Involvement of RbAp48 in erythroid differentiation of murine erythroleukemia cells induced by sodium butyrate.

Normal mammalian terminal erythroid differentiation is a precisely regulated process during which the progenitor cells execute particular programs to form a mature erythrocytic phenotype. In the present study, it was found that RbAp48, a histone-binding protein associated with retinoblastoma protein, was upregulated during terminal erythroid maturation in vivo and in vitro. This indicated that RbAp48, at least in part, participated in the regulation of murine erythropoiesis. Following sodium butyrate (SB) induction, murine erythroleukemia (MEL) cells began to re-enter erythroid differentiation and the ratio of differentiated cells reached ~80% at 72 h. The erythroid maturation-related mRNA expression of α-globin, ß-globin and glycophorin A (GPA) was increased markedly, which indicated that SB induced MEL differentiation. During MEL differentiation, the RbAp48 level showed a 1.5-fold increase at 72 h, and the globin transcription factor (GATA)-1 level was also upregulated in the early stage of differentiation. By contrast, the c-Myc level was gradually downregulated in MEL differentiation. Using an immunofluorescence assay, the results of the study directly showed that the average fluorescence intensity of RbAp48 in each cell reached an almost 1.7-fold increase at 72 and 96 h. This was consistent with the western blot results of RbAp48 during MEL differentiation. In addition, reduced expression of RbAp48 by RNA inference decreased SB-induced MEL differentiation by ~20%, indicating that a high level of RbAp48 was essential for MEL differentiation. Taken together, these results established a functional link between RbAp48 and erythroid differentiation.

Outer membrane protein OmpW of Escherichia coli is required for resistance to phagocytosis.

Eight-stranded ß-barrel outer membrane proteins can confer bacterial virulence via resistance to host innate defenses. This resistance function of OmpW, which was recently identified as an eight-stranded ß-barrel protein, was investigated in this study. Our results demonstrated that upregulation of OmpW correlated with increased bacterial survival during phagocytosis. Bacterial mutants harboring a deletion of ompW exhibited a significantly increased phagocytosis rate. Both observations suggest that the OmpW protein protects bacteria against host phagocytosis. In addition, expression of ompW is regulated by iron, which implies that the resistance provided by OmpW may be an important factor in iron-related infectious diseases. Furthermore, OmpW has been identified as a protective antigen that protects mice against bacterial infection and is therefore a promising target for vaccine development against infectious diseases.

Different regulatory pathways are involved in the proliferative inhibition of two types of leukemia cell lines induced by paclitaxel.

Paclitaxel, one of the broadest-spectrum anticancer agents, is currently being used in the treatment of patients with solid tumors. In the present study, we compared the effect of paclitaxel on two types of leukemia cells. Our results showed that paclitaxel could inhibit the proliferation of MEL and K562 cells in a dose- and time-dependent manner. The mechanism of proliferative inhibition in K562 cells treated by paclitaxel was related to the cell cycle arrest in the G2/M phase, as well as the induction of apoptosis. By contrast, MEL cells treated by paclitaxel showed significant characteristics of necrosis, which indicated that the mode of cell death induced by paclitaxel in these two types of leukemia cells differed. Advances in research of the cell cycle, apoptosis and necrosis will extend our understanding of the mechanisms of paclitaxel-induced cell death, particularly in leukemia cells. Further elucidation of the mechanisms of necrosis in MEL cells may expedite the development of improved paclitaxel-based regimens for cancer therapy.

Novel cytotoxic exhibition mode of antimicrobial peptide anoplin in MEL cells, the cell line of murine Friend leukemia virus-induced leukemic cells.

Anoplin is a recently discovered antimicrobial peptide (AMP) isolated from the venom sac of the spider wasp Anoplius samariensis, and it is one of the shortest α-helical AMP found naturally to date consisting of only ten amino acids. Previous results showed that anoplin exhibits potent antimicrobial activity but little hemolytic activity. In this study, we synthesized anoplin, studied its cytotoxicity in Friend virus-induced leukemia cells [murine erythroleukemia (MEL) cells], and proposed its possible mechanism. Our results showed that anoplin could inhibit the proliferation of MEL cells in a dose-dependent and time-dependent manner via disrupting the integrity of cell membrane, which indicated that anoplin exerts its cytotoxicity efficacy. In addition, the cell cycle distribution of MEL cells was arrested in the G0/G1 phase significantly. However, anoplin could not induce obvious apoptosis in MEL cells, as well as anoplin could not induce visible changes on morphology and quantity in the bone marrow cells isolated from normal mice. All of these results indicate that anoplin, as generally believed, is a selective AMP, a value characteristic in the design of safe therapeutic agents. The cytotoxicity of anoplin on MEL cells was mainly attributable to the plasma membrane perturbation and also to the intracellular events such as the arrest of cell cycle. Although this is an initial study that explored the activity of anoplin in vitro rather than in vivo, with the increasing resistance of conventional chemotherapy, there is no doubt that anoplin has desirable feature to be developed as a novel and selective anticancer agent.

Study on the distribution sites and the molecular mechanism of analgesia after intracerebroventricular injection of rat/mouse hemokinin-1 in mice.

Hemokinin-1 is a peptide encoded by Pptc, which belongs to the family of mammalian tachykinins. Our previous results showed that rat/mouse hemokinin-1 (r/m HK-1) produced striking analgesia after intracerebroventricular (i.c.v.) injection in mice, and the analgesia could be blocked by the NK1 receptor antagonist and the opioid receptor antagonist, respectively. However, the precise distribution sites and the molecular mechanism involved in the analgesic effect after i.c.v. administration of r/m HK-1 are needed to be further investigated deeply. Using the fluorescence labeling method, our present results directly showed that r/m HK-1 peptides were mainly distributed at the ventricular walls and several juxta-ventricular structures for the first time. Our results showed that the mRNA expressions of NK1 receptor, PPT-A, PPT-C, KOR, PDYN, DOR and PENK were not changed markedly, as well as the protein expression of NK1 receptor was hardly changed. However, both the transcripts and proteins of MOR and POMC were up-regulated significantly, indicating that the analgesic effect induced by i.c.v. administration of r/m HK-1 is related to the activation of NK1 receptor first, then it is related to the release of endogenous proopiomelanocortin, as well as the increased expression level of µ opioid receptor. These results should facilitate further the analysis of the analgesia of r/m HK-1 in the central nerval system in acute pain and may open novel pharmacological interventions.

Identification of galectin-7 as a potential biomarker for esophageal squamous cell carcinoma by proteomic analysis.

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is one of the most common malignancies. Early diagnosis is critical for guiding the therapeutic management of ESCC. It is imperative to find more effective biomarkers of ESCC. METHODS: To identify novel biomarkers for esophageal squamous cell carcinoma (ESCC), specimens from 10 patients with ESCC were subjected to a comparative proteomic analysis. The proteomic patterns of ESCC samples and normal esophageal epithelial tissues (NEETs) were compared using two-dimensional gel electrophoresis. And differentially expressed proteins were identified using MALDI-TOF-MS/MS. For further identification of protein in selected spot, western blotting and immunohistochemistry were employed. RESULTS: Twelve proteins were up-regulated and fifteen proteins were down-regulated in the ESCC samples compared with the NEET samples. Up-regulation of galectin-7 was further confirmed by western blotting and immunohistochemistry. Furthermore, immunohistochemical staining of galectin-7 was performed on a tissue microarray containing ESCC samples (n = 50) and NEET samples (n = 10). The expression levels of galectin-7 were markedly higher in the ESCC samples than in the NEET samples (P = 0.012). In addition, tissue microarray analysis also showed that the expression level of galectin-7 was related to the differentiation of ESCC. CONCLUSIONS: The present proteomics analysis revealed that galectin-7 was highly expressed in ESCC tissues. The alteration in the expression of galectin-7 was confirmed using a tissue microarray. These findings suggest that galectin-7 could be used as a potential biomarker for ESCC.

In vitro unfolding of insulin: characterization of intermediates and putative unfolding pathway.

The in vitro insulin unfolding had been studied using the "equilibrium unfolding" method where protein is unfolded by reducing reagents in the presence of trace amounts of oxidants such as oxidized glutathione. Nine intermediates were captured in the unfolding process, named as P1A, P2A, P3A, P4A, P3B, P4B, P5B, P6B, and P7B, which were all either A chain derivatives or B chain derivatives. No intermediate with inter-A-B chain disulfide was captured. Based on the character of the intermediates, their distribution during the unfolding process and the hypothetic "transient" intermediates, an in vitro putative unfolding pathway of insulin had been proposed. Besides, the comparison of the intermediates captured in unfolding with the intermediates captured in the refolding process of insulin revealed that both unfolding/refolding processes of insulin shared common intermediates. Based on these observations we suggested that the unfolding pathway of insulin was similar to the refolding pathway but flowed in the opposite direction.

Comparative proteomic analysis of colon cancer cells in response to oxaliplatin treatment.

Colon cancer is one of the most common malignancies in the world. Oxaliplatin, a third-generation platinum compound, is widely used in clinical chemotherapy of colon cancer. Although the mechanisms of the antitumor effect of Oxaliplatin have been investigated in recent years, the proteomic changes that are associated with the cellular response to this compound are poorly understood. In this study, we performed a comparative proteomic analysis to survey the global changes in protein expression levels after Oxaliplatin treatment in three colon cancer cell lines: HT29, SW620, and LoVo. Two-dimensional gel electrophoresis coupled with MALDI-TOF/TOF mass spectrometry revealed 57, 48, and 53 differentially expressed proteins in the three cell lines (HT29, SW620 and LoVo, respectively) after Oxaliplatin treatment. Of these proteins, 21 overlapped among all three cell lines. These overlapping proteins participate in many cellular processes, such as apoptosis, signal transduction, transcription and translation, cell structural organization, and metabolism. Additionally, the expression levels of ezrin (EZRI), heat-shock protein beta-1 (HSPB1), translationally controlled tumor protein (TCTP), and cell division control protein 2 homolog (CDC2) were confirmed by immunoblotting. This is the first direct proteomic analysis of Oxaliplatin-treated colon cancer cells. Several interesting proteins that we found warrant further investigation owing to their potential significant functions in the antitumor effect of Oxaliplatin.

Ferritin heavy chain-mediated iron homeostasis and subsequent increased reactive oxygen species production are essential for epithelial-mesenchymal transition.

The epithelial-mesenchymal transition (EMT) plays a critical role in tumor progression. To obtain a broad view of the molecules involved in EMT, we carried out a comparative proteomic analysis of transforming growth factor-beta1 (TGF-beta1)-induced EMT in AML-12 murine hepatocytes. A total of 36 proteins with significant alterations in abundance were identified. Among these proteins, ferritin heavy chain (FHC), a cellular iron storage protein, was characterized as a novel modulator in TGF-beta1-induced EMT. In response to TGF-beta1, there was a dramatic decrease in the FHC levels, which caused iron release from FHC and, therefore, increased the intracellular labile iron pool (LIP). Abolishing the increase in LIP blocked TGF-beta1-induced EMT. In addition, increased LIP levels promoted the production of reactive oxygen species (ROS), which in turn activated p38 mitogen-activated protein kinase. The elimination of ROS inhibited EMT, whereas H2O2 treatment rescued TGF-beta1-induced EMT in cells in which the LIP increase was abrogated. Overexpression of exogenous FHC attenuated the increases in LIP and ROS production, leading to a suppression of EMT. We also showed that TGF-beta1-mediated down-regulation of FHC occurs via 3' untranslated region-dependent repression of the translation of FHC mRNA. Moreover, we found that FHC down-regulation is an event that occurs between the early and highly invasive advanced stages in esophageal adenocarcinoma and that depletion of LIP or ROS suppresses the migration of tumor cells. Our data show that cellular iron homeostasis regulated by FHC plays a critical role in TGF-beta1-induced EMT.

Comparative proteomic analysis of cell cycle-dependent apoptosis induced by transforming growth factor-beta.

Transforming growth factor-beta (TGF-beta) can induce G2/M phase-dependent apoptosis and G1/S phase-dependent epithelial-mesenchymal transition (EMT) in hepatocytes, but the underlying mechanism remains poorly understood. In this study, we investigated alterations in the global proteome using two dimensional gel electrophoresis of AML-12 murine hepatocyte cells after treatment with TGF-beta at several time points after synchronization in the G2/M or G1/S phase. Upon TGF-beta treatment, the expression levels of 44 proteins were found to be significantly changed in cells synchronized in the G2/M phase. These proteins were identified by MALDI-TOF/TOF and classified into seven categories according to function. In addition, TGF-beta induced downregulation of glutamine synthetase in cells in G2/M but not G1/S phase, and this was further confirmed by immunoblotting. Moreover, exogenous glutamine completely blocked TGF-beta-induced apoptosis in G2/M and non-synchronized cells, whereas it had no effect on EMT, suggesting that the downregulation of glutamine synthetase is involved in G2/M phase-dependent apoptosis. These results provide new insight into the mechanism of the multifunctional effects of TGF-beta and how apoptosis and EMT are regulated in the same type of cells.

Comparative proteomic analysis of proteins involved in cell aggregation during neural differentiation of P19 mouse embryonic carcinoma cells.

Cell-cell interactions play a crucial role during embryogenesis and are enhanced during cell aggregation. P19 mouse embryonic carcinoma cells can differentiate into neural cells by the addition of retinoic acid (RA) or by overexpression of the Wnt1 gene, with both processes dependent on cell aggregation. To identify molecules involved in the cell aggregation process, two-dimensional gel electrophoresis (2DE) was used to establish the cell aggregation-associated protein profiles. MALDI-TOF/TOF was used to identify 71 protein spots with differential expression patterns. Among these spots, 54 were differentially expressed in both P19 and Wnt1-overexpressing P19 (Wnt1/P19) cell aggregates, with 42 proteins up-regulated and 12 proteins down-regulated. The other 17 spots were differentially expressed only in Wnt1/P19 cells. The expression patterns of 5 cell aggregation-associated proteins, N-myc downstream-regulated gene 1 (NDRG1), 14-3-3 epsilon, 14-3-3 gamma, acid calponin and cell division control protein 2 homologue (Cdc2), were confirmed by immunoblot and RT-PCR. To further investigate the relationship between cell aggregation and neural differentiation, NDRG1 expression was inhibited by RNA interference during P19 cell aggregation. Silencing of NDRG1 reduced the size of cell aggregates and the expression of N-cadherin, and it also impaired the RA-induced P19 cell neural differentiation. In conclusion, this study provides new clues for the possible mechanism underlying cell aggregation during pluripotent stem cell neural differentiation.

Stain efficiency and MALDI-TOF MS compatibility of seven visible staining procedures.

Visible stain is still the most popular protein staining method used in proteomic approaches. However, most published data have been derived from comparisons between visible dyes and fluorescent dyes. In this work, we have focused on seven widely used visible staining procedures--Neuhoff CCB, blue silver, and five silver stains (LKB SN, He SN, Yan SN, Vorum SN, and Blum SN)--and studied their stain efficiencies and MALDI-TOF MS compatibilities on 1-D and 2-D PAGE. It was concluded that blue silver is slightly better in terms of stain efficiency than Neuhoff CCB, but it presented worse MS compatibility. Neuhoff CCB presented better MS compatibility and superior linearity but worse sensitivity than silver stains. Among the five silvering procedures, He SN showed the best MS compatibility and a reasonable staining efficiency; Yan SN lowered the chances of obtaining the protein identity by PMF but gave the best stain efficiency; Vorum SN gave a very clear background and a great contrast, while Blum SN was the worst in this respect. The implications of these results for the selection of a convenient stain are discussed according to specific objectives as well as practical aspects.

Molecular cloning and characterization of two novel cellulase genes from the mollusc Ampullaria crossean.

Cellulase genes have been reported not only from fungi, bacteria and plant, but also from some invertebrate animals. Here, two cellulase (endo-beta-1,4-glucanase, EC 3.2.1.4) genes, eg27I and eg27II, were cloned from the freshwater snail Ampullaria crossean cDNA using degenerate primers. The nucleotide sequences of the two genes shared 94.5% identity. The open reading frames of both genes consisted of 588 bp, encoding 195 amino acids. Both EG27I and EG27II belong to the glycoside hydrolase family 45, and each lacks a carbohydrate-binding module. The presence of introns demonstrated a eukaryotic origin of the EG27 gene, and, in addition, successful cloning of EG27 cDNA supported endogenous production of EG27 cellulase by Ampullaria crossean. Investigation of the EG27 cDNA from A. crossean will provide further information on GHF45 cellulases.

Identification of candidate prostate cancer biomarkers in prostate needle biopsy specimens using proteomic analysis.

Although serum prostate specific antigen (PSA) is a well-established diagnostic tool for prostate cancer (PCa) detection, the definitive diagnosis of PCa is based on the information contained in prostate needle biopsy (PNBX) specimens. To define the proteomic features of PNBX specimens to identify candidate biomarkers for PCa, PNBX specimens from patients with PCa or benign prostatic hyperplasia (BPH) were subjected to comparative proteomic analysis. 2-DE revealed that 52 protein spots exhibited statistically significantly changes among PCa and BPH groups. Interesting spots were identified by MALDI-TOF-MS/MS. The 2 most notable groups of proteins identified included latent androgen receptor coregulators [FLNA(7-15) and FKBP4] and enzymes involved in mitochondrial fatty acid beta-oxidation (DCI and ECHS1). An imbalance in the expression of peroxiredoxin subtypes was noted in PCa specimens. Furthermore, different post-translationally modified isoforms of HSP27 and HSP70.1 were identified. Importantly, changes in FLNA(7-15), FKBP4, and PRDX4 expression were confirmed by immunoblot analyses. Our results suggest that a proteomics-based approach is useful for developing a more complete picture of the protein profile of PNBX specimen. The proteins identified by this approach may be useful molecular targets for PCa diagnostics and therapeutics.

Molecular and biochemical characterization of Ba-EGA, a cellulase secreted by Bacillus sp. AC-1 from Ampullaria crosseans.

A novel gene (Ba-ega) of Bacillus sp. AC-1, encoding an endoglucanase (Ba-EGA), was cloned and expressed in Escherichia coli. Ba-ega, containing a 1,980-bp open reading frame (ORF), encoded a protein of 659 amino acids and had a molecular mass of 74.87 kDa. Ba-EGA was a modular enzyme composed of a family-9 glycosyl hydrolase catalytic module (CM9) and a family-3 carbohydrate-binding module (CBM3). To investigate the functions of the CBM3 and CM9, a number of truncated derivatives of Ba-EGA were constructed, and all were active. The catalytic module (rCM9) alone was less stable at high temperature than the recombinant Ba-EGA (rBa-EGA). The temperature stability for the complex of rCM9 and rCBM3 was still lower than rBa-EGA, but higher than rCM9 alone. These observations indicated the existence of a non-covalent interaction between CM9 and CBM3 that might strengthen the stability of CM9. However, this interaction is not strong enough to mimic the protective effect of the CBM in the wild-type enzyme.

Purification and characterization of two endo-beta-1,4-glucanases from mollusca, Ampullaria crossean.

Two novel endo-beta-1,4-glucanases, EG45 and EG27, were isolated from the gastric juice of mollusca, Ampullaria crossean, by anion exchange, hydrophobic interaction, gel filtration and a second round of anion exchange chromatography. The purified proteins EG45 and EG27 appeared as a single band on sodium dodecylsulfate polyacrylamide gel electrophoresis with a molecular mass of 45 kDa and 27 kDa, respectively. The optimum pH for CMC activity was 5.5 for EG45 and 4.4-4.8 for EG27. The optimum temperature range for EG27 was broad, between 50 degrees and 60 degrees; for EG45 it was 50 degrees. The analysis on the stability of these two endo-beta-1,4-glucanases showed that EG27 was acceptably stable at pH 3.0-11.0 even when the incubation time was prolonged to 24 h at 30 degrees, whereas EG45 remained relatively stable at pH 5.0-8.0. About 85% of the activity of EG27 could be retained upon incubation at 60 degrees for 24 h. However, less than 10% residual activity of EG45 was detected at 50 degrees. Among different kinds of substrates, both enzymes showed a high preference for carboxymethyl cellulose. EG45, in particular, showed a carboxymethyl cellulose hydrolytic activity of 146.5 IU/mg protein. Both enzymes showed low activities to xylan (from oat spelt) and Sigmacell 101, and they were inactive to p-nitrophenyl-beta-D-cellobioside, salicin and starch.

Differential display of proteins involved in the neural differentiation of mouse embryonic carcinoma P19 cells by comparative proteomic analysis.

Mouse embryonic carcinoma P19 cell has been used extensively as a model to study molecular mechanisms of neural differentiation in vitro. After retinoic acid (RA) treatment and aggregation, P19 cells can differentiate into neural cells including neurons and glial cells. In this study, comparative proteomic analysis is utilized to approach the protein profiles associated with the RA-induced neural differentiation of P19 cells. Image analysis of silver stained two-dimensional gels indicated that 28 protein spots had significantly differential expression patterns in both quantity and quality. With mass spectrometry analysis and protein functional exploration, many proteins demonstrated an association with distinct aspects of neural differentiation. These proteins were gag polyprotein, rod cGMP-specific 3',5'-cyclic phosphodiesterase, 53 kDa BRG1-associated factor A, N-myc downstream regulated 1, Vitamin D receptor associated factor 1, stromal cell derived factor receptor 1, phosphoglycerate mutase, Ran-specific GTPase-activating protein, and retinoic acid (RA)-binding protein. While some cytoskeleton-related proteins such as beta cytoskeletal actin, gamma-actin, actin-related protein 1, tropomyosin 1, and cofilin 1 are related to cell migration and aggregation, other proteins have shown a relationship with distinct aspects of neural differentiation including energy production and utilization, protein synthesis and folding, cell signaling transduction, and self-protection. The differential expression patterns of these 28 proteins indicate their different roles during the neural differentiation of P19 cells. As an initial step toward unveiling the regulations involved in the commitment of pluripotent cells to a neural fate, information from this study may be helpful to uncover the molecular mechanisms of neural differentiation.

pH-dependent stability of EGX, a multi-functional cellulase from mollusca, Ampullaria crossean.

The cellulase activity and stability of EGX, a multi-functional cellulase previously purified from the mollusca Ampullaria crossean, was systematically studied under different pH. The pH induced con-formation and stability change of EGX have been investigated by using the intrinsic fluorescence, ANS fluorescence and CD spectrum. It has been found that the conformation and activity of this cellulase were strongly dependent on the pH. EGX was stable for both the enzyme activity and the conformation from pH 5.6 to pH 7.4. As shown by intrinsic and ANS fluorescence, no red shift of emission maximum occurred and a negligible intensity change was observed at pH 5.6-7.4. The activity of EGX remained about 80% in pH 5.6-7.4 and obviously decreased out of side the pH range. Urea-induced changes in EGX at pH 5.4 and pH 8.0 were measured by intrinsic fluorescence and CD spectrum. At pH 5.4, a significantly red shift of emission maximum occurred when the concentration of urea was 5 M compared to the concentration was 3 M at pH 8.0. The alpha-helix at pH 5.4 was 40.51% in the absence of urea and 31.04% in the presence of 4 M urea. At pH 8.0 the alpha-helix was 7.23% in the presence of 4 M urea. The data indicated that EGX was much susceptible to urea-induced unfolding at pH 8.0 and much stable at pH 5.4. The greater pH dependent stability of EGX may allow the enzyme to adequately catalyze the hydrolysis of cellulosic materials under natural or industrial extreme conditions.

Proteomic analysis of differential protein expression in a human hepatoma revertant cell line by using an improved two-dimensional electrophoresis procedure combined with matrix assisted laser desorption/ionization-time of flight-mass spectrometry.

The proteomic profiles of a human hepatoma revertant, CL1, and its original cell line, SMMC7721, were compared by using an improved two-dimensional electrophoresis (2-DE) procedure, with multi-IPGstrips gels (length

[Analysis of differentially expressed lung metastasis-associated proteins in adenoid cystic carcinoma cell lines].

OBJECTIVE: To analyze differentially expressed metastasis-associated proteins in Adenoid cystic carcinoma cell lines of human salivary gland by proteomics. METHODS: Protein expression alterations of ACC-2 and ACC-M cells were described by 2-D gels. After image analysis by software, proteins of interest were excised from the gels and identified by matrix assisted laser desorption ionization time-of-flight mass spectrometer. RESULTS: 12 protein spots showed significantly differential expression patterns between two cell lines. In the identified protein candidates, transketolase, modulator recognition factor 2, Dim1p homolog, splicing factor (arginine/serine-rich 9) and v-Ha-ras l oncogene were all lowly expressed in the poorly metastatic ACC-2 cell and significantly upregulated in highly metastatic ACC-M cell, while type I collagen pro alpha and tumor necrosis factor (ligand) superfamily member 4 showed a high expression in ACC-2 cells and a low expression in ACC-M cells. Pirin (spot 6) just appears in ACC-2 cell and was not detectable in ACC-M cell, while retinal homeobox protein was just detected in ACC-M cell and did not appear in ACC-2 cell. CONCLUSIONS: The proteins may be involved in the adenoid cystic carcinoma lung metastasis through different mechanisms. Our work may contribute to discover diagnostic markers and therapeutic targets.