Removal of Serum Lipids and Lipid-Derived Metabolites to Investigate Breast Cancer Cell Biology.

The use of cultured cells has been instrumental in studying biochemical, molecular, and cellular processes. The composition of serum that cells are maintained in can have a profound impact on important cellular checkpoints. Cell growth and apoptosis are analyzed in an estrogen receptor positive breast cancer cell line in the presence of serum that have been treated to remove steroids or lipids, as well-described in the literature. It is shown that maintaining cells in the presence of charcoal-dextran-treated serum causes reduced growth rate, which can be reversed by the addition of estradiol. Silica-treated-serum also slows down cell growth and induces apoptosis. In order to investigate the role of lipids in these phenotypes, the levels of a wide range of lipids in different sera are investigated. It is shown that silica-treatment significantly depletes phosphatidylcholines and cholesterol. It is also shown that lipogenesis is stimulated when cells are cultured with silica-treated-serum and this is reversed by the addition of exogenous lipids, which also restores growth rate and apoptosis. The results show that cultured cells are sensitive to different serum, most likely due to the differences in levels of structural and signaling metabolites present in their growth environment.

The Role of p38 MAPK in Triacylglycerol Accumulation during Apoptosis.

Lipids are emerging as key regulators of apoptosis. Specific lipid species are associated with apoptosis with important functional roles, but the understanding of the regulation of these lipid species is still limited. It has been previously shown by our laboratory that polyunsaturated triacylglycerols accumulate and get stored within lipid droplets during apoptosis via activated glycerolipid biosynthesis. In this work, the biochemical mechanisms that result in the activation of glycerolipid biosynthesis and, consequently, triacylglycerol and lipid droplet accumulation during apoptosis are investigated. The transcriptomes of control and apoptotic HCT-116 cells are compared and gene enrichment analysis revealed the upregulation of p38 mitogen-activated protein kinase (MAPK). It is shown that p38 MAPK regulates triacylglycerol biosynthesis through diacylglycerol acyltransferase1 during apoptosis. Perilipin 2 and cytosolic phospholipase A2delta are also shown to be involved in lipid droplet and polyunsaturated triacylglycerol accumulation in this process. Overall, the results provide new insights into the upregulation of glycerolipid synthesis during apoptosis.

A Protective Role for Triacylglycerols during Apoptosis.

Triacylglycerols (TAGs) are one of the major constituents of the glycerolipid family. Their main role in cells is to store excess fatty acids, and they are mostly found within lipid droplets. TAGs contain acyl chains that vary in length and degree of unsaturation, resulting in hundreds of chemically distinct species. We have previously reported that TAGs containing polyunsaturated fatty acyl chains (PUFA-TAGs) accumulate via activation of diacylglycerol acyltransferases during apoptosis. In this work, we show that accumulation of PUFA-TAGs is a general phenomenon during this process. We further show that the accumulated PUFA-TAGs are stored in lipid droplets. Because membrane-residing PUFA phospholipids can undergo oxidation and form reactive species under increased levels of oxidative stress, we hypothesized that incorporation of PUFAs into PUFA-TAGs and their localization within lipid droplets during apoptosis limit the toxicity during this process. Indeed, exogenous delivery of a polyunsaturated fatty acid resulted in a profound accumulation of PUFA phospholipids and rendered cells more sensitive to oxidative stress, causing reduced viability. Overall, our results support the concept that activation of TAG biosynthesis protects cells from lipid peroxide-induced membrane damage under increased levels of oxidative stress during apoptosis. As such, targeting triacylglycerol biosynthesis in cancer cells might represent a new approach to promoting cell death during apoptosis.

Noncanonical Roles of Lipids in Different Cellular Fates.

Lipids are a diverse class of biomolecules. The biosynthesis and transport of these molecules are controlled by a considerable number of proteins, which facilitate spatiotemporal regulation of lipids during different fundamental cellular processes. Although lipids are traditionally considered as molecules for energy storage and as structural components of membranes, they are being increasingly recognized for their signaling roles. There is a growing appreciation of lipids' chemical diversity, which approaches that of proteins. In this Perspective, we discuss recent studies that suggest novel functions for distinct lipid species during different cellular processes. In particular, we discuss findings from our laboratory that illuminate the involvement of ceramides, polyunsaturated triacylglycerols, and very long chain fatty acids in different cellular fates. We also highlight recent innovative methods that have enabled the recognition of previously unknown lipid classes and/or roles of these molecules in different biological processes. We envision that advances in lipid identification, visualization, and perturbation will pave the way for broader investigations into this fascinating and influential class of biomolecules.

CBR1 rs9024 genotype status impacts the bioactivation of loxoprofen in human liver.

Loxoprofen is an anti-inflammatory drug that requires bioactivation into the trans-OH metabolite to exert pharmacological activity. Evidence suggests that carbonyl reductase 1 (CBR1) is important during the bioactivation of loxoprofen. This study examined the impact of the functional single nucleotide polymorphism CBR1 rs9024 on the bioactivation of loxoprofen in a collection of human liver samples. The synthesis ratios of trans-OH loxoprofen/cis-OH loxoprofen were 33% higher in liver cytosols from donors homozygous for the CBR1 rs9024 G allele in comparison with the ratios in samples from donors with heterozygous GA genotypes. Complementary studies examined the impact of CBR1 rs9024 on the bioactivation of loxoprofen in lymphoblastoid cell lines. CBR1 rs9024 genotype status impacts the synthesis of the bioactive trans-OH metabolite of loxoprofen in human liver.

Rapid Light-Triggered Drug Release in Liposomes Containing Small Amounts of Unsaturated and Porphyrin-Phospholipids.

Prompt membrane permeabilization is a requisite for liposomes designed for local stimuli-induced intravascular release of therapeutic payloads. Incorporation of a small amount (i.e., 5 molar percent) of an unsaturated phospholipid, such as dioleoylphosphatidylcholine (DOPC), accelerates near infrared (NIR) light-triggered doxorubicin release in porphyrin-phospholipid (PoP) liposomes by an order of magnitude. In physiological conditions in vitro, the loaded drug can be released in a minute under NIR irradiation, while liposomes maintain serum stability otherwise. This enables rapid laser-induced drug release using remarkably low amounts of PoP (i.e., 0.3 molar percent). Light-triggered drug release occurs concomitantly with DOPC and cholesterol oxidation, as detected by mass spectrometry. In the presence of an oxygen scavenger or an antioxidant, light-triggered drug release is inhibited, suggesting that the mechanism is related to singlet oxygen mediated oxidization of unsaturated lipids. Despite the irreversible modification of lipid composition, DOPC-containing PoP liposome permeabilization is transient. Human pancreatic xenograft growth in mice is significantly delayed with a single chemophototherapy treatment following intravenous administration of 6 mg kg(-1) doxorubicin, loaded in liposomes containing small amounts of DOPC and PoP.

Very Long Chain Fatty Acids Are Functionally Involved in Necroptosis.

Necroptosis is a form of regulated cell death that is linked to various human diseases. Distinct membrane-related, thus lipid-dependent, alterations take place during necroptosis. However, little is known about the roles of specific lipids in this process. We used an untargeted LC-MS-based approach to reveal that distinct lipid species are regulated at the molecular level during necroptosis. We found that ceramides and very long chain fatty acids accumulate during this process. Intrigued by the specificity of very long chain fatty acid accumulation, we focused on characterizing their involvement during necroptosis. Biochemical characterizations suggested that activated fatty acid biosynthesis and elongation could be responsible for these accumulations. We further showed that inhibition of fatty acid biosynthesis and depletion of very long chain fatty acids prevented loss of plasma membrane integrity and cell death, strongly suggesting that very long chain fatty acids are functionally involved in necroptosis.

Vessel-Targeted Chemophototherapy with Cationic Porphyrin-Phospholipid Liposomes.

Cationic liposomes have been used for targeted drug delivery to tumor blood vessels, via mechanisms that are not fully elucidated. Doxorubicin (Dox)-loaded liposomes were prepared that incorporate a cationic lipid; 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), along with a small amount of porphyrin-phospholipid (PoP). Near-infrared (NIR) light caused release of entrapped Dox via PoP-mediated DOTAP photo-oxidation. The formulation was optimized to enable extremely rapid NIR light-triggered Dox release (i.e., in 15 seconds), while retaining reasonable serum stability. In vitro, cationic PoP liposomes readily bound to both MIA PaCa-2 human pancreatic cancer cells and human vascular endothelial cells. When administered intravenously, cationic PoP liposomes were cleared from circulation within minutes, with most accumulation in the liver and spleen. Fluorescence imaging revealed that some cationic PoP liposomes also localized at the tumor blood vessels. Compared with analogous neutral liposomes, strong tumor photoablation was induced with a single treatment of cationic PoP liposomes and laser irradiation (5 mg/kg Dox and 100 J/cm2 NIR light). Unexpectedly, empty cationic PoP liposomes (lacking Dox) induced equally potent antitumor phototherapeutic effects as the drug loaded ones. A more balanced chemo- and phototherapeutic response was subsequently achieved when antitumor studies were repeated using higher drug dosing (7 mg/kg Dox) and a low fluence phototreatment (20 J/cm2 NIR light). These results demonstrate the feasibility of vessel-targeted chemophototherapy using cationic PoP liposomes and also illustrate synergistic considerations. Mol Cancer Ther; 16(11); 2452-61. ©2017 AACR.

Multifunctional Liposomes for Image-Guided Intratumoral Chemo-Phototherapy.

Intratumoral (IT) drug injections reduce systemic toxicity, but delivered volumes and distribution can be inconsistent. To improve IT delivery paradigms, porphyrin-phospholipid (PoP) liposomes are passively loaded with three hydrophilic cargos: sulforhodamine B, a fluorophore; gadolinium-gadopentetic acid, a magnetic resonance (MR) agent; and oxaliplatin, a colorectal cancer chemotherapeutic. Liposome composition is optimized so that cargo is retained in serum and storage, but is released in less than 1 min with exposure to near infrared light. Light-triggered release occurs with PoP-induced photooxidation of unsaturated lipids and all cargos release concurrently. In subcutaneous murine colorectal tumors, drainage of released cargo is delayed when laser treatment occurs 24 h after IT injection, at doses orders of magnitude lower than systemic ones. Delayed light-triggering results in substantial tumor shrinkage relative to controls a week following treatment, although regrowth occurs subsequently. MR imaging reveals that over this time frame, pools of liposomes within the tumor migrate to adjacent regions, possibly leading to altered spatial distribution during triggered drug release. Although further characterization of cargo loading and release is required, this proof-of-principle study suggests that multimodal theranostic IT delivery approaches hold potential to both guide injections and interpret outcomes, in particular when combined with chemo-phototherapy.

Specific Triacylglycerols Accumulate via Increased Lipogenesis During 5-FU-Induced Apoptosis.

Lipids are emerging as key regulators of fundamental cellular processes including cell survival, division, and death. Apoptosis, a form of programmed cell death, is accompanied by numerous membrane-related phenotypic changes. However, we have an incomplete understanding of the involvement of specific lipid structures during this process. Here, we report that triacylglycerols are regulated at the molecular level during 5-fluorouracil-induced apoptosis in HCT-116. Mass-spectrometry-based global lipid profiling shows that specific triacylglycerols accumulate during apoptosis. Expression levels and activities of enzymes that are responsible for the biosynthesis and metabolic processing of triacylglycerols suggest that triacylglycerol biosynthesis is responsible for these accumulations. Based on our data, we propose that regulation of triacylglycerols at the molecular level happens downstream of p53 activation and potentially is a mechanism to prevent lipid oxidation during apoptosis.

Differential Regulation of Specific Sphingolipids in Colon Cancer Cells during Staurosporine-Induced Apoptosis.

Apoptosis is accompanied by distinct morphological changes at the plasma and organelle membrane level. Involvement of certain lipids in apoptosis has been established; however, we have limited understanding of the specific lipid structures that participate in this process. We used untargeted comparative lipidomics to study the changes in lipid composition during staurosporine-induced apoptosis in HCT-116. Our results revealed that ceramides, dihydroceramides, and sphingomyelins, with defined acyl chains, constitute the majority of changes in the lipidome. Expression levels and activities of enzymes responsible for the biosynthesis of lipids that change suggest that de novo synthesis causes these specific changes. Further analysis of the lipidome during apoptosis in other cancer and non-cancer cell lines suggested that accumulation of ceramides and dihydroceramides is specific to cancer cells. Taken together, our data propose that these molecules are regulated at the lipid-specific level during apoptosis and that this regulation differs between cancer and non-cancer cells.

Simultaneous determination of ß-lactam antibiotics and ß-lactamase inhibitors in bovine milk by ultra performance liquid chromatography-tandem mass spectrometry.

An ultra performance liquid chromatography-tandem mass spectrometric (UPLC-MS/MS) method has been developed for the simultaneous determination of four ß-lactam antibiotics (amoxicillin, ampicillin, cefotaxime, and cefoperazone) and two ß-lactamase inhibitors (tazobactam, sulbactam) in bovine milk. The analytes were extracted with water from bovine milk and purified with Oasis HLB solid phase extraction (SPE) cartridges. The analytes were determined in less than 3min by UPLC-MS/MS in positive and negative electrospray ionization (ESI) modes, separately. The method was linear over the range of 1-100µg/L for tazobactam, sulbactam, ampicillin, and cefoperazone, and 2-100µg/L for amoxicillin and cefotaxime. The recoveries for all six analytes in bovine milk ranged from 82.5 to 98.3%. The limits of detection and the limits of quantitation were 0.1-0.2µg/L and 0.3-0.5µg/L, respectively. The intra- and inter-day precisions were less than 6% for each compound.