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Exosomes are deliverers of critically functional proteins, capable of transforming target cells in numerous cancers, including hepatocellular carcinoma (HCC). We hypothesize that the motility of HCC cells can be featured by comparative proteome of exosomes. Hence, we performed the super-SILAC-based MS analysis on the exosomes secreted by three human HCC cell lines, including the non-motile Hep3B cell, and the motile 97H and LM3 cells. More than 1400 exosomal proteins were confidently quantified in each MS analysis with highly biological reproducibility. We justified that 469 and 443 exosomal proteins represented differentially expressed proteins (DEPs) in the 97H/Hep3B and LM3/Hep3B comparisons, respectively. These DEPs focused on sugar metabolism-centric canonical pathways per ingenuity pathway analysis, which was consistent with the gene ontology analysis on biological process enrichment. These pathways included glycolysis I, gluconeogenesis I and pentose phosphate pathways; and the DEPs enriched in these pathways could form a tightly connected network. By analyzing the relative abundance of proteins and translating mRNAs, we found significantly positive correlation between exosomes and cells. The involved exosomal proteins were again focusing on sugar metabolism. In conclusion, motile HCC cells tend to preferentially export more sugar metabolism-associated proteins via exosomes that differentiate them from non-motile HCC cells.

Odoroside A (OA) is an active ingredient extracted from the leaves of Nerium oleander Linn. (Apocynaceae). This study aims to examine the anticancer bioactivity of OA against CRC cells and to investigate the action mechanisms involved. As a result, OA can significantly inhibit cellular ability and induce apoptosis of CRC cells in a concentration-dependent manner without any obvious cytotoxicity in normal colorectal epithelial cells. Then, quantitative proteomics combined with bioinformatics is adopted to investigate the alterations of proteins and signaling pathways in response to OA treatment. As suggested by the proteomic analysis, flow cytometry and Western blotting analyses validate that exposure of CRC cells to OA causes cell cycle arrest and apoptosis, accompanied with the activation of the ROS/p53 signaling pathway. This observation demonstrates that OA, as a natural product, can induce oxidative stress to suppress tumor cell growth, implicating a novel therapeutic agent against CRC without obvious side effects.

Andrographolide-lipoic acid conjugate (AL-1) is a new in-house synthesized chemical entity, which was derived by covalently linking andrographolide with lipoic acid. However, its anti-cancer effect and cytotoxic mechanism remains unknown. In this study, we found that AL-1 could significantly inhibit cell viability of human leukemia K562 cells by inducing G2/M arrest and apoptosis in a dose-dependent manner. Thirty-one AL-1-regulated protein alterations were identified by proteomics analysis. Gene ontology and ingenuity pathway analysis revealed that a cluster of proteins of oxidative redox state and apoptotic cell death-related proteins, such as PRDX2, PRDX3, PRDX6, TXNRD1, and GLRX3, were regulated by AL-1. Functional studies confirmed that AL-1 induced apoptosis of K562 cells through a ROS-dependent mechanism, and anti-oxidant, N-acetyl-L-cysteine, could completely block AL-1-induced cytotoxicity, implicating that ROS generation played a vital role in AL-1 cytotoxicity. Accumulated ROS resulted in oxidative DNA damage and subsequent G2/M arrest and mitochondrial-mediated apoptosis. The current work reveals that a novel andrographolide derivative AL-1 exerts its anticancer cytotoxicity through a ROS-dependent DNA damage and mitochondrial-mediated apoptosis mechanism.

Genistein exerts its anticarcinogenic effects by inducing G2/M arrest and apoptosis of cancer cells. However, the precise molecular mechanism of action of genistein has not been completely elucidated. In this study, we used quantitative proteomics to identify the genistein-induced protein alterations in gastric cancer cells and investigate the molecular mechanism responsible for the anti-cancer actions of genistein. Total 86 proteins were identified to be regulated by genistein, most of which were clustered into the regulation of cell division and G2/M transition, consistent with the anti-cancer effect of genistein. Many proteins including kinesin family proteins, TPX2, CDCA8, and CIT were identified for the first time to be regulated by genistein. Interestingly, five kinesin family proteins including KIF11, KIF20A, KIF22, KIF23, and CENPF were found to be simultaneously downregulated by genistein. Significantly decreased KIF20A was selected for further functional studies. The silencing of KIF20A inhibited cell viability and induced G2/M arrest, similar to the effects of genistein treatment in gastric cancer. And the silencing of KIF20A also increased cancer cell sensitivity to genistein inhibition, whereas overexpression of KIF20A markedly attenuated genistein-induced cell viability inhibition and G2/M arrest. These observations suggested that KIF20A played an important role in anti-cancer actions of genistein, and thus may be a potential molecular target for drug intervention of gastric cancer.

We previously used proteomics technology to globally identify gastric cancer-associated proteins and found that gastrokine 1 (GKN1) was dramatically underexpressed in gastric cancer tissues. Here, we further showed that GKN1 could inhibit cell growth and induce cell cycle arrest in gastric cancer cells. The activity of protein kinase PKCδ/θ was inhibited by GKN1, whereas the activity of ERK1/2 and JNK1/2 was increased by GKN1, suggesting that GKN1 induced growth inhibition of gastric cancer cells by synergistically regulating the activity of these protein kinases. Seventy-four proteins were found to be regulated by GKN1 by proteomics analysis, including α-enolase (ENO1) and Cathepsin D. Interestingly, ENO1 is an important hub in the protein-protein interaction network of the 74 differential proteins. Silencing of ENO1 resulted in growth inhibition and cell cycle arrest of gastric cancer cells, similar to the effect of GKN1 overexpression in cells, whereas ENO1 overexpression blocked GKN1-induced growth inhibition and cell cycle arrest. These observations suggested that ENO1 downregulation played an important role in GKN1-induced growth inhibition of gastric cancer cells.

Subcellular proteomics was used to compare the protein profiles between human lung adenocarcinoma A549 cells and human bronchial epithelial (HBE) cells. In total, 106 differential proteins were identified and the altered expression levels of partial identified proteins were confirmed by Western blot analysis. Importantly, pathway analysis and biological validation revealed epithelial-mesenchymal transition (EMT) phenotype shift in A549 cells as compared with HBE cells. The EMT phenotype of A549 cells can be increased by self-producing TGF-β1 and significantly decreased by silencing heterogeneous nuclear ribonucleoprotein (hnRNPK) expression. As EMT has been considered as an important event during malignant tumor progression and metastasis, investigating EMT and deciphering the related pathways may lead to more efficient strategies to fight lung cancer progression. By integrating the subcellular proteomic data with EMT-related functional studies, we revealed new insights into the EMT progress of lung carcinogenesis, providing clues for further investigations on the discovery of potential therapeutic targets.

MIP-T3 (microtubule-interacting protein associated with TRAF3) is a microtubule-interacting protein that evolutionarily conserved from worms to humans, but whose cellular functions remains unknown. To get insight into the functions of MIP-T3, we set out to identify MIP-T3 interacting proteins by immunoprecipitation in human embryonic kidney 293 cells and MS analysis. As the results, a total of 34 proteins were identified and most of them were novel MIP-T3 putative partners. The MIP-T3-associated proteins could be grouped into nine clusters based on their molecule functions, including cytoskeleton, chaperone, nucleic acid binding, kinase and so on. Three MIP-T3-interacted proteins - actin, HSPA8 and tubulin - were further confirmed by reciprocal coimmunoprecipitations and colocalization analysis. The interaction of MIP-T3 with both actin filaments and microtubule suggested that MIP-T3 may play an important role in regulation of cytoskeleton dynamics in cells. Our results therefore not only uncover a large number of MIP-T3-associated proteins that possess a variety of cellular functions, but also provide new research directions for the study of the functions of MIP-T3.

Genistein is a natural protein tyrosine kinase inhibitor that exerts anti-cancer effect by inducing G2/M arrest and apoptosis. However, the phosphotyrosine signaling pathways mediated by genistein are largely unknown. In this study, we combined tyrosine phosphoprotein enrichment with MS-based quantitative proteomics technology to globally identify genistein-regulated tyrosine phosphoproteins aiming to depict genistein-inhibited phosphotyrosine cascades. Our experiments resulted in the identification of 213 phosphotyrosine sites on 181 genistein-regulated proteins. Many identified phosphoproteins, including nine protein kinases, eight receptors, five protein phosphatases, seven transcriptical regulators and four signal adaptors, were novel inhibitory effectors with no previously known function in the anti-cancer mechanism of genistein. Functional analysis suggested that genistein-regulated protein tyrosine phosphorylation mainly by inhibiting the activity of tyrosine kinase EGFR, PDGFR, insulin receptor, Abl, Fgr, Itk, Fyn and Src. Core signaling molecules inhibited by genistein can be functionally categorized into the canonial Receptor-MAPK or Receptor-PI3K/AKT cascades. The method used here may be suitable for the identification of inhibitory effectors and tyrosine kinases regulated by anti-cancer drugs.

Increasing evidence shows that calpain-mediated proteolytic processing of a selective number of proteins plays an important role in neuronal apoptosis. Study of calpain-mediated cleavage events and related functions may contribute to a better understanding of neuronal apoptosis and neurodegenerative diseases. We, therefore, investigated the role of calpain substrates in potassium deprivation-induced apoptosis of cerebellar granule neurons (CGNs). Twelve previously known and seven novel candidates of calpain substrates were identified by 2-D DIGE and MALDI-TOF/TOF MS analysis. Further, the identified novel calpain substrates were validated by Western blot analysis. Moreover, we focused on the collapsin response mediator proteins (CRMP-1, -2, -3 and -4 isoforms) and found that CRMPs were proteolytically processed by calpain but not by caspase, both in vivo and in vitro. To clarify the properties of the calpain-mediated proteolysis of CRMPs, we constructed the deletion mutants of CRMPs for additional biochemical studies. In vitro cleavage assays revealed that CRMP-1, -2 and -4 were truncated by calpain at the C-terminus, whereas CRMP-3 was cleaved at the N-terminus. Finally, we assessed the role of CRMPs in the process of potassium deprivation-triggered neuronal apoptosis by overexpressing the truncated CRMPs in CGNs. Our data clearly showed that the truncated CRMP-3 and -4, but not CRMP-1 and -2, significantly induced neuronal apoptosis. These findings demonstrated that calpain-truncated CRMP-3 and -4 act as pro-apoptotic players when CGNs undergo apoptosis.

Chromium (Cr) has been widely used in industry for more than one century. Exposure to hexavalent Cr compounds is strongly associated with increasing risk of lung cancer. Extensive researches at DNA level indicated that generation of ROS from the reduction of Cr(VI) leading to DNA damage is the major cause of the toxicity and carcinogenicity of Cr(VI). The present study in cellular and protein levels confirmed that Cr(VI) induced apoptosis of lung epithelial cells (LEC) via ROS generation. To view the differentially expressed proteins in the process of Cr(VI) reduction, subcellular proteomics was applied and allowed the identification of more than 30 proteins with expression alteration. Most of those proteins are correlated with ROS-elicited responses, which were further validated by Western blotting analysis, induction of p53 pathway and antioxidative treatment. The current findings provided additional evidence in protein level to support the claim that ROS generated during the process of Cr(VI) reduction are involved in the Cr(VI)-induced toxicity and carcinogenesis.

There has been increasing interest in the potential applications of gold (III) complexes as anticancer drugs with higher cytotoxicity and fewer side effects than existing metal anticancer drugs. Our previous findings demonstrated that gold (III) porphyrin 1a preferentially induced apoptosis in a cancer cell line (SUNE1). In this study, we identified differentially expressed proteins related to the drug's cytotoxic action by comparing the protein alterations induced by gold (III) porphyrin 1a and cisplatin treatments. Several clusters of altered proteins were identified, including cellular structure and stress-related chaperone proteins, proteins involved in reactive oxygen species and enzyme proteins, translation factors, proteins that mediate cell proliferation or differentiation, and proteins participating in the internal degradation systems. Our results indicated that multiple factors leading to apoptosis were involved in drug cytotoxicity in SUNE1 cells. The balance between pro-apoptotic and anti-apoptotic signals determined the final fate of cancer cells.

A murine monoclonal antibody (mAb), CLD3 (IgG(1),kappa), was generated against hepatocellular carcinoma (HCC). Both immunofluorescence and immunohistochemical assays indicated the reactivity of CLD3 mAb localized at the nucleus and/or cytoplasm of tumorigenic HCC cell lines as well as in liver cancer tissues. By immunoprecipitation and using the matrix-assisted laser desorption/ionization-time of flight mass spectrometry approach, the antigenic specificity of CLD3 was determined to be heterodimeric Ku70 and Ku80 autoantigen, which was confirmed by Western blotting.

Severe acute respiratory syndrome (SARS) is a new infectious disease with a global impact. Understanding its pathogenesis and developing specific diagnostic methods for its early diagnosis are crucial for the effective management and control of this disease. By using proteomic technology, truncated forms of alpha(1)-antitrypsin (TF-alpha(1)-AT) were found to increase significantly and consistently in sera of SARS patients compared to control subjects. The result showed a sensitivity of 100% for SARS patients and a specificity of 92.8% for controls. Furthermore, the levels of these proteins significantly correlated with certain clinico-pathological parameters. The dramatic increase in TF-alpha(1)-AT may be the result of degradation of alpha(1)-AT. As alpha(1)-AT plays an important role in the protection of lung function, its degradation may be an important factor in the pathogenesis of SARS. These findings indicate that increased TF-alpha(1)-AT may be therapeutically relevant, and may also be a useful biological marker for the diagnosis of SARS.

Oral tongue carcinoma is an aggressive tumor that particularly affects chronic smokers, drinkers and betel squid chewers. Patients often present symptoms at a late stage, and there is a high recurrence rate after treatment. In this article, we report the first proteomic analysis of oral tongue carcinoma to globally search for tumor related proteins. Apart from helping us to understand the molecular pathogenesis of the carcinoma, these proteins may also have potential clinical applications as biomarkers, enabling the tumor to be identified at an early stage in high risk individuals, treatment response to be predicted, and residual or recurrent carcinoma to be detected sooner after treatment. The protein expression profiles of ten oral tongue squamous cell carcinomas and their matched normal mucosal resection margins were examined by two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization-time of flight mass spectroscopy. A number of tumor-associated proteins including heat shock protein (HSP)60, HSP27, alpha B-crystalline, ATP synthase beta, calgranulin B, myosin, tropomyosin and galectin 1 were consistently found to be significantly altered in their expression levels in tongue carcinoma tissues, compared with their paired normal mucosae. The expression profile portrays a global protein alteration that appears specific to oral tongue cancer. The potential of utilizing these tumor related proteins for screening cancer and monitoring recurrence warrants further investigation.

Hepatitis B virus (HBV), a serious infectious and widespread human pathogen, represents a major health problem worldwide. Chronic HBV infection has a very high risk of evolving into hepatocellular carcinoma. Although considerable progress was made during the recent past, the pathogenesis of HBV infection is still elusive and a definite diagnosis of HBV infected liver information still relies on biopsy histological test. In this report, we used proteomics technology to globally examine HBV infected serum samples aiming at searching for disease-associated proteins that can be used as serological biomarkers for diagnosis and/or target proteins for pathogenetic study. By comparing with normal and HBV negative serum samples, we found that at least seven proteins were significantly changed in HBV infected sera. These greatly altered proteins were identified to be haptoglobin beta and alpha2 chain, apolipoprotein A-I and A-IV, alpha1-antitrypsin, transthyretin and DNA topoisomerase IIbeta. The alteration of these proteins is displayed not only in quantity but also in patterns (or specificity), which can be correlated with necroinflammatory scores. In particular, apolipoprotein A-I presents heterogeneous change in expression level with different isoforms and alpha1-antitrypsin produces evidently different fragments implying diverse cleavage pathways. These unique phenomena appear specific to HBV infection. A combination simultaneously considering the quantities and isoforms of these proteins could be a useful serum biomarker (or index) for HBV diagnosis and therapy.

Isolated from Elephantopus scaber L., a Chinese medicinal herb that is widely used to prevent and treat cancers in China, isodeoxyelephantopin (ESI) exerted antitumor effects on several cancer cells. However, its antitumor mechanism is still not clear. In this study, we found that ESI could induce G2/M arrest and subsequently stimulate cell apoptosis in dose- and time-dependent manners. We used SILAC quantitative proteomics to identify ESI-regulated proteins in cancer cells, and found that 124 proteins were significantly altered in expression. Gene ontology and Ingenuity Pathway Analysis revealed that these proteins were mainly involved in the regulation of oxidative stress and inflammation response. Functional studies demonstrated that ESI induced G2/M arrest and apoptosis by inducing ROS generation, and that antioxidant N-acetyl-l-cysteine could block the ESI-induced antitumor effects. Accumulated ROS resulted in DNA breakage, subsequent G2/M arrest and mitochondrial-mediated apoptosis. ESI upregulated the expression of anticancer inflammation factors IL-12a, IFN-α, and IFN-β through ROS-dependent and independent pathways. The current work reveals that ESI exerts its antitumor effects through ROS-dependent DNA damage, mitochondrial-mediated apoptosis mechanism and antitumor inflammation factor pathway.

Increasing evidence shows that protein phosphorylation on serine (Ser), threonine (Thr) and tyrosine (Tyr) residues is a major regulatory post-translational modification in the bacteria. To reveal the phosphorylation state in the Gram-negative pathogenic bacterium Helicobacter pylori, we carried out a global and site-specific phosphoproteomic analysis based on TiO 2 -phosphopeptide enrichment and high-accuracy LC-MS/MS determination. Eighty-two phosphopeptides from 67 proteins were identified with 126 phosphorylation sites, among which 79 class I sites were determined to have a distribution of 42.8:38.7:18.5% for the Ser/ Thr/Tyr phosphorylation, respectively. The H. pylori phosphoproteome is characterized by comparably big size, high ratio of Tyr phosphorylation, high abundance of multiple phosphorylation sites in individual phosphopeptides and over-representation of membrane proteins. An interaction network covering 28 phosphoproteins was constructed with a total of 163 proteins centering on the major H. pylori virulence factor VacA, indicating that protein phosphorylation in H. pylori may be delicately controlled to regulate many aspects of the metabolic pathways and bacterial virulence.

Although microRNAs (miRNAs) have been reported to play an important role in carcinogenesis, their molecular mechanism remains largely unknown because of our limited understanding of miRNA target genes. miR-373 was found to be capable of promoting breast cancer invasion and metastasis, but only a target gene was experimentally identified on the basis of mRNA expression analysis. In this study, we used SILAC-based quantitative proteomics to globally identify the genes regulated by miR-373. Totally, 3666 proteins were identified, and 335 proteins were found to be regulated by miR-373. Among the 192 proteins that were downregulated by miR-373, 27 (14.1%) were predicted to have at least one potential match site at their 3'-UTR for miR-373 seed sequence. However, miR-373 did not affect the mRNA level of the five selected candidate targets, TXNIP, TRPS1, RABEP1, GRHL2 and HIP1, suggesting that the protein expressions were regulated by miR-373 via translational inhibition instead of mRNA degradation. Luciferase and mutation assays validated that TXNIP and RABEP1 were the direct target genes of miR-373. More than 30 proteins reported to be involved in cancer invasion and metastasis were found to be regulated by miR-373 in breast cancer for the first time.

The aim of metalloproteomics is to identify and characterize putative metal-binding proteins and metal-binding motifs. In this study, we performed a systematical metalloproteomic analysis on Streptococcus pneumoniae through the combined use of efficient immobilized metal affinity chromatography enrichment and high-accuracy linear ion trap-Orbitrap MS to identify metal-binding proteins and metal-binding peptides. In total, 232 and 166 putative metal-binding proteins were respectively isolated by Cu- and Zn-immobilized metal affinity chromatography columns, in which 133 proteins were present in both preparations. The putative metalloproteins are mainly involved in protein, nucleotide and carbon metabolisms, oxidation and cell cycle regulation. Based on the sequence of the putative Cu- and Zn-binding peptides, putative Cu-binding motifs were identified: H(X)mH (m=0-11), C(X)(2) C, C(X)nH (n=2-4, 6, 9), H(X)iM (i=0-10) and M(X)tM (t=8 or 12), while putative Zn-binding motifs were identified as follows: H(X)mH (m=1-12), H(X)iM (i=0-12), M(X)tM (t=0, 3 and 4), C(X)nH (n=1, 2, 7, 10 and 11). Equilibrium dialysis and inductively coupled plasma-MS experiments confirmed that the artificially synthesized peptides harboring differential identified metal-binding motifs interacted directly with the metal ions. The metalloproteomic study presented here suggests that the comparably large size and diverse functions of the S. pneumoniae metalloproteome may play important roles in various biological processes and thus contribute to the bacterial pathologies.

Polyphyllin D (PD) is a potent cytotoxic saponin found in Paris polyphylla. In the present study, bioinformatic, proteomic and transcriptomic analyses were performed to study the mechanisms of action of PD on human nonsmall cell lung cancer (NSCLC) cell line (NCI-H460). Using a gene expression-based bioinformatic tool (connectivity map), PD was identified as a potential ER stress inducer. Our proteomic and transcriptomic analyses revealed that PD treatment led to upregulation of typical ER stress-related proteins/genes including glucose-regulated protein 78 (BiP/GRP78) and protein disulfide isomerase (PDI). In particular, elevated expression of C/EBP homologous transcription factor (chop) and activation of caspase-4 occurred at early time point (8 h) of PD treatment, signifying an initial ER stress-mediated apoptosis. Induction of tumor suppressor p53, disruption of mitochondrial membrane, activation of caspase-9 and caspase-3 were detected upon prolonged PD treatment. Collectively, these data revealed that PD induced the cytotoxic effect through a mechanism initiated by ER stress followed by mitochondrial apoptotic pathway. The ability of activating two major pathways of apoptosis makes PD an attractive drug lead for anticancer therapeutics.

Dioscin, extracted from the root of Polygonatum zanlanscianense pamp, exhibits cytotoxicity towards human myeloblast leukemia HL-60 cells. Proteomic analysis revealed that the expression of mitochondrial associated proteins was substantially altered in HL-60 cells corresponding to the dioscin treatment, suggesting that mitochondria are the major cellular target of dioscin. Mitochondrial functional studies validated that mitochondrial apoptotic pathway was initiated by dioscin treatment. Changes in proteome other than mitochondrial related proteins implicate that other mechanisms were also involved in dioscin-induced apoptosis in HL-60 cells, including the activity impairment in protein synthesis, alterations of phosphatases in cell signaling, and deregulation of oxidative stress and cell proliferation. Current study of protein alterations in dioscin-treated HL-60 cells suggested that dioscin exerts cytotoxicity through multiple apoptosis-inducing pathways.

Ranking as the fourth commonest cancer, esophageal squamous cell carcinoma (ESCC) represents one of the leading causes of cancer death in China. One of the main reasons for the low survival rate is that neoplasms in esophagus are not detected until they have invaded into surrounding tissues or spread throughout the body at advanced stages. A better understanding of the malignant mechanism and early diagnosis are important for fighting ESCC. In this study, we used proteomics to analyze ESCC tissues, aiming at defining the proteomic features implicated in the multistage progression of esophageal carcinogenesis. Proteins that exhibited significantly different expressions were identified by peptide mass fingerprinting and validated by Western blotting and reverse transcriptase-polymerase chain reaction. The protein changes were then correlated to the different grades of disease differentiation. Compared to those in adjacent normal epitheliums, the expression of 15 proteins including enolase, elongation factor Tu, isocitrate dehydrogenase, tubulin alpha-1 chain, tubulin beta-5 chain, actin (cytoplasmic 1), glyceraldehyde-3 phosphate dehydrogenase, tropomyosin isoform 4 (TPM4), prohibitin, peroxiredoxin 1 (PRX1), manganese-containing superoxide dismutase (MnSOD), neuronal protein, and transgelin was up-regulated; and the expression of five proteins including TPM1, squamous cell carcinoma antigen 1 (SCCA1), stratifin, peroxiredoxin 2 isoform a, and alpha B crystalline was down-regulated in cancer tissues with a statistical significance (p < 0.05). In addition, the differential expression of SCCA1, PRX1, MnSOD, TPM4, and prohibitin can be observed in precancerous lesions of ESCC. The expression of stratifin, prohibitin, and SCCA1 dropped with increasing dedifferentiation of ESCC. These data may suggest that these proteins contribute to the multistage process of carcinogenesis, tumor progression, and invasiveness of ESCC.

Squamous cell carcinoma (SCC) of the buccal mucosa is an aggressive oral cancer. It mainly occurs in Central and Southeast Asia, and is closely related to the practice of tobacco smoking and betel squid chewing. The high recurrence and low survival rates of buccal SCC require our continued efforts to understand the pathogenesis of the disease for designing better therapeutic strategies. We used proteomic technology to analyze buccal SCC tissues aiming at identifying tumor-associated proteins for the utilization as biomarkers or molecular targets. With the exception of alpha B-crystallin being substantially reduced, a number of proteins were found to be significantly over-expressed in cancer tissues. These increased proteins included glycolytic enzymes, heat-shock proteins, tumor antigens, cytoskeleton proteins, enzymes involved in detoxification and anti-oxidation systems, and proteins involved in mitochondrial and intracellular signaling pathways. These extensive protein variations indicate that multiple cellular pathways were involved in the process of tumorigenesis, and suggest that multiple protein molecules should be simultaneously targeted as an effective strategy to counter the disease. At least, SCC antigen, G protein, glutathione S-transferase, manganese superoxide dismutase, annexins, voltage-dependent anion channel, cyclophilin A, stratifin and galectin 7 are candidates for targeted proteins. The present findings also demonstrated that rich protein information can be produced by means of proteomic analysis for a better understanding of the oncogenesis and pathogenesis in a global way, which in turn is a basis for the rational designs of diagnostic and therapeutic methods.