Literature DB >> 25733813

Proteomic response to 5,6-dimethylxanthenone 4-acetic acid (DMXAA, vadimezan) in human non-small cell lung cancer A549 cells determined by the stable-isotope labeling by amino acids in cell culture (SILAC) approach.

Shu-Ting Pan1, Zhi-Wei Zhou2, Zhi-Xu He3, Xueji Zhang4, Tianxin Yang5, Yin-Xue Yang6, Dong Wang7, Jia-Xuan Qiu1, Shu-Feng Zhou8.   

Abstract

5,6-Dimethylxanthenone 4-acetic acid (DMXAA), also known as ASA404 and vadimezan, is a potent tumor blood vessel-disrupting agent and cytokine inducer used alone or in combination with other cytotoxic agents for the treatment of non-small cell lung cancer (NSCLC) and other cancers. However, the latest Phase III clinical trial has shown frustrating outcomes in the treatment of NSCLC, since the therapeutic targets and underlying mechanism for the anticancer effect of DMXAA are not yet fully understood. This study aimed to examine the proteomic response to DMXAA and unveil the global molecular targets and possible mechanisms for the anticancer effect of DMXAA in NSCLC A549 cells using a stable-isotope labeling by amino acids in cell culture (SILAC) approach. The proteomic data showed that treatment with DMXAA modulated the expression of 588 protein molecules in A549 cells, with 281 protein molecules being up regulated and 306 protein molecules being downregulated. Ingenuity pathway analysis (IPA) identified 256 signaling pathways and 184 cellular functional proteins that were regulated by DMXAA in A549 cells. These targeted molecules and signaling pathways were mostly involved in cell proliferation and survival, redox homeostasis, sugar, amino acid and nucleic acid metabolism, cell migration, and invasion and programed cell death. Subsequently, the effects of DMXAA on cell cycle distribution, apoptosis, autophagy, and reactive oxygen species (ROS) generation were experimentally verified. Flow cytometric analysis showed that DMXAA significantly induced G1 phase arrest in A549 cells. Western blotting assays demonstrated that DMXAA induced apoptosis via a mitochondria-dependent pathway and promoted autophagy, as indicated by the increased level of cytosolic cytochrome c, activation of caspase 3, and enhanced expression of beclin 1 and microtubule-associated protein 1A/1B-light chain 3 (LC3-II) in A549 cells. Moreover, DMXAA significantly promoted intracellular ROS generation in A549 cells. Collectively, this SILAC study quantitatively evaluates the proteomic response to treatment with DMXAA that helps to globally identify the potential molecular targets and elucidate the underlying mechanism of DMXAA in the treatment of NSCLC.

Entities:  

Keywords:  DMXAA; SILAC; apoptosis; autophagy; cell cycle; non-small cell lung cancer

Mesh：

Substances：

Year:  2015        PMID： 25733813      PMCID： PMC4338781          DOI： 10.2147/DDDT.S76021

Source DB:  PubMed          Journal:  Drug Des Devel Ther        ISSN： 1177-8881            Impact factor:   4.162


Introduction

Lung cancer is the most common cancer and the leading cause of cancer-related death in humans worldwide.1,2 There were about 1.8 million new cases diagnosed with lung cancer in 2012, accounting for 12.9% of the total cases of cancer.1,2 Small-cell lung cancer and non-small cell lung cancer (NSCLC) are the two major types of lung cancer. NSCLC is the most common type, accounting for 70%–85% of all cases of lung cancer. In the USA, there were 207,339 new cases of lung cancer and 156,953 deaths resulting from lung cancer in 2011,3 and it is estimated that there were 224,210 new cases of lung cancer and 159,260 deaths due to lung cancer in 2014.4 In the People’s Republic of China, lung cancer is the most common cancer and the leading cause of cancer-related death, with a skyrocketing increase in incidence and mortality rates.2,5 In 2009, the incidence rate for lung cancer was about 53.57/100,000, accounting for 18.74% of overall new cases of cancer; the mortality rate for lung cancer was about 45.57/100,000, accounting for 25.24% of cancer-related deaths.2,5 Current therapies for lung cancer include surgery, chemotherapy, radiotherapy, immunotherapy, and targeted therapy, which are used alone or in combination. However, the therapeutic outcome for lung cancer is often disappointing, in particular for advanced NSCLC,6,7 due to the poor response to current therapeutics, drug resistance, and severe side effects, which highlights an urgent need for discovery of efficacious and safe new agents for the treatment of NSCLC. 5,6-Dimethylxanthenone-4-acetic acid (DMXAA, Figure 1), also known as vadimezan and ASA404, is a vascular-disrupting agent that reduces the blood supply to tumoral tissue, resulting in tumor regression.8,9 However, the molecular targets and exact mechanisms of action of DMXAA are elusive so far. DMXAA shows inhibitory effects against several protein kinases, with the most potent effects being on the vascular endothelial growth factor receptor tyrosine kinase family.10,11 DMXAA is a potent inducer of tumor necrosis factor-α and activates host immune effectors that assist in killing cancer cells.10 DMXAA induces rapid vascular collapse and subsequent tumor hemorrhagic necrosis via induction of apoptosis in tumor vascular endothelial cells and indirect vascular effects induced by various cytokines, in particular, tumor necrosis factor-α, serotonin, and nitric oxide.10 The pharmacokinetics of DMXAA has also be investigated. In cancer patients, DMXAA concentration-time profiles are well described by a three-compartment model with saturable elimination.12 Body surface area and sex are significant covariates on the volume of distribution of the central compartment and the maximum elimination rate, respectively.12 DMXAA is extensively metabolized in human liver microsomes and cancer patients. There are two major metabolites of DMXAA, ie, DMXAA acyl glucuronide and 6-hydroxymethyl-5-methylxanthenone-4-acetic acid (6-OH-MXAA). Cytochrome P450 1A2 is responsible for the conversion of DMXAA to 6-OH-MXAA, with an apparent Km of 6.2 μM and a Vmax of 0.014 nmol/minute/mg.13 DMXAA is also extensively metabolized by uridine 5′-diphospho-glucuronosyltransferase 1A2 (UGT1A2) and UGT2B7, with a greater contribution from UGT2B7.14 DMXAA has been tested mainly in the treatment of NSCLC, and also in prostate cancer and human epidermal growth factor receptor 2-negative breast cancer.15–20 In these clinical studies, DMXAA is used alone and more often in combination with other cytotoxic drugs. A Phase II clinical trial showed that DMXAA in combination with carboplatin and paclitaxel had a potent anticancer effect in NSCLC patients.15 This triple combination therapy prolonged the survival of about 5 months when compared with the monotherapy.15 However, the Phase III clinical trial conducted by Lara et al showed that the triple chemotherapy of DMXAA with carboplatin and paclitaxel failed to improve the efficacy of the monothera-py.16 This may be due to the complexity of the mechanisms of action of DMXAA. DMXAA has been shown to target the stimulator of interferon gene (STING) pathway and this effect is only observed in mice but not in humans.21–23 However, this cannot provide a convincing explanation for the failure of DMXAA in the Phase III trial in NSCLC patients. Therefore, it is of great importance to globally understand and uncover the molecular targets and related signaling pathways involved in the anticancer effect of DMXAA and DMXAA-based combination therapies.
Figure 1

Chemical structure of 5,6-dimethylxanthenone 4-acetic acid (DMXAA).

So far, there are many studies on the mechanisms of action of DMXAA in the treatment of NSCLC, showing that DMXAA can activate STING-dependent innate immune pathways and mitogen-activated protein kinases and inhibit vascular endothelial growth factor receptor.11,21–23 However, there is a lack of evidence to depict the global molecular targets and related signaling pathways for the NSCLC cell killing effects of DMXAA, such as cell proliferation, programmed cell death, and cell migration and invasion. Notably, targeting cell cycle progression, apoptosis, autophagy, and epithelial to mesenchymal transition (EMT) has been proposed for treatment of NSCLC.24 Therefore, an approach that can evaluate cellular proteomic responses to the DMXAA is important for the optimal treatment of NSCLC. Stable-isotope labeling by amino acids in cell culture (SILAC) is a practical and powerful approach to uncovering the global proteomic response to drug treatment and other interventions.25 In particular, it can be used to systemically and quantitatively assess the target network of drugs, to evaluate drug toxicity, and to identify new biomarkers for the diagnosis and treatment of important diseases, including NSCLC.25–27 In this regard, we investigated the molecular targets of DMXAA in A549 cells using a combination of proteomic and functional approaches, with a focus on cell cycle distribution, apoptosis, autophagy, and redox homeostasis.

Materials and methods

Chemicals and reagents

DMXAA (purity ≥ 98%), 13C6-L-lysine, L-lysine, 13C 156 N4-L-arginine, L-arginine, RNase A, propidium iodide, Dulbecco’s phosphate-buffered saline (PBS), heat-inactivated fetal bovine serum (FBS), dialyzed FBS, and Roswell Park Memorial Institute (RPMI)-1640 medium for SILAC were purchased from Sigma-Aldrich Co. (St Louis, MO, USA). The 5-(and 6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-H2DCFDA) was sourced from Invitrogen Inc. (Carlsbad, CA, USA). A FASP™ protein digestion kit was purchased from Protein Discovery Inc. (Knoxville, TN, USA). RPMI-1640 medium for general cultural use was obtained from Corning Cellgro Inc. (Herndon, VA, USA). The polyvinylidene difluoride membrane was purchased from EMD Millipore Inc. (Bedford, MA, USA). Proteomic quantitation kits for acidification, desalting, and digestion, ionic detergent compatibility reagent, a Pierce bicinchoninic acid protein assay kit, and Western blotting substrate were obtained from Thermo Fisher Scientific Inc. (Waltham, MA, USA). Primary antibodies against human cytochrome c, cleaved caspase 3, microtubule-associated protein 1A/1B-light chain 3-I (LC3-I), LC3-II, and beclin 1 were all purchased from Cell Signaling Technology Inc. (Beverly, MA, USA). The antibody against human β-actin was obtained from Santa Cruz Biotechnology Inc. (Dallas, TX, USA).

Cell line and cell culture

The A549 NSCLC cell line was obtained from American Type Culture Collection (Manassas, VA, USA) and cultured in RPMI-1640 medium supplemented with 10% heat-inactivated FBS. The cells were maintained at 37°C in a 5% CO2/95% air humidified incubator. DMXAA was dissolved in dimethyl sulfoxide at a stock concentration of 20 mM and stored at −20°C. It was freshly diluted to predetermined concentrations with culture medium. The final concentration of dimethyl sulfoxide was 0.05% (v/v). The control cells received the vehicle only.

Quantitative proteomic study using SILAC

Quantitative proteomic experiments were performed using a SILAC-based approach as described previously.25,26,28 Briefly, A549 cells were cultured in RPMI-1640 medium (for SILAC) with (heavy) or without (light) stable isotope-labeled amino acids (13C6 L-lysine and 13C6 15N4 L-arginine) and 10% dialyzed FBS. A549 cells cultured in heavy medium were treated with 10 μM DMXAA for 24 hours after six cell doubling times. After treatment with DMXAA, A549 cell samples were harvested and lysed with hot lysis buffer (100 mM Tris base, 4% sodium dodecyl sulfate [SDS], and 100 mM dithiothreitol). The cell lysate was denatured at 95°C for 5 minutes and then sonicated for 3 seconds with six pulses. The samples were then centrifuged at 15,000× g for 20 minutes at room temperature and the supernatant was collected. The protein concentration was determined using ionic detergent compatibility reagent. Subsequently, equal amounts of heavy and light protein samples were combined to reach a total volume of 30–60 μL containing 300–600 μg protein. The combined protein sample was digested using an filter-aided sample prep (FASP™) protein digestion kit. After digestion, the resulting sample was acidified to a pH of 3 and desalted using a C18 solid-phase extraction column. The samples were then concentrated using a vacuum concentrator at 45°C for 120 minutes, and the peptide mixtures (5 μL) were subjected to the hybrid linear ion trap (LTQ Orbitrap XL™, Thermo Fisher Scientific Inc.). Liquid chromatography-tandem mass spectrometry was performed using a 10 cm long, 75 μm (inner diameter) reversed-phase column packed with 5 μm diameter C18 material having a pore size of 300 Å (New Objective Inc., Woburn, MA, USA) with a gradient mobile phase of 2%–40% acetonitrile in 0.1% formic acid at 200 μL per minute for 125 minutes. The Orbitrap full mass spectrometry scanning was performed at a mass (m/z) resolving power of 60,000, with positive polarity in profile mode (M + H+). The peptide SILAC ratio was calculated using MaxQuant version 1.2.0.13. The SILAC ratio was determined by averaging all peptide SILAC ratios from peptides identified of the same protein. The proteins were identified using Scaffold 4.3.2 (Proteome Software Inc., Portland, OR, USA) and the pathway was analyzed using ingenuity pathway analysis (IPA) from QIAGEN Inc. (Redwood City, CA, USA).

Cell cycle analysis using flow cytometry

The effect of treatment with DMXAA on the cell cycle was determined by flow cytometry as described previously.29 Briefly, A549 cells were treated with DMXAA at concentrations of 0.1, 1, and 10 μM for 24 hours. In separate experiments, A549 cells were treated with 10 μM DMXAA over a 72-hour period. The cells were suspended, fixed in 70% ethanol, washed in PBS, and resuspended in 1 mL of PBS containing 1 mg/mL RNase A and 50 μg/mL propidium iodide. The cells were incubated in the dark for 30 minutes at room temperature. Next, the cells were subject to cell cycle analysis using a flow cytometer (BD LSR II Analyzer; Becton Dickinson Immunocytometry Systems, San Jose, CA, USA). The flow cytometer collected 10,000 events for analysis.

Measurement of intracellular reactive oxygen species (ROS) levels

Intracellular levels of ROS were measured by a fluorometer using CM-H2DCFDA according to the manufacturer’s instructions. Cell-permeant CM-H2DCFDA passively diffuses into cells and is retained in the cells after cleavage by intracellular esterases. Upon oxidation by ROS, the nonfluorescent CM-H2DCFDA is converted to highly fluorescent CM-DCF. Briefly, A549 cells were seeded into a 96-well plate at a density of 1×104 cells/well. After treatment with DMXAA at 0.1, 1, and 10 μM for 48 hours, the cells were incubated with CM-H2DCFDA at 5 μM in PBS for 30 minutes. The fluorescence intensity was detected at wavelengths of 485 nm (excitation) and 530 nm (emission). The control cells were treated with vehicle only (0.05% dimethyl sulfoxide, v/v).

Western blotting analysis

A549 cells were washed with pre-cold PBS after 24-hour treatment with DMXAA at 0.1, 1, and 10 μM, lysed with radioimmunoprecipitation (RIPA) buffer containing the protease inhibitor and phosphatase inhibitor cocktails, and centrifuged at 3,000× g for 10 minutes at 4°C. Protein concentrations were measured using a Pierce bicinchoninic acid protein assay kit. An equal amount of protein sample (30 μg) was resolved by SDS polyacrylamide gel electrophoresis (PAGE) sample loading buffer and electrophoresed on 12% SDS-PAGE minigel after thermal denaturation at 95°C for 5 minutes. The proteins were transferred onto an Immobilon polyvinylidene difluoride membrane at 400 mA for 1 hour at 4°C. Membranes were blocked with skim milk and probed with the indicated primary antibody overnight at 4°C and then blotted with appropriate horseradish peroxidase-conjugated secondary anti-mouse or anti-rabbit antibody. Visualization was performed using a ChemiDoc™ XRS system (Bio-Rad, Hercules, CA, USA) with enhanced chemiluminescence substrate, and the blots were analyzed using Image Lab 3.0 (Bio-Rad). The protein level was normalized to the matching densitometric value of the internal control β-actin.

Statistical analysis

The data are presented as the mean ± standard deviation (SD). Comparisons of multiple groups were evaluated by one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison procedure. Values of P<0.05 were considered to be statistically significant. Assays were performed at least three times independently.

Results

Overview of proteomic response to DMXAA treatment in A549 cells

To reveal the potential molecular targets of DMXAA in the treatment of NSCLC, we conducted proteomic experiments to evaluate the interactome of DMXAA in A549 cells. There were 588 protein molecules identified as potential molecular targets of DMXAA in A549 cells, with 281 protein molecules being upregulated and 306 protein molecules being downregulated (Table 1). Subsequently, these proteins were subjected to IPA. The results showed that 256 signaling pathways and 184 cellular functional proteins were regulated by DMXAA in A549 cells (Tables 2 and 3). These functional proteins were involved in a number of important cellular processes, including cell proliferation, redox homeostasis, cell metabolism, cell migration and invasion, cell survival, and cell death. The signaling pathways included the G1 and G2 checkpoint regulation pathways, the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR)signaling pathway, the 5′-AMP-activated protein kinase (AMPK) signaling pathway, the nuclear factor erythroid 2-related factor 2 (Nrf2)-mediated oxidative stress response pathway, the epithelial adherens junction signaling pathway, regulation of the epithelial-mesenchymal transition signaling pathway, the nuclear factor-κB signaling pathway, and the apoptosis signaling pathway. The IPA results showed that the top ten targeted signaling pathways were the eukaryotic initiation factor (eIF) 2 signaling pathway, mTOR signaling pathway, eIF4 and p70S6K signaling pathway, epithelial adherens junction signaling pathway, remodeling of epithelial adherens junctions pathway, Nrf2-mediated oxidative stress response signaling pathway, RhoA signaling pathway, integrin signaling pathway, Rho-mediated regulation of actin-based motility signaling pathway, and Fcγ receptor-mediated phagocytosis signaling pathway (Table 2).
Table 1

The 588 protein molecules regulated by DMXAA (5,6-dimethylxanthenone 4-acetic acid) in A549 cells

Protein ID/symbolMolecular weight (kDa)Normalized heavy/light ratio
Q5VXJ593.1150.036825
P3C2G165.710.052938
K1C1058.8260.090984
K1C962.0640.11756
CB06773.2350.14108
1433F28.2180.22201
K1C1451.5610.33079
K2C166.0380.3831
F5H2G252.9090.43495
K2C6B60.0660.4391
MANF20.70.52102
F8VVM236.1610.56761
APMAP32.1610.59766
KRT8555.8020.59966
B4DS1364.8050.60022
F5H6E318.4670.62881
E9PEU447.2040.65669
E9PF8025.30.65828
F5H3I434.5570.66102
AL3A254.8470.71801
RAB1B22.1710.71899
F5GY6528.4940.71909
ISG1517.8870.71998
ACACA257.240.72374
H90B458.2640.72926
K22E65.4320.73358
F5H0X650.4350.73677
F8VSA65.86680.73947
AK1C136.7880.74328
RLA111.5140.74644
B4DKS837.2550.75044
TMOD339.5940.75127
BASP122.6930.75558
COX225.5650.77367
MARCS31.5540.77387
F8WD96300.77581
F5GX1126.5050.77779
K2C853.7040.78215
K1C1848.0570.7824
FKB1A11.9510.78264
B4E1K733.3370.7835
Q5TCU6258.080.784
RL2917.7520.78622
F8W7P772.7160.78997
CHM4B24.950.79348
E9PNH113.1630.79972
C9JW378.83330.79997
CLCC139.8370.80061
B4E24114.2030.80205
E9PH2925.8380.80339
NNMT29.5740.80458
E7ESM6101.210.80816
RL3612.2540.80826
ETFB27.8430.81589
F5GWR226.630.81598
SC61B9.97430.81767
ALDR35.8530.81844
AK1C236.7350.81875
GRHPR35.6680.81892
PON237.9960.82002
B7Z25447.8370.82473
FRIL20.0190.82759
ATP5H18.4910.82832
B4DNJ543.3680.82967
HINT113.8020.83028
D6RDN34.12860.83128
S10A411.7280.83154
CUL4B102.30.83195
IDHC46.6590.83275
E9PDQ841.4380.83332
F8W91437.1440.83563
ANXA435.8820.83679
S38A245.1780.837
F5H3T852.3050.84001
CDC3744.4680.84082
A6NKT156.360.84176
B4E0X866.2310.84218
SMRD255.2380.84281
F8WF8114.2750.84585
RRBP1108.630.84762
SFPQ76.1490.85165
B4DRT417.3260.85394
B4DT4330.0250.85426
KHDR144.0270.8553
E9PPQ520.7230.85913
D6RFI021.5040.8613
CDC4221.2580.86162
COX4119.5760.86224
EF1A250.470.86292
SCMC151.3540.86294
C9J1T29.44590.86457
GBG128.00610.8649
E9PKD147.4310.86946
C9JPV112.0630.86969
BLVRB22.1190.86996
F5H4L532.7340.87025
K2C751.3850.87164
FKBP325.1770.87335
F5H1J138.7390.87421
ECH135.8160.87427
EFTU49.5410.87443
AL1A154.8610.8745
RAB5C23.4820.8745
D6RFH414.8460.87485
HSPB122.7820.8751
G6PD59.2560.87609
RB11A24.3930.87617
HCD226.9230.8781
CALX67.5670.87892
B1AHC764.0750.87955
F5H7G757.3420.88015
CAZA132.9220.88192
S100P10.40.88323
AK1C336.8530.88425
CALR48.1410.88565
NOP288.9720.88741
HMGA111.6760.88955
F8W6V853.1160.88964
MIF12.4760.89038
USMG56.45750.89172
PSB222.8360.89303
CBR130.3750.8935
SF3B344.6050.89353
PRDX517.0310.89389
LRRF182.6880.89483
B4DQJ851.8720.89714
CCNK41.2930.9026
FSCN154.5290.90332
CS02175.3560.90345
B4E02262.8780.90614
G3V5P416.9160.90639
PDIA472.9320.90743
KYNU52.3510.90767
PRS6B43.5070.90881
D6RF6237.1110.90967
S10A610.180.91026
F5GYB819.4710.91104
B3KUK219.730.91118
PPIB23.7420.91132
D6REM688.3580.91208
F2Z3A540.1270.91256
R13AX12.1340.91339
SRSF122.460.91352
GRP7872.3320.91353
F2Z2J928.850.91353
DNJA245.7450.91354
RS1916.060.91521
RL23A17.6950.91665
B7Z2V637.7510.91676
TMED927.2770.91682
IMUP8.51150.91694
ENPL92.4680.917
LKHA457.2990.91952
SERC35.1880.91971
B1AM7713.4750.92027
F5GYG976.2330.92196
D6RF4412.640.92222
B4E0R6109.360.9266
MDHM35.5030.92738
RS1517.040.92751
AL3A150.3940.92795
ATPB56.5590.92872
Q5T8U321.5450.93039
LGUL19.0430.93096
A8K31859.1770.93162
CH1010.9320.93233
CYTB11.1390.9324
C8KIM047.2670.93263
G3V5Q127.1250.93343
RS1416.2730.93344
A6NN0112.1460.93348
B7Z6M165.6320.93372
B3KUB431.8570.93524
NUDC38.2420.9354
FAS273.420.93581
F5GWY258.6150.93612
SMD213.5270.93633
MDHC36.4260.93699
B4DIT768.6480.93774
D6RFJ847.2910.9383
E9PRQ615.1590.93879
FIS116.9370.93901
SAHH47.7160.93913
RLA211.6650.93935
AATM47.5170.93946
INF2134.620.93976
ATPA59.750.93996
HNRPG42.3310.94001
EIF112.7320.94212
C9JPM414.5530.94439
ARF120.6970.94471
FLNB275.660.945
NSF1C28.5220.94549
CDV322.0790.94575
E7EWF145.4980.94724
NDKB30.1370.94913
B1AH7716.7750.95
H2A1J13.9360.95043
GDIR123.2070.95062
LDHB36.6380.95153
PA1B325.7340.95246
HNRPU88.9790.95295
G3V3I116.6450.95297
F2Z3F829.2450.95333
B4DVU388.1610.95514
HSP7494.330.9556
B4DKM527.4780.95717
SPRE28.0480.9575
B4DEM757.6450.95821
F5GX3913.6310.95862
F5GZ2785.640.96103
PRDX122.110.96121
PTBP157.2210.96124
F5H66783.2670.9617
C9JLU116.9960.96193
B4DR7044.8120.96206
SMD113.2810.9621
E7EU1294.8630.96289
E7ETK530.6110.96294
B4DXW142.0030.9639
RCN138.890.96394
PABP161.180.96407
ANXA138.7140.96409
PARK719.8910.96461
H1221.3640.96469
B7Z6A414.8540.96516
F8VNT914.2650.96517
E9PKZ022.3890.96545
F5H1S218.8450.96676
LRC5934.930.96685
UGDH55.0230.96807
TPIS26.6690.96808
ROA237.4290.96842
VDAC130.7720.96869
KPYM57.9360.96936
ANXA535.9360.96942
UBE2N17.1380.96961
ENOA47.1680.97022
S10AA11.2030.97031
HN116.0140.97056
PPIA18.0120.9708
RL1217.8180.97093
F5GZ1699.2710.97167
TCPA60.3430.97176
B4DRF436.430.97231
ILF243.0620.9728
PDCD514.2850.97482
XPO222.670.97527
RLA034.2730.9763
B4DZP445.0040.97635
F8W81050.5290.97677
B3KQT954.1020.97689
E5RJR518.720.97695
CAP151.9010.97716
A8K3Z344.7840.97817
B4DUR855.6740.9785
C9JV5713.0250.97853
E7EMJ631.4550.97864
RS17L15.550.97906
F5H8J375.1770.97909
RS922.5910.97946
LDHA36.6880.97949
HNRDL27.1910.97974
B7Z4V272.40.98075
CKAP466.0220.9808
PDIA157.1160.98205
AT2A2109.730.98343
LPPRC157.90.98408
TALDO37.540.98454
PHB233.2960.98512
NBAS254.810.9852
F5GY5032.8950.98539
RS4X29.5970.98585
B4DPJ854.8670.98615
SYEP170.590.98633
HNRPC27.8210.98649
EF1A150.140.98718
D6RG1325.6080.98723
AK1BA36.0190.98779
EF1G50.1180.98818
NAMPT55.520.98886
F8VTY841.7460.98925
C9JTK612.3020.9895
TCPE59.670.98993
RS1817.7180.99091
FKBP451.8040.99111
MOES67.8190.99187
ARP244.760.99233
RS522.8760.99268
RL1120.1240.99323
F8VRG36.43520.99333
C9JZ2022.270.99398
C9JB507.8880.99449
DX39A49.1290.9946
F5GYN428.050.99464
B7Z79547.7090.99535
B7Z4T954.8040.99554
TERA89.3210.99599
TCP414.3950.99621
B4DGN546.5750.99641
DBPA31.9470.99662
ADT232.8520.99664
CALM16.8370.99736
PGK144.6140.99895
ECHM31.3870.99951
MARE129.9990.99998
RS722.1271
API549.4961.0009
1433G28.3021.0011
IQGA1189.251.0015
B4DDF764.1811.0018
LETM183.3531.0024
E7EWI934.1021.0027
D6RAS323.3831.0035
B4DLR822.7931.0053
PROF115.0541.0054
ALO17118.431.0056
RS326.6881.0064
RAB1022.5411.0066
XRCC582.7041.0076
UBA1117.851.0076
CH6061.0541.0081
A6NLM816.2451.0083
F8W0A940.7931.0086
NONO54.2311.0087
C9J7S319.9691.0092
C1QBP31.3621.0099
D6RAN420.8741.0103
PDC6I96.0221.0107
B4E3C217.0941.0115
MAP485.2511.0117
GSTP123.3561.0129
RL10A24.8311.0129
ACTS42.0511.0141
DHX9140.961.0146
PCBP137.4971.0146
F5H1W055.9071.0147
H3315.3281.0156
RS628.681.0164
G3V2798.20331.0169
D6RBT823.8621.0179
PRDX625.0351.0193
CAND1136.371.0201
TCTP19.5951.0203
B4DDG114.1211.0204
C9JCK518.6071.0216
PSB322.9491.022
MAP1B270.631.0222
CN16628.0681.0223
ACTN4104.851.0228
E9PQD725.2111.0232
E7EX5315.7221.0241
B7ZAT252.7171.026
PAIRB42.4261.0261
E9PCY747.0871.0262
HNRPK48.5621.0268
B8ZZQ611.7581.0272
ACOC98.3981.0274
IF5A116.8321.0284
B4E33539.2261.0286
PCNA28.7681.0288
A6NL939.53551.0294
FLNA280.011.0304
Q5JR9521.8791.0308
C9J9K329.5051.0309
E7ETA034.0281.0312
TBB4B49.831.0315
COF118.5021.0318
CLIC126.9221.0322
RINI49.9731.0325
1433E29.1741.033
C9JF496.65661.0331
RS1317.2221.0332
GUAA76.7151.0333
AN32B22.2761.0342
E9PGI820.181.0342
CLCA23.6621.0345
DYHC1532.41.035
E9PMD728.8981.035
1433T27.7641.0354
E9PEN329.3531.0367
CLH1187.891.0371
E9PFH841.8241.0377
G3V3T321.1261.0378
GDIB50.6631.0384
PALLD73.3211.04
B1ALW19.45191.0404
A8MUD924.4331.0407
NDKA17.1491.0422
Q2YDB716.5411.0422
S10AB11.741.0434
QCR248.4421.0437
RS1018.8981.0446
B7Z9L052.331.0449
G3P36.0531.046
E7EQR465.5781.046
GBLP35.0761.0464
1433B27.851.0465
F5H0N037.4061.0468
G6PI63.1461.0478
E7EUG171.1781.0499
EF1B24.7631.0504
H411.3671.0504
BTF317.6991.0505
PGRC223.8181.0508
F8W11824.6941.051
H3215.3881.0514
DYL210.351.0516
E9PEC032.8191.0516
ALDOA39.421.0522
EF295.3371.0523
TBA1C49.8951.0528
IPYR32.661.054
F8VZJ215.0161.0547
PRS1044.1721.0559
F8W7K3282.141.0567
H2B1L13.9521.0571
G3V5B319.1541.058
HSP7170.0511.0588
B2REB830.9921.0591
B4DR6341.1671.0595
RL2214.7871.0599
ACTN1103.061.06
B4E2S739.8111.0605
G3V5M39.20441.0608
C9JU5613.3361.061
B7Z1N635.4231.0625
CAN279.9941.0631
F8VWC518.0911.0641
SODC15.9361.0644
TBA1B50.1511.0646
RS1616.4451.065
C9JD329.66941.0658
VINC116.721.0668
RS2513.7421.0671
A4D17720.8111.0685
Q5HYE720.421.0694
GLRX337.4321.0699
B4E3E871.3541.0717
LEG114.7161.0729
A8K8G022.9641.0736
TPM428.5211.0744
F5H89774.2671.0744
E9PJH815.4611.0749
E9PNR918.2971.0749
F8W18125.8921.0756
AIBP20.431.0758
HSP7C70.8971.076
CPNS128.3151.0764
ANXA238.6041.0765
1433Z27.7451.077
HS90A84.6591.0771
C9JKI315.9281.0779
HNRPL64.1321.0786
NPM29.4641.0788
AHNK629.091.079
F8VYX648.461.0796
EF1D31.1211.0797
GTF2I107.971.0801
D6R9P330.3021.0806
PSME128.7231.0815
Q8N1C059.5491.0838
ADT332.8661.0863
B4DY6619.1521.087
PLEC531.781.0874
E7EX8165.8811.0877
C9J7129.79821.088
F6RFD515.3971.0883
PSME227.4011.09
HS90B83.2631.0909
F5H4D631.4511.0914
B4DR3158.1621.0921
RS1214.5151.0928
TADBP27.9721.0948
B6EAT937.2771.0972
RS15A14.8391.0986
RS2013.3731.1055
B4E3S041.6031.108
RS287.84091.1109
LMNA74.1391.1139
ROA129.3861.1144
PGRC121.6711.1155
C9JHS65.65641.1161
Q5TA0120.9381.1186
TIF1B79.4731.1193
HNRPM73.621.1205
RL1923.4661.1208
TXNL132.2511.1218
RAN24.4231.1235
F8W7C618.5921.1241
G3XAI4119.771.1248
IF4A146.1531.1252
VIME53.6511.1263
F5H0T159.7771.1272
C9JFR711.3331.1279
E7EPB314.5581.1287
DDX569.1471.1298
F5H6Q213.7891.1306
ITB187.4451.1347
B4DP2429.4851.135
TAGL222.3911.1361
B3KS3141.9521.1361
SPTB2251.391.1366
G3V53144.061.1371
PYGB96.6951.141
PRP8273.61.1413
DNJA144.8681.1421
TBB2B49.9531.1428
HNRPQ58.7351.1433
B7Z9C435.2531.1444
IMB197.1691.1455
F8W0G416.6371.1455
KINH109.681.1465
F8WBE58.88091.1471
METK243.661.1482
PTRF33.3621.1512
K6PP85.5951.1514
B7Z2S560.0211.1531
B2RDM236.1771.1541
B4DSC014.5031.1554
ECHA82.9991.1555
F8VPF314.4361.1565
B0V3J026.1011.159
F8VR509.72611.1619
B3KTM628.0441.1649
BAF10.0581.1652
IF2A36.1121.1676
Q5H90755.7951.1682
TPM132.8761.1693
IF626.5991.1727
SYTC83.4341.1736
Q75MH112.9851.1814
B4DZI899.0451.1821
Q5T4L47.35641.1834
Q5T09313.4391.1877
H3115.4041.1882
Q5VU5927.1741.1982
TBB4A49.5851.1987
MYH9226.531.2006
IMA257.8611.2074
GLSK65.4591.2081
Q5T7C418.3111.2156
C9JFK934.7591.2186
G3V15370.3531.223
PSA720.1931.2257
E5RHS526.6351.2275
1433S27.7741.2337
F2Z3D08.11111.238
PSA429.4831.2384
B7Z8D314.7611.2419
B4E2Z355.9391.2474
E9PP2116.941.25
ROA030.841.2609
B4DMT533.241.2628
C9K0U814.1311.2706
UBP15112.421.2708
ML12A19.7941.2917
LASP129.7171.2975
A8MUB148.3281.2985
APOL237.0921.304
HMOX132.8181.3048
PSA327.6471.312
TYB105.02561.3172
Q5T4B65.11271.3249
C9IZ4117.0541.3258
F5H4W044.9331.331
LAT155.011.3337
PSB126.4891.335
B4DPJ617.4731.34
F5H0C834.7621.3474
TBB350.4321.3583
TBAL345.5171.4041
DUT17.7481.4353
F8WC1547.8191.4365
PLIN328.1571.4972
F5GWR922.2181.499
C9JL855.70441.5138
E7ERP831.8011.5322
AAAT56.5981.6533
RBM317.171.7162
B4DP1116.4761.8007
MYOF179.551.9046
SRP1414.571.9407
PLP216.691NaN
HNF4A52.784NaN
YLPM1241.64NaN
E7EWZ6111.13NaN
Q5TCU332.814NaN
Table 2

Signaling pathways for target proteins regulated by DMXAA (5,6-dimethylxanthenone 4-acetic acid) in A549 cells

Ingenuity canonical pathways−LogPMolecules
eIF2 signaling2.11E01EIF3C, AKT2, RPS3A, RPS27, RPS2, RPL17, RPS8, RPL23, EIF3E, RPL9, RPL7, RPL15, RPL14, RPL8, EIF3F, RPL7A, RPL28, RPS26, RPL5, RPL10, PPP1CA, RPL31, RPL18, RPL13, RPSA
mTOR signaling7.52E00AKT2, EIF3C, RPS3A, RPS27, RPS2, RPS8, EIF3E, EIF3F, PPP2R1A, RPS26, RHOA, RPSA, EIF4B
Regulation of eIF4 and p70S6K signaling6.82E00AKT2, EIF3F, PPP2R1A, EIF3C, RPS3A, RPS26, RPS27, RPS2, RPS8, EIF3E, RPSA
Epithelial adherens junction signaling5.87E00AKT2, ACTR3, TUBB6, MYL6, ACTB, RHOA, TUBA4A, CTNNA1, ARPC3, ZYX
Remodeling of epithelial adherens junctions5.43E00ACTR3, TUBB6, ACTB, TUBA4A, CTNNA1, ARPC3, ZYX
Nrf2-mediated oxidative stress response5.05E00GSR, SOD2, STIP1, ACTB, NQO1, CCT7, TXN, PTPLAD1, TXNRD1, GSTO1
RhoA signaling4.66E00ACTR3, CFL2, MYL6, EZR, ACTB, RHOA, PFN2, ARPC3
Integrin signaling4.62E00RAC2, AKT2, ACTR3, ARF4, ACTB, RHOA, CAV1, ARPC3, ZYX, TLN1
Regulation of actin-based motility by Rho4.59E00RAC2, ACTR3, MYL6, ACTB, RHOA, PFN2, ARPC3
Fcγ receptor-mediated phagocytosis in macrophages and monocytes4.53E00RAC2, AKT2, ACTR3, EZR, ACTB, ARPC3, TLN1
Actin cytoskeleton signaling4.35E00RAC2, ACTR3, CFL2, MYL6, EZR, ACTB, RHOA, PFN2, ARPC3, TLN1
Axonal guidance signaling4.17E00DPYSL2, RAC2, AKT2, MYL6, PDIA3, TUBA4A, ACTR3, TUBB6, CFL2, RHOA, RTN4, ARPC3, PFN2, PSMD14
Purine nucleotides de novo biosynthesis II3.88E00IMPDH2, PAICS, ATIC
Germ cell-Sertoli cell junction signaling3.83E00RAC2, TUBB6, CFL2, ACTB, RHOA, TUBA4A, CTNNA1, ZYX
Protein ubiquitination pathway3.77E00PSMA6, PSMC1, UBE2L3, HSPA9, PSMD14, PSMA1, UBC, PSMA2, SKP1, PSMC5
RhoGDI signaling3.6E00ACTR3, CFL2, MYL6, EZR, ACTB, RHOA, CD44, ARPC3
Inosine-5′-phosphate biosynthesis II3.57E00PAICS, ATIC
Vitamin C transport3.55E00TXN, TXNRD1, GSTO1
Huntington’s disease signaling3.44E00TGM2, CTSD, AKT2, CYCS, CPLX2, HSPA9, POLR2H, UBC, PSME3
RAN signaling3.28E00TNPO1, XPO1, IPO5
Thioredoxin pathway2.88E00TXN, TXNRD1
Superoxide radical degradation2.88E00SOD2, NQO1
Gluconeogenesis I2.78E00ENO2, ME1, ALDOC
Integrin linked kinase signaling2.69E00AKT2, PPP2R1A, CFL2, MYL6, ACTB, RHOA, NACA
Aryl hydrocarbon receptor signaling2.65E00TGM2, CTSD, NEDD8, NQO1, PTGES3, GSTO1
Leukocyte extravasation signaling2.54E00RAC2, MYL6, EZR, ACTB, RHOA, CD44, CTNNA1
Rac signaling2.5E00ACTR3, CFL2, RHOA, CD44, ARPC3
Gap junction signaling2.43E00AKT2, TUBB6, PDIA3, ACTB, CAV1, TUBA4A
Pentose phosphate pathway2.33E00PGD, TKT
Caveolar-mediated endocytosis signaling2.31E00ARCN1, ACTB, CAV1, COPB2
Tight junction signaling2.28E00AKT2, PPP2R1A, MYL6, ACTB, RHOA, CTNNA1
Ephrin receptor signaling2.19E00RAC2, AKT2, ACTR3, CFL2, RHOA, ARPC3
Ceramide signaling2.15E00CTSD, AKT2, PPP2R1A, CYCS
Signaling by Rho family GTPases2.15E00ACTR3, CFL2, MYL6, EZR, ACTB, RHOA, ARPC3
Sertoli cell-Sertoli cell junction signaling2.13E00AKT2, TUBB6, ACTB, CAV1, TUBA4A, CTNNA1
Virus entry via endocytic pathways1.99E00RAC2, ACTB, CAV1, TFRC
Antioxidant action of vitamin C1.86E00PDIA3, TXN, TXNRD1, GSTO1
Semaphorin signaling in neurons1.86E00DPYSL2, CFL2, RHOA
Actin nucleation by ARP-WASP complex1.79E00ACTR3, RHOA, ARPC3
Glutamate biosynthesis II1.72E00GLUD1
Glutamate degradation X1.72E00GLUD1
Glycolysis I1.63E00ENO2, ALDOC
Mitochondrial dysfunction1.62E00GSR, PRDX3, SOD2, CYCS, VDAC2
14-3-3-mediated signaling1.59E00AKT2, TUBB6, PDIA3, TUBA4A
Pyrimidine ribonucleotide interconversion1.57E00CMPK1, CTPS1
Ascorbate recycling (cytosolic)1.55E00GSTO1
Glutathione redox reactions II1.55E00GSR
Thyroid hormone biosynthesis1.55E00CTSD
4-aminobutyrate degradation I1.55E00SUCLG2
Pigment epithelium derived factor signaling1.52E00AKT2, SOD2, RHOA
Pyrimidine ribonucleotide biosynthesis de novo1.51E00CMPK1, CTPS1
Clathrin-mediated endocytosis signaling1.5E00ACTR3, ACTB, TFRC, ARPC3, UBC
Ephrin B signaling1.49E00RAC2, CFL2, RHOA
Breast cancer regulation by stathmin11.45E00PPP2R1A, TUBB6, RHOA, TUBA4A, PPP1CA
Arsenate detoxification I (glutaredoxin)1.43E00GSTO1
Uracil degradation II (reductive)1.43E00DPYSL2
2-ketoglutarate dehydrogenase complex1.43E00DLST
Thymine degradation1.43E00DPYSL2
Cell cycle regulation by BTG family proteins1.36E00PPP2R1A, PRMT1
tRNA splicing1.36E00TSEN34, APEX1
Pentose phosphate pathway (oxidative branch)1.33E00PGD
Glutamate degradation III (via 4-aminobutyrate)1.33E00SUCLG2
Focal adhesion kinase signaling1.3E00AKT2, ACTB, TLN1
Docosahexaenoic acid signaling1.28E00AKT2, CYCS
tRNA charging1.28E00RARS, DARS
Arginine biosynthesis IV1.25E00GLUD1
Pentose phosphate pathway (nonoxidative branch)1.25E00TKT
Citrulline-nitric oxide cycle1.25E00CAV1
Death receptor signaling1.24E00ACIN1, CYCS, ACTB
Mechanisms of viral exit from host cells1.24E00ACTB, XPO1
Telomerase signaling1.17E00AKT2, PPP2R1A, PTGES3
Hypoxia-inducible factor-1α signaling1.13E00AKT2, CAV1, APEX1
Cdc42 signaling1.12E00ACTR3, CFL2, MYL6, ARPC3
Ephrin A signaling1.12E00CFL2, RHOA
Wnt/β-catenin signaling1.11E00AKT2, PPP2R1A, CD44, UBC
Prostanoid biosynthesis1.08E00PTGES3
Sucrose degradation V (mammalian)1.08E00ALDOC
Ketolysis1.08E00ACAT1
Sphingosine-1-phosphate signaling1.07E00AKT2, PDIA3, RHOA
Retinoic acid receptor activation1.06E00AKT2, RPL7A, PSMC5, PRMT1
Endometrial cancer signaling1.06E00AKT2, CTNNA1
Ketogenesis1.04E00ACAT1
Production of nitric oxide and reactive oxygen species in macrophages1.03E00AKT2, PPP2R1A, RHOA, PPP1CA
Lymphotoxin β receptor signaling1.03E00AKT2, CYCS
Hereditary breast cancer signaling1.02E00AKT2, POLR2H, UBC
Glutaryl-CoA degradation1E00ACAT1
Gα12/13 signaling9.98E-01AKT2, MYL6, RHOA
Glioma invasiveness signaling9.91E-01RHOA, CD44
Regulation of cellular mechanics by calpain protease9.91E-01EZR, TLN1
CD28 signaling in T-helper cells9.9E-01AKT2, ACTR3, ARPC3
ERK/MAPK signaling9.87E-01RAC2, PPP2R1A, TLN1, PPP1CA
Glucocorticoid receptor signaling9.85E-01HMGB1, AKT2, HSPA9, POLR2H, PTGES3
p70S6K signaling9.82E-01AKT2, PPP2R1A, PDIA3
Myc-mediated apoptosis signaling9.79E-01AKT2, CYCS
High-mobility group box 1 signaling9.74E-01HMGB1, AKT2, RHOA
Thrombin signaling9.63E-01AKT2, MYL6, PDIA3, RHOA
Induction of apoptosis by human immunodeficiency virus-19.54E-01CYCS, SLC25A3
Xenobiotic metabolism signaling9.34E-01PPP2R1A, CES1, NQO1, PTGES3, GSTO1
Cell cycle: G1/S checkpoint regulation9.08E-01RPL5, SKP1
Cellular effects of sildenafil (Viagra®)9.05E-01MYL6, PDIA3, ACTB
Isoleucine degradation I9.03E-01ACAT1
Urate biosynthesis/inosine 5′-phosphate degradation9.03E-01IMPDH2
Mevalonate pathway I9.03E-01ACAT1
Hypoxia signaling in the cardiovascular system8.97E-01UBE2L3, NQO1
Cardiac β-adrenergic signaling8.76E-01PPP2R1A, PPP1CA, APEX1
Chondroitin sulfate degradation (metazoa)8.75E-01CD44
Superpathway of citrulline metabolism8.75E-01CAV1
Agrin interactions at neuromuscular junction8.55E-01RAC2, ACTB
Granzyme B signaling8.49E-01CYCS
Dermatan sulfate degradation (metazoa)8.49E-01CD44
Methionine degradation I (to homocysteine)8.49E-01PRMT1
Parkinson’s signaling8.49E-01CYCS
Renal cell carcinoma signaling8.35E-01AKT2, UBC
Small-cell lung cancer signaling8.35E-01AKT2, CYCS
Endothelial nitric oxide synthase signaling8.22E-01AKT2, HSPA9, CAV1
Synaptic long-term depression8.16E-01PPP2R1A, PDIA3, CAV1
Leptin signaling in obesity8.07E-01AKT2, PDIA3
Glutathione redox reactions I8.02E-01GSR
Superpathway of geranylgeranyldiphosphate biosynthesis I (via mevalonate)8.02E-01ACAT1
Cysteine biosynthesis III (mammalia)8.02E-01PRMT1
Glioblastoma multiforme signaling7.91E-01AKT2, PDIA3, RHOA
DNA damage-induced 14-3-3σ signaling7.8E-01AKT2
Cyclins and cell cycle regulation7.71E-01PPP2R1A, SKP1
Dopamine receptor signaling7.71E-01PPP2R1A, PPP1CA
Granzyme A signaling7.6E-01APEX1
Purine nucleotide degradation II (aerobic)7.6E-01IMPDH2
Tryptophan degradation III (eukaryotic)7.6E-01ACAT1
C-X-C-motif chemokine receptor-4 signaling7.55E-01AKT2, MYL6, RHOA
Polyamine regulation in colon cancer7.23E-01PSME3
Pyrimidine deoxyribonucleotide biosynthesis I de novo7.23E-01CMPK1
Phospholipase C signaling7.15E-01TGM2, PEBP1, MYL6, RHOA
Thyroid hormone receptor/retinoid X receptor activation7.14E-01AKT2, ME1
Tricarboxylic acid cycle II (eukaryotic)7.05E-01DLST
Dopamine-DARPP32 feedback in cAMP signaling7.05E-01PPP2R1A, PDIA3, PPP1CA
Cytotoxic T-lymphocyte antigen 4 signaling in cytotoxic T lymphocytes6.91E-01AKT2, PPP2R1A
Gβγ signaling6.91E-01AKT2, CAV1
Ultraviolet-induced MAPK signaling6.91E-01CYCS, PDIA3
Interleukin-22 signaling6.89E-01AKT2
Tumoricidal function of hepatic natural killer cells6.89E-01CYCS
Crosstalk between dendritic cells and natural killer cells6.84E-01ACTB, TLN1
p21-activated kinase signaling6.84E-01CFL2, MYL6
Apoptosis signaling6.84E-01ACIN1, CYCS
Triacylglycerol degradation6.73E-01CES1
Vascular endothelial growth factor signaling6.7E-01AKT2, ACTB
Lipid antigen presentation by CD16.58E-01PDIA3
Antiproliferative role of TOB in T-cell signaling6.58E-01SKP1
cAMP response element-binding protein signaling in neurons6.54E-01AKT2, PDIA3, POLR2H
Salvage pathways of pyrimidine ribonucleotides6.49E-01AKT2, CMPK1
Interleukin-15 production6.44E-01TWF1
B-cell receptor signaling6.3E-01RAC2, AKT2, CFL2
Glutathione-mediated detoxification6.3E-01GSTO1
Amyotrophic lateral sclerosis signaling6.22E-01CYCS, SSR4
Calcium signaling6.21E-01MYL6, TPM3, ASPH
Superpathway of cholesterol biosynthesis6.16E-01ACAT1
CDK5 signaling6.16E-01PPP2R1A, PPP1CA
Nitric oxide signaling in the cardiovascular system6.16E-01AKT2, CAV1
Interleukin-8 signaling5.98E-01RAC2, AKT2, RHOA
Paxillin signaling5.98E-01ACTB, TLN1
Superpathway of methionine degradation5.91E-01PRMT1
Cytotoxic T lymphocyte-mediated apoptosis of target cells5.8E-01CYCS
Molecular mechanisms of cancer5.74E-01RAC2, AKT2, CYCS, RHOA, CTNNA1
Agranulocyte adhesion and diapedesis5.73E-01MYL6, EZR, ACTB
Nerve growth factor signaling5.68E-01AKT2, RHOA
N-formyl-l-methionyl-l-leucyl-phenylalanine signaling in neutrophils5.63E-01ACTR3, ARPC3
Fc epsilon RI signaling5.57E-01RAC2, AKT2
TNF-related weak inducer of apoptosis signaling5.57E-01CYCS
Inhibition of angiogenesis by thrombospondin 15.57E-01AKT2
Retinol biosynthesis5.57E-01CES1
Natural killer cell signaling5.52E-01RAC2, AKT2
Role of tissue factor in cancer5.52E-01AKT2, CFL2
Triacylglycerol biosynthesis5.47E-01ELOVL1
Stearate biosynthesis I (animals)5.47E-01ELOVL1
Nucleotide excision repair pathway5.47E-01POLR2H
Antigen presentation pathway5.26E-01PDIA3
Protein kinase A signaling5.21E-01MYL6, PDIA3, RHOA, PPP1CA, APEX1
CCR3 signaling in eosinophils5.15E-01CFL2, RHOA
Phosphatase and tensin homolog signaling5.1E-01RAC2, AKT2
Netrin signaling5.07E-01RAC2
Synaptic long-term potentiation5.05E-01PDIA3, PPP1CA
P2Y purigenic receptor signaling pathway5.05E-01AKT2, PDIA3
Sperm motility5.01E-01TWF1, PDIA3
Role of protein kinase R in interferon induction and antiviral response4.98E-01CYCS
PI3K/Akt signaling4.86E-01AKT2, PPP2R1A
Melanoma signaling4.81E-01AKT2
Estrogen receptor signaling4.68E-01POLR2H, HNRNPD
PI3K signaling in B lymphocytes4.64E-01AKT2, PDIA3
Ovarian cancer signaling4.51E-01AKT2, CD44
Cardiac hypertrophy signaling4.49E-01MYL6, PDIA3, RHOA
Role of Oct4 in mammalian embryonic stem cell pluripotency4.49E-01PHB
Macrophage stimulating protein-RON signaling pathway4.49E-01ACTB
Insulin receptor signaling4.39E-01AKT2, PPP1CA
Relaxin signaling4.35E-01AKT2, APEX1
5′-AMP-activated protein kinase signaling4.35E-01AKT2, PPP2R1A
TNF receptor-1 signaling4.27E-01CYCS
Cell cycle: G2/M DNA damage checkpoint regulation4.27E-01SKP1
Assembly of RNA polymerase II complex4.2E-01POLR2H
Amyloid processing4.14E-01AKT2
CD27 signaling in lymphocytes4.07E-01CYCS
Interleukin-2 signaling4.01E-01AKT2
Gαq signaling3.89E-01AKT2, RHOA
Role of checkpoint kinase 1 proteins in cell cycle checkpoint control3.89E-01PPP2R1A
Epidermal growth factor signaling3.83E-01AKT2
Nur77 signaling in T lymphocytes3.77E-01CYCS
Phospholipases3.77E-01PDIA3
Aldosterone signaling in epithelial cells3.72E-01PDIA3, HSPA9
Tec kinase signaling3.53E-01ACTB, RHOA
Estrogen-dependent breast cancer signaling3.5E-01AKT2
Granulocyte-monocyte colony stimulating factor signaling3.5E-01AKT2
Retinoic acid mediated apoptosis signaling3.4E-01CYCS
Interleukin-17A signaling in airway cells3.4E-01AKT2
Pyridoxal 5′-phosphate salvage pathway3.4E-01AKT2
Non-small cell lung cancer signaling3.35E-01AKT2
Interleukin-15 signaling3.3E-01AKT2
Angiopoietin signaling3.3E-01AKT2
Mitotic roles of polo-like kinase3.3E-01PPP2R1A
Role of PI3K/Akt signaling in the pathogenesis of influenza3.3E-01AKT2
Pregnane X receptor/9-cis retinoic acid receptor activation3.25E-01AKT2
Erythropoietin signaling3.25E-01AKT2
Gamma aminobutyric acid receptor signaling3.25E-01UBC
Role of MAPK signaling in the pathogenesis of influenza3.21E-01AKT2
Macropinocytosis signaling3.21E-01RHOA
Acute phase response signaling3.2E-01AKT2, SOD2
Role of NFAT in regulation of immune response3.15E-01AKT2, XPO1
Endothelin-1 signaling3.12E-01PDIA3, CAV1
Melatonin signaling3.12E-01PDIA3
Interleukin-3 signaling3.07E-01AKT2
Chemokine signaling3.07E-01RHOA
Interleukin-17 signaling3.03E-01AKT2
Janus kinase/Stat signaling3.03E-01AKT2
Nuclear factor kappaB activation by viruses2.99E-01AKT2
FLT3 signaling in hematopoietic progenitor cells2.95E-01AKT2
Toll-like receptor signaling2.95E-01UBC
Dendritic cell maturation2.94E-01AKT2, PDIA3
Role of NFAT in cardiac hypertrophy2.94E-01AKT2, PDIA3
Triggering receptor expressed on myeloid cells 1 signaling2.91E-01AKT2
HER-2 signaling in breast cancer2.87E-01AKT2
VEGF family ligand-receptor interactions2.87E-01AKT2
Interleukin-4 signaling2.87E-01AKT2
Acute myeloid leukemia signaling2.83E-01AKT2
Platelet-derived growth factor signaling2.83E-01CAV1
Regulation of the epithelial to mesenchymal transition pathway2.81E-01AKT2, RHOA
1,25(OH)2D/retinoid X receptor2.79E-01PSMC5
Role of macrophages, fibroblasts, and endothelial cells in rheumatoid arthritis2.68E-01AKT2, PDIA3, RHOA
Prostate cancer signaling2.65E-01AKT2
Fibroblast growth factor signaling2.55E-01AKT2
Neuregulin signaling2.45E-01AKT2
RANK signaling in osteoclasts2.45E-01AKT2
Chronic myeloid leukemia signaling2.3E-01AKT2
Stress-activated protein/Janus kinase signaling2.27E-01RAC2
Glioma signaling2.24E-01AKT2
Mouse embryonic stem cell pluripotency2.24E-01AKT2
Insulin-like growth factor 1 signaling2.19E-01AKT2
p53 signaling2.16E-01AKT2
Neuropathic pain signaling in dorsal horn neurons2.11E-01PDIA3
Cholecystokinin/gastrin-mediated signaling2.08E-01RHOA
Hepatocyte growth factor signaling1.98E-01AKT2

Abbreviations: ARP, actin-related protein; cAMP, cyclic adenosine monophosphate; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; PI3K, phosphatidylinositol-4,5-bisphosphate 3-kinase; NFAT, nuclear factor of activated T-cells; TNF, tumor necrosis factor; VEGF, vascular endothelial growth factor; WASP, Wiskott-Aldrich syndrome protein.

Table 3

The 184 direct targets of DMXAA (5,6-dimethylxanthenone 4-acetic acid) in A549 cells analyzed by ingenuity pathway analysis

Protein IDSymbolEntrez gene nameLocationType(s)Fold change
Q5VXJ5SYCP1Synaptonemal complex protein 1NucleusOther−27.155
F8VVM2SLC25A3Solute carrier family 25 (mitochondrial carrier; phosphate carrier), member 3CytoplasmTransporter−1.762
B4DS13EIF4BEukaryotic translation initiation factor 4BCytoplasmTranslation regulator−1.666
E9PEU4ARCN1Archain 1CytoplasmOther−1.523
F5H3I4ACTR1AARP1 actin-related protein 1 homolog A (yeast)CytoplasmOther−1.513
F5GY65SLC25A11Solute carrier family 25 (mitochondrial carrier; oxoglutarate carrier), member 11CytoplasmTransporter−1.391
F8VSA6NEDD8Neural precursor cell expressed, developmentally down-regulated 8NucleusEnzyme−1.352
B4DKS8HNRNPFHeterogeneous nuclear ribonucleoprotein FNucleusOther−1.333
F8WD96CTSDCathepsin DCytoplasmPeptidase−1.289
F5GX11PSMA1Proteasome (prosome, macropain) subunit, α type, 1CytoplasmPeptidase−1.286
B4E1K7STOML2Stomatin (EPB72)-like 2Plasma membraneOther−1.276
Q5TCU6TLN1Talin 1Plasma membraneOther−1.276
F8W7P7WDHD1WD repeat and HMG-box DNA binding protein 1NucleusOther−1.266
E9PNH1GANABα-Glucosidase; neutral ABCytoplasmEnzyme−1.250
C9JW37PSMD14Proteasome (prosome, macropain) 26S subunit, non-ATPase, 14CytoplasmPeptidase−1.250
B4E241SRSF3Serine/arginine-rich splicing factor 3NucleusOther−1.247
E9PH29PRDX3Peroxiredoxin 3CytoplasmEnzyme−1.245
B7Z254PDIA6Protein disulfide isomerase family A, member 6CytoplasmEnzyme−1.213
B4DNJ5RPN1Ribophorin ICytoplasmEnzyme−1.205
D6RDN3CPLX2Complexin 2CytoplasmOther−1.203
E9PDQ8SUCLG2Succinate-CoA ligase, GDP-forming, β subunitCytoplasmEnzyme−1.200
F8W914RTN4Reticulon 4CytoplasmOther−1.197
F5H3T8RARSArginyl-tRNA synthetaseCytoplasmEnzyme−1.190
B4E0X8FUBP1Far upstream element (FUSE) binding protein 1NucleusTranscription regulator−1.187
F8WF81DDB1Damage-specific DNA binding protein 1, 127 kDaNucleusOther−1.182
B4DT43ETFAElectron-transfer-flavoprotein, α polypeptideCytoplasmTransporter−1.171
B4DRT4PEBP1Phosphatidylethanolamine binding protein 1CytoplasmOther−1.171
E9PPQ5CHORDC1Cysteine and histidine-rich domain (CHORD) containing 1OtherOther−1.164
D6RFI0SFXN1Sideroflexin 1CytoplasmTransporter−1.161
C9J1T2RHOARas homolog family member ACytoplasmEnzyme−1.157
C9JPV1SLC6A6Solute carrier family 6 (neurotransmitter transporter), member 6Plasma membraneTransporter−1.150
D6RFH4CYB5BCytochrome b5 type B (outer mitochondrial membrane)CytoplasmEnzyme−1.143
B4DQJ8PGDPhosphogluconate dehydrogenaseCytoplasmEnzyme−1.115
B4E022TKTTransketolaseCytoplasmEnzyme−1.104
G3V5P4CFL2Cofilin 2 (muscle)Extracellular spaceOther−1.103
D6RF62PAICSPhosphoribosylaminoimidazole carboxylase, phosphoribosylaminoimidazole succinocarboxamide synthetaseCytoplasmEnzyme−1.099
B3KUK2SOD2Superoxide dismutase 2, mitochondrialCytoplasmEnzyme−1.097
D6REM6MATR3Matrin 3NucleusOther−1.096
B7Z2V6ATP6V1AATPase, H+ transporting, lysosomal 70 kDa, V1 subunit APlasma membraneTransporter−1.091
B1AM77STOMStomatinPlasma membraneOther−1.087
D6RF44HNRNPDHeterogeneous nuclear ribonucleoprotein DNucleusTranscription regulator−1.084
B4E0R6IPO5Importin 5NucleusTransporter−1.079
Q5T8U3RPL7ARibosomal protein L7aCytoplasmOther−1.075
A8K318PRKCSHProtein kinase C substrate 80K-HCytoplasmEnzyme−1.073
C8KIM0GSRGlutathione reductaseCytoplasmEnzyme−1.072
G3V5Q1APEX1APEX nuclease (multifunctional DNA repair enzyme) 1NucleusEnzyme−1.071
A6NN01H2AFVH2A histone family, member VNucleusOther−1.071
B7Z6M1PLS3Plastin 3CytoplasmOther−1.071
B3KUB4CA12Carbonic anhydrase XIIPlasma membraneEnzyme−1.069
F5GWY2ATIC5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/inosine 5′-monophosphate cyclohydrolaseCytoplasmEnzyme−1.068
B4DIT7TGM2Transglutaminase 2CytoplasmEnzyme−1.066
E9PRQ6ACAT1Acetyl-CoA acetyltransferase 1CytoplasmEnzyme−1.065
C9JPM4ARF4ADP-ribosylation factor 4CytoplasmOther−1.059
B1AH77RAC2Ras-related C3 botulinum toxin substrate 2 (rho family, small GTP binding protein Rac2)CytoplasmEnzyme−1.053
G3V3I1PSMA6Proteasome (prosome, macropain) subunit, α type, 6CytoplasmPeptidase−1.049
B4DVU3EIF3CEukaryotic translation initiation factor 3, subunit COtherTranslation regulator−1.047
B4DKM5VDAC2Voltage-dependent anion channel 2CytoplasmIon channel−1.045
B4DEM7CCT8Chaperonin containing TCP1, subunit 8 (theta)CytoplasmEnzyme−1.044
F5GX39TMED2Transmembrane emp24 domain trafficking protein 2CytoplasmTransporter−1.043
F5GZ27LONP1Lon peptidase 1, mitochondrialCytoplasmPeptidase−1.041
F5H667ASPHAspartate β-hydroxylaseCytoplasmEnzyme−1.040
C9JLU1POLR2HPolymerase (RNA) II (DNA-directed) polypeptide HNucleusEnzyme−1.040
B4DR70FUSFused in sarcomaNucleusTranscription regulator−1.039
E7ETK5IMPDH2Inosine 5′-monophosphate dehydrogenase 2CytoplasmEnzyme−1.038
B4DXW1ACTR3ARP3 actin-related protein 3 homolog (yeast)Plasma membraneOther−1.037
F8VNT9CD63CD63 moleculePlasma membraneOther−1.036
E9PKZ0RPL8Ribosomal protein L8OtherOther−1.036
B7Z6A4SURF4Surfeit 4CytoplasmOther−1.036
F5H1S2RPL13Ribosomal protein L13CytoplasmOther−1.034
B4DRF4PTPLAD1Protein tyrosine phosphatase-like A domain containing 1CytoplasmOther−1.028
B4DZP4DYNC1LI2Dynein, cytoplasmic 1, light intermediate chain 2CytoplasmOther−1.024
B3KQT9PDIA3Protein disulfide isomerase family A, member 3CytoplasmPeptidase−1.024
E5RJR5SKP1S-phase kinase-associated protein 1NucleusTranscription regulator−1.024
C9JV57BZW1Basic leucine zipper and W2 domains 1CytoplasmTranslation regulator−1.022
B4DUR8CCT3Chaperonin containing TCP1, subunit 3 (γ)CytoplasmOther−1.022
A8K3Z3PSMC5Proteasome (prosome, macropain) 26S subunit, ATPase, 5NucleusTranscription regulator−1.022
F5H8J3CLPTM1Cleft lip and palate associated transmembrane protein 1Plasma membraneOther−1.021
B7Z4V2HSPA9Heat shock 70 kDa protein 9 (mortalin)CytoplasmOther−1.020
F5GY50PTGR1Prostaglandin reductase 1CytoplasmEnzyme−1.015
B4DPJ8CCT6AChaperonin containing TCP1, subunit 6A (ζ 1)CytoplasmOther−1.014
D6RG13RPS3ARibosomal protein S3ANucleusOther−1.013
C9JTK6OLA1Obg-like ATPase 1CytoplasmOther−1.011
F8VRG3TWF1Twinfilin actin-binding protein 1CytoplasmKinase−1.007
C9JZ20PHBProhibitinNucleusTranscription regulator−1.006
C9JB50RPL28Ribosomal protein L28CytoplasmOther−1.006
B7Z795CES1Carboxylesterase 1CytoplasmEnzyme−1.005
F5GYN4OTUB1OTU deubiquitinase, ubiquitin aldehyde binding 1CytoplasmEnzyme−1.005
B7Z4T9CCT7Chaperonin containing TCP1, subunit 7 (eta)CytoplasmOther−1.004
B4DGN5GLUD1Glutamate dehydrogenase 1CytoplasmEnzyme−1.004
B4DDF7PPP2R1AProtein phosphatase 2, regulatory subunit A, alphaCytoplasmPhosphatase1.002
B4DLR8NQO1NAD(P)H dehydrogenase, quinone 1CytoplasmEnzyme1.005
A6NLM8SSR4Signal sequence receptor, deltaCytoplasmOther1.008
C9J7S3DARSAspartyl-tRNA synthetaseCytoplasmEnzyme1.009
D6RAN4RPL9Ribosomal protein L9CytoplasmOther1.010
B4E3C2RPL17Ribosomal protein L17CytoplasmOther1.012
G3V279ERHEnhancer of rudimentary homolog (Drosophila)NucleusOther1.017
B4DDG1UBE2L3Ubiquitin-conjugating enzyme E2L 3NucleusEnzyme1.020
C9JCK5PSMA2Proteasome (prosome, macropain) subunit, alpha type, 2CytoplasmPeptidase1.022
E9PQD7RPS2Ribosomal protein S2CytoplasmOther1.023
E7EX53RPL15Ribosomal protein L15CytoplasmOther1.024
B7ZAT2CCT2Chaperonin containing TCP1, subunit 2 (β)CytoplasmKinase1.026
E9PCY7HNRNPH1Heterogeneous nuclear ribonucleoprotein H1 (H)NucleusOther1.026
B8ZZQ6PTMAα-prothymosinNucleusOther1.027
B4E335ACTBβ-actinCytoplasmOther1.029
A6NL93HMGN1High mobility group nucleosome binding domain 1NucleusTranscription regulator1.029
Q5JR95RPS8Ribosomal protein S8CytoplasmOther1.031
C9J9K3RPSARibosomal protein SACytoplasmTranslation regulator1.031
C9JF49XPO1Exportin 1NucleusTransporter1.033
E9PGI8CMPK1Cytidine monophosphate (UMP-CMP) kinase 1, cytosolicNucleusKinase1.034
E9PMD7PPP1CAProtein phosphatase 1, catalytic subunit, alpha isozymeCytoplasmPhosphatase1.035
G3V3T3ACIN1Apoptotic chromatin condensation inducer 1NucleusEnzyme1.038
B1ALW1TXNThioredoxinCytoplasmEnzyme1.040
A8MUD9RPL7Ribosomal protein L7NucleusTranscription regulator1.041
Q2YDB7PPIFPeptidylprolyl isomerase FCytoplasmEnzyme1.042
B7Z9L0CCT4Chaperonin containing TCP1, subunit 4 (δ)CytoplasmOther1.045
E7EQR4EZREzrinPlasma membraneOther1.046
F8W118NAP1L1Nucleosome assembly protein 1-like 1NucleusOther1.051
F8VZJ2NACANascent polypeptide-associated complex α subunitCytoplasmTranscription regulator1.055
G3V5B3ERO1LERO1-like (Saccharomyces cerevisiae)CytoplasmEnzyme1.058
B4E2S7LAMP2Lysosomal-associated membrane protein 2Plasma membraneEnzyme1.060
B4DR63PSMC1Proteasome (prosome, macropain) 26S subunit, ATPase, 1NucleusPeptidase1.060
G3V5M3DLSTDihydrolipoamide S-succinyltransferaseCytoplasmEnzyme1.061
C9JU56RPL31Ribosomal protein L31CytoplasmOther1.061
B7Z1N6ALDOCAldolase C, fructose-bisphosphateCytoplasmEnzyme1.062
F8VWC5RPL18Ribosomal protein L18CytoplasmOther1.064
C9JD32RPL23Ribosomal protein L23CytoplasmOther1.066
A4D177CBX3Chromobox homolog 3NucleusTranscription regulator1.068
Q5HYE7CRYZζ-Crystallin, (quinone reductase)CytoplasmEnzyme1.069
B4E3E8DDX3XDEAD (Asp-Glu-Ala-Asp) box helicase 3, X-linkedCytoplasmEnzyme1.072
A8K8G0HDGFHepatoma-derived growth factorExtracellular spaceGrowth factor1.074
F5H897TRAP1Tumor necrosis factor receptor-associated protein 1CytoplasmEnzyme1.074
E9PNR9PRMT1Protein arginine methyltransferase 1NucleusEnzyme1.075
E9PJH8SERPINH1Serpin peptidase inhibitor, clade H (heat shock protein 47), member 1Extracellular spaceOther1.075
C9JKI3CAV1Caveolin 1, caveolae protein, 22 kDaPlasma membraneTransmembrane receptor1.078
D6R9P3HNRNPABHeterogeneous nuclear ribonucleoprotein A/BNucleusEnzyme1.081
Q8N1C0CTNNA1Catenin (cadherin-associated protein), α1, 102 kDaPlasma membraneOther1.084
B4DY66SAE1SUMO1 activating enzyme subunit 1CytoplasmEnzyme1.087
F6RFD5DSTNDestrin (actin depolymerizing factor)CytoplasmOther1.088
C9J712PFN2Profilin 2CytoplasmOther1.088
F5H4D6G3BP1GTPase activating protein (SH3 domain) binding protein 1NucleusEnzyme1.091
B4DR31DPYSL2Dihydropyrimidinase-like 2CytoplasmEnzyme1.092
B6EAT9CD44CD44 molecule (Indian blood group)Plasma membraneEnzyme1.097
B4E3S0CORO1CCoronin, actin binding protein, 1CCytoplasmOther1.108
C9JHS6AKT2V-akt murine thymoma viral oncogene homolog 2CytoplasmKinase1.116
Q5TA01GSTO1Glutathione S-transferase ω1CytoplasmEnzyme1.119
F8W7C6RPL10Ribosomal protein L10CytoplasmOther1.124
F5H0T1STIP1Stress-induced phosphoprotein 1CytoplasmOther1.127
C9JFR7CYCSCytochrome c, somaticCytoplasmTransporter1.128
E7EPB3RPL14Ribosomal protein L14CytoplasmOther1.129
F5H6Q2UBCUbiquitin CCytoplasmEnzyme1.131
B4DP24ELOVL1ELOVL fatty acid elongase 1CytoplasmEnzyme1.135
B3KS31TUBB6Tubulin, β6 class VCytoplasmOther1.136
B7Z9C4CTPS1CTP synthase 1NucleusEnzyme1.144
F8W0G4PCBP2Poly(rC) binding protein 2NucleusOther1.146
F8WBE5TFRCTransferrin receptorPlasma membraneTransporter1.147
B7Z2S5TXNRD1Thioredoxin reductase 1CytoplasmEnzyme1.153
B2RDM2TXNDC5Thioredoxin domain containing 5 (endoplasmic reticulum)CytoplasmEnzyme1.154
B4DSC0TNPO1Transportin 1NucleusTransporter1.155
F8VPF3MYL6Myosin, light chain 6, alkali, smooth muscle and non-muscleCytoplasmOther1.156
B0V3J0TSEN34TSEN34 tRNA splicing endonuclease subunitNucleusEnzyme1.159
F8VR50ARPC3Actin related protein 2/3 complex, subunit 3, 21 kDaCytoplasmOther1.162
B3KTM6RPL5Ribosomal protein L5CytoplasmOther1.165
Q5H907MAGED2Melanoma antigen family D, 2Plasma membraneOther1.168
Q75MH1RPS26Ribosomal protein S26CytoplasmOther1.181
B4DZI8COPB2Coatomer protein complex, subunit beta 2 (β prime)CytoplasmTransporter1.182
Q5T4L4RPS27Ribosomal protein S27CytoplasmOther1.183
Q5T093RER1Retention in endoplasmic reticulum sorting receptor 1CytoplasmOther1.188
Q5VU59TPM3Tropomyosin 3CytoplasmOther1.198
Q5T7C4HMGB1High mobility group box 1NucleusOther1.216
C9JFK9BAG3Bcl2-associated athanogene 3CytoplasmOther1.219
G3V153CAPRIN1Cell cycle associated protein 1Plasma membraneOther1.223
E5RHS5EIF3EEukaryotic translation initiation factor 3, subunit ECytoplasmOther1.228
B7Z8D3PSME3Proteasome (prosome, macropain) activator subunit 3 (PA28 γ; Ki)CytoplasmPeptidase1.242
B4E2Z3SLC3A2Solute carrier family 3 (amino acid transporter heavy chain), member 2Plasma membraneTransporter1.247
E9PP21CSRP1Cysteine and glycine-rich protein 1NucleusOther1.250
B4DMT5EIF3FEukaryotic translation initiation factor 3, subunit FCytoplasmTranslation regulator1.263
C9K0U8SSBP1Single-stranded DNA binding protein 1, mitochondrialCytoplasmOther1.271
A8MUB1TUBA4Aα-tubulin, 4aCytoplasmOther1.298
C9IZ41ZYXZyxinPlasma membraneOther1.326
F5H4W0ME1Malic enzyme 1, NADP+-dependent, cytosolicCytoplasmEnzyme1.331
B4DPJ6TPD52L2Tumor protein D52-like 2CytoplasmOther1.340
F5H0C8ENO2γ-enolase 2 (neuronal)CytoplasmEnzyme1.347
C9JL85MTPNMyotrophinNucleusTranscription regulator1.514
B4DP11PTGES3Prostaglandin E synthase 3 (cytosolic)CytoplasmEnzyme1.801

DMXAA modulates networked signaling pathways in A549 cells

As seen in Figures 2 and 3, DMXAA showed an ability to regulate a number of networked signaling pathways that have critical roles in the regulation of cellular processes. IPA classified the top ten networks of signaling pathways responding to DMXAA in A549 cells (Table 4). These signaling networks have important roles in pathophysiological functions and the development of many important diseases. They included gene expression, DNA replication, recombination and repair, protein synthesis, small molecule biochemistry, carbohydrate metabolism, lipid metabolism, energy production, cellular response to therapeutics, connective tissue development and function, cellular assembly and organization, cellular compromise, cell morphology, free radical scavenging, cell death and survival, neurological disease, skeletal and muscular disorders, cardiac damage, cardiac fibrosis, development and function of cardiovascular system, and development of cancer.
Figure 2

Proteomic analysis revealed the molecular interactome regulated by DMXAA in A549 cells.

Notes: A549 cells were treated with DMXAA 10 μM for 24 hours and the protein samples were subjected to quantitative SILAC-based proteomic analysis. There were 588 protein molecules regulated by DMXAA in A549 cells, with 281 protein molecules being increased and 306 protein molecules being decreased. Red indicates upregulation; green indicates downregulation; brown indicates predicted activation, and blue indicates predicted inhibition. The intensity of green and red molecule colors indicates the degree of downregulation and upregulation, respectively. The solid arrow indicates direct interaction and the dashed arrow indicates indirect interaction.

Abbreviations: DMXAA, 5,6-dimethylxanthenone 4-acetic acid; SILAC, stable-isotope labeling by amino acids in cell culture.

Figure 3

Proteomic analysis revealed a network of signaling pathways regulated by DMXAA in A549 cells.

Note: The network of signaling pathways was analyzed by ingenuity pathway analysis according to the 588 protein molecules regulated by DMXAA in A549 cells.

Abbreviation: DMXAA, 5,6-dimethylxanthenone 4-acetic acid; mTOR, mammalian target of rapamycin; ILK, integrin-linked kinase; Nrf2, nuclear factor erythroid 2-related factor 2; eIF, eukaryotic initiation factor; S6K, p70S6 kinase.

Table 4

Networks of potential molecular targets regulated by DMXAA (5,6-dimethylxanthenone 4-acetic acid) in A549 cells

IDMolecules in networkScoreFocus moleculesTop diseases and functions
1ASPH, ATIC, BZW1, CHORDC1, CLPTM1, CMPK1, CRYZ, CYB5B, DYNC1LI2, ELOVL1, GANAB, H2AFV, ME1, OLA1, PAICS, PDIA6, PGD, PRKCSH, PTGR1, RER1, SAE1, SFXN1, SLC25A3, SLC25A11, SLC6A6, SSBP1, STOM, SUCLG2, SURF4, SYCP1, TMED2, TPD52L2, TSEN34, TXNDC5, UBC7735Carbohydrate metabolism, small molecule biochemistry, lipid metabolism
260S ribosomal subunit, ACIN1, Akt, CD63, DDX3X, EIF3, EIF3C, EIF3E, EIF3F, EIF4B, histone H1, HNRNPF, β-importin, IPO5, MTORC2, NEDD8, Rar, RPL5, RPL7, RPL9, RPL10, RPL13, RPL14, RPL15, RPL17, RPL18, RPL23, RPL28, RPL31, RPL7A, SLC3A2, thymidine kinase, TNPO1, TPM3, TRAP15227Gene expression, protein synthesis, cancer
3APEX1, ARF4, ARPC3, collagen type I, cytochrome C, ERK, ETFA, HDGF, HISTONE, HMGB1, HMGN1, HNRNPD, Hsp27, mitochondrial complex 1, NACA, NAP1L1, PHB, PLS3, PP2A, PPP2R1A, PRDX3, PRMT1, PTMA, ribosomal 40s subunit, RNR, RPS2, RPS8, RPS26, RPS27, RPS3A, RPSA, STOML2, TCF, VDAC2, XPO14725Cellular response to therapeutics, connective tissue development and function, carbohydrate metabolism
419S proteasome, 20s proteasome, 26s proteasome, α-tubulin, ATPase, BAG3, β-tubulin, CCT2, CCT3, CCT4, CCT7, CCT8, CCT6A, FUBP1, GSR, IκB, immunoproteasome Pa28/20s, LAMP2, LONP1, MTPN, NF-κB (complex), NQO1, PEBP1, proteasome PA700/20s, PSMA, PSMA1, PSMA2, PSMA6, PSMC1, PSMC5, PSMD14, PSME3, PTPLAD1, TUBB6, ubiquitin4123Cell death and survival, DNA replication, recombination, and repair, energy production
5ACTB, actin, ACTR1A, α-actinin, CAPRIN1, CFL2, cofilin, DARS, DLST, DPYSL2, DSTN, ERK1/2, Erm, EZR, F-actin, gilamin, G-actin, G3BP1, GLUD1, Na+, K+-ATPase, PCBP2, PFN2, PKG, profilin, proinsulin, Rho GDI, Rock, RPL8, RTN4, SRSF3, thioredoxin reductase, TLN1, TWF1, TXNRD1, ZYX3420Cellular assembly and organization, cellular compromise, cellular function and maintenance
6ALDOC, ATP6V1A, ATP6V1E2, BIN3, BTF3L4, CORO1C, DDB1, DNAJB3, DNAJC16, DNAJC22, DNAJC28, ERH, GRPEL2, GTF2H2C_2, HNRNPAB, HNRNPH1, HSPA9, MATR3, OTUB1, PAGE1, PCDHAC2, POLR2H, POLR2J2/POLR2J3, PPIF, RDM1, RPN1, SERPINH1, SPAG7, SSR4, STON1-GTF2A1L, TARBP1, TBP, TMEM106B, tubulin (complex), UBC2516Gene expression, cell morphology, cellular assembly and organization
7ACAT1, ADCY, AMPK, CA12, calmodulin, caspase, CK2, Creb, CSRP1, CTSD, cyclin A, CYCS, ERO1L, estrogen receptor, FUS, Hsp70, Hsp90, IL1, IMPDH2, insulin, Lh, MYL6, NOS, PDIA3, PI3K (complex), PLC, PTGES3, Rb, RNA polymerase II, STIP1, TGM2, thyroid hormone receptor, TUBA4A, tubulin (family), TXN2315Neurological disease, skeletal and muscular disorders, cell death and survival
8ACTR3, α-catenin, ARCN1, CBX3, CD3, COPB2, CTNNA1, CTPS1, hemoglobin, histone H3, histone H4, IFN-β, IgG, IL12 (complex), immunoglobulin, interferon-α, MAGED2, MAPK, mediator, NMDA receptor, p38 MAPK, PKA, PKC(s), PLC-γ, RARS, Ras homolog, SFK, SRC (family), STAT5a/b, TFRC, TNF (family), trypsin, VEGF, WDHD11410Infectious disease, cardiac damage, cardiac fibrosis
9AKT2, AP1, BCR (complex), calpain, CAV1, CD44, collagen(s), ENO2, fibrinogen, focal adhesion kinase, IGM, integrin, JNK, laminin, LDH (complex), LDL, LFA-1, MEK, NADPH oxidase, NFAT (family), p85 (PI3K), PDGF (complex), PDGF BB, PI3K (family), Pld, Rac, RAC2, Ras, RHOA, SOD, SOD2, SOS, TGF-β, TKT, tyrosine kinase108Free radical scavenging, cell morphology, cellular assembly and organization
10APP, CENPI, CEP250, CES1, CHCHD6, CMAS, CPLX2, DDX10, ERBB2, FBXL7, FBXL20, FBXW9, FSH, GNLY, GNRH2, GPR12, GSK3, GSPT2, GSTO1, MTORC1, NDP, PCIF1, PDXP, PPFIA4, PPP1CA, PPP1R32, PRR16, RGL1, SH3BGRL3, SKP1, SSH3, TSKS, UBE2L3, ZC3HC1, ZFYVE176Cell morphology, cellular function and maintenance, cardiovascular system development and function

DMXAA modulates important regulators involved in cell cycle distribution in A549 cells

It has been reported that regulation of cell cycle distribution is an effective approach in the treatment of lung cancer,24 and that vascular-disrupting agents exhibit modulating effects on cell cycle distribution. However, it has not been fully uncovered the molecular targets and underlying mechanisms of DMXAA. Therefore, in order to explore the effect and potential molecular targets of DMXAA on cell cycle distribution in A549 cells, we treated A549 cells with 10 μM DMXAA for 24 hours and then subjected samples of the cells to quantitative proteomic analysis. The proteomic results showed that DMXAA had an effect on the regulation of cyclins and the cell cycle distribution at G1/S and G2/M DNA damage checkpoints in A549 cells with the involvement of a number of functional proteins, such as PPP2R1A, RPL5, and SKP1 (Table 2). These findings suggest that DMXAA may modulate cell cycle distribution, contributing to its anticancer effect.

DMXAA regulates apoptosis and autophagy in A549 cells

Apoptosis and autophagy are two predominant programmed cell death pathways.30 Manipulating apoptosis and autophagy has been considered to be a promising strategy in the treatment of cancer via the regulation of mitochondria-dependent/-independent pathways.31–35 As shown in Table 2, DMXAA regulated the apoptotic signaling pathway, mitochondrial function, and death receptor signaling pathway, involving a number of functional proteins. These included ACIN1, CYCS, ACTB, AKT2, GSR, PRDX3, SOD2, and VDAC2 (Table 2). Further, the mTOR signaling pathway plays a pivotal role in the regulation of autophagy, and has been proposed to be a promising target in the treatment of NSCLC.36 Vascular disruption combined with mTOR inhibition showed an enhanced anticancer effect when compared with monotherapy.37 As shown in Table 2 and Figure 4, DMXAA showed an ability to modulate the mTOR signaling pathway in A549 cells. The results showed that DMXAA decreased the expression of EIF3C, EIF4B, RHOA, and RPS3A, but increased the expression of AKT2, EIF3E, EIF3F, PPP2R1A, RPS2, RPS8, RPS26, RPS27, and RPSA in A549 cells (Table 2), suggesting that modulation of mTOR signaling may play an important role in the cancer cell killing effect of DMXAA in A549 cells. Taken together, the results suggest that the regulatory effects of DMXAA on apoptosis, mitochondrial function, and mTOR signaling pathway contribute, at least in part, to the anticancer effect of this drug in the treatment of NSCLC.
Figure 4

DMXAA modulates mTOR signaling pathway in A549 cells.

Notes: A549 cells were treated with DMXAA 10 μM for 24 hours and the protein samples were subject to quantitative proteomic analysis. Red indicates upregulation; green indicates downregulation; brown indicates predicted activation. The intensity of green and red molecule colors indicates the degree of downregulation and upregulation, respectively. The solid arrow indicates direct interaction and the dashed arrow indicates indirect interaction.

Abbreviations: DMXAA, 5,6-dimethylxanthenone 4-acetic acid; mTOR, mammalian target of rapamycin; eIF, eukaryotic initiation factor; AKT, protein kinase B; PKC, protein kinase C; TSC, tuberous sclerosis complex; VEGF, vascular endothelial growth factor; HIF, hypoxia-inducible factor; DAG, diacylglycerol; ATG, autophagy-associated protein; PMA, phorbol myristate acetate; PIP2, phosphatidylinositol 4,5-bisphosphate; IRS1, insulin receptor substrate-1; RTK, receptor tyrosine kinase; LKB1, liver kinase B1; REDD1, protein regulated in development and DNA damage response 1; PI3K, phosphatidylinositide 3-kinase; 4EBP, eukaryotic translation initiation factor 4E binding protein 1; INSR, insulin receptor; AMPK, AMP-activated protein kinase.

DMXAA regulates redox homeostasis involving ROS-mediated and Nrf2-mediated signaling pathways in A549 cells

Induction of ROS plays a critical role in the production of cytokines, contributing to the cancer cell killing effect of DMXAA.38 However, the regulatory effect of DMXAA on ROS generation-related molecules and signaling pathways is not fully understood. In this study, we observed that DMXAA regulated several critical signaling pathways related to ROS generation and redox homeostasis in A549 cells. Our quantitative proteomic study showed that treatment with DMXAA regulated oxidative phosphorylation, Nrf2-mediated oxidative stress response, and superoxide radical degradation in A549 cells (Table 2 and Figure 5). A number of functional proteins were found to be involved in these pathways, including ACTB, CCT7, GSR, GSTO1, NQO1, PTPLAD1, SOD2, STIP1, TXN, and TXNRD1 (Table 2). Of note, Nrf2-mediated signaling pathways have a critical role in the maintenance of intracellular redox homeostasis in response to various stimuli via regulating antioxidant responsive elements.39,40 The quantitative proteomic data suggest that modulation of the expression of functional proteins involved in Nrf2-mediated signaling pathways may contribute to the anticancer effect of DMXAA in the treatment of NSCLC.
Figure 5

DMXAA regulates Nrf2-mediated signaling pathways in A549 cells.

Notes: A549 cells were treated with DMXAA 10 μM for 24 hours and the protein samples were subjected to quantitative proteomic analysis. Red indicates upregulation and green indicates downregulation. The intensity of green and red molecule colors indicates the degree of downregulation and upregulation, respectively. The solid arrow indicates direct interaction and the dashed arrow indicates indirect interaction.

Abbreviation: DMXAA, 5,6-dimethylxanthenone 4-acetic acid; ER, endoplasmic reticulum; ROS, reactive oxygen species; Nrf2, nuclear factor erythroid 2-related factor 2; SOD, superoxide dismutase; HO-1, heme oxygenase 1; GST, glutathione S-transferase; UGT, uridine 5’-diphospho-glucuronosyltransferase; CCT7, chaperonin containing TCP1, subunit 7; CAT, catalase; FMO, flavin-containing monooxygenase; MRP, multi-drug resistance associated protein; PSM, proteasome; VCP, valosin containing protein; UBB, ubiquitin B; HIP2, huntingtin interacting protein 2; TXN, thioredoxin; FTL, ferritin, light polypeptide; ATF4, activating transcription factor 4; BACH1, BTB and CNC homology 1, basic leucine zipper transcription factor 1; ERK, extracellular signal-regulated kinase; HSP, heat shock protein; PMF-1, polyamine-modulated factor 1; NRPB, nuclear restricted protein, BTB domain-like; MAF, v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog; CYP, cytochrome P450; KEAP1, Kelch-like ECH-associated protein 1; MEK2, MAP kinase kinase 2; ASK1, apoptosis signal regulating kinase 1; TAK1, testicular receptor 4; GSK3, glycogen synthase kinase 3; JNK, c-Jun N-terminal kinase 1; CUL3, cullin 3; MEKK, MAP kinase kinase kinase.

Taken together, our quantitative proteomic study revealed a number of important functional proteins and associated signaling pathways that are regulated in A549 cells in response to treatment with DMXAA. These cellular signaling pathways play a pivotal role in the regulation of the cell cycle, apoptosis, autophagy, and oxidative stress. In our subsequent validation experiments, we confirmed the effect of DMXAA on cell cycle distribution, apoptosis, autophagy, and ROS generation in A549 cells.

Verification of molecular targets of DMXAA in A549 cells

The quantitative proteomic studies described above showed that DMXAA can modulate a number of functional protein molecules and related signaling pathways involved in cell proliferation, invasion and migration, death, and survival. In order to verify the quantitative proteomic data further, we investigated how DMXAA affected cell cycle distribution, apoptosis, autophagy, and redox homeostasis in A549 cells.

DMXAA induces G1 arrest in A549 cells

To validate the effect of DMXAA on cell growth, the cell cycle distribution was determined in A549 cells using flow cytometric analysis. As shown in Figure 6, a concentration-dependent increase in the cell number in G1 phase was observed after incubation of A549 cells with DMXAA at 0.1, 1, and 10 μM for 24 hours, with a 1.1-fold, 1.1-fold, and 1.4-fold increase in the number of cells arrested in G1 phase, respectively, compared with control cells treated with vehicle only (P<0.001 by one-way ANOVA, Figure 6A and B). In contrast, there was a marked decrease in the number of cells in S and G2/M phases in A549 cells treated with DMXAA at 0.1, 1, and 10 μM for 24 hours (P<0.01 or P<0.001 by one-way ANOVA, Figure 6A and B). Taken together, the results show that DMXAA can regulate the cell cycle distribution, contributing to its anticancer effect in A549 cells. Moreover, the inducing effect of DMXAA on cell cycle arrest further verifies the regulatory action of DMXAA on G1 and G2 checkpoints as determined by our proteomic study.
Figure 6

DMXAA induces G1 phase arrest in A549 cells.

Notes: Cell cycle distribution of A549 cells when treated with DMXAA 0.1, 1, or 0 μM for 24 hours. (A) Representative DNA fluorescence histograms showing the effect of DMXAA on cell cycle distribution of A549 cells and (B) bar graphs showing the percentage of A549 cells in G1, S, and G2 phases. Data are shown as the mean ± SD of three independent experiments. **P<0.01 and ***P<0.001 by one-way analysis of variance.

Abbreviation: DMXAA, 5,6-dimethylxanthenone 4-acetic acid.

DMXAA induces apoptosis and autophagy in A549 cells

As stated in our proteomic results, treatment with DMXAA induced apoptotic and autophagic responses in A549 cells involving several important signaling pathways. In the mitochondria/cytochrome c-mediated apoptotic pathway, release of cytochrome c from the mitochondria to the cytosol and resultant activation of the caspase cascade are key steps in the apoptosis process.41,42 Beclin 1 and LC3-I/II are two important markers in the initiation and progression of autophagy and are critical for formation of autophagosomes.43,44 During the autophagy process, LC3/Atg8 is cleaved at its C-terminus by Atg4 to generate cytosolic LC3-I.45 LC3-I is subsequently conjugated to phosphatidylethanolamine, then proteolytically cleaved and lipidated by Atg3 and Atg7 to form LC3-II, which attaches to the membrane of the autophagosome. To verify the proteomic response to DMXAA with regard to cell death, we performed Western blotting assays to examine the expression of cytochrome c, caspase 3, beclin 1, and LC3-I/II in A549 cells treated with DMXAA. Incubation of A549 cells with DMXAA at 0.1, 1, and 10 μM markedly increased the cytosolic level of cytochrome c by 1.2-, 1.6-, and 1.6-fold, respectively, compared with the control cells (P<0.05 or P<0.01 by one-way ANOVA, Figure 7A and B). Cleaved caspase 3 was increased by DMXAA in A549 cells in a concentration-dependent manner. Incubation of A549 cells with DMXAA at 0.1, 1, and 10 μM significantly increased the level of cleaved caspase 3 by 1.3-, 1.4-, and 2.0-fold, respectively, compared with control cells (P<0.05 by one-way ANOVA, Figure 7A and B). These results indicate that DMXAA induces a marked increase in the cytosolic level of cytochrome c and activation of caspase 3, eventually leading to apoptotic death in A549 cells.
Figure 7

DMXAA increases the cytosolic level of cytochrome c and activation of caspase 3, and promotes expression of beclin 1, LC3-I, and LC3-II in A549 cells.

Notes: A549 cells were treated with DMXAA 0.1, 1, or 10 μM for 24 hours and protein samples were subjected to Western blotting assay. (A) Representative blots of cytosolic cytochrome c, cleaved caspase 3, beclin 1, LC3-I, and LC3-II in A549 cells and (B) bar graphs showing the relative levels of cytosolic cytochrome c, cleaved caspase 3, beclin 1, LC3-I, and LC3-II in A549 cells. Data are shown as the mean ± SD of three independent experiments. *P<0.05 and **P<0.01 by one-way analysis of variance.

Abbreviations: DMXAA, 5,6-dimethylxanthenone 4-acetic acid; LC3, microtubule-associated protein 1A/1B-light chain 3.

We further examined the effect of DMXAA on beclin 1 and LC3-I/II expression levels. Treatment of A549 cells with DMXAA for 24 hours significantly increased the expression of beclin 1. There was a 1.5-, 2.1-, and 1.9-fold increase in beclin 1 in A549 cells treated with DMXAA 0.1, 1, and 10 μM, respectively, for 24 hours (P<0.05 by one-way ANOVA, Figure 7A and B). Upon activation of LC3-I/II, our Western blotting analysis revealed two clear bands of LC3-I and II in A549 cells after 24 hour treatment with DMXAA (Figure 7A). Incubation of DMXAA at 0.1, 1, and 10 μM markedly increased the expression of LC3-I and LC3-II (Figure 7A and B). In comparison with the control cells, there was a 1.6-, 1.9-, and 1.6-fold increase in the level of LC3-I, and a 2.1-, 3.3-, and 2.9-fold increase in the level of LC3-II in A549 cells treated with DMXAA 0.1, 1, and 10 μM, respectively, for 24 hours. In addition, the ratio of LC3-II to LC3-I was markedly increased by 1.3-, 1.8-, and 1.8-fold in A549 cells treated with DMXAA 0.1, 1, and 5 μM, respectively (P<0.05 or P<0.01 by one-way ANOVA, Figure 7A and B). Taken together, the proteomic and Western blotting results show that DMXAA induces apoptosis and autophagy in A549 cells, contributing to the anticancer effects of DMXAA in the treatment of NSCLC.

DMXAA induces generation of intracellular ROS in A549 cells

As shown in the proteomic results, treatment with DMXAA can regulate intracellular redox homeostasis in A549 cells, which may contribute to the apoptosis-inducing and autophagy-inducing effects of DMXAA. Thus, we determined the effect of DMXAA on intracellular ROS levels in A549 cells. The intracellular levels of ROS were increased 1.1-, 1.1-, and 1.3-fold in a concentration-dependent manner when A549 cells were treated with DMXAA 0.1, 1, and 10 μM for 48 hours (P<0.001 by one-way ANOVA, Figure 8). The ROS-inducing effect of DMXAA further confirms its regulatory effect on intracellular redox homeostasis in A549 cells.
Figure 8

DMXAA induces intracellular ROS generation in A549 cells.

Notes: Intracellular ROS level in A549 cells treated with DMXAA 0.1, 1, or 10 μM for 48 hours. Data are shown as the mean ± SD of three independent experiments. ***P<0.01 by one-way analysis of variance.

Abbreviation: DMXAA, 5,6-dimethylxanthenone 4-acetic acid; ROS, reactive oxygen species.

Discussion

NSCLC remains a devastating cancer, with the highest incidence and mortality rate, and treatment of the disease remains a major challenge due to the poor efficacy and severe side effects of both standard and new chemotherapeutic agents. There is an increasing interest in new agents and therapies for the treatment of lung cancer. DMXAA, a flavonoid tumor vascular-disrupting agent, has been found to have anticancer activity in vitro and in vivo in the treatment of NSCLC when used alone or in combination. It targets the established tumor blood vessels and inhibits tumor blood flow, resulting in necrosis of solid tumors. It has also been reported that DMXAA can regulate multiple signaling pathways involved in cell cycle progression, apoptosis, autophagy, and ROS generation.11,46–51 However, the global potential molecular targets and the possible mechanisms involved are not fully identified as yet. In the present study, we showed a comprehensive network of signaling pathways responding to treatment with DMXAA in A549 cells using a quantitative SILAC-based proteomic approach. The network of signaling pathways was mainly involved in cell cycle distribution, cell invasion and migration, redox homeostasis, and cell death. We verified that DMXAA arrested A549 cells in G1 phase, promoted apoptosis, induced marked autophagy, and triggered ROS generation. The SILAC-based proteomic approach can quantitatively and comprehensively evaluate the effect of a given compound and identify its potential molecular targets and related signaling pathways.52–54 Previous studies have used this approach in A549 cells and tried to explore the potential molecular targets and possible mechanism for NSCLC therapy.55–63 In our study, we used a quantitative SILAC-based proteomic approach to evaluate the responses of A549 cells to treatment with DMXAA. This approach showed that DMXAA regulates a number of functional proteins and molecular signaling pathways involved in cell cycle progression, apoptosis, autophagy, and redox homeostasis in A549 cells, such as PPP2R1A, RPL5, SKP1, ACIN1, CYCS, ACTB, AKT2, GSR, PRDX3, SOD2, VDAC2, EIF3C, EIF4B, RHOA, RPS3A, AKT2, EIF3E, EIF3F, PPP2R1A, RPS2, RPS8, RPS26, RPS27, RPSA, ACTB, CCT7, GSR, GSTO1, NQO1, PTPLAD1, STIP1, TXN, and TXNRD1. The proteomic results suggest that DMXAA may target these molecules to elicit its anticancer effects in the treatment of NSCLC. Notably, we went on to validate the proteomic responses to DMXAA in A549 cells. We found that DMXAA arrested A549 cells in G1 phase in a concentration-dependent manner, and speculated that the possible mechanism of DMXAA with regard to G1 arrest in A549 cells might involve a number of key regulators, including p21 Waf1/Cip1, p53, cyclins and cyclin-dependent kinases. p21 is a cyclin-dependent kinase inhibitor regulated by p53, and can bind to the Cdc2-cyclin B1 complex, thereby inducing cell cycle arrest.64 Further, cell cycle progression is tightly regulated by cyclins and cyclin-dependent kinases.65 Cyclins have no catalytic activity and are inactive in the absence of a partner cyclin. The complex formed by the association of Cdc2 and cyclin B1 plays a major role in the entry of cells into mitosis. Phosphorylation of Cdc2 at Thr161 by cyclin-dependent kinase–activating kinases is essential for the activity of Cdc2 kinase. Phosphorylation of Cdc2 at Thr14 and Tyr15 is catalyzed by Wee1 and Myt1 protein kinases, resulting in inhibition of Cdc2.65 During G2/M transition, Cdc2 is rapidly converted into the active form by dephosphorylation of Tyr14 and Tyr15, catalyzed by Cdc25 phosphatase. Thus, taking the proteomic and flow cytometric results into consideration, DMXAA-induced cell cycle arrest may occur via regulation of key modulators controlling the G1 and G2 checkpoints in A549 cells. The present proteomic study also shows that DMXAA regulated mitochondrial function and cell death. Mitochondrial disruption and subsequent release of cytochrome c initiates the process of apoptosis, with the latter being initiated by proapoptotic members of the Bcl-2 family but antagonized by antiapoptotic members of this family.66,67 Antiapoptotic members of Bcl-2 can be inhibited by post-translational modification and/or by increased expression of PUMA, which is an essential regulator of p53-mediated cell apoptosis.68 In addition, cytochrome c released from the mitochondria can activate caspase 9, which then activates caspase 3 and caspase 7.69 In our study, we observed that the cytosolic level of cytochrome c was significantly increased and that caspase 3 was markedly activated after treatment with DMXAA. The activated caspase 3 ultimately induced apoptosis, with a decrease in the Bcl-2 level. Further, the proteomic results show that DMXAA has a modulating effect on the mTOR signaling pathway. Under optimal growth conditions, activated mTORC1 inhibits autophagy by direct phosphorylation of Atg13 and ULK1 at Ser757.70–72 This phosphorylation inhibits ULK1 kinase activity and subsequent autophagosome formation. When the kinase activity of mTORC1 is suppressed, the autophagic machinery is initiated. In the present study, DMXAA induced autophagy in A549 cells as indicated by the increased expression of beclin 1 and the ratio of LC3-II over LC3-I. The amount of LC3-II or the ratio between LC3-II and LC3-I correlates well with the number of autophagosomes. Taken together, the autophagy-inducing effect of DMXAA may contribute to its anticancer activity via regulation of the mTOR signaling pathway. In addition, our proteomic study showed that DMXAA regulates the Nrf2-mediated signaling pathway, which controls the basal and induced expression of a wide array of antioxidant response element-dependent genes to regulate the physiological and pathophysiological outcomes of exposure to oxidants.40,73,74 We found a significant inducing effect of DMXAA on ROS generation in A549 cells. However, the mechanism of how DMXAA induces ROS generation is unclear. Nrf2 is a nuclear transcription factor that plays a pivotal role in regulation of oxidative stress by modulating the transcription of antioxidant response elements.40 It indicates that DMXAA may induce oxidative stress via the Nrf2-mediated signaling pathway. Our results suggest that ROS may have an important role in DMXAA-induced apoptosis and autophagy in A549 cells. However, further studies are needed to elucidate how DMXAA induces generation of ROS and modulates redox homeostasis. In summary, the quantitative SILAC-based proteomic approach used in this study showed that DMXAA inhibited cell proliferation, predominantly activated the mitochondria-dependent apoptotic pathway and induced autophagy, and increased intracellular levels of ROS in human A549 cells involving a number of key functional proteins and related molecular signaling pathways. This study may provide a clue enabling full identification of the molecular targets and elucidate the underlying mechanisms of DMXAA in the treatment of NSCLC, resulting in an improved therapeutic effect and fewer side effects in the clinical setting.
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