Functional Lipidomics: Palmitic Acid Impairs Hepatocellular

A MERICAN A SSOCIATION FOR T HE STUDY OF LIVER D I S E ASES

HEPATOLOGY, VOL. 66, NO. 2, 2017

Functional Lipidomics: Palmitic Acid Impairs Hepatocellular Carcinoma Development by Modulating Membrane Fluidity and Glucose Metabolism Ling Lin,1,2* Ying Ding,1,3* Yi Wang,1,2,4 Zhenxin Wang,1 Xuefei Yin,1,4 Guoquan Yan,4 Lei Zhang,2 Pengyuan Yang,1,2,4 and Huali Shen1,2,4 Lipids are essential cellular components and energy sources of living organisms, and altered lipid composition is increasingly recognized as a signature of cancer. We performed lipidomic analysis in a series of hepatocellular carcinoma (HCC) cells and identified over 1,700 intact lipids originating from three major lipid categories. Comparative lipidomic screening revealed that 93 significantly changed lipids and decreased palmitic acyl (C16:0)–containing glycerophospholipids were positively associated with metastatic abilities of HCC cells. Furthermore, both in vitro and in vivo experiments demonstrated that C16:0 incubation specifically reduced malignant cell proliferation, impaired cell invasiveness, and suppressed tumor growth in mouse xenograft models. Biochemical experiments demonstrated that C16:0 treatment decreased cell membrane fluidity and limited glucose metabolism. A phosphoproteomics approach further revealed such C16:0 incubation attenuated phosphorylation levels of mammalian target of rapamycin (mTOR) and signal transducer and activator of transcription 3 (STAT3) pathway proteins. Multiple reaction monitoring analysis of 443 lipid molecules showed 8 reduced C16:0-containing lipids out of total 10 altered lipids when cancer tissues were compared with adjacent nontumor tissues in a cohort of clinical HCC specimens (P < 0.05). Conclusion: These data collectively demonstrate the biomedical potential of using altered lipid metabolism as a diagnostic marker for cancerous cells and open an opportunity for treating aggressive HCCs by targeting altered C16:0 metabolism. (HEPATOLOGY 2017;66:432-448).

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ipids are small molecules with diverse chemical structures that fulfill multiple distinct yet critical roles in nearly all aspects of cellular function. Increased lipogenesis has been recognized as an important early hallmark of the rewired metabolism of cancerous cells.(1,2) Of all the lipogenic enzymes in the fatty acid (FA) synthesis pathway, most efforts have been focused on pharmacologically inhibiting acetyl

coenzyme A carboxylase, FA synthase, or sterol regulatory element binding proteins to devise novel therapeutic strategies for metabolically treating and preventing cancer.(3-5) These compounds have shown significant antitumor activities with concomitant suppression of FA synthesis, whereas they have also exhibited unsatisfactory pharmacokinetics or unexpected side effects like weight loss or even target-related

Abbreviations: ADP, adenosine diphosphate; ATP, adenosine triphosphate; C16:0, palmitic acid; C18:1, oleic acid; FA, fatty acid; GLUT, glucose transporter; GP, glycerophospholipid; HCC, hepatocellular carcinoma; HPLC, high-performance liquid chromatography; MRM, multiple reaction monitoring; MS, mass spectrometry; mTOR, mammalian target of rapamycin; PC, phosphatidylcholine; PE, phosphatidylethanolamine; SD, standard deviation; STAT, signal transducer and activator of transcription. Received June 10, 2016; accepted December 26, 2016. Additional Supporting Information may be found at onlinelibrary.wiley.com/doi/10.1002/hep.29033/suppinfo. *These authors contributed equally to this work. Supported by grants from the National Key Basic Research Program of China (2013CB910503, 2013CB910802 [Correction added July 17, 2017, after original online publication: second National Key Basic Research Program of China grant number was corrected.]), the Major Program of the Ministry of Health (2012ZX10002012-006), the MOST Program (2014AA020902), the Shanghai Science and Technology Research Project (16ZR1402400, 15DZ2291100), and the Zhuo Xue Program of Fudan University. C 2017 by the American Association for the Study of Liver Diseases. Copyright V View this article online at wileyonlinelibrary.com. DOI 10.1002/hep.29033 Potential conflict of interest: Nothing to report.

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toxicities. Hence, there is an urgent need for the discovery of specific lipid signatures and additional optimal therapeutic targets in cancer. Continuous technological advances in mass spectrometry (MS) have enabled qualitative and quantitative analyses of a broad range of lipid molecules.(6-8) Painstaking research is seeking specialized lipid alterations which might be involved in the onset of cancer and its development, such as breast cancer,(9-11) prostate cancer,(11,12) lung cancer,(11) colon cancer,(13) and brain tumors.(14) Despite the explosion of information in the field of lipids, studies have not been able to systematically evaluate changes in lipid metabolism that can affect numerous aspects of tumorigenesis, nor have they fully elucidated the function of various lipids or the metabolic enzymes controlling lipid composition. Herein, we attempted to identify altered lipid molecules and aberrant lipid metabolites using a shotgun lipidomic approach in a series of hepatocellular carcinoma (HCC) cells and further elucidated the potential causal roles of those disordered lipids in cancer development.

Materials and Methods REAGENTS AND MATERIALS High-performance liquid chromatography (HPLC)– grade CHCl3, methanol, isopropyl alcohol, hexane, and NH4OAc were purchased from Merck Millipore (Billerica, MA). Analytical-grade formic acid, acetone, acetonitrile, dithiothreitol, iodoacetamide, ammonium bicarbonate, and Tris base were purchased from SigmaAldrich (St. Louis, MO). Bioxtra-grade palmitic acid (C16:0), oleic acid (C18:1), stearic acid, and cisplatin were also purchased from Sigma-Aldrich. Lipid internal

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standard was purchased from Avanti Polar Lipids (Alabaster, AL). Other regents were analytical grade.

CELL LINES AND CLINICAL SPECIMENS As research models, Hep3B cells were purchased from American Type Culture Collection, while SW480, SW620, AGS, BGC-823, and HGC-27 cells were purchased from the cell bank of Chinese Academy of Sciences (Shanghai, China). These cell lines were obtained within 6 months before being used in this study. L02, MHCC97H (97H), and HCCLM3 (LM3) cells were obtained from Liver Cancer Institute, Zhongshan Hospital of Fudan University, among which 97H and LM3 cells were established at this institute.(15) These three cell lines were authenticated by short tandem repeat validation analysis during the study period. Detailed descriptions of these cell lines and cell culture methods can be found in the Supporting Information. Clinical HCC specimens were obtained from a cohort of 10 patients. All patients were diagnosed with primary HCC, and none had received any preoperative cancer treatment. Ethical approval from the Zhongshan Hospital Research Ethics Committee and patient written informed consent were obtained. The clinicopathological characteristics are presented in Supporting Table S7.

LIPID EXTRACTION FOR MS ANALYSIS Total lipid was extracted from 2 3 107 cells (at least 0.1 g), using a modified method of Bligh and Dyer.(16) An internal standard cocktail (Avanti Lipids Polar) was added at an amount of 10 lL to each sample according to 1 mg of extracted tissue protein during

ARTICLE INFORMATION: From the 1Department of Systems Biology for Medicine, School of Basic Medical Sciences, 2Institutes of Biomedical Sciences of Shanghai Medical School, Fudan University, Shanghai, China; 3Shanghai Institute for Food and Drug Control, Shanghai, China; 4Department of Chemistry, Fudan University, Shanghai, China.

ADDRESS CORRESPONDENCE AND REPRINT REQUESTS TO: P.-Y. Yang, Ph.D., or H.-L. Shen, Ph.D. Institutes of Biomedical Sciences of Shanghai Medical School, Fudan University 131 Dong’an Road

Shanghai 200032, China E-mail: [email protected] or [email protected] Tel: 86-21-54237664

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lipid extraction (Supporting Table S8). Lipid extracts were subjected to triple-quadrupole MS (QTRAP 4000 and 6500; SCIEX, Framingham, MA). Both negative and positive electrospray ionization modes were used, and precursor ion scans and neutral loss scans were run in each mode.(17) Each experiment was repeated at least three times. Lipid identification was based on MS data and assisted by the bioinformatics tool Lipid MS Predict (http://www.lipidmaps.org/). Quantitation was done by one internal standard per lipid class. Detailed descriptions of the lipid extraction method can be found in the Supporting Information.

LIPIDOMIC DATA ANALYSIS Peak identifications were performed using Analyst software (version 1.6; SCIEX). Only peaks that comprised >2% intensity and a signal-to-noise ratio >5 were identified. The relative signal intensities were processed by normalization to total signal intensities and cell numbers. Peak changes between samples were confirmed by manual quantification. Multivariate statistical analysis and cluster analysis were accomplished using MetaboAnalyst 3.0 (www.metaboanalyst.ca).(18,19) A detailed description of data analysis can be found in the Supporting Information.

MULTIPLE REACTION MONITORING ANALYSIS BY NORMAL PHASE HPLC/MS Lipid compositions were separated on the Shimadzu LC-20AB HPLC system (Tokyo, Japan). Then, QTRAP 4000 MS was operated in multiple reaction monitoring (MRM) mode. Detailed descriptions of the chromatographic elution gradient and the precursor/product ion pairs can be found in the Supporting Information.

PROLIFERATION ASSAY Cell proliferation was determined over a 7-day period by Cell Counting Kit-8 (Dojindo, Kumamoto, Japan). After a 24-hour serum withdrawal, 2 3 103 cells were plated in 96-well plates in 0.1 mL growth medium with C16:0 of different concentrations (0, 50, 100, 200 lM). Cell Counting Kit-8 solution was added 10 lL/well, and cells were subsequently incubated for 2 hours at 378C. Then the plates were read on a multiwell scanning spectrophotometer (Multiskan

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MK3; Thermo Fisher Scientific) at 450 nm, with wavelength correction at 630 nm.

CELL MIGRATION AND INVASION A wound-healing assay was used to evaluate cell migration ability, and cell invasive ability was determined using transwell chambers coated with extracellular matrix gel (BD Biosciences, San Jose, CA). Invasive activity was quantified by counting the number of trespassed cells per five high-power fields (magnification 3100) from three independent experiments (mean 6 standard deviation [SD]), and a t test was used to show significant differences (P < 0.05). Detailed descriptions of these two experiment can be found in the Supporting Information.

QUANTITATIVE IMAGING OF MEMBRANE FLUIDITY Plasma membrane fluidity was evaluated by the laser scanning platform Leica TCS-SP5 (Leica Microsystems, Wetzlar, Germany).(20) Living cells with or without 200 lM C16:0 treatment were labeled with the polarity-sensitive membrane probe laurdan (Sigma-Aldrich). Laurdan intensity images were obtained under excitation at 405 nm with a multiphoton laser system. Generalized polarization images were pseudocolored in ImageJ software. Detailed descriptions of the imaging equipment setup and generalized polarization value calibration can be found in the Supporting Information.

GLUCOSE UPTAKE AND LACTATE PRODUCTION ASSAY Glucose uptake of each sample was measured using the Glucose Uptake Colorimetric Assay Kit (K676100; BioVision, Milpitas, CA). Cells were seeded with a 4-point dosage course (0, 50, 100, 200 lM) of C16:0 treatment. Glucose levels in culture media were measured according to the recommended protocol and normalized to cell number. Lactate production in the culture medium of each sample was determined by the Lactate Colorimetric Assay Kit (BioVision; K607100). Detailed descriptions of each experiment can be found in the Supporting Information.


MRM ANALYSIS OF ADENOSINE TRIPHOSPHATE AND ADENOSINE DIPHOSPHATE LEVELS Cellular adenosine triphosphate (ATP) and adenosine diphosphate (ADP) levels were determined on the Shimadzu LC-20AB HPLC system coupled with the SCIEX QTRAP 4000 linear ion trap MS system. The MS was operated in negative ion and MRM mode. For ADP, MRM transitions were 426/79 and 426/ 134. For ATP, peaks from top to bottom represent transitions 506/159, 506/408, and 506/273.(21) Detailed descriptions can be found in the Supporting Information.

IMMUNOFLUORESCENCE STAINING Immunostaining was carried out using the recommended protocol, and imaging was performed on a Leica TCS-SP5 confocal microscope. Briefly, cells were fixed with 4% paraformaldehyde and then blocked in 5% goat serum (Biotech Well, Shanghai, China), followed by primary antibody incubation overnight at 48C. Primary antibodies included polyclonal rabbit anti–glucose transporter 1 (GLUT1; 1:100; Millipore) and monoclonal mouse anti-GLUT4 (1:200; Abcam, Cambridge, UK). Following primary antibody incubation and washes, cells were incubated with fluorescein isothiocyanate–conjugated goat antirabbit immunoglobulin G and Cy3-conjugated goat antimouse immunoglobulin G, respectively (Jackson ImmunoResearch, West Grove, PA) for 30 minutes in the dark at room temperature. 4’,6-Diamidino-2-phenylindole (Sigma) was used for nuclear identification. Cells were observed using a Leica TCS-SP5 confocal microscope after staining.

WESTERN BLOTS Total cell lysates were prepared by radio immunoprecipitation assay buffer (Pierce, Rockford, IL) containing protease and phosphatase inhibitors (Roche, Basel, Switzerland). Protein concentrations were determined by bicinchoninic acid assay (Pierce). Proteins (30 lg) were separated on sodium dodecyl sulfatepolyacrylamide gel electrophoresis gels (Novex, Carlsbad, CA) and transferred to polyvinylidene fluoride membranes (Merck Millipore). Membranes were then blocked in 5% fat-free milk and immunoblotted with

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appropriate antibodies. Detailed descriptions of the antibodies and methods can be found in the Supporting Information.

IN VIVO ASSAYS FOR TUMOR GROWTH AND METASTASIS All experimental animal procedures were approved by the Animal Care and Use Committee of Fudan University and performed under supervision (P.Y. and H.S.). Male athymic nude mice, 5-7 weeks old (Shanghai Laboratory Animal Center), were maintained in individually ventilated caging systems. LM3 cells (5 3 106) were injected subcutaneously into the right flank of each mouse. Tumor-bearing mice were randomized into four groups (n 5 8) 5 days later, and each group was closely matched before treatment. Mice were treated with C16:0 (10.26 mg/kg/day) by gavage. The positive control group received 2.2 mg/kg of intraperitoneal cisplatin twice a week, while the negative control group received 9 g/L NaCl solution daily. Antitumor effects of C16:0 (5.13 mg/kg) in combination with cisplatin (1.1 mg/kg) on HCC were also examined. Tumor volumes were evaluated every 3 days. Blood was collected through the orbital artery and vein and centrifuged at 425 g for 15 minutes at 48C to obtain serum. Then, animals were killed on day 26, and tumor weights were determined after dissection. Subcutaneous tumors were removed and dissected into 1-mm3 fragments, which were incubated into the liver of nude mice to establish orthotopic implantation models.(22) C16:0 was intragastrically administrated (10.26 mg/kg/day) a week later, and the control group received 9 g/L NaCl solution. Mice were sacrificed 7 weeks later, and tumor wet weights were taken at the end of experiment. At autopsy, tumors, livers, and lungs were resected and processed for routine gross and microscopic examination for metastasis assay. Lung metastases were evaluated independently by two pathologists. The formula of tumor volume, descriptions of the analytes in mice serum, and criteria of pathological staging in lung metastases can be found in the Supporting Information.

DIMETHYL LABELING AND PHOSPHOPEPTIDE ENRICHMENT Lysates containing 5 mg of total proteins from cells treated either with or without C16:0 were digested

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with sequencing-grade modified trypsin (Promega, Madison, WI) at 378C for 16 hours. Untreated and treated samples were subjected to “light” and the “heavy” labeling reactions separately. Labeled mixtures were combined and desalted on a 500-mg Sep-Pak C18 cartridge (Waters, Milford, MA). Peptides were separated on an SCX column (200 3 9.4 mm, 5 lM particle size, 200 Å pore size; PolyLC, Columbia, MD). Phosphopeptides were finally enriched by TiO2 (GL Science, Saitama, Japan) and dissolved in 0.1% formic acid for liquid chromatographic–MS analysis.(23) Detailed procedures of the dimethyl labeling reaction, SCX separation, and TiO2 enrichment can be found in the Supporting Information.

LIQUID CHROMATOGRAPHIC– MS ANALYSIS AND PHOSPHOPROTEOMIC DATA PROCESSING Experiments were performed on a nanoACQUITY ultra-HPLC system (Waters) connected to a Q Exactive Orbitrap MS (Thermo Fisher Scientific) equipped with an online nano-electrospray ion source. Proteome Discoverer software (version 1.4; Thermo Fisher Scientific) with the Mascot search engine (version 2.3.2; Matrix Science, London, UK) was used to identify and quantify phosphoproteins. The database was Human UniProtKB/Swiss-Prot (release 2014-04-10). PhosphoRS 3.0 was further used to calculate the phosphorylation site probability. Detailed descriptions of the searching parameters can be found in the Supporting Information.

NETWORK ANALYSIS To determine which pathways were markedly altered by C16:0 treatment, lists of differentially expressed phosphoproteins compared with nontreatment controls were imported into Ingenuity Pathway Analysis (www.ingenuity.com; Ingenuity Systems, Redwood City, CA). Core analyses, upstream analyses, and regulator effects analyses were performed.

STATISTICAL ANALYSIS All in vitro experiments were conducted in triplicate and carried out on three separate occasions. Numerical data were expressed as means 6 SD. Statistically significant differences between the means for the different

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groups were determined by two-tailed unpaired Student t test and defined as P < 0.05.

Results MS-BASED LIPIDOMIC ANALYSIS IN HUMAN HCC CELLS Using shotgun lipidomic analysis, we provided comprehensive identification and quantification of lipid compositions in a series of HCC cell lines: nonmetastatic hepatoma cell line Hep3B, low metastatic 97L, and high metastatic LM3 cells. A total of 1,701 unique lipids were identified through the integration of positive and negative electrospray ionization tandem MS (Supporting Table S1), including 1,221 glycerophospholipids (GPs), 313 glycerolipids, and 167 sphingolipids (Fig. 1A; Supporting Table S2). To obtain an overview of the association between lipidomic composition and the different metastatic potentials in HCCs, we performed relative quantifications to the major lipid classes and FA distributions. Most GP species (phosphatidylcholine [PC], phosphatidic acid, phosphatidylglycerol, and phosphatidylserine) had decreased trends in metastatic cell lines 97L and LM3 compared with Hep3B, whereas significantly increased d18:1-containing sphingolipids and sulfatide were observed (P < 0.05, t test; Fig. 1B; Supporting Table S3). Triacylglycerol detected in positive-ion mode showed a >4.5 times increase in metastatic 97L and LM3 cells compared to Hep 3B (P < 0.05, t test; Supporting Fig. S1A). Total FA distributions of the lipids were analyzed by precursor ion scanning in negative mode or neutral loss scanning in positive mode. The C16, C18, and C20 series fatty acyl chains were the main fractions of lipids. C16:0 and C14:0 were statistically decreased, while C20:5 was up-regulated by approximately 1.5-fold in metastatic cell lines 97L and LM3 compared with the nonmetastatic Hep3B (P < 0.05, t test). C18:3 and C20:4 were elevated with increasing metastatic potential of HCCs, but there was no significant difference (Fig. 1C; Supporting Table S4). Gas chromatographic–MS analysis was further used to validate the decreased C16:0-containing GPs in metastatic 97L and LM3 cells compared with Hep3B (Supporting Fig. S1B). Relative quantifications were next applied to all the identified lipids in the three HCC cell lines, and 93 significantly changed lipids are presented in Supporting Table S5. Metabolomic data analyses were next


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FIG. 1

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performed using MetaboAnalyst 3.0.(18,19) Significant differences for the variables between Hep3B, 97L, and LM3 are depicted as a heatmap in Fig. 1D, which was ranked by analysis of variance. Each row represents an individual lipid, and each column represents an individual sample. Interestingly, the correlative values between the 93 lipids revealed two distinct clusters: cluster 1 was the Hep3B group only, whereas cluster 2 contained both LM3 and 97L. Systematic lipidomic changes occurring in different groups were then assessed by two widely used multivariate methods: principal component analysis and partial least squaresdiscriminant analysis. The first principal component accounted for 62.5% of the total variance and separated nonmetastatic Hep3B cells from the two metastatic cell lines 97L and LM3. The second principal component accounted for 13.1% of the variance in the data (Fig. 1E). Partial least squares-discriminant analysis was used to enhance the separation between the groups (Fig. 1F). Phosphatidylethanolamine (PE; 18:1/22:2) with the highest variable importance of projection value was the most powerful group discriminator in differentiating metastatic 97L and LM3 from nonmetastatic Hep3B (Supporting Fig. S1C). Collectively, our lipidomic data unraveled a strikingly consistent separation between nonmetastatic and metastatic HCC cells. It is interesting to note the decreased saturated fatty acyl–containing GPs (such as C16:0 and C14:0) and the increased unsaturated fatty acyl–containing GPs (such as C20:4 and C20:5) in metastatic cells compared with nonmetastatic ones.

C16:0 INHIBITS HCC CELL PROLIFERATION, MIGRATION, AND INVASION IN VITRO To explore the role of C16:0-containing GPs, we first performed cell proliferation experiments by treating HCC cells with C16:0 of different concentrations


(0, 50, 100, 200 lM). The growth of Hep3B and 97L cells was inhibited gradually, while cell proliferation of high metastatic LM3 was suppressed remarkably with C16:0 supply, in a dose-dependent manner (Fig. 2A). To exclude the lipotoxic effect of C16:0 accumulation in HCC cells, we evaluated the proliferation of nontransformed hepatic L02 cells with the same treatment condition. Interestingly, the proliferation rate of L02 remained unchanged when cells were exposed to C16:0 (Fig. 2A). Meanwhile, induction of C18:1 or stearic acid did not inhibit cell proliferation in LM3 cells (Supporting Fig. S2A,B), indicating selective inhibition of C16:0 on metastatic HCC cell growth. We further examined cell growth of colon cancer cell lines and gastric cancer cell lines to prove that the growth suppression role of C16:0 is not universal even for cancer cells. Exposure of SW480 and SW620 cells to C16:0 severely suppressed cell proliferation (Supporting Fig. S3A,B). When gastric cancer cell lines (e.g., AGS and BGC-823) were exposed to C16:0, cell growth was inhibited; however, the metastatic cell line HGC-27 remained unchanged (Supporting Fig. S3CE). These experiments indicated that C16:0 exhibited varying degrees of inhibition in different cancer cell lines. Because increased cell migration and invasion are the determinant hallmarks of metastatic tumor cells, we explored the influence of C16:0 treatment on aggressive phenotypes. Exposure of Hep3B or LM3 cells to C16:0 severely suppressed cell migration in a wound-healing experiment (Supporting Fig. S4A,B). The wound closures were measured, and quantification analyses are presented in Fig. 2B, normalized to the initial width when the wound was created. Moreover, the number of invaded cells was markedly reduced when LM3 cells were treated with multiple doses of C16:0 (282 versus 632; P 5 0.006) (Fig. 2C), suggesting that C16:0 treatment inhibits cell migration and invasion activity in vitro.

FIG. 1. Lipid profiles of HCC cell lines and metabolomic analysis of the significantly changed lipids. (A) Lipid classes in nonmetastatic Hep3B, lowly metastatic 97L, and highly metastatic LM3 cells. (B) Relative MS intensity of major lipid species and (C) FA distributions in three HCC cell lines (blue, Hep3B; red, 97L; green, LM3). Each column represents the mean 6 SD of three independent experiments. *Statistically significant difference (P < 0.05). (D) Systematic lipidomic changes occurring in different HCC cell lines were clustered using hierarchical clustering analysis. Clustering results shown as heatmap with distance measured using Pearson and clustering algorithm using average. Pseudocolors indicated positive (orange) or negative (blue) correlation values or missing values (gray). The scale represents the correlation values from 2 to –2. Principal component analysis (E) and partial least squaresdiscriminant analysis (F) two-dimensional scores plot were used for exploratory data analysis through dimensional reduction and data visualization. Principal component 1 differentiates between nonmetastatic Hep3B cells and the two metastatic HCC cell lines, while component 1 further enhanced the separation between the groups. Abbreviations: DG, diacylglycerol; PA, phosphatidic acid; PI, phosphatidylinositol; PC1/PC2, principal components 1 and 2; PG, phosphatidylglycerol; PS, phosphatidylserine; SL, sulfatides; SP, sphingolipid; TG, triacylglycerol.

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FIG. 2. C16:0 modulates the phenotype of HCC cells in vitro. (A) Growth of C16:0-treated cells was analyzed using Cell Counting Kit-8. *Significant difference from nontreatment controls (P < 0.05) for a mean 6 SD of five samples per condition. Error bars represent 6SD. (B) In a wound-healing experiment, the migration status of C16:0-treated Hep3B and LM3 cells was determined by measuring the movement of cells into a scraped area at indicated times. Wound widths were measured, and quantification analyses are presented (*P < 0.05, **P < 0.001). (C) Cell invasive activity after C16:0 administration in LM3 cells was measured using transwell assay. Mean 6 SD of triplicate experiments is plotted (*P < 0.05).

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C16:0 IMPAIRS TUMORIGENICITY AND LUNG METASTASIS IN VIVO Then we investigated the in vivo antitumor effect of C16:0 in nude mice bearing LM3 cell carcinoma xenografts. Tumor growth was observed, and macroscopic images show isolated tumors (Fig. 3A). C16:0 treatment alone (10.26 mg/kg/day) or cisplatin alone (1.1 mg/kg) caused huge suppression of tumor growth in both size and weight, while combined therapy with the two further reduced the tumor volume slightly compared to C16:0 or cisplatin alone, which statistically differed from the negative control (P < 0.05; Fig. 3B,C). We measured six routine laboratory analytes in mouse serum from four treatment groups (n 5 8); alanine aminotransferase and creatinine were increased significantly in both the cisplatin group and the combined group (*P < 0.05), while no statistically significant changes were detected between the C16:0 treatment group and the control group, indicating that C16:0 treatment was tolerated well with no signs of toxicity during 6 weeks of daily treatment (Supporting Fig. S5). To further determine the effects of C16:0 on in vivo tumor metastasis, LM3 tumor xenografts were isolated from the foregoing subcutaneous tumor specimens and implanted into the liver to establish orthotopic models.(22) In this study, implanted fragments survived and all mice formed tumor nodules at the liver (Fig. 3D). The average wet weight of orthotopic tumor in the C16:0 treatment group (10.26 mg/kg/day) was lighter than that in the negative control group, but statistical significance was not reached (Fig. 3E). Microphotographs of intrahepatic and pulmonary metastatic lesions are shown by hematoxylin and eosin staining (Fig. 3F,G). Lung metastases were visible in 4 (50%) mice of the treatment group, while the incidence in the control group was 62.5%. The total number and grade of lung metastatic lesions in the C16:0 treatment group was much lower than those in controls (P < 0.05) (Fig. 3H). These results show the antitumor effects of C16:0 on in vivo tumor growth and metastasis of HCC xenografts.

C16:0 DECREASES CELL MEMBRANE FLUIDITY, LIMITS GLUCOSE METABOLISM, AND REDUCES ATP LEVEL To elucidate the antitumor mechanism of C16:0 in HCC, we examined whether C16:0 incubation could

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restore the amount of C16:0-containing lipids in LM3 cells. MRM analysis showed elevated C16:0 amounts and decreased C18:1 levels when LM3 cells were treated with C16:0, using Hep3B for normalization (Supporting Fig. S6). In detail, C16:0-containing GPs were increased in LM3 treatment cells compared with nontreatment ones, such as PE (16:0/16:1) and PE (16:0/17:1); meanwhile, reduced C18:1 was simultaneously detected comprising PE (18:1/18:1), PE (18:0/18:1), and phosphatidylserine (18:1/18:0). Down-regulated C18:1-containing sphingolipids like Cer (d18:1/16:0) and Cer (d18:1/24:0) in LM3 treatment cells were also detected. However, there was decreased PC (16:0/15:1) as an exception after C16:0 induction. Lipid bilayer membranes control a variety of cellular processes. Alterations in lipid composition can change membrane properties and thus influence membrane functions, such as material exchanges and signal transduction.(24) We first speculated that saturated C16:0 supplementation or deprivation would influence physical membrane fluidity. The merged hue-saturationbrightness image had both membrane fluidity (generalized polarization) and structural (intensity) information, providing an excellent tool for membrane fluidity visualization (Fig. 4A). Generalized polarization values ranged from –1, corresponding to the highest fluidity, to 11 for the lowest fluidity. Generalized polarization distribution curves were plotted corresponding to LM3 cells with or without C16:0 treatment, by fitting used gaussian functions for asymmetric curves (Fig. 4B). With C16:0 treatment, LM3 cells had higher generalized polarization values than nontreated controls; thus, the peak value of the curve was shifted from 0.302 to 0.396, indicating that membrane fluidity was decreased. It is widely accepted that decreased membrane fluidity will affect the functional activity of membrane and membrane protein dynamics. Experiments were carried out to quantify one important membrane function, glucose uptake, which is used by tumor cells to fuel cell proliferation and invasiveness and the following lactate production in the culture medium of each sample. Increasing C16:0 concentration (from 0 to 200 lM) reduced cellular glucose uptake as well as lactate production in a dose-dependent fashion (from 137.3 to 68.5 pmol and from 56.07 to 36.02 mM), respectively (Fig. 4C,D). Glucose metabolism of Hep3B did not show a statistical difference in response to C16:0 treatment (data not shown).


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FIG. 3

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Relative to mitochondrial oxidative phosphorylation, cancer cells derive the bulk of their metabolic energy from glucose through glycolysis. ATP is the primary energy transporter in almost every cell process, and the turnover rate of ATP to ADP and vice versa can be used as an indicator for energy metabolism.(25) The ATP/ADP ratios obtained from LM3 cells were decreased significantly after 24 hours of C16:0 treatment (P < 0.05) (Fig. 4E). The ATP/ADP levels of Hep3B cells remained unchanged when exposed to C16:0 (200 lM), but the ATP/ADP ratios were reduced in the 97L treatment group (P < 0.05) (Supporting Fig. S7). To compensate for the >10-fold lower efficiency of ATP production afforded by glycolysis, cancer cells may up-regulate the expression or activation of glucose transporters, which substantially increases glucose import from the extracellular space into the cytoplasm.(26,27) We examined the expression of glucose transporters, such as GLUT1 and GLUT4, with or without C16:0 treatment. Immunofluorescent staining revealed a strong membranous signal of GLUT1 in LM3 cells, and C16:0 (200 lM) did not alter GLUT1 expression or location. Additionally, confocal images showed membranous and cytoplasmic locations of GLUT4 in LM3 cells; however, GLUT4 expression was slightly lower in the C16:0 treatment group compared with the control (Fig. 4F). Western blot analysis comfirmed the unchanged expression of total GLUT1 and decreased expression of GLUT4 in LM3 cells with the induction of C16:0 in a dose-dependent manner (Fig. 4G). Taken together, we concluded that restoration of C16:0-containing GPs in HCC cells by palmitic acid incubation caused a dose-dependent inhibition of membrane fluidity, glucose use, and ATP generation.

PHOSPHOPROTEOMIC ANALYSIS OF C16:0-TREATED CELLS Reversible protein phosphorylation, an essential posttranslational modification, is intimately involved in


almost every cellular process including cell proliferation, apoptosis, cellular metabolism, and signal transduction pathways.(28) We applied a quantitative phosphoproteomics approach combined with dimethyl labeling to systematically investigate how C16:0 treatment could change global cellular protein phosphorylation and consequently contribute to HCC aggressiveness (Supporting Fig. S8A). A signal intensity ratio of >2.0 (log ratio >1) or