Victoria Lladó

2-Hydroxyoleate, a nontoxic membrane binding anticancer drug, induces glioma cell differentiation and autophagy

Despite recent advances in the development of new cancer therapies, the treatment options for glioma remain limited, and the survival rate of patients has changed little over the past three decades. Here, we show that 2-hydroxyoleic acid (2OHOA) induces differentiation and autophagy of human glioma cells. Compared to the current reference drug for this condition, temozolomide (TMZ), 2OHOA combated glioma more efficiently and, unlike TMZ, tumor relapse was not observed following 2OHOA treatment. The novel mechanism of action of 2OHOA is associated with important changes in membrane-lipid composition, primarily a recovery of sphingomyelin (SM) levels, which is markedly low in glioma cells before treatment. Parallel to membrane-lipid regulation, treatment with 2OHOA induced a dramatic translocation of Ras from the membrane to the cytoplasm, which inhibited the MAP kinase pathway, reduced activity of the PI3K/Akt pathway, and downregulated Cyclin D-CDK4/6 proteins followed by hypophosphorylation of the retinoblastoma protein (RB). These regulatory effects were associated with induction of glioma cell differentiation into mature glial cells followed by autophagic cell death. Given its high efficacy, low toxicity, ease of oral administration, and good distribution to the brain, 2OHOA constitutes a new and potentially valuable therapeutic tool for glioma patients.

Minerval induces apoptosis in Jurkat and other cancer cells.

Minerval is an oleic acid synthetic analogue that impairs lung cancer (A549) cell proliferation upon modulation of the plasma membrane lipid structure and subsequent regulation of protein kinase C localization and activity. However, this mechanism does not fully explain the regression of tumours induced by this drug in animal models of cancer. Here we show that Minerval also induced apoptosis in Jurkat T‐lymphoblastic leukaemia and other cancer cells. Minerval inhibited proliferation of Jurkat cells, concomitant with a decrease of cyclin D3 and cdk2 (cyclin‐dependent kinase2). In addition, the changes that induced on Jurkat cell membrane organization caused clustering (capping) of the death receptor Fas (CD95), caspase‐8 activation and initiation of the extrinsic apoptosis pathway, which finally resulted in programmed cell death. The present results suggest that the intrinsic pathway (associated with caspase‐9 function) was activated downstream by caspase‐8. In a xenograft model of human leukaemia, Minerval also inhibited tumour progression and induced tumour cell death. Studies carried out in a wide variety of cancer cell types demonstrated that apoptosis was the main molecular mechanism triggered by Minerval. This is the first report on the pro‐apoptotic activity of Minerval, and in part explains the effectiveness of this non‐toxic anticancer drug and its wide spectrum against different types of cancer.

Pivotal role of dihydrofolate reductase knockdown in the anticancer activity of 2-hydroxyoleic acid

α-Hydroxy-9-cis-octadecenoic acid, a synthetic fatty acid that modifies the composition and structure of lipid membranes. 2-Hydroxyoleic acid (HOA) generated interest due to its potent, yet nontoxic, anticancer activity. It induces cell cycle arrest in human lung cancer (A549) cells and apoptosis in human leukemia (Jurkat) cells. These two pathways may explain how HOA induces regression of a variety of cancers. We showed that HOA repressed the expression of dihydrofolate reductase (DHFR), the enzyme responsible for tetrahydrofolate (THF) synthesis. Folinic acid, which readily produces THF without the participation of DHFR, reverses the antitumor effects of HOA in A549 and Jurkat cells, as well as the inhibitory influence on cyclin D and cdk2 in A549 cells, and on DNA and PARP degradation in Jurkat cells. This effect was very specific, because either elaidic acid (an analog of HOA) or other lipids, failed to alter A549 or Jurkat cell growth. THF is a cofactor necessary for DNA synthesis. Thus, impairment of DNA synthesis appears to be a common mechanism involved in the different responses elicited by cancer cells following treatment with HOA, namely cell cycle arrest or apoptosis. Compared with other antifolates, such as methotrexate, HOA did not directly inhibit DHFR but rather, it repressed its expression, a mode of action that offers certain therapeutic advantages. These results not only demonstrate the effect of a fatty acid on the expression of DHFR, but also emphasize the potential of HOA to be used as a wide-spectrum drug against cancer.

Normalization of sphingomyelin levels by 2-hydroxyoleic acid induces autophagic cell death of SF767 cancer cells.

The very high mortality rate of gliomas reflects the unmet therapeutic need associated with this type of brain tumor. We have discovered that the plasma membrane fulfills a critical role in the propagation of tumorigenic signals, whereby changes in membrane lipid content can either activate or silence relevant pathways. We have designed a synthetic fatty acid, 2-hydroxyoleic acid (2OHOA), that specifically activates sphingomyelin synthase (SGMS), thereby modifying the lipid content of cancer cell membranes and restoring lipid levels to those found in normal cells. In reverting, the structure of the membrane by activating SGMS, 2OHOA inhibits the RAS-MAPK pathway, which in turn fails to activate the CCND (Cyclin D)-CDK4/CDK6 and PI3K-AKT1 pathways. The overall result in SF767 cancer cells, a line that is resistant to apoptosis, is the sequential induction of cell cycle arrest, cell differentiation and autophagy. Such effects are not observed in normal cells (MRC-5) and thus, this specific activation of programmed cell death infers greater efficacy and lower toxicity to 2OHOA than that associated with temozolomide (TMZ), the reference drug for the treatment of glioma.

G protein-membrane interactions II: Effect of G protein-linked lipids on membrane structure and G protein-membrane interactions

G proteins often bear myristoyl, palmitoyl and isoprenyl moieties, which favor their association with the membrane and their accumulation in G Protein Coupled Receptor-rich microdomains. These lipids influence the biophysical properties of membranes and thereby modulate G protein binding to bilayers. In this context, we showed here that geranylgeraniol, but neither myristate nor palmitate, increased the inverted hexagonal (HII) phase propensity of phosphatidylethanolamine-containing membranes. While myristate and palmitate preferentially associated with phosphatidylcholine membranes, geranylgeraniol favored nonlamellar-prone membranes. In addition, Gαi1 monomers had a higher affinity for lamellar phases, while Gβγ and Gαβγ showed a marked preference for nonlamellar prone membranes. Moreover, geranylgeraniol enhanced the binding of G protein dimers and trimers to phosphatidylethanolamine-containing membranes, yet it decreased that of monomers. By contrast, both myristate and palmitate increased the Gαi1 preference for lamellar membranes. Palmitoylation reinforced the binding of the monomer to PC membranes and myristoylation decreased its binding to PE-enriched bilayer. Finally, binding of dimers and trimers to lamellar-prone membranes was decreased by palmitate and myristate, but it was increased in nonlamellar-prone bilayers. These results demonstrate that co/post-translational G protein lipid modifications regulate the membrane lipid structure and that they influence the physico-chemical properties of membranes, which in part explains why G protein subunits sort to different plasma membrane domains. This article is part of a Special Issue entitled: Membrane Lipid Therapy: Drugs Targeting Biomembranes edited by Pablo V. Escribá.

G protein–membrane interactions I: Gαi1 myristoyl and palmitoyl modifications in protein–lipid interactions and its implications in membrane microdomain localization

G proteins are fundamental elements in signal transduction involved in key cell responses, and their interactions with cell membrane lipids are critical events whose nature is not fully understood. Here, we have studied how the presence of myristic and palmitic acid moieties affects the interaction of the Gαi1 protein with model and biological membranes. For this purpose, we quantified the binding of purified Gαi1 protein and Gαi1 protein acylation mutants to model membranes, with lipid compositions that resemble different membrane microdomains. We observed that myristic and palmitic acids not only act as membrane anchors but also regulate Gαi1 subunit interaction with lipids characteristics of certain membrane microdomains. Thus, when the Gαi1 subunit contains both fatty acids it prefers raft-like lamellar membranes, with a high sphingomyelin and cholesterol content and little phosphatidylserine and phosphatidylethanolamine. By contrast, the myristoylated and non-palmitoylated Gαi1 subunit prefers other types of ordered lipid microdomains with higher phosphatidylserine content. These results in part explain the mobility of Gαi1 protein upon reversible palmitoylation to meet one or another type of signaling protein partner. These results also serve as an example of how membrane lipid alterations can change membrane signaling or how membrane lipid therapy can regulate the cells physiology.