Hyuck Jun Mok

Autotaxin Regulates Maintenance of Ovarian Cancer Stem Cells through Lysophosphatidic Acid‐Mediated Autocrine Mechanism

Ovarian cancer shows high mortality due to development of resistance to chemotherapy and relapse. Cancer stem cells (CSCs) have been suggested to be a major contributor in developing drug resistance and relapse in ovarian cancer. In this study, we isolated CSCs through sphere culture of A2780, SKOV3, OVCAR3 epithelial ovarian cancer cells and primary ovarian cancer cells from patients. We identified heat‐stable factors secreted from ovarian CSCs stimulated migration and proliferation of CSCs. Mass spectrometry and ELISA analysis revealed that lysophosphatidic acid (LPA) was significantly elevated in CSC culture media compared with non‐CSC culture media. Treatment of CSCs with LPA resulted in augmented CSC characteristics such as sphere‐forming ability, resistance to anticancer drugs, tumorigenic potential in xenograft transplantation, and high expression of CSC‐associated genes, including OCT4, SOX2, and aldehyde dehydrogenase 1. Treatment of CSCs with LPA receptor 1‐specific inhibitors or silencing of LPA receptor 1 expression abrogated the LPA‐stimulated CSC properties. Autotaxin, an LPA‐producing enzyme, is highly secreted from ovarian CSCs, and pharmacological inhibition or knockdown of autotaxin markedly attenuated the LPA‐producing, tumorigenic, and drug resistance potentials of CSCs. Clinicopathological analysis showed a significant survival disadvantage of patients with positive staining of autotaxin. In addition, we further identified that AKT1 activity was upregulated in ovarian CSCs through an LPA‐dependent mechanism and silencing of AKT1 expression led to suppression of CSC characteristics. These results suggest that autotaxin‐LPA‐LPA receptor 1‐AKT1 signaling axis is critical for maintaining CSC characteristics through an autocrine loop and provide a novel therapeutic target for ovarian CSCs. Stem Cells 2016;34:551–564

Phospholipids as cancer biomarkers: Mass spectrometry‐based analysis

Lipids, particularly phospholipids (PLs), are key components of cellular membrane. PLs play important and diverse roles in cells such as chemical-energy storage, cellular signaling, cell membranes, and cell-cell interactions in tissues. All these cellular processes are pertinent to cells that undergo transformation, cancer progression, and metastasis. Thus, there is a strong possibility that some classes of PLs are expected to present in cancer cells and tissues in cellular physiology. The mass spectrometric soft-ionization techniques, electrospray ionization (ESI), and matrix-assisted laser desorption/ionization (MALDI) are well-established in the proteomics field, have been used for lipidomic analysis in cancer research. This review focused on the applications of mass spectrometry (MS) mainly on ESI-MS and MALDI-MS in the structural characterization, molecular composition and key roles of various PLs present in cancer cells, tissues, blood, and urine, and on their importance for cancer-related problems as well as challenges for development of novel PL-based biomarkers. The profiling of PLs helps to rationalize their functions in biological systems, and will also provide diagnostic information to elucidate mechanisms behind the control of cancer, diabetes, and neurodegenerative diseases. The investigation of cellular PLs with MS methods suggests new insights on various cancer diseases and clinical applications in the drug discovery and development of biomarkers for various PL-related different cancer diseases. PL profiling in tissues, cells and body fluids also reflect the general condition of the whole organism and can indicate the existence of cancer and other diseases. PL profiling with MS opens new prospects to assess alterations of PLs in cancer, screening specific biomarkers and provide a basis for the development of novel therapeutic strategies.

Proteomic analysis of extracellular vesicles derived from propionibacterium acnes

Extracellular vesicle (EV) has been reported to conduct critical pathophysiological functions as an emerging mode of communication in bacteria. Recently, Propionibacterium acnes, an anaerobic Gram‐positive human commensal found in the skin and gastrointestinal tract, has drawn increasing attention as an underestimated pathogen in a variety of diseases.

Age-Associated Lipidome Changes in Metaphase II Mouse Oocytes

The quality of mammalian oocytes declines with age, which negatively affects fertilization and developmental potential. The aging process often accompanies damages to macromolecules such as proteins, DNA, and lipids. To investigate if aged oocytes display an altered lipidome compared to young oocytes, we performed a global lipidomic analysis between oocytes from 4-week-old and 42 to 50-week-old mice. Increased oxidative stress is often considered as one of the main causes of cellular aging. Thus, we set up a group of 4-week-old oocytes treated with hydrogen peroxide (H2O2), a commonly used oxidative stressor, to compare if similar lipid species are altered between aged and oxidative-stressed oocytes. Between young and aged oocytes, we identified 26 decreased and 6 increased lipids in aged oocytes; and between young and H2O2-treated oocytes, we identified 35 decreased and 26 increased lipids in H2O2-treated oocytes. The decreased lipid species in these two comparisons were overlapped, whereas the increased lipid species were distinct. Multiple phospholipid classes, phosphatidic acid (PA), phosphatidylinositol (PI), phosphatidylserine (PS), and lysophosphatidylserine (LPS) significantly decreased both in H2O2-treated and aged oocytes, suggesting that the integrity of plasma membrane is similarly affected under these conditions. In contrast, a dramatic increase in diacylglycerol (DG) was only noted in H2O2-treated oocytes, indicating that the acute effect of H2O2-caused oxidative stress is distinct from aging-associated lipidome alteration. In H2O2-treated oocytes, the expression of lysophosphatidylcholine acyltransferase 1 increased along with increases in phosphatidylcholine. Overall, our data reveal that several classes of phospholipids are affected in aged oocytes, suggesting that the integrity of plasma membrane is associated with maintaining fertilization and developmental potential of mouse oocytes.

Analysis of the phospholipid profile of metaphase II mouse oocytes undergoing vitrification.

Oocyte freezing confers thermal and chemical stress upon the oolemma and various other intracellular structures due to the formation of ice crystals. The lipid profiles of oocytes and embryos are closely associated with both, the degrees of their membrane fluidity, as well as the degree of chilling and freezing injuries that may occur during cryopreservation. In spite of the importance of lipids in the process of cryopreservation, the phospholipid status in oocytes and embryos before and after freezing has not been investigated. In this study, we employed mass spectrometric analysis to examine if vitrification has an effect on the phospholipid profiles of mouse oocytes. Freshly prepared metaphase II mouse oocytes were vitrified using copper grids and stored in liquid nitrogen for 2 weeks. Fresh and vitrified-warmed oocytes were subjected to phospholipid extraction procedure. Mass spectrometric analyses revealed that multiple species of phospholipids are reduced in vitrified-warmed oocytes. LIFT analyses identified 31 underexpressed and 5 overexpressed phospholipids in vitrified mouse oocytes. The intensities of phosphatidylinositol (PI) {18∶2/16∶0} [M−H]− and phosphatidylglycerol (PG) {14∶0/18∶2} [M−H]− were decreased the most with fold changes of 30.5 and 19.1 in negative ion mode, respectively. Several sphingomyelins (SM) including SM {d38∶3} [M+H]+ and SM {d34∶0} [M+K]+ were decreased significantly in positive ion mode. Overall, the declining trend of multiple phospholipids demonstrates that vitrification has a marked effect on phospholipid profiles of oocytes. These results show that the identified phospholipids can be used as potential biomarkers of oocyte undergoing vitrification and will allow for the development of strategies to preserve phospholipids during oocyte cryopreservation.

UPLC-MS/MS-Based Profiling of Eicosanoids in RAW264.7 Cells Treated with Lipopolysaccharide.

While both the pro- and anti-inflammatory effects of several eicosanoids have been widely studied, the degree of inflammation in cells that results from various eicosanoids has yet to be comprehensively studied. The objective of this study was to assess the effect of lipopolysaccharide (LPS) treatment on eicosanoid content in RAW264.7 cells. An Ultra performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS)-based profiling method was used to analyze the eicosanoid contents of RAW264.7 cells treated with different LPS concentrations. The profiling data were subjected to statistical analyses, such as principal component analysis (PCA) and hierarchical clustering analysis. LPS treatment increased nitric oxide production and secretion of pro-inflammatory cytokines, such as tumor necrosis factor-α and interleukin-6, in a concentration-dependent manner. In total, 79 eicosanoids were identified in the cells. RAW264.7 cells treated with different LPS concentrations were well differentiated in the PCA score plot. A heatmap was used to identify the eicosanoids that were up- or down-regulated according to the degree of inflammation and LPS concentration. Thirty-nine eicosanoids were upregulated and seven were down-regulated by LPS treatment in a concentration-dependent manner. Our novel UPLC-MS/MS technique can profile eicosanoids, and can evaluate the correlations between inflammation and eicosanoid metabolism.

Detailed characterization of alterations in the lipid profiles during autophagic cell death of leukemia cells

In this study, a lipidomics approach based on UPLC-QqQ/MS was applied to profile various lipids in human leukemia cells undergoing autophagic cell death (ACD). Our previous study indicated that AIC-47, a 3-decenoic acid derivative, induced ACD in cancer cells that was associated with lipophagy. To understand the altered metabolism of lipids during ACD, 23 lipid classes were profiled in AIC-47 – treated cancer cells. Using the optimal UPLC conditions, individual lipid species were well separated in 30 min. By multiple reaction monitoring the 397 individual lipid species were successfully identified and quantified. 14 classes of lipid—TG, DG, PS, PG, PI, PA, LPC, LPE, LPS, LPG, LPI, Cer, Sa, and Cer1P—were upregulated and 3 classes—ChE, PC, and LPA—were downregulated in the ACD-induced cells compared to the control (P ≤ 0.05 by t-test). Other classes, such as PE, SM, dCer, So, So1P, and Sa1P, showed no changes. These results indicate that lipid metabolism of ACD is related to the mechanism of autophagy.

UPLC-QqQ/MS-Based Lipidomics Approach To Characterize Lipid Alterations in Inflammatory Macrophages

In this study, UPLC-QqQ/MS-based lipidomics was applied to profile various lipids from RAW264.7 macrophages treated with different concentrations of lipopolysaccharide (LPS). The degree of inflammation increased with the LPS concentration. To elucidate the altered lipid metabolism of inflammatory macrophages, we targeted to analyze 25 lipid classes from LPS-treated RAW264.7 cells. As a result, 523 lipid species were successfully profiled by using the optimal UPLC and MRM. Statistical data analyses such as PCA, PLS-DA, and HCA differentiated five RAW264.7 cells treated with different concentrations of LPS. VIP plot, heat map, and bar plot also provided lists of up- or down-regulated lipids according to the LPS concentration. From the results, 11 classes of lipids, TG, DG, ChE, PE, PS, PI, PA, LyPC, LyPE, Cer, and dCer, were increased, and three classes, cholesterol, PC, and LyPA, were decreased in an LPS concentration-dependent manner. Furthermore, the treatment of an anti-inflammatory compound recovered the levels of PC, PE, PI, PA, LyPE, LyPA, and Cer from the activated macrophages. Finally, these results demonstrate the correlation between inflammation and lipid metabolism in macrophages. The differentially regulated lipids also have the potential to be used as biomarkers for inflammation.

A rapid and sensitive profiling of free fatty acids using liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) after chemical derivatization

Free fatty acids (FFAs) have diverse roles in cellular energy and signaling and they are critical molecules in various biological states. Due to the poor ionization efficiency of FFAs under electrospray ionization mass spectrometry (ESI-MS) conditions, it is a challenging aspect to construct a robust platform for profiling of various FFAs in biological samples using liquid chromatography ESI-MS. In the present study, we applied trimethylsilyldiazomethane (TMSD) derivatization to improve ionization efficiencies in the profiling of FFAs. Multiple reaction monitoring (MRM) was used for the selective quantification of methylated FFAs. The optimal TMSD methylation was validated for a reliable FFA profiling. Furthermore, the high-throughput analysis of FFAs was successfully performed in short analysis and derivatization times. To verify the utility and effectiveness of the developed method, we compared both methylation and nonmethylation (intact FFA) data in the profiling of FFAs in mice liver and plasma. It is noteworthy that the methylation derivatization provided better results in FFA profiling. Further, we performed statistical data analysis where HBV and mock mice tissues were discriminated when the methylated FFAs data were used. In the lipidomics field, the present method can also be applied for the profiling of FFAs in biological samples for biomarker discovery. The present validated LC/ESI-MS/MS assay method may also be used for FFA profiling modeling studies in other biomedical samples.

Serum lipidomic analysis for the discovery of biomarkers for major depressive disorder in drug-free patients.

Lipidomic analysis can be used to efficiently identify hundreds of lipid molecular species in biological materials and has been recently established as an important tool for biomarker discovery in various neuropsychiatric disorders including major depressive disorder (MDD). In this study, quantitative targeted serum lipidomic profiling was performed on female subjects using liquid chromatography-mass spectrometry. Global lipid profiling of pooled serum samples from 10 patients currently with MDD (cMDD), 10 patients with remitted MDD (rMDD), and 10 healthy controls revealed 37 differentially regulated lipids (DRLs). DRLs were further verified using multiple-reaction monitoring (MRM) in each of the 25 samples from the three groups of independent cohorts. Using multivariate analysis and MRM data we identified serum biomarker panels of discriminatory lipids that differentiated between pairs of groups: lysophosphatidic acid (LPA)(16:1), triglycerides (TG)(44:0), and TG(54:8) distinguished cMDD from controls with 76% accuracy; lysophosphatidylcholines(16:1), TG(44:0), TG(46:0), and TG(50:1) distinguished between cMDD and rMDD at 65% accuracy; and LPA(16:1), TG(52:6), TG(54:8), and TG(58:10) distinguished between rMDD and controls with 60% accuracy. Our lipidomic analysis identified peripheral lipid signatures of MDD, which thereby provides providing important biomarker candidates for MDD.