolomide (TMZ) is currently the most efficient chemotoxic drug for GBM therapy; however, many patients develop resistance to TMZ. Combining TMZ with another agent could present an improved treatment option if it could overcome TMZ resistance and avoid side effects. Sphingosine kinase inhibitors (SKIs) have emerged as anticancer agents. Sphingosine kinases are often overexpressed in tumors where their activity of phosphorylating sphingosine (Sph) contributes to tumor growth and migration. They control the levels of the pro-apoptotic ceramide (Cer) and Sph and of the pro-survival sphingosine-1 phosphate. In the present work, TMZ was combined with a specific SKI, and the cytotoxic effect of each drug alone or in combination was tested on GBM cell lines. The combination of sublethal doses of both agents resulted in the cell death potentiation of GBM cell lines without affecting astrocyte viability. It triggered a caspase-3-dependent cell death that was preceded by accumulation of dihydrosphingosine (dhSph) and dihydroceramide (dhCer), oxidative stress, endoplasmic reticulum stress, and autophagy. Autophagy was identified as the crucial switch that facilitated induction of this cell death potentiation. The sublethal dose of the inhibitor induced these stress events, whereas that of TMZ induced the destructive autophagy switch. Remarkably, neither Cer nor Sph, but rather the Cer intermediates, dhSph and dhCer, was involved in the cytotoxicity from the combination. Cell lines sensitive to the combination expressed low levels of the antioxidant enzyme glutathione peroxidase-1, indicating this enzyme as a potential marker of sensitivity to such treatment. This work shows for the first time a strong interaction between a SKI and TMZ, leading to a tumor cell-specific death induction. It further demonstrates the biological relevance of dihydrosphingolipids in cell death mechanisms and emphasizes the potential of drugs that affect sphingolipid metabolism for cancer therapy.

Topics: Antineoplastic Agents; Apoptosis; Autophagy; Brain Neoplasms; Cell Death; Cell Line, Tumor; Ceramides; Dacarbazine; Drug Resistance, Neoplasm; Drug Therapy, Combination; Endoplasmic Reticulum Stress; Enzyme Inhibitors; Glioblastoma; Humans; Phosphotransferases (Alcohol Group Acceptor); Sphingosine; Temozolomide

Melanoma differentiation associated gene-7(mda-7) encodes IL-24, a cytokine that can selectively trigger apoptosis in transformed cells. Recombinant mda-7 adenovirus (Ad.mda-7) effectively kills glioma cells, offering a novel gene therapy strategy to address deadly brain tumors. In this study, we defined the proximal mechanisms by which Ad-mda-7 kills glioma cells. Key factors implicated included activation of the endoplasmic reticulum stress kinase protein kinase R-like endoplasmic reticulum kinase (PERK), Ca(++) elevation, ceramide generation and reactive oxygen species (ROS) production. PERK inhibition blocked ceramide or dihydroceramide generation, which were critical for Ca(++) induction and subsequent ROS formation. Activation of autophagy and cell death relied upon ROS formation, the inhibition of which ablated Ad.mda-7-killing activity. In contrast, inhibiting TRX induced by Ad.MDA-7 enhanced tumor cytotoxicity and improved animal survival in an orthotopic tumor model. Our findings indicate that mda-7/IL-24 induces an endoplasmic reticulum stress response that triggers production of ceramide, Ca(2+), and ROS, which in turn promote glioma cell autophagy and cell death.

Topics: Animals; Autophagy; Blotting, Western; Calcium; Cell Line; Cell Line, Tumor; Cell Survival; Ceramides; eIF-2 Kinase; Glioblastoma; Humans; Interleukins; Mass Spectrometry; Mice; Mutation; Oxidoreductases; Reactive Oxygen Species; RNA Interference; Superoxide Dismutase; Thioredoxins; Transfection; Xenograft Model Antitumor Assays