Protein composition is the giant arm of the post-genetic age, and many researchers have investigated the pathogenesis of cancer through proteomics analysis. A previous study40 demonstrated that all the proteins might be expressed in the tumor cells and tumor microenvironment. Surprisingly, cluster analysis revealed that diffuse gastric cancer could not only be divided into three molecular subtypes (PX1–3), but also was closely related to the survival prognosis and chemotherapy sensitivity. Sato et al.41 found that the use of CKAP4 as a biomarker changes the current treatment of lung cancer patients. In addition, the combination of CKAP4 and conventional markers can significantly improve diagnostic accuracy.
Although there have been extensive studies on LL-37, the phosphorylation analysis of LL-37 and its derived peptides in cells has not yet been reported. In this study, we implemented a quantitative proteomics analysis to determine how protein phosphorylation systematically advances apoptosis of lung cancer A549 cells treated with antimicrobial peptide merecidin.
Next, we used to quantitative proteomics research strategy via TMT labeling, phosphorylation modification enrichment techniques, and high-resolution liquid chromatography-mass spectrometry. The localization probability was > 0.75. The data were filtered to identify 8,317 phosphorylation sites on 2,900 proteins, of which 7,937 sites for 2,787 proteins provided quantitative information. We identified that 485 sites in the drug group vs mock comparison group were upregulated, and 860 sites were downregulated. The identified proteins were annotated regarding GO, protein domains, KEGG pathways, and subcellular structure localization to describe the biological process, cellular component, and molecular function. The protein motif is closely related to the biological functions of proteins, which was used to analyze the regularity of amino acid sequences before and after all phosphorylation modification sites in the sample. The data suggested that in the biological process, the protein motif is mainly involved in cell process, biological regulation, and signal transduction. The cell composition mainly involves the cytoplasm, cell membrane, and organelle, and the molecular function involves protein molecule binding, activation, and molecular function regulation. In terms of subcellular composition, the composition of the nucleus, organelles, cytoplasm, and mitochondria are involved. The functional enrichment results showed that differentially expressed proteins are involved in the regulation of the whole process from DNA replication to RNA transcription and translation, as well as protein-peptide chain formation, plait, and domain formation. The enrichment of the KEGG pathway revealed the proteins, such as GnRH, RIG-I-like, Ras, ErbB, PI3K-Akt, mTOR, and AMPK in signaling pathways. In summary, merecidin-treated A549 cells affect the cell functions from the nucleus to the cytoplasm and organelles. In addition, the COG/KOG functional classification statistics identified cytoskeleton proteins, intracellular material transport, secretion, and vesicle transport.
Historically, we found that the antimicrobial peptide merecidin induced apoptosis in lung cancer A549 cells34, 35. We also screened an array of proteins from the identification protein interaction network (Fig. 5) as follows: MAPK1, also known as ERK2, is associated with tumor proliferation42, differentiation, and invasion, and its upregulation is associated with the occurrence and development of various tumors43.Mitogen-activated protein kinase 3(MAPK3),knows as ERK1,whereas ERK pathway mediates cellular survival and growth,as well as leads to increased apoptosis44༌the study suggests that MAPK3 has close relationship with cardiomyocyte apoptosis in IRI45. MAPK14 gene, known as p38α, is one of the four family members of MAP kinase which exert versatile functions in diverse cellular processes in cancer: cell cycle regulation, proliferation, survival and motility46,as well as MAPK14 (P38) can be involved in a variety of cellular stresses, including internal metabolic stress, DNA damage, external growth factor pathway, cell-matrix interaction and intercellular communication47, 48.Mesquita et al have showed p38α inhibition Cellular migration was impaired and decreased the gastric cancer cell proliferation by provoking cell cycle arrest and cell death49.In this study ,the phosphorylation site T180/Y182 was downregulated by merecidin .Ephrin type-B receptor 2(EPHB2) is associated with the occurrence and development of A variety of diseases.EPHB2 has been shown to be associated with cells apoptosis in many studies50, 51. 52Dong et al have suggested that EphB/ephrinB reverse signaling is involved in retinal ganglion cell (RGC) apoptosis in experimental glaucoma.In our study ,the phosphorylation site S776/S575of EphB2 Upregulated. There are also a number of identified apoptosis-related proteins that have been published in the previous by our research group53.Furthermore, we speculated that merecidin induction of A540 cells enriched the KEGG-identified classic apoptosis-related signaling pathways: ErbB54, PI3K/AKT55, and AMPK-Akt56.
We suspected that merecidin might induce autophagy of A549 cells. Based on the literature, we selected the following proteins from the protein interaction network. ATG2B was mainly involved in the formation of autophagosomes and fused with lysosomes before degradation57. ATG2A and ATG2B genes are involved in autophagosome formation in HeLa cells58. In this study, the phosphorylation was upregulated at sites S240/S255 of ATG2B. ATG9A is involved in autophagosome and cytoplasmic vacuolar transport (Cvt) and is the only transmembrane protein. Atg9 plays a crucial role in the formation of autophagosomes59. After the treatment of A549 cells with merecidin, the phosphorylation at site S656/738/735 is upregulated. ATG1360 is involved in autophagy and targets the mTOR kinase signaling pathways by controlling the ATG13-ULK1 phosphorylation status and ATG13-ULK1 to regulate autophagy. Human serine/threonine protein kinase ULK1 is one of the major regulatory factors of apoptosis. ULK1/2 induces proliferation61, autophagy in ULK complex, and controls autophagosome biogenesis via early signaling pathways62. The S738/S735 of ATG13 and S479/S477 of ULK1 was upregulated in this study. RAB7A is a key regulator of lysosomal transport and critical for autophagosome-lysosome fusion63. As a vital protein in the FOXO1/RAB7 signaling pathway, RAB7A induces the autophagy by regulating RAB7 upregulation. The phosphorylation at S72 site in RAB7 is also upregulated in this study. MAPK164 harbors several signal integration points that participate in a variety of cellular processes, such as proliferation, differentiation, transcription, and development. The upstream kinase phosphorylation activation MAPK1/ERK2 was applied for the stimulation of the nucleus, and the inhibition of phosphorylation of MAPK1 activates autophagy65, 66. The phosphorylation site T185/Y187 of MAPK1 is upregulated after treatment with merecidin. EIF4B is also known as cap-binding protein, one of the vital components of the eIF4F complex. The high expression can lead to tumor proliferation, invasion, and metastasis. Zhang et al.67 found that inhibition of EIF4B expression enhances autophagic death, and S579 of EIF4B was found to be upregulated in this study. The EGFR signaling pathway regulates the autophagy process; inhibition of the expression of EGFR elevates autophagy, leading to autophagic death68. In this study, TSC2 was found to activate autophagy by activating the TSC2/mTOR signaling pathway69, 70, and the phosphorylation site S1132/S1452 of TSC2 is downregulated. PAK4 is highly expressed in a variety of tumor tissues and cells and is related to cytological behaviors, such as cell proliferation, cycle, and migration. Low expression of PAK4 can upregulate p53, inhibit mTOR or Akt/mTOR signaling pathway, inhibits cell migration, and activate autophagy71, or the signaling pathway72 can be downregulated, thus activating autophagy73, 74. The phosphorylation site S103 of PAK4 downregulated after merecidin treatment. AKT1 regulates metabolism, proliferation, cell survival, growth, and angiogenesis, inhibits growth, and induces apoptosis of non-SCLC by directly regulating Akt1/229, mediated by phosphorylation of serine and/or threonine by a series of downstream substrates. In addition, Akt1/Foxo3a75 and PI3K/AKT/mTOR76 signaling pathways are associated with autophagy, and the phosphorylation at site S124 of AKT1 is downregulated after merecidin treatment. Additionally, we found that the autophagy-related pathways mTOR and PI3K-Akt were selected from KEEG pathway enrichment data. In conclusion, we hypothesized that merecidin induces autophagy in A549 cells and by mTOR and/or PI3K-Akt signaling pathway.