Roles of immunohistochemical staining in diagnosing pulmonary squamous cell carcinoma.

BACKGROUND: Differentiating morphologic features based on hematoxylin-eosin (HE) staining is the most common method to classify pathological subtypes of non-small-cell lung cancer (NSCLC). However, its accuracy and inter-observer reproducibility in pathological diagnosis of poorly differentiated NSCLC remained to be improved. MATERIALS AND METHODS: We attempted to explore the role of immunohistochemistry (IHC) staining in diagnosing pulmonary squamous cell carcinoma (SQCC) with poorly differentiated features by HE staining or with elevated serum adenocarcinoma-specific tumor markers (AD-TMs). We also compared the difference of epidermal growth factor receptor (EGFR) mutation rate between patients with confirmed SQCC and those with revised pathological subtype. Logistic regression analyses were used to test the association between different factors and diagnostic accuracy. RESULTS: A total of 132 patients who met the eligible criteria and had adequate specimens for IHC confirmation were included. Pathological revised cases in poor differentiated subgroup, biopsy samples and high-level AD-TMs cases were more than those with high/moderate differentiation, surgical specimens and normal-level AD-TMs. Moreover, biopsy sample was a significant factor decreasing diagnostic accuracy of pathological subtype (OR, 4.037; 95% CI 1.446-11.267, p=0.008). Additionally, EGFR mutation rate was higher in patients with pathological diagnostic changes than those with confirmed SQCC (16.7% vs 4.4%, p=0.157). CONCLUSIONS: Diagnosis based on HE staining only might cause pathological misinterpretation in NSCLC patients with poor differentiation or high-level AD-TMs, especially those with biopsy samples. HE staining and IHC should be combined as pathological diagnostic standard. The occurrence of EGFR mutations in pulmonary SQCC might be overestimated.

ABO blood group, Epstein-Barr virus infection and prognosis of patients with non-metastatic nasopharyngeal carcinoma.

BACKGROUND: A prior study showed blood type A/AB to be associated with an increased risk of nasopharyngeal carcinoma (NPC) compared to subjects with blood type O. However, the relationship between ABO blood groups and prognosis of NPC patients is still questionable. In addition, whether Epstein-Barr virus (EBV) infection is associated with prognosis of NPC patients with different ABO blood groups is unclear. MATERIALS AND METHODS: We conducted univariate and multivariable Cox regression analyses based on a consecutive cohort of 1,601 patients to investigate the above issues. RESULTS: There was no significant difference in overall survival (OS) between different ABO blood groups (p=0.629), neither between A vs. non-A blood groups (p=0.895) nor AB vs. non-AB blood group (p=0.309) in univariate analyses and after adjusting for other factors. Interaction tests revealed that high immunoglobulin A against Epstein-Barr virus viral capsid antigen (VcA-IgA) level was associated with a favorable prognosis in male patients with UICC stage II disease who had an A blood type (p=0.008), compared with those with non-A blood type. In addition, male patients with an A blood group with a high blood lymphocyte level showed a tendency towards better survival in UICC stage III (p=0.096). CONCLUSIONS: ABO blood group status is not associated with the prognosis of patients with NPC. Additionally, blood group A male NPC patients with high VcA-IgA level or high blood lymphocyte counts might be correlated with a favorable prognosis in UICC stage II or III, respectively.

A novel genetically encoded fluorescent protein as a Cu(I) indicator.

We constructed the copper(I)-binding domain of Mycobacterium tuberculosis (Mtb) CDC 1551 (residues 1-162) between cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP), and formed a novel genetically encoded fluorescent copper(I) responsive protein (PMtb). The sensitivity and selectivity to copper(I) of the PMtb was sought. The experiments showed that the copper(I)-binding domain of the PMtb was highly sensitive and selective towards copper(I).

Differentially expressed proteins of MCF-7 human breast cancer cells affected by Zilongjin, a complementary Chinese herbal medicine.

PURPOSE: Zilongjin, a complementary Chinese herbal medicine, has been used to alleviate the adverse effects of chemotherapeutic drugs in cancer therapy. However, the mechanisms of anti-cancer activity of Zilongjin are still largely unkonwn. EXPERIMENTAL DESIGN: First, the proteomic approach of combined 2-DE and ESI-MS/MS was used to investigate the effect of Zilongjin on the protein expression in MCF-7 cells. Then, the differential expression of some proteins was confirmed by Western blot, cytoimmunofluoresecnce, and quantitative real-time RT-PCR analysis. RESULTS: The identified proteins with differential expression, involved in such events as protein translation, cellular signal transduction, cytoskeleton formation and transportation, include seven downregulating proteins, such as Eukaryotic translation initiation factor 3 subunit I, Eukaryotic translation initiation factor 1A Y-chromosomal, Ran-specific GTPase-activating protein, Ubiquitin-conjugating enzyme E2 N, Tropomodulin-3, Macrophage-capping protein, and Tumor protein D52, as well as two upregulating proteins, HSP ß-1 and keratin18. Moreover, the differential expression of three proteins was confirmed. CONCLUSIONS AND CLINICAL RELEVANCE: (i) These results provide a new insight into the molecular mechanisms of Zilongjin on therapy for breast cancer. (ii) The application of the proteomic approaches will result in the more extended appreciation of Chinese medicine than those known at present.

[Identification and clinical evaluation of lung cancer serum biomarker L-lactate dehydrogenase B].

OBJECTIVE: To identify potential biomarkers related with lung cancer metastasis. METHODS: Conditional media proteins collected from a primary non-small cell lung cancer cell line (NSCLC) NCI-H226 and its brain metastatic subline H226Br were analyzed by SDS-PAGE and MALDI-TOF-MS. Twelve biomarkers were identified, of which LDHB was significantly up-regulated in H226Br cell and was further validated using ELISA in sera including 105 lung cancer samples, 41 pneumonia and pulmonary tuberculosis samples and 65 healthy samples. RESULTS: The levels of LDHB were specifically elevated in NSCLC sera [A value 0.485 (0 - 1.415)] compared with pneumonia and pulmonary tuberculosis [A value 0.187 (0 - 0.609), P < 0.01] and healthy group [A value, 0.159 (0 - 0.524), P < 0.01] and were progressively increased with the clinical stage. At the cutoff point 0.260 (A value) on the ROC curve, the sensitivity, specificity and total accuracy of LDHB were 81%, 70% and 76% respectively. CONCLUSION: These findings demonstrated that secretome could open up a possibility to identify novel biomarkers related with cancer occurrence and metastasis.

Involvement of the 90-kDa heat shock protein (Hsp-90) in CB2 cannabinoid receptor-mediated cell migration: a new role of Hsp-90 in migration signaling of a G protein-coupled receptor.

The endocannabinoid 2-arachidonoylglycerol (2-AG) enhances cell migration through the CB2 cannabinoid receptor. In this study, using an immunoprecipitation and mass spectrometry-based proteomic approach, we first identified the 90-kDa heat shock protein (Hsp90), a chaperone protein with novel signaling functions, as a CB2-interacting protein. The CB2/Hsp90 interaction was confirmed in human embryonic kidney 293 cells expressing transfected CB2 and in differentiated HL-60 cells expressing endogenous CB2, by coimmunoprecipitation and Western blot experiments, as well as by treatment with geldanamycin (GA), a specific Hsp90 inhibitor. Disruption of the CB2/Hsp90 interaction by treatment with GA or reducing Hsp90 levels with specific short interfering RNAs markedly inhibited 2-AG-induced cell migration, demonstrating that Hsp90 is crucial for 2-AG-induced cell migration. 2-AG treatment resulted in a CB2-mediated stimulation of Rac1 activity, and treatment with GA blocked 2AG-induced activation of Rac1. It is noteworthy that expression of the dominant-negative form of Rac1 reduced 2-AG-induced cell migration. These data demonstrate that 2-AG-induced activation of Rac1 is essential for 2-AG-induced cell migration, and the CB2/Hsp90 interaction is needed for 2-AG-induced activation of Rac1. Furthermore, 2-AG-induced Rac1 activation was sensitive to pertussis toxin treatment, hence involving G(i) proteins. In addition, treatment with GA significantly inhibited the CB2/Galpha(i2) interaction. As a whole, our data indicate that Hsp90 may serve as scaffold to keep the CB2 receptor and its signaling components, including Galpha(i2), in proximity, thus facilitating CB2-mediated signaling to cell migration through the G(i)-Rac1 pathway. By demonstrating that Hsp90 is essential for CB2-mediated signaling to cell migration, this study reveals a novel role of Hsp90 in the signaling events mediated by a G protein-coupled receptor.

Protein profile of human lung squamous carcinoma cell line NCI-H226.

OBJECTIVE: To construct a database of human lung squamous carcinoma cell line NCI-H226 and to facilitate discovery of novel subtypes markers of lung cancer. METHOD: Proteomic technique was used to analyze human lung squamous carcinoma cell line NCI-H226. The proteins of the NCI-H226 cells were separated by two-dimensional gel electrophoresis and identified by mass spectrometry. RESULTS: The results showed that a good reproducibility of the 2-D gel pattern was attained. The position deviation of matched spots among three 2-D gels was 1.95 +/- 0.53 mm in the isoelectric focusing direction, and 1.73 +/- 0.45 mm in the sodium dodecyl sulfate-polyacrylamide gel electrophoresis direction. One hundred and twenty-seven proteins, including enzymes, signal transduction proteins, structure proteins, transport proteins, etc. were characterized, of which, 29 identified proteins in NCI-H226 cells were reported for the first time to be involved in lung cancer carcinogenesis. CONCLUSION: The information obtained from this study could provide some valuable clues for further study on the carcinogenetic mechanism of different types of lung cancer, and may help us to discover some potential subtype-specific biomarkers of lung cancer.

Serum amyloid A protein: a potential biomarker correlated with clinical stage of lung cancer.

OBJECTIVE: To identify serum diagnosis or progression biomarkers in patients with lung cancer using protein chip profiling analysis. METHOD: Profiling analysis was performed on 450 sera collected from 213 patients with lung cancer, 19 with pneumonia, 16 with pulmonary tuberculosis, 65 with laryngeal carcinoma, 55 with laryngopharyngeal carcinoma patients, and 82 normal individuals. A new strategy was developed to identify the biomarkers on chip by trypsin pre-digestion. RESULTS: Profiling analysis demonstrated that an 11.6 kDa protein was significantly elevated in lung cancer patients, compared with the control groups (P < 0.001). The level and percentage of 11.6 kDa protein progressively increased with the clinical stages I-IV and were also higher in patients with squamous cell carcinoma than in other subtypes. This biomarker could be decreased after operation or chemotherapy. On the other hand, 11.6 kDa protein was also increased in 50% benign diseases of lung and 13% of other cancer controls. After trypsin pre-digestion, a set of new peptide biomarkers was noticed to appear only in the samples containing a 11.6 kDa peak. Further identification showed that 2177 Da was a fragment of serum amyloid A (SAA, MW 11.6 kDa). Two of the new peaks, 1550 Da and 1611 Da, were defined from the same protein by database searching. This result was further confirmed by partial purification of 11.6 kDa protein and MS analysis. CONCLUSION: SAA is a useful biomarker to monitor the progression of lung cancer and can directly identify some biomarkers on chip.

[Application of serum surface-enhanced laser desorption/ionization proteomic patterns in distinguishing non-small cell lung cancer patients from healthy people].

OBJECTIVE: To explore the application of serum surface-enhanced laser desorption/ionization (SELDI) marker patterns in distinguishing non-small cell lung cancer patients from healthy people by protein chip technology. METHODS: One hundred and sixty-three serum samples (123 patients with lung cancer and 40 healthy persons), were randomly divided into a training set [94 cases, 53 non-small cell lung cancer (NSCLC), 21 small cell lung cancer and 20 healthy persons] and a blinded test set (69 cases), were included for analysis by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS). Five protein peaks at 11,493, 6,429, 8,245, 5,336 and 2,536 were automatically chosen for the system training and the development of a decision classification tree model (marker pattern). The accuracy of the model was tested with the blinded test set (an independent set of masked serum samples from 49 patients with NSCLC and 20 healthy persons). RESULTS: The model differentiated the patients with NSCLC from the healthy people with a sensitivity of 95.9% (71/74) and a specificity of 90.0% (18/20) in the training set and a sensitivity of 83.7%, and a specificity of 80.0% in the blinded set respectively. CONCLUSION: SELDI-TOF-MS technique can correctly distinguish NSCLC patients from healthy people, and it has the potential for the development of a screening test for the detection of NSCLC.

Application of surface-enhanced laser desorption/ionization time-of-flight-based serum proteomic array technique for the early diagnosis of prostate cancer.

AIM: To identify the serum biomarkers of prostate cancer (PCa) by protein chip and bioinformatics. METHODS: Serum samples from 83 PCa patients and 95 healthy men were taken from a mass screening in Changchun, China. Protein profiling was carried out using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS). The data of spectra were analyzed using two bioinformatics tools. RESULTS: Eighteen serum differential proteins were identified in the PCa group compared with the control group (P < 0.01). There were four proteins at the higher serum level and 14 proteins at the lower serum level in the PCa group. A decision tree classification algorithm that used an eight-protein mass pattern was developed to correctly classify the samples. A sensitivity of 92.0% and a specificity of 96.7% for the study group were obtained by comparing the PCa and control groups. CONCLUSION: We identified new serum biomarkers of PCa. SELDI-TOF MS coupled with a decision tree classification algorithm will provide a highly accurate and innovative approach for the early diagnosis of PCa.

Application of serum SELDI proteomic patterns in diagnosis of lung cancer.

BACKGROUND: Currently, no satisfactory biomarkers are available to screen for lung cancer. Surface-Enhanced Laser Desorption/ionization Time-of-Flight Mass Spectrometry ProteinChip system (SELDI-TOF-MS) is one of the currently used techniques to identify biomarkers for cancers. The aim of this study is to explore the application of serum SELDI proteomic patterns to distinguish lung cancer patients from healthy individuals. METHODS: A total of 208 serum samples, including 158 lung cancer patients and 50 healthy individuals, were randomly divided into a training set (including 11 sera from patients with stages I/II lung cancer, 63 from patients with stages III/IV lung cancer and 20 from healthy controls) and a blinded test set (including 43 sera from patients with stages I/II lung cancer, 41 from patients with stages III/IV lung cancer and 30 from healthy controls). All samples were analyzed by SELDI technology. The spectra were generated on weak cation exchange (WCX2) chips, and protein peaks clustering and classification analyses were made using Ciphergen Biomarker Wizard and Biomarker Pattern software, respectively. We additionally determined Cyfra21-1 and NSE in the 208 serum samples included in this study using an electrochemiluminescent immunoassay. RESULTS: Five protein peaks at 11493, 6429, 8245, 5335 and 2538 Da were automatically chosen as a biomarker pattern in the training set. When the SELDI marker pattern was tested with the blinded test set, it yielded a sensitivity of 86.9%, a specificity of 80.0% and a positive predictive value of 92.4%. The sensitivities provided by Cyfra21-1 and NSE used individually or in combination were significantly lower than that of the SELDI marker pattern (P < 0.005 or 0.05, respectively). Based on the results of the test set, we found that the SELDI marker pattern showed a sensitivity of 91.4% in the detection of non-small cell lung cancers (NSCLC), which was significantly higher than that in the detection of small cell lung cancers (P < 0.05); The pattern also had a sensitivity of 79.1% in the detection of lung cancers in stages I/II. CONCLUSION: These results suggest that serum SELDI protein profiling can distinguish lung cancer patients, especially NSCLC patients, from normal subjects with relatively high sensitivity and specificity, and the SELDI-TOF-MS is a potential tool for the screening of lung cancer.

[Coordination inhibition of the proliferation of MCF-7 by enhancing expression of p53,p21 and decreasing expression of c-myc].

To study the coordination inhibition of the proliferation of breast cancer cell line MCF-7 by enhancing expression of p53,p21 and decreasing expression of c-myc, as well as the relationship among these genes when they express in the cells, eukaryotic expression plasmids of sense p53,p21 and antisense c-myc were constructed first, different concentrations of three plasmids were determined and the combinations were designed according to factorial design. Cells were transfected by the combination plasimids, then the inhibition rate of proliferation of the transfected cells were tested. The results were analyzed using the methods of statistics including Jin's Q-test, Least significant difference (LSD), cluster analysis. It was shown that all of them (Sense p21,p53, antisense c-myc) could inhibit the proliferation of MCF-7 in different levels of concentration, while the degrees of inhibition were different among them. In the aspect of coordination inhibition, the proliferation of MCF-7 was inhibited much strongly by p21 coordination with antisense c-myc and p53 coordination with antisense c-myc, but p53 coordination with p21 did not show inhibition effect. Rusults of cluster analysis showed that the first combination of three genes showed the best coordination, the ninth combination had the highest rate of inhibition. In conclusion, we suppose that when p53 as a cancer suppressor gene and p21 as a CDK suppressor gene express highly, and the expression of c-myc as a cancer gene is inhibited meantime could obviously enhance the inhibition on the proliferation of MCF-7 cells by coordination effect.

[Proteomic analysis of prostate cancer using surface enhanced laser desorption/ionization mass spectrometry].

OBJECTIVE: To identify the serum biomarkers of prostate cancer by using protein chip and bioinformatics. METHODS: Eighty three prostate cancer (PCA) patients and ninety five healthy people from mass screen in Changchun were detected by surface-enhanced laser desorption/ionization mass spectrometry (SELDI-MS). The data of spectra were analyzed by bioinformatics tools-Biomarker Wizard and Biomarker Pattern. RESULTS: Compared with the spectra of healthy people, there were 18 potential markers detected in the spectra of the PCA patients, the protein expression was high in 4 of which and low in the 10 of which. The softwares Biomarkerwizard and Biomarker Pattern automatically, under given conditions, selected 8 biomarker proteins to be used to establish a five layer decision tree differentiate to diagnose PCA and differentiate PCA from healthy people with a specificity of 92.632% and a sensitivity of 96.386%. CONCLUSION: New serum biomarkers of PCA have been identified, and this SELDI mass spectrometry coupled with decision tree classification algorithm will provide a highly accurate and innovative approach for the early diagnosis of PCA.

[The active expression of CenpB, a constitutive protein in the centromeres of chromosomes, in breast cancer tissues].

At present, a point that cell biologists and medicine scientists focus their close attention on is the mechanisms of cell proliferation and carceration. Breast cancer, one of the frequently occurring cancers, is often been studied intensively. Centromere constitutive molecules, related to various regulatory factors, play an important role in cell proliferation check point regulation. Cell cycle engine molecules, oncogenes, anti-oncogenes and other molecules conform a cell proliferation network. The basic courses of all tumors are associated to this network. However, there are still many problems to be resolved in the analyses of cancer related genes which cause tumors and tumor gene markers. In the current study, using Northern blot, 31 samples of breast cancer tissues and their normal (not cancerous) tissues a little far away from them in the same individuals showed that, in the majority of the tests (87.1%), the mRNA of centromere protein CenpB over expressed in breast cancer tissues, and moreover, tissue in situ hybridization also revealed that all of the CenpB-over-expressed cancer tissues, having identified with Northern blot, over expressed CenpB mRNA. Analyzing the same samples by means of Western blot, the result was highly consistent to the studies in the RNA level. A conclusion was drawn that the over expression of CenpB gene probably relates to malignant cell proliferation in breast gland. It has been testified by researchers that a few of CenpB homogenous proteins are co-operative, the loss of their genes resulting in chromosomes' separating abnormally and cell growth's slowing down. Having transfected HeLa (Tet-Off) cells with anti-sense Cenp in a previous experiment, we ever got a result that cellular duplicating time was prolonged for another 32.8 h, and together with the inhibition of centromere assembly, the mitotic index dropped sharply. In another research, we drew a conclusion that CenpG may be related to cancer, and its differential expressing probably relates to malignant cell proliferation. Combined with these researches, the results obtained from the current study are beneficial to further recognition of the mechanism of cancer.

A preliminary analysis of non-small cell lung cancer biomarkers in serum.

OBJECTIVE: To identify potential serum biomarkers that could be used to discriminate lung cancers from normal. METHODS: Proteomic spectra of twenty-eight serum samples from patients with non-small cell lung cancer and twelve from normal individuals were generated by SELDI (Surfaced Enhanced Laser Desorption/Ionization) Mass Spectrometry. Anion-exchange columns were used to fractionate the sera into 6 designated pH groups. Two different types of protein chip arrays, IMAC-Cu and WCX2, were employed. Samples were examined in PBSII Protein Chip Reader (Ciphergen Biosystem Inc) and the discriminatory profiling between cancer and normal samples was analyzed with Biomarker Pattern software. RESULTS: Five distinct potential lung cancer biomarkers with higher sensitivity and specificity were found, with four common biomarkers in both IMAC-Cu and WCX2 chip; the remaining biomarker occurred only in WCX2 chip. Two biomarkers were up-regulated while three biomarkers were down-regulated in the serum samples from patients with non-small cell lung cancer. The sensitivities provided by the individual biomarkers were 75%-96.43% and specificities were 75%-100%. CONCLUSIONS: The preliminary results suggest that serum is a capable resource for detecting specific non-small cell lung cancer biomarkers. SELDI mass spectrometry is a useful tool for the detection and identification of new potential biomarker of non-small cell lung cancer in serum.

[Studies of the gene expression and structure alteration of vimentin during differentiation of rat preadipocytes].

Observations on structure and distribution of vimentin as determined by indirect immunofluorescence were showed that the arrangement of vimentin intermediate filments (IFs) changed from an extended fibrillar state to a complex cage formation tightly associated with the forming lipid globules during adipose conversion of rat preadipocytes. The effects of differentiation of rat preadipocytes into adipocytes on vimentin mRNA levels as determined by in situ hybridization (ISH) using oligonucleotides probes to vimentin mRNA labeled by digoxigenin (DIG), and on vimentim protein expression as determined by Western blotting were researched. The results suggested that the expressions of vimentin with molecular weight of about 57 kD at the levels of both mRNA and protein was existed throughout the predipocyte differentiation and downregulation of vimentin is required for adipose conversion. The specificity of the association of lipid globules with vimentin IFs during adipose conversion was taken as an example of a possible IF function in relation to a preadipocytes differentiation process, especially discussed as a functional relationship in supporting adiposgenesis.