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

Department of Applied Chemistry, College Yongin, 446-701, Republic of Korea. E-mail: Department of Herbal Crop Research, Natio Science, RDA, Eumseong 369-873, Republic United Graduate School of Drug Discovery University, 1-1 Yanagido, Gifu 501-1193, Ja The Institute of Natural Science, College o Yongin, 446-701, Republic of Kore † Electronic supplementary informa 10.1039/c6ra01965j Cite this: RSC Adv., 2016, 6, 29512


Introduction
For effective treatment of cancer it is critical to investigate specic molecules with anticancer effects. 1,210-Hydroxy-2decenoic acid, which is a component of royal jelly, shows anticancer activity. 3In our previous study, various decenoic acid derivatives were synthesized and screened. 4As a result, several 3-decenoic acid derivatives were found to show potent anticancer activity; in particular, AIC-47 induced autophagic cell death (ACD) in human leukemia K562 cells.As several anticancer drugs have been reported to induce ACD in cancer cells 5,6 it is important to understand the mechanism of ACD.
Cancer cells have oen been found to have lower autophagic capacity than their normal counterparts. 7Autophagy in cancer cells is activated in response to various cellular stresses such as nutrient and growth factor starvation, 8 and by therapeutic treatment. 9In such circumstances the dying cells display a large-scale accumulation of autophagic vacuoles, and this phenomenon led to the classication of ACD, which is a form of programmed cell death. 10However, this denition is under debate in different scientic communities because autophagy is generally thought of as a survival mechanism. 11orphologically, the autophagosomes in cancer cells treated with AIC-47 contain lipid droplets (LDs). 4Several studies have characterized the roles of LDs as cellular stores of neutral lipids as well as a lipid source to support autophagic membrane formation. 12,13LDs are also associated with a specic type of autophagy called lipophagy that regulates lipid metabolism. 14,15n particular, lipophagy has the potential to regulate cellular energy homeostasis as well as lipid content. 16n this study, we performed lipid proling of ACD-induced cancer cells to investigate the correlation between lipids and ACD.Lipid proling is a critical method to study lipid functions in various biological samples. 17,18The roles of several lipids in the control of autophagy have been previously reported; 19 however, there are still many other lipids that have not yet been characterized in autophagic cells.Furthermore, this study is the rst evaluation of lipid alterations in cancer cells in the state of ACD induced by a decenoic acid derivative.

Cell culture and treatment
The human leukemia cell line K562 was cultured in RPMI-1640 medium (Invitrogen, Carlsbad, CA, USA) containing 10% (v/v) FBS and 1% penicillin in controlled cell culture conditions of 5% CO 2 and 37 C.For treatment with AIC-47, 1 mL of AIC-47 was dissolved in 300 mL of dimethyl sulfoxide (DMSO), resulting in a 10 mM solution.This solution was dissolved in 1 mL culture medium to give a nal concentration of 10 mM AIC-47.ACD was induced aer 48-72 h.Aer treatment, the cell pellet was collected from each culture dish and the number of cells was counted using a hemocytometer.The total number of cells was 2.55 Â 10 6 for AIC-47 treated and 4.45 Â 10 6 for non-treated cells.Lipid extraction was performed using 1.275 Â 10 6 cells from each group.

TMSD methylation
A solution of trimethylsilyldiazomethane (TMSD) (2 mol L À1 ) in hexane was added to the lipid extracts from the cell lines and the lipid standard samples dissolved in methanol to obtain yellow-colored solutions.Aer vortexing for 30 s, methylation was performed at 37 C for 15 min.Addition of glacial acetic acid (6 mL) quenched the methylation and yielded colorless samples.The samples were subjected to LC/MS.

UPLC-QqQ/MS analysis
Quantitative lipid proling was performed by 6490 Accurate-Mass Triple Quadrupole (QqQ) LC-MS coupled to a 1200 series HPLC system (Agilent Technologies, Wilmington, DE, USA) with a Hypersil GOLD column (2.1 Â 100 mm ID; 1.9 mm, Thermo Science).This technique provides high sensitivity by iFunnel technology that consists of three components: Agilent Jet Stream technology, a hexabore capillary, and a dual ion funnel.The temperature of the column oven and sample tray was set to 40 C and 4 C, respectively.Solvent A consisted of an acetonitrile-methanol-water mixture

Result and discussion
Construction of a lipid proling method based on UPLC/QqQ-MS For the proling of various lipids, we constructed a MRM method based on UPLC-QqQ/MS.First, 23 lipid standards were used to optimize the MRM conditions for TG, DG, ChE, PC, PE, PS, PG, PI, PA, LPC, LPE, LPS, LPG, LPI, LPA, SM, Cer, dCer, So, Sa, Cer1P, So1P, and Sa1P.In a previous study, TMSD methylation was applied for the sensitive proling of several highly acidic lipids. 21 Next, validation of lipid analysis based on MRM was performed to estimate the performance of lipid quantication.Each lipid standard was analyzed six times with an internal standard (IS), PC (12 : 0-12 : 0), and the relative standard deviation (RSD) (%) was calculated.The RSDs (%) of the relative retention times (RTs) and relative peak areas were smaller than 1.5% and 8.9%, respectively.The method showed high reproducibility, and the correlation (R 2 ) in each lipid analysis was at least 0.9803, indicating high reliability.The limits of detection (LODs) of each lipid standard were also listed (Table S2 †). 22pid proling of control and AIC-47treated leukemia cells Next, we performed lipid proling of control and AIC-47treated leukemia cells.The ability of AIC-47 to induce ACD has already been demonstrated in a previous study. 4To characterize in detail the lipid content of ACD-induced leukemia cells, we analyzed three neutral lipids (TG, DG, ChE), six phospholipids (PC, PE, PS, PG, PI, PA), six lysophospholipids (LPC, LPE, LPS, LPG, LPI, LPA), and eight sphingolipids (SM, Cer, dCer, So, Sa, Cer1P, So1P, Sa1P).Neutral lipids have functions in energy storage 23,24 and phospholipids, lysophospholipids, and sphingolipids have roles in cellular membrane components and signaling transduction. 25,26Thus, detailed proling of these lipids is useful to not only to estimate the lipid content but also to understand their functional roles in ACD.Using the optimal UPLC-QqQ/MS conditions, various lipids including 71 TGs, 19 DGs, 11 ChEs, 59 PCs, 36 PEs, 29 PSs, 21 PGs, 19 PIs, 33 PAs, 15 LPCs, 11 LPEs, 2 LPSs, 6 LPGs, 6 LPIs, 17 LPAs, 14 SMs, 13 Cers, 3 dCers, 2 So, Sa, 7 Cer1Ps, So1P, and Sa1P were successfully analyzed in AIC-47treated leukemia cells (Table S3 †).The total ion chromatogram of lipids is represented in Fig. S1.† Phospholipids, lysophospholipids, sphingolipids, and DGs were eluted from 0 to 15 min, whereas TGs, ChEs, and methylated PIs were eluted from 15 to 21 min.Using an octadecylsilyl silica column lipid species were separated according to their fatty acid composition, including different carbon numbers and double bond numbers.Each IS was used to conrm the RT of lipids.Each lipid species was assigned according to its RT in the chromatogram.

Detailed characterization of altered lipids in ACD-induced cancer cells
In the ACD-induced leukemia cells and controls, a total of 397 individual lipid species were analyzed by UPLC-QqQ/MS.First, we applied principal component analysis (PCA) to differentiate ACD-induced leukemia cells and controls.In the PCA score plot, the two groups were separated well with 77.2% total variances including principal component (PC) 1 (64.5%) and PC 2 (12.7%) (Fig. 1A).This indicated that ACD induced the alteration of various lipids in leukemia cells.In the volcano plot, 56 lipid species were selected as the differently regulated lipids with fold change >2 and p value < 0.05 (Fig. 1B, Table 1).We found that several lipids were up or down regulated by ACD.
Next, we performed the quantication of each lipid class to nd the lipid alterations induced by ACD.From the MRM data, the peak area of each lipid was calculated and normalized to the IS peak area (lipid species peak area/IS peak area).The quantied values of each species were then summed to obtain the total amount of each lipid class.For example, in the case of TG, the normalized values of 71 individual TG species were summed to calculate the total amount of TG in the sample.Next, we applied the t-test for the comparison of ACD-induced leukemia cells and controls (signicance at P # 0.05).As a result, the quantitative alteration of 23 lipid classes was in the ACD-induced leukemia cells (Table 2).Compared to the control, 14 classes-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 leukemia cells.Other classes, such as PE, SM, dCer, So, So1P, and Sa1P, showed no changes.These lipid alterations might be associated with the autophagy that triggers ACD.Therefore, we tried to match our lipid proling data of ACD-induced cells with previous reports about the various roles of lipids in autophagy.
In our previous study we found that AIC-47treated cancer cells showed LDs together with autophagosomes. 4Several lipids, including TG, DG, ChE, PC, and PE, were critical components of these LDs.In the treated cells, TG and DG were upregulated, and ChE was downregulated.It was previously reported that AIC-47 induces a switch from glycolysis to the TCA cycle through switching from pyruvate kinase M (PKM)2 to PKM1, suggesting that increased generation of citric acid promotes synthesis of fatty acid and TG. 27Furthermore, ChE is known to inhibit autophagy by activating p38 MAPK in macrophages. 28Thus, AIC-47 may inactivate p38 MAPK and induce downregulation of ChE, leading to autophagy.The LDs generated by AIC-47 may consist of mainly TG and DG.
PC and PE, which are the main components of the LD surface, 29 were not upregulated.The downregulation of PC and no change of PE may be related to the PA/DG/protein kinase C (PKC) signaling cascade and elongation of isolation membranes, respectively.It was previously reported that PKC signaling has a critical role in generation of autophagy. 30,31In general, phospholipase D (PLD), which is a positive modulator of autophagy, hydrolyzes PC to PA. DG is produced from PA by PA phosphatase (PAP) and mediates the downstream stimulation of PKC, which can in turn activate autophagy by dissociating the Bcl-2 and Beclin 1 complex and by stimulating NADPH oxidase. 32Our results also showed downregulation of PC and upregulation of PA and DG in the AIC-47treated cells.This might indicate that lipid metabolism for PKC signaling is related to autophagy (Fig. 2A).During autophagy, PE has critical roles in the elongation and closure of the isolation membrane 19 and an articial increase in PE levels increases cellular autophagic ux. 33PE is generally produced by decarboxylation of PS.The upregulated PS in the AIC-47treated cells might be used to produce PE for the isolation membrane.Furthermore, PE might show no changes because it may be dissociated from the surface of autophagosome membranes (Fig. 2B).Upregulated PI is also related to PI3K class III signaling, which is essential for induction of autophagy.In our previous study we observed downregulation of Bcl-2 and upregulation of Beclin-1, indicating that PI3K class III signaling is activated by treatment with AIC-47. 27PI-3-phosphate (PI3P) generated from PI is also reported to be an important inducer of autophagy. 19In neutrophils, LPS activates reactive oxygen species (ROS) 34 production through interaction with PKC.Upregulated LPS by AIC-47 may be associated with ROS generation and autophagy if the ROS production mechanism in leukemia cells is the same as that in neutrophils.Furthermore, downregulated LPA is known to inhibit autophagy. 35revious studies have also reported that Cer can activate autophagy by inhibiting the phosphorylation of Akt, reducing the activation of mTOR, and upregulating Beclin 1 function. 36,37nclusions A lipidomics approach based on UPLC-QqQ/MS was used to characterize the altered metabolism of various lipids in ACDinduced human leukemia cells.Using the optimal UPLC and MRM, 397 individual species of 23 lipid classes were successfully analyzed.In the comparison of ACD-induced cells and controls, statistical data analysis indicated that ACD causes altered lipid metabolism in cancer cells.Upregulation or downregulation of lipids that have known roles in autophagy  demonstrated that the lipid metabolism of ACD is correlated to autophagy.Furthermore, the induction of simultaneous alteration of other lipids by ACD is well described.Finally, the novel technique based on UPLC-QqQ/MS shows promising applications for comprehensive lipid proling as well as for revealing and elucidating lipid alterations in a certain biological system such as ACD.
Therefore, we used TMSD methylation for the analysis of PS, PI, PA, LPS, LPI, LPA, Cer1P, So1P, and Sa1P.By MS scan, the adducted ions of each lipid were conrmed to construct the MRM transition (precursor m/z [Q1] > product m/z [Q3]).As a result, in the positive ion mode of ESI, † each lipid was detected with two adducted ions, a hydrogen ion (H + ; m/z 1.01) and an ammonium ion (NH 4 + ; m/z 18.03).For example, TG, DG, ChE, and PG were detected as [M + NH 4 ] + ions.Furthermore, PC, PE, LPC, LPE, LPG, SM, Cer, dCer, So, Sa, and several methylated lipids such as PS, PI, PA, LPS, LPI, LPA, Cer1P, So1P, and Sa1P were detected as [M + H] + ions.By the product ion scan, m/z of one product ion with high intensity was determined as the Q3 of transition, and the MS/MS collision energy was optimized (Table S1 †).

Fig. 1
Fig. 1 Principal component analysis score plot of controls (red) and ACD-induced leukemia cells (green) (A) and the volcano plot representing differently regulated lipids in ACD-induced leukemia cells compared to controls (fold change > 2, p value < 0.05) (B).

Fig. 2
Fig. 2 The lipid metabolism of autophagic cell death.(A) Quantitative alteration of PC, PA, and DG related to PKC signaling.(B) PE, synthesized from PS, has critical roles in autophagosome formation and closure.PA, phosphatidic acid; PC, phosphatidylcholine; DG, diacylglycerol; PKC, protein kinase C; PE, phosphatidylethanolamine; PS, phosphatidylserine.

Table 1
The list of differently regulated lipids in ACD-induced leukemia cells

Table 2
Quantitative alteration of 23 lipids in ACD-induced leukemia cells