First, we attempt to validate the activation of gluconeogenesis by citreoviridin in lung cancer cells. The expression of other gluconeogenic enzymes, including mitochondrial pyruvate carboxylase, cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C) and glucose 6-phosphatase, is needed to be checked. The transcription of a key regulator of gluconeogenic genes, the peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α) should be measured. Enzymes that modulate glycolysis and gluconeogenesis, PFK-1 and PFK-2/FBPase-2, have bifunctional activities on the same protein and are controlled by phosphorylation. Hence, the phosphorylation and the activity of PFK-1 and PFK-2/FBPase-2 should also be determined.
For verifying the glycogen synthesis from glucose, the phosphorylation states of glycogen synthase and glycogen phosphorylase can be determined to confirm the activities of these two enzymes. Furthermore, whether glucose is converted to other compounds is required to be investigated. The contents of glycogen, myo-inositol or sorbitol in lung cancer cells treated with citreoviridin can be measured. The links between gluconeogenesis and tumorigenesis needs substantial evidences.
It is still unclear what signaling pathway is induced by targeting ectopic ATP synthase and affects the glucose metabolism in cancer cells. The signaling pathways
64
controlling glucose metabolism may provide some implication for the issue. The PI3K/AKT pathway, LKB1/AMPK pathway, MYC, PKA and p53 are suggested to regulate glucose metabolism. Determination of the activation of these pathways by citreoviridin may help to clarify the relationship between targeting ectopic ATP synthase and metabolic regulations in lung cancer.
65
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Figures
83
Figure 1. The structure and molecular formula of citreoviridin. Citreoviridin is a
polyenic α-pyrone derivative produced by molds of genera Penicillium and Aspergillus.
Molecular weight = 402.5
C 23 H 30 O 6
84
Figure 2. Schematic workflow of shotgun proteomics. 1) Proteins are first digested into
peptides, and 2) the resulting peptides are separated by on-line high-performance liquid chromatography (HPLC). 3) Peptides eluted from the HPLC column are converted into gas phase ions by electrospray and delivered into the mass spectrometer. 4) Peptide ions are scanned by the first mass spectrometer to generate precursor ion spectrum. 5) Next, several peptide precursor ions are selected from the precursor ion spectrum to collision-induced dissociation (CID) for fragmentation. The resulting fragment ions are detected by the second mass spectrometer, which generates fragment ion spectrum. 6) Fragment ion spectra are assigned to their corresponding peptides by sequence database search algorithm. 7) At last, set of peptides are assigned to the most likely protein for85
Figure 3. Workflow of isobaric tags for relative and absolute quantitation (iTRAQ).
Proteins are extracted from samples and digested into peptides. iTRAQ reagent consists of an isobaric tag and a peptide reactive group. The isobaric tag includes a reporter collision-induced dissociation (CID) during the MS/MS event. The MS/MS generates a fragment ion spectrum consisted of iTRAQ reporter ion signals and fragmented peptide ion signals. The iTRAQ reporter ion signals in the low mass range area (m/z 114-117) are used for quantitation, while the signals of peptide fragment ions are used for peptide sequence identification.
86
Reproducibility assessment by iTRAQ duplicate experiment (3.1)
iTRAQ Duplicate
Differential proteomic profiling by quantitative analysis (3.3-3.5)
LC-MS/MS Data
Figure 4. The overall experimental design of this study. This study is divided into four
parts. First, reproducibility of the experiments was checked. Next, proteomic profiling of citreoviridin-treated tumors were acquired by iTRAQ experiments. In the third part, proteins were quantified and cut-off values were calculated for selecting differentially expressed proteins. Finally, we performed bioinformatics analysis to elucidate the pathway regulated by citreoviridin, which was validated by Western blotting.87
Figure 5. The sources of tumor samples. Tumor samples were from human lung cancer
mouse xenograft models. Cultured CL1-0 lung adenocarcinoma cells49 were subcutaneously injected into NOD.CB17-Prkdcscid/NcrCrl mice. DMSO (control) or citreoviridin (citreoviridin treatment) were intraperitoneally injected when tumor size reached 1000 mm3. Mice were sacrificed until the tumor volume reached 1000 mm3 and tumor samples were collected.
88
Figure 6. Workflow of iTRAQ quantitative proteomic experiment. Total proteins were
extracted from tumor samples followed by reduction, alkylation and trypsin digestion.
The resulting peptides from different samples were labeled with iTRAQ 114, 115, 116 and 117 tags, respectively. Labeled peptides were combined and analyzed by LC-MS/MS directly in duplicate and small-scale experiment. In large-scale experiment, combined iTRAQ-labeled peptides were fractioned by strong cation exchange (SCX) chromatography and each fraction was individually analyzed by LC-MS/MS.
Tumor tissue
89
Figure 7. Workflow of data processing and analysis. Raw data from LC-MS/MS
analysis were first searched against database combining Swiss-Prot human database and Swiss-Prot mouse database by Mascot search engine for protein identification. We used Multi-Q software48 to detect iTRAQ signature ions and select qualified peptides for quantitation. The peak intensity of each iTRAQ signature ion was first normalized and the relative protein abundance ratio of each protein was calculated.
Data
90
Figure 8. Workflow of bioinformatics analysis of differentially expressed human
proteins. First, differentially expressed proteins identified from small-scale and large-scale experiments were combined, and human proteins were selected for further analysis. We used DAVID for Gene Ontology analysis and MetaCore for pathway map analysis and network analysis.91
Figure 9. The experimental design and data analysis process of iTRAQ duplicate
experiment. Tumor samples from control and citreoviridin treatment mice were both separated into two samples and labeled with different iTRAQ tags, respectively. For reproducibility assessment, the intensities of iTRAQ signature ions of selected peptides were plotted.
Duplicate experiment
Control Citreoviridin treatment
Sample C1 Sample T1
iTRAQ 114 iTRAQ 115 iTRAQ 116 iTRAQ 117
Half Half Half Half
92
Figure 10. iTRAQ quantitative proteomic experiments showed high reproducibility and accuracy. (A) Scattering plot of two replicate control
tumor samples, iTRAQ 114-labeled and iTRAQ 115-labeled . (B) Scattering plot of two replicate citreoviridin-treated tumor samples, iTRAQ 116-labeled and iTRAQ 117-labeled .iTR A Q 11 5 in tens it y (ion c oun t 10
3)
iTRAQ 114 intensity (ion count 10
3)
iTR A Q 11 7 in tens it y (ion c oun t 10
3)
iTRAQ 116 intensity (ion count 10
3)
A B
93
Figure 11. The experimental design and data analysis process of iTRAQ small-scale
experiment and large-scale experiment. Two biological replicates of both control andSample C1 Sample C2 Sample T1 Sample T2
SCX chromatography
94
citreoviridin-treated tumor samples were labeled with different iTRAQ tags. For small-scale experiment, combined iTRAQ-labeled peptides were directly analyzed by LC-MS/MS. For large-scale experiment, combined iTRAQ-labeled peptides were first fractioned by SCX chromatography and each fraction was individually analyzed by LC-MS/MS. The cut-off values for selecting differentially expressed proteins were calculated from the S values acquired from the large-scale experiment. Subsequently, the protein abundance ratios were used for calculating R value and selecting differentially expressed proteins by comparing with the cut-off values.
95
Figure 12. Strong cation exchange (SCX) chromatography. In large-scale experiment,
combined iTRAQ-labeled peptides were fractioned by SCX chromatography. At pH 3.0, PolySulfoethyl A was negatively charged and peptides were positively charged, so peptides were bonded to the column. Gradient salt was used for eluting peptides.Peptides with fewer positive charges were eluted first. SCX chromatography fractions peptides based on the positive charges they carry.
Peptides
Polysulfoethyl A-bonded silica particles
(5 μm, 200 Å-pore size)
Silica PolySULFOETHYL A TM
2.1 × 20 0 mm
𝑇 1
𝐶 1 𝑇 2
𝐶 2
96