Alkylglyceronephosphate Synthase ( AGPS ) Alters Lipid Signaling Pathways and Supports Chemotherapy Resistance of Glioma and Hepatic Carcinoma Cell Lines

The hallmarks of cancer are keys for cancer treatment. The most fundamental hallmark of cancer cells is their ability to sustain proliferation (Marquardt et al., 2012). This trait is the target of chemotherapy agents, an old but still widely used class of cancer therapeutic medicine. These drugs could be divided into several types. One type is called alkylating agents, the typical drug is cisplatin (Srivastava et al., 2010). Another type is topoisomerase inhibitors, with doxorubicin is the typical one (Pommier et al., 2010). Cisplatin and doxorubicin are extensively used to treatment different types of cancer. However, resistance to these drugs is rather common. Reprogramming of energy metabolism is an emerging hallmark of cancer in the last decade. Cancer cells reprogram the cellular energy metabolism in order to support continuous cell growth and proliferation (Morandi et al., 2014). One well known such alteration is the Warburg effect (Vander et al., 2009). Recent research suggests that alterations in metabolism could not be viewed as just a response to cell proliferation, but


Introduction
The hallmarks of cancer are keys for cancer treatment.The most fundamental hallmark of cancer cells is their ability to sustain proliferation (Marquardt et al., 2012).This trait is the target of chemotherapy agents, an old but still widely used class of cancer therapeutic medicine.These drugs could be divided into several types.One type is called alkylating agents, the typical drug is cisplatin (Srivastava et al., 2010).Another type is topoisomerase inhibitors, with doxorubicin is the typical one (Pommier et al., 2010).Cisplatin and doxorubicin are extensively used to treatment different types of cancer.However, resistance to these drugs is rather common.
Reprogramming of energy metabolism is an emerging hallmark of cancer in the last decade.Cancer cells reprogram the cellular energy metabolism in order to support continuous cell growth and proliferation (Morandi et al., 2014).One well known such alteration is the Warburg effect (Vander et al., 2009).Recent research suggests that alterations in metabolism could not be viewed as just a response to cell proliferation, but RESEARCH ARTICLE Alkylglyceronephosphate Synthase (AGPS) Alters Lipid Signaling Pathways and Supports Chemotherapy Resistance of Glioma and Hepatic Carcinoma Cell Lines Yu Zhu 1& *, Xing-Jun Liu 2& , Ping Yang 1& , Meng Zhao 3& , Li-Xia Lv 1 , Guo-Dong Zhang 1 , Qin Wang 1 , Ling Zhang 1 metabolites themselves can be oncogenic by altering cell signaling and blocking cellular differentiation (Weinstein et al., 2014).Targeting cancer cell metabolism is hence an interesting strategy.Here we focus on the abnormal lipids metabolism.Lipids are not just structural components of cell but can also have important roles in signaling.Deregulation of lipide metabolism is common in cancer and can affect numerous cellular processes, including cell growth and proliferation (Prabhu et al., 2013).Many lipid synthesis related genes have been considered involved in tumor procedure, for example that prostaglandinendoperoxide synthase 2 (PTGS2) and phospholipase A2 group IIA (PLA2G2A) genes encode enzymes involved in arachidonic acid and prostaglandin biosynthesis, which is associated with carcinogenesis (Liu et al., 2014).Recently, alkylglyceronephosphate synthase (AGPS), an enzyme that catalyzes the critical step in ether lipids synthesis, is shown to up-regulate in multiple types of cancer cell and primary tumor.AGPS alters the balance of structural and signaling lipids to fuel cancer pathogenicity (Benjamin et al., 2013).Nonetheless, whether AGPS is involved in chemotherapy resistance has remained unclear.
In this study, we showed that AGPS elevated in glioma U87MG/DDP and hepatic carcinoma HepG2/ADM cell lines.Knockdown of AGPS restored the drug sensitivity in these cells.We also showed that silencing of AGPS led to reduced cellular level of lysophosphatidic acid (LPA), lysophosphatidic acid-ether (LPAe) and prostaglandin E2 (PGE2), which could fuel aggressive and progress of cancer (Hull et al., 2004;Brindley et al., 2013).Knockdown of AGPS also results in down-regulation of multi-drug resistance proteins and anti-apoptosis proteins.

Cell culture and establishment of drug-resistant cell clones
U87MG and HepG2 cell lines were obtained from Cell Bank of Chinese Academy of Science (Shanghai, China) 5% CO 2 .The culture medium was DMEM supplemented with 10% fetal bovine serum (FBS) for HepG2 and U87MG.Cisplatin-resistant U87MG/DDP cell line and Doxorubicin-resistant HepG2/ADM cell line were established by continuing exposure of the parental cells to gradient increasing concentration of respective drug.

MTS assay to determine the drug sensitivity of the cells
0.1 ml of cells suspension (HepG2/ADM: 1×10 5 /ml, U87MG/DDP: 3×10 4 /ml) were seeded into each well of the 96-well microplate, and incubated overnight to get the cells to adherence.Then different concentration of Cisplatin or Doxorubicin was added to each well and the cells were cultured for another 72hrs.CellTiter 96 Aqueous One Solution Reagent (Promega) was used according to the manufacturer's instructions.4hrs later the cell viability was determined by measuring the absorbance at 490nm using a microplate reader.

Flow cytometry to determine intracellular Rhodamine-123, based assay to determine activity of caspase-3/8
About 1×10 6 of the cells were collected and resuspended in 0.1 ml culture medium.The cells were 1 hr and the intracellular concentration was determined analysis, PI (BD Biosciences) was used to stain DNA.For then Annexin V-FITC/PI dual stain (BD Biosciences) was employed.For caspase-3/8 activation analysis, for HepG2/ADM was added for 24h, then activity of caspase-3/8 was measured by Caspase 3 and Caspase 8 activity assay kits (Promega) .

Real-time PCR analysis
About 3×10 6 of cells were harvested for RT-PCR analysis.The total mRNA was extracted from the cells by the Dynabeads mRNA Direct Kit (Invitrogen) according to the manual instruction.Total mRNA was then reverse oligo (dT)20, 25 units of RNase inhibitor, and 20 units of avian myeloblastosis virus reverse transcriptase (Roche Diagnostics).The transcription level of target proteins was detected by semiquantitative real-time PCR using the icycler iQ detection system (Bio-Rad).The PCR condition full details were shown in Table 1.

Extraction and quantization of LPA and LPAe
Cancer cells were starved in serum-free media for 4 h to minimize the contribution of serum-derived metabolites.About 1×10 6 cells were washed twice by PBS and harvested by scraping and centrifugation.Cell pellets were extracted and analyzed by LC-MS/MS as described mixture of 2 (chloroform): 1 (methanol): 1 PBS with C12:0 dodecylglycerol (10 nmol) and pentadecanoic acid (10 nmol) as internal standards.Organic and aqueous layers were separated by centrifugation.The aqueous layer by chloroform.The organic layer was dried by N2 and analyzed by Agilent 6430 LC-MS/MS.

Analysis of PGE2 by enzyme immunoassay
0.1 ml of cancer cells (HepG2/ADM: 1×10 6 /ml, U87MG/DDP: 4×10 5 /ml) were plated in 96-well plates and cultured overnight.The next day, cells were treated with before collecting the culture medium.PGE2 levels in the medium were determined by PGE2 enzyme immunoassay kits (Cayman Chemical).

Measurement of PLD activity
Cancer cells were seeded into 24-well plate and allowed to reach to near 90% fusion.Cells were washed 50 mM (pH 8.0) was add to each well and the cells were broken by three freeze and thaw cycles.Samples were collected, mixed with an equal volume of the Amplex Red reaction buffer (Amplex Red Phospholipase D assay kit, Life Technologies), and the PLD activity was estimated Streptomyces chromofuscus (Sigma-Aldrich).Data are given as the mean of six determinations performed on three different cell preparations.

Stimulation of EP receptors by PEG2 and respective agonists
Cancer cells were seeded into 96-well plate as MTS agonist; L-902,688, 100nM, EP4 receptor agonist; Cayman Chemical) were added to each well 1h before adding different concentration of drugs to stimulate EP receptors.72hrs later, CellTiter 96 Aqueous One Solution Reagent (Promega) was employed to determine the cell viability.

Statistical analyses
Data were expressed as means±SD., statistical analysis was carried out using One Way ANOVA and p<0.05

Results
The HepG2/ADM and U87MG/DDP cells were resistant to chemotherapy drug and over-expressed AGPS Clones of drug resistant cancer cells were established by continuing exposure to chemotherapy agents.drug resistant cancer cells compared to the drug sensitive HepG2 and U87MG cells (parent group).
The drug resistant clones were transfected with The FCM results showed that there was an increased intracellular accumulation of Rhodamine-123 in human hepatic carcinoma HepG2/ADM and glioma U87MG/DDP p>0.05, compared to the control group; *p<0.05,compared to the control group.B) The FCM results showed that there was an increased G0/G1 phase in human hepatic carcinoma HepG2/ p>0.05, compared to the control group; *p<0.05,compared to the control group.C) The FCM results showed that there was an increased apoptosis rate in human hepatic carcinoma HepG2/ADM and glioma U87MG/DDP AGPS silencing groups compared with HepG2/ADM and U87MG/DDP cell lines.
p>0.05, compared to the control group; *p<0.05,compared to the control group.D) was an increased caspase-3/8 activity in human hepatic carcinoma HepG2/ADM and glioma U87MG/DDP AGPS silencing groups compared with p>0.05, compared to the control group; *p<0.05,compared to the control group. A) different sequences of AGPS-shRNA (shRNA #1 and shRNA #2) in order to produce two groups of cells with different AGPS silencing level.Both HepG2/ADM and U87MG/DDP cells transfected with AGPS-shRNA resulted in down-regulation of AGPS mRNA expression in shRNA #1 and shRNA #2 groups compared to the HepG2/ ADM and U87MG/DDP cells (control group) (p<0.05).
The blank vector HepG2/ADM and U87MG/DDP cells (negative group) had no effect on AGPS mRNA expression when compared to the HepG2/ADM and U87MG/DDP control groups (p>0.05),meanwhile, the AGPS protein expression was showed the similar tendency (Figure 1).

AGPS silencing restored the drug sensitivity
After knockdown of the AGPS, we examined the drug sensitivity of each cell clone by MTS assay.As shown in Figure 2, we could see the cell proliferation rate of the AGPS silencing cells were lower than that of the control group.The negatively transfected cells were nearly unaffected.The IC 50 of shRNA#1 and shRNA#2 sensitive compared with the control group (IC 50 was p<0.05).The reversal factor was 4.5 and 8.3, respectively.The IC 50 of shRNA#1 and shRNA#2 groups compared with the control group (IC 50 (p<0.05).The reversal factor was 3.0 and 5.0, respectively.These results indicated that AGPS play a positive role in Restore of drug sensitivity was accompanied with an intracellular accumulation of Rhodamine-123, cell cycle arrest and increase of apoptosis rate and caspase-3/8 activities The FCM analysis showed that AGPS silenced cells meant that more Rh-123 was retained in the cancer cells.Meanwhile, cell cycle analysis showed that in the AGPS attenuated cancer cells, more cells were distributed in G0/G1 phase, and the percentages of S phase and G2/M phase cells were reduced.After treatment with cisplatin or doxorubicin, the apoptosis rates of AGPS silenced cells cells.Further, the activiy of caspase-3/8 was increased control group as shown in Figure 3.

AGPS modulated the expression of MDR1, MRP1,
We investigated whether expression of transporter proteins from ABC family, such as MDR1, MRP1 and ABCG2, which have the potency to transport both Rh-123 and chemotherapy agents, were modulated.Meanwhile, its interaction with TCF-4 in the nucleus (Castellone et al., 2005).Bcl-2 and survivin have been implicated in antiapoptosis, and is also thought to be involved in resistance to conventional cancer treatment (Mita et al., 2008;Aust et al., 2013).As shown in Figure 4, the expression of was down regulated in AGPS silencing groups compared with control group.The RT-PCR results were consistent to the results from western blotting (Figure 4).

AGPS modulated drug resistance through LPA, LPAe and PGE2 mediated lipid signaling pathways
LPA, LPAe and PGE2 are several lipids that have been found to be involved in AGPS mediated cancer pathogenicity.Here we demonstrated that in all the three drug resistant cancer cells, LPA, LPAe and PGE2 levels increased when compared to the non-resistant cancer cells.Silencing of AGPS resulted in reduction of LPA, LPAe and PGE2.Since both LPA, LPAe and PLD binds to LPA receptors to drive multiple aspect of cancer (Blackburn et al., 2012;Madan et al., 2013), we tested the downstream signaling activities of LPA receptor.We found that both level of LPA, LPAe and PLD in control group of HepG2/ ADM and U87MG/DDP was increased compared to the HepG2 and U87MG cells (parent group) (p<0.05),control and negative group of HepG2/ADM and U87MG/ DDP (p>0.05).Further, both level of LPA, LPAe and PLD in shRNA#1 and shRNA#2 group of HepG2/ADM and U87MG/DDP was decreased to compared to the control group of HepG2/ADM and U87MG/DDP (p>0.05)(Figure 5).
Furthermore, the phosphorylaion of AKT was downregulated as well, which meat the activity of PI3K/AKT pathway was attenuated.PI3K/AKT is a key signaling pathway downstream LPA receptor.To confirm the role of PEG2 in AGPS mediated drug resistance, we supplemented the AGPS silenced cells with PEG2 and EP receptor agonists and found that the drug sensitivity established by AGPS shRNA#1 and shRNA#2 was reversed (Figure 6).HepG2/ADM and glioma U87MG/DDP cell lines compared with HepG2 and U87MG cell lines, meanwhile, there was an increased drug sensitivity in human hepatic carcinoma HepG2/ADM and glioma U87MG/DDP AGPS silencing groups compared with HepG2/ ADM and U87MG/DDP cell lines.Further, there was a decreased drug sensitivity in human hepatic carcinoma HepG2/ADM and p<0.05, compared to the parent group; #p>0.05,compared to the control group; *: p<0.05, compared to the control group; *p<0.05,compared to the AGPS-shRNA#1 group;

Discussion
In order to investigate the role of AGPS in chemotherapy resistance, we used lentivirus to knockdown AGPS in several drug-resistant cancer cell lines in this study.In consistent with previous study that indicate AGPS play a role in the cancer pathogenesis, our MTS assay showed that drug sensitivity of the resistant cancer cells could be restored after AGPS silencing.Further investigations showed that after AGPS silencing, intracellular accumulation of Rh-123 increased, more cells were distributed to G0/G1 phase, and apoptosis rate increased after chemotherapy agent treatment.Therefore, we following these clues to explore the possible mechanism of AGPS mediated resistance.
One major mechanism that involved in drugs resistance is increased expression of multi-drug resistant MDR1, MRP1 and so on (Louisa et al., 2014).The increase of intracellular concentration of Rh-123 means silencing.Western blotting and RT-PCR showed that the expression of ABC family transporters MDR1, MRP1 and ABCG2 were down-regulated by AGPS silencing.Other mechanisms involve cell cycle promotion and antiapoptosis effects.Cell cycle analysis showed that AGPS silencing resulted in more cancer cells distributing to G0/G1 phase.We found that a key cell cycle modulator western blotting and RT-PCR showed that the expression of Bcl-2 and survivin, which are involved in cell apoptosis, were modulated by AGPS as well.The mechanisms that AGPS mediated the expression of these proteins could be through the PEG2 pathway as described below.
AGPS is an enzyme that catalyzes the synthesis of lipids.It has been reported that AGPS affects the level of LPA and LPAe, which could promote the cancer.
Here we showed that after AGPS silencing, level of LPA and LPAe were all down-regulated in HepG2/ play a role in the drug resistance of our cancer cells, we determined the downstream pathway.LPA and LPAe could activate the LPA receptor and further activate the PI3K/AKT pathway.Our western blotting assay showed that phosphorylation of AKT was downregulated with AGPS silencing.Furthermore, the activity of PLD, a phospholipase that transduces the activity of LPA receptor, was down-regulated as well.It has been reported that PLD plays a role in the drug resistance of cancer.LPA receptor stimulates PLD activation and then accumulates intracellular PA (phosphatidate).Elevation of PA could activate several pathways such as SK-1/S1P, Raf/ERK, survival.
Another lipid-related substrate that is affected by AGPS is PEG2.Our EIA assay showed that intracellular PEG2 was down-regulated after AGPS silencing.Moreover, when supplementing the AGPS silenced cancer cells with PEG2, the drug resistance reemerged, which further valid the role of PGE2 in AGPS mediated drug resistance.In cancer cells, PEG2 could bind to respective receptors to activate the downstream signaling pathway.There are 4 2009).It has been reported that in human hepatocellular carcinoma cells EP1 receptor transactivates EGFR/MET receptor tyrosine kinases and enhances invasiveness (Han et al., 2006;Gillibert-Duplantier et al., 2007).U87MG has been reported to express both EP2 and EP4 (Kambe et al., cancer cells, we used selective agonist to stimulate the respective receptor.Selective EP1 and EP3 agonist 19(R)-hydroxy PGE1 could restore drug resistance of HepG2/ADM, while selective EP2 agonist CAY10399 and selective EP4 agonist L-902,688 had similar effect on U87MG.PGE2 is the key in COX-2 regulating the express of multidrug resistance proteins in cancer cells (Woodward et al., 1993;Tani et al., 2001;Takaoka et al., 2008;Henderson et al., 2011;Hu et al., 2013;Kashiwagi et al., 2013).COX-2/PGE2 pathway could enhance the cancer cell cycle.Furthermore, PGE2 could regulate the expression of Bcl-2 and survivin to prevent cancer cells from apoptosis (Krysan et al., 2004).These literature reports are consistent with our observation mentioned above.PI3K/AKT is key pathway downstream EP receptors as well.Hence, the downregulation of Akt phosphorylation mentioned above might be the joint action of modulation to LPA receptor and EP receptor.
In summary, we found that AGPS overexpression could be a causation of chemotherapy agent resistance of cancer cells.AGPS silencing could lead to drug uptake and cell cycle arrest and apoptotic cell death.Our study suggested that lipid signaling pathways was critical for AGPS mediated drug resistance.

Figure 1 .
Figure 1.The Expression of AGPS in Human Gliomaand Hepatic Carcinoma Cell Lines.A) Western blotting results showed that there was an increased protein expression of AGPS in human hepatic carcinoma HepG2/ADM and glioma U87MG/DDP cell lines compared with HepG2 and U87MG cell lines, meanwhile, there was a decreased protein expression of AGPS in human hepatic carcinoma HepG2/ADM and glioma U87MG/DDP AGPS silencing groups compared with internal control.B) Realtime PCR results showed that there was an increased mRNA expression of AGPS in human hepatic carcinoma HepG2/ADM and glioma U87MG/DDP cell lines compared with HepG2 and U87MG cell lines, meanwhile, there was a decreased mRNA expression of AGPS in human hepatic carcinoma HepG2/ADM and glioma U87MG/DDP AGPS silencing groups compared with HepG2/ADM and U87MG/DDP p<0.05, compared to the parent group; #p>0.05,compared to the control group; *p<0.05,compared to the control group

Figure 4 .
Figure 4.The effect of AGPS Silencing on Expression of Drug Resistance Related Gene in Human Glioma and hepatic Carcinoma Cell lines.A) Western blotting results showed that there was a decreased protein expression of MDR1, p-AKT in human hepatic carcinoma HepG2/ADM and glioma U87MG/DDP B) Realtime p-AKT in human hepatic carcinoma HepG2/ADM and glioma U87MG/DDP AGPS silencing groups compared with HepG2/ADM and p<0.05, compared to the control group

Figure 6 .
Figure 6.The Effect of AGPS Silencing on Drug Sensitivity Mediated by PEG2 and EP receptor in Human Glioma and Hepatic Carcinoma Cell Lines.The results showed that there was a decreased drug sensitivity in human hepatic carcinoma