Aiphanol, a multi-targeting stilbenolignan, potently suppresses mouse lymphangiogenesis and lymphatic metastasis.

The high incidence of lymphatic metastasis is closely related to poor prognosis and mortality in cancers. Potent inhibitors to prevent pathological lymphangiogenesis and lymphatic spread are urgently needed. The VEGF-C-VEGFR3 pathway plays a vital role in driving lymphangiogenesis and lymph node metastasis. In addition, COX2 in tumor cells and tumor-associated macrophages (TAMs) facilitates lymphangiogenesis. We recently reported that aiphanol, a natural stilbenolignan, attenuates tumor angiogenesis by repressing VEGFR2 and COX2. In this study, we evaluated the antilymphangiogenic and antimetastatic potency of aiphanol using in vitro, ex vivo and in vivo systems. We first demonstrated that aiphanol directly bound to VEGFR3 and blocked its kinase activity with an half-maximal inhibitory concentration (IC50) value of 0.29 µM in an in vitro ADP-GloTM kinase assay. Furthermore, we showed that aiphanol (7.5-30 µM) dose-dependently counteracted VEGF-C-induced proliferation, migration and tubular formation of lymphatic endothelial cells (LECs), which was further verified in vivo. VEGFR3 knockdown markedly mitigated the inhibitory potency of aiphanol on lymphangiogenesis. In 4T1-luc breast tumor-bearing mice, oral administration of aiphanol (5 and 30 mg· kg-1 ·d-1) dose-dependently decreased lymphatic metastasis and prolonged survival time, which was associated with impaired lymphangiogenesis, angiogenesis and, interestingly, macrophage infiltration. In addition, we found that aiphanol decreased the COX2-dependent secretion of PGE2 and VEGF-C from tumor cells and macrophages. These results demonstrate that aiphanol is an appealing agent for preventing lymphangiogenesis and lymphatic dissemination by synergistically targeting VEGFR3 and inhibiting the COX2-PGE2-VEGF-C signaling axis.

A multi-targeting natural product, aiphanol, inhibits tumor growth and metastasis.

Cancer is one of the main causes of death in humans worldwide, the development of more effective anticancer drugs that can inhibit the malignant progression of cancer cells is of great significance. Aiphanol is a natural product identified from the seeds of Arecaceae and the rhizome of Smilax glabra Roxb. Our preliminary studies revealed that it had potential antiangiogenic and antilymphangiogenic activity by directly targeting VEGFR2/3 and COX2 in endothelial cells. However, the influence of aiphanol on cancer cells per se remains largely undefined. In this study, the effects and related mechanisms of aiphanol on cancer growth and metastasis were evaluated in vitro and in vivo. Acute toxicity assay and pharmacokinetic analysis were utilized to investigate the safety profile and metabolism characteristics of aiphanol. We revealed that aiphanol inhibited the proliferation of various types of cancer cells and the growth of xenograft tumors in mice and zebrafish models. The possible mechanism was associated with the inactivation of multiple kinases, including FAK, AKT and ERK, and the upregulation of BAX and cleaved caspase-3 to promote cancer cell apoptosis. Aiphanol significantly inhibited cancer cell migration and invasion, which was related to the inhibition of epithelial-mesenchymal transition (EMT) and F-actin aggregation. Aiphanol effectively attenuated the metastasis of several types of cancer cells in vivo. In addition, aiphanol exerted no significant toxicity and had fast metabolism. Collectively, we demonstrated the anticancer effects of aiphanol and suggested that aiphanol has potential as a safe and effective therapeutic agent to treat cancer.

Combined expression of metastasis related markers Naa10p, SNCG and PRL-3 and its prognostic value in breast cancer patients.

Combinations of multiple biomarkers representing distinct aspects of metastasis may have better prognostic value for breast cancer patients, especially those in late stages. In this study, we evaluated the protein levels of N-α-acetyltransferase 10 protein (Naa10p), synuclein-γ (SNCG), and phosphatase of regenerating liver-3 (PRL-3) in 365 patients with breast cancer by immunohistochemistry. Distinct prognostic subgroups of breast cancer were identified by combination of the three biomarkers. The Naa10p+SNCG-PRL-3- subgroup showed best prognosis with a median distant metastasis-free survival (DMFS) of 140 months, while the Naa10p-SNCG+PRL-3+ subgroup had the worst prognosis with a median DMFS of 60.5 months. Multivariate analysis indicated Naa10p, SNCG, PRL-3, and the TNM classification were all independent prognostic factors for both DMFS and overall survival (OS). The three biomarker combination of Naa10p, SNCG and PRL-3 performed better in patients with lymph node metastasis, especially those with more advanced tumors than other subgroups. In conclusion, the combined expression profile of Naa10p, SNCG and PRL-3, alone or in combination with the TNM classification system, may provide a precise estimate of prognosis of breast cancer patients.

[Establishment of drug resistant cell line of MGC-803 and analysis of differential secretome].

OBJECTIVE: To identify chemoresistance-associated secretory proteins by proteomic approaches, and to provide the basis for selecting suitable chemotherapy in gastric cancer treatment. METHODS: Drug resistant cell lines were established by gradient drug treatment with MGC-803 gastric cancer cells. The secreted proteins of MGC-803 parental and resistant cells were collected from the conditional medium without serum and separated by two-dimensional gel electrophoresis (2-DE).The proteins were analyzed by PD Quest 7.1.0 software and identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Real-time RT-PCR was performed to confirm the difference of expression on the mRNA level. RESULTS: The 5-fluorouracil (5FU), paclitaxel (TA) and cisplatin (DDP)-resistant gastric cancer cell lines with the resistance indexes of 110.6, 70.0 and 13.3 respectively, were established successfully. DDP-resistant cells had strong cross-resistance to 5FU and TA, and the resistance indexes were 23.5 and 114.0. 5FU-resistant cells also had strong cross-resistance to TA with the resistance index 70.0. The 2-DE patterns of protein component spectra from the conditional medium were obtained with 18 proteins whose abundances were increased in all chemoresistance cells for more than 2-fold, 13 of which were identified by mass spectrometry, including protease and proteins involved in signal transduction. Compared with the parental cell MGC-803, SLMAP, TOP3A, DYNC1H1, RHPN1, PUF60 and SIAH1 were significantly up-regulated in three drug resistant cells, IFT172 and FILIP were up-regulated in 5FU-resistant and TA-resistant cells, PLVAP and LMNA were up-regulated in TA- and DDP-resistant cells. Further validation revealed that SIAH1 protein was enriched in cell lysates and the conditional medium from all the drug resistant cells. CONCLUSION: By establishing the 5FU-,TA- and DDP-resistant gastric cancer cell lines and assisted by 2-DE and mass spectrometry, we demonstrated the different secretory protein profiling and found that SIAH1 had significantly increased in both cell lysates and the conditional medium of the drug-resistant cells, which are potential candidates for developing chemoresistance markers in sera from gastric cancer patients.

Chemical constituents from the rhizomes of Smilax glabra and their antimicrobial activity.

Six new phenolic compounds, named smiglabrone A (1), smiglabrone B (2), smilachromanone (3), smiglastilbene (4), smiglactone (5), smiglabrol (6), together with fifty-seven known ones 7-63 were isolated from the rhizomes of Smilax glabra. Their structures were elucidated on the basis of extensive spectroscopic analyses, as well as by comparison with literature data. Twenty-seven of these compounds were obtained from and identified in the genus Smilax for the first time. The absolute configuration of (2S)-1,2-O-di-trans-p-coumaroylglycerol (43) was determined for the first time using the exciton-coupled circular dichroism (ECCD) method. Thirty isolated compounds were evaluated for their antimicrobial activity against three Gram-negative bacteria, three Gram-positive bacteria and one fungus, and the corresponding structure-activity relationships were also discussed. Eighteen compounds were found to be antimicrobial against the microorganisms tested and the minimum inhibitory concentrations (MIC) were in the range of 0.0794-3.09 mM. Among them, compound 1 showed antimicrobial activity against Canidia albicans with MIC value of 0.146 mM, which was stronger than cinchonain Ia with an MIC of 0.332 mM. Compounds 3 and 4 exhibited inhibitory activity against Staphylococcus aureus with MIC values of 0.303 and 0.205 mM, respectively. The results indicated that these antimicrobial constituents of this crude drug might be responsible for its clinical antimicrobial effect.

[Value of serum P53 antibody as a tumor marker surveillance of colorectal cancer within follow-up after curative resection].

P53 gene mutations and the abnormal P53 protein can introduce the production to P53 antibody. A large number of studies showed that serum levels of P53 antibody had the correlation with the prognosis of patients with different cancers, the lymph node invasion and metastasis of colorectal cancer, and its recurrence after the curative resection. And it is possible for its application in predicting the early recurrence and metastasis in colorectal cancer after the curative resection.However, there are still a lot of work needed to be done before its use in the clinical settings.

HIWI expression profile in cancer cells and its prognostic value for patients with colorectal cancer.

BACKGROUND: HIWI is a member of PIWI gene family and its expression is found in various tumors, indicating that it may play a pivotal role in tumor development. This study was designated to examine HIWI protein expression profile in several cancer cell lines and its prognostic value for patients with colorectal cancer. METHODS: Totally 270 patients who underwent surgical resection of primary colorectal cancer between January 1999 and December 2002 with a median follow-up time of 33 months were registered in the study. Formalin-fixed and paraffin-embedded specimens from these patients and 236 matched adjacent non-cancerous normal colorectal tissues were collected. Anti-HIWI monoclonal antibodies were generated and used for evaluating HIWI protein expression. χ(2) tests were conducted to determine the association between HIWI expression and the other variables. Survival curves were estimated using the Kaplan-Meier method and compared by the log-rank test. Multivariate analysis was performed by using the Cox regression model. RESULTS: By generating antibodies specific for HIWI, we examined HIWI protein expression in several cancer cell lines and demonstrated positive expression of HIWI in 69 out of 270 (25.6%) colorectal cancer tissues; 15 of 236 (6.4%) matched adjacent non-cancerous tissues were also positive for HIWI. Patients with positive HIWI expression in adjacent non-cancerous tissue had statistically lower overall survival (OS) and disease free survival (DFS) compared with negative patients (OS: 10.4% vs. 55.5%, P = 0.009; DFS: 10.4% vs. 55.1%, P = 0.015). For early stage group (stages I and II), patients with positive HIWI expression had significantly lower OS and DFS (OS: 57.4% vs. 79.5%, P = 0.014; DFS: 56.7% vs. 80.5%, P = 0.010). In lymph node negative group, patients with positive HIWI expression had statistically lower OS and DFS (OS: 53.0% vs. 73.5%, P = 0.037; DFS: 52.2% vs. 74.6%, P = 0.025). Multivariate analysis revealed that HIWI over-expression was a significant prognostic factor for OS (95%CI: 1.132 - 2.479, P = 0.010). CONCLUSION: HIWI could be a potential prognostic biomarker for the patients with colorectal cancer, especially for those at early stages or without lymph node metastasis.

[Application of biotherapy in recurrent or metastatic gastric cancer].

Post-operative recurrence and metastasis remain the leading causes of death for patients with gastric cancer. The major determinants of recurrence and metastasis are the biological characteristics of cancer cells and the immunological status of the patients. In recent years, due to the target-specificity, biotherapy has yielded efficacious responses in diverse clinical applications for cancer treatment, partially for the treatment of recurrence and metastasis of gastric cancer. However, because of the high diversities in clinical manifestations, patients' condition, and tumor's characteristics, there is no ideal strategy of biotherapy established for the prevention and treatment of recurrence and metastasis in gastric cancer. Therefore, a lot of work need to be done in basic research and clinical trial to make the biotherapy effective in treatment of gastric cancer recurrence.

[Construction and expression of PRL-3 plasmid with C104S point mutation and CAAX deletion].

OBJECTIVE: To construct PRL-3 gene C104S point mutation and CAAX deletion mutants: pcDNA3-myc-PRL-3 (C104S), pEGFP-PRL-3 (C104S), pcDNA3-myc-PRL-3 (DeltaCAAX) and pEGFP-PRL-3 (DeltaCAAX), and express these plasmids in eukaryotic cells. METHODS: Recombinant plasmids were mutated with pcDNA3-myc-PRL-3 plasmid as template and specific primers. Mutants were identified by restriction enzyme digestion and DNA sequencing. Then these recombinant plasmids were transfected into LoVo cells. The expression of fusion proteins were detected by western blotting and the localization of fusion proteins were examined by GPF fluorescence labelling. RESULTS: The mutants were successfully constructed and expressed in eukaryotic cells. PRL-3 (DeltaCAAX) relocates from plasma membrane/early endosome to the cytoplasm and/or nucleus, which provides a structural insight of PRL-3 protein. CONCLUSION: Construction of eukaryotic expression recombinant plasmids of PRL-3 gene C104S point mutation and CAAX deletion mutants provide a useful tool for the study of PRL-3's role in cancer.

[Identification of antigenic epitope regions recognized by anti-synuclein-gamma monoclonal antibodies].

OBJECTIVE: To identify the antigenic epitope regions recognized by anti-synuclein-gamma monoclonal antibodies. METHODS: Six expression vectors of truncated SNCG were constructed and the truncated SNCG-GST fusion proteins were expressed in E.coli. The binding activities of anti-synuclein-gamma (SNCG) monoclonal antibodies (mAbs) with truncated SNCG were analyzed by ELISA and Western blot. According to the speculative epitope regions, expression vectors of potential epitopes were constructed and the GST-epitope fusion proteins were expressed in E.coli. And the fusion proteins were analyzed by ELISA and Western blot with mAbs to confirm the epitope regions. RESULTS: Epitope regions recognized by eleven anti-SNCG mAbs were identified. The epitopes recognized by 9#, 42#, and 3E4 were located in the region from 1st to 21st amino acids of SNCG, 11#, 22# in the region from 78th to 92nd amino acids, 1#, 14#, 18#, 31#, 36# in the region from 93rd to 110th amino acids, and 6C8 in the region from 111th to 127th amino acids. CONCLUSION: The epitope regions recognized by eleven anti-SNCG mAbs were identified. It will benefit the research on SNCG and application of anti-SNCG monoclonal antibodies.

Expression of phosphatase regenerating liver 3 is an independent prognostic indicator for gastric cancer.

AIM: To investigate the prognostic significance of phosphatase regenerating liver 3 (PRL-3) protein expression in gastric cancer. METHODS: PRL-3 expression in paraffin-embedded tumor specimens from 293 patients with gastric cancer was studied retrospectively by immunohistochemistry. Monoclonal antibody specifically against PRL-3, 3B6, was obtained with hybridoma technique. RESULTS: Positive PRL-3 expression was detected in 43.3% (127 of 293) of gastric cancer cases. High expression of PRL-3 was positively correlated with tumor size, depth of invasion, vascular/lymphatic invasion, lymph node metastasis, high TNM stage and tumor recurrence. Patients with positive PRL-3 expression had a significantly lower 5-year survival rate than those with negative expression (28.3% vs 51.9%, P < 0.0001). Patients who received curative surgery, and with positive PRL-3 expression had a significant shorter overall survival and disease-free disadvantage over patients with negative expression (hazard ratio of 16.7 and 16.6, respectively; P < 0.0001 for both). Multivariate analysis revealed that PRL-3 expression was an independent prognostic indicator for overall and disease-free survival of gastric cancer patients, particularly for survival in TNM stage III patients. CONCLUSION: PRL-3 expression is a new independent prognostic indicator to predict the potential of recurrence and survival in patients with gastric cancer at the time of tumor resection.

[Effect of trastuzumab on tumor cell lines shedding high or low level of HER-2 ECD].

OBJECTIVE: To examine the effect of trastuzumab on cell proliferation, colony formation and changes of HER-2 proteins in human breast cancer cell line SKBR3 and human ovarian cancer cell line SKOV3 cells which overexpress p185 HER-2 but shed high or low HER-2 extracellular domain (ECD) levels. METHODS: SKBR3 cells and SKOV3 cells were treated with or without trastuzumab. Cell number and the rate of colony formation were calculated. Western blot analysis was used to detect p185 HER-2, HER-2 ECD and phospho-HER-2. Two-site ELISA assay was used for the detection of HER-2 ECD. RESULTS: Trastuzumab inhibited cell proliferation, colony formation, and decreased or eliminated the levels of two uncharacterized phospho-proteins (molar weight about 90 000 and 40 000) in SKBR3 cells shedding high level of HER-2 ECD expression. These responses were not observed in SKOV3 cells shedding low level of HER-2 ECD expression. But total p185, phospho-p185 and phospho-p95 proteins did not appear to change in SKBR3 and SKOV3 cells after treatment with trastuzumab. Trastuzumab reacts not only with proteolytic cleavage HER-2 ECD containing HER-2 ECD I , II , III and IV subdomains of p185 HER-2 extracellular domain, but also with the secreted autoinhibitor p68/ECD III a specifying 340 residues, identical to subdomains I and II from the extracellular domain of p185 HER-2, followed by a unique C-terminal sequence of 79 aa encoded by intron 8, which suggested that there may be a trastuzumab binding site on p68/ECD III a protein. Comparing with HER-2 ECD levels of the same number of SKBR3 cells, there was no significant decrease of HER-2 ECD shedding level after treatment with or without trastuzumab for 4 days in serum-free medium. CONCLUSION: Antitumor effects of trastuzumab may be related to the two uncharacterized phospho-p90 and/or phospho-p40 proteins. There is probably a trastuzumab epitope on p68/ECD III a. The decrease of HER-2 ECD levels may be positively correlated with the number of SKBR3 cells.

[Inhibitory effect of Adp53 and F56 on growth and metastasis of transplantation of the breast tumor in mice].

OBJECTIVE: To investigate the effects of Adp53 and F56 on the growth and lung metastasis of breast cancer. METHODS: The BICR-H1 cells were inoculated into the mammary fatty pad of BALB/C nude mice and NOD/SCID mice to establish breast cancer model. Then the nude mice with xenograft tumor were randomized into group Adp53+F56, Adp53, F56 and control. The NOD/SCID mice with xenograft tumor were randomized into group Adp53+F56, Adp53, F56, Adlacz and control. They were treated for 3 weeks according to the plan, diversity of the volume and histopathology of xenograft tumor of nude mice was observed and the expressions of p53 and VEGF gene, and microvessel density (MVD) were detected by immunohistochemistry. Lung metastasis of breast cancer in NOD/SCID mice was observed. RESULTS: (1) Intratumoral injections of Adp53, F56, and their combination resulted in an inhibition on the growth of xenograft tumor of BICR-H1 cells. The ultimate relative growth volumes of groups Adp53+F56, Adp53, F56 and control were 2.47,4.37,4.69 and 12.49 respectively. (2) After treatment, P53 positive rate of group Adp53+F56, Adp53 increased 9.4%, 6.3% than before respectively, but compared with control group, the difference is not significant (P=0.693); VEGF protein of group Adp53+F56, Adp53 and F56 decreased 21.9%, 9.4% and 3.1% than before respectively, but compared with control group, the difference was not significant (P=0.284). Necrosis and decrease of vessel in the tumor and morphological change of endothelium were observed under light microscope in the groups Adp53+F56, Adp53 and F56. MVD estimated by FVIII-RA staining of group Adp53+F56, Adp53 and F56 were 14.50+/-2.54, 16.28+/-3.44 and 18.06+/-7.66, compared with control group(24.93+/-6.53), the difference is significant (P=0.000). (3) The average number of lung metastasis of NOD/SCID mice in group Adp53+F56, Adp53 and F56 were 1.143+/-0.378, 2.750+/-0.886 and 3.375+/-0.518 respectively, lower than Adlacz group(5.000+/-0.816) and control group (5.670+/-0.817) obviously (P=0.000). CONCLUSION: Adp53 combined with F56 can greatly inhibit growth and metastasis of breast cancer in vivo. The mechanism of anti-tumor effects of Adp53 and F56 may be related to the anti-angiogenesis effect on malignant tumor through inhibiting the expression and activity of VEGF.

Lipoprotein p37 from Mycoplasma hyorhinis inhibiting mammalian cell adhesion.

p37 protein is a membrane lipoprotein of Mycoplasma hyorhinis, and our previous work showed that there was high ratio of M. hyorhinis infection in human gastric carcinoma. To investigate the possible functions of p37 in cancer development, the nucleotide sequence of p37 gene was modified and expressed well in transfected cells. We found that p37 localized at the Golgi apparatus and could be secreted out of the cell. Human gastric cancer cells AGS, after being transfected with the p37 gene, were smaller, more spherical and easy to detach from each other. Their adhesion to matrix was also diminished and cytoskeleton in these stable p37 AGS cell was rearranged and transcription co-factor beta-actin was transferred to nucleolus with down-regulation of ICAM-1 and integrin beta1. These findings will be helpful for us to elucidate the effects of p37 on eukaryotic cells as well as to better understand the potential relationship between cancer and mycoplasma infection.

[Establishment of a stable and regulable 293 cell line expressing mycoplasma hyorhinis protein P37].

OBJECTIVE: To establish a stable cell line, which can express P37 protein of mycoplasma hyorhinis and be regulated by tetracycline, for investigating the effect of p37 on phenotype of cells and its mechanism. METHODS: Recombinant plasmid PcDNA5/FRT/TO-p37 was constructed and cotransfected with pOG44 into Flp-In-T-REx-293 cells by lipofectamine. Positive clones were screened with Hygromycin and Blasticidin. RT-PCR and Western blot were used to exam the mRNA and protein expression in selected clones. The expression level at different inducing times and concentrations of tetracycline were examined. MTT assay was used to observe the effect of P37 on proliferation of 293 cells. RESULTS: P37 protein, which is 43.5x10(3), was expressed in the selected clone as well as secreted from cells. Tetracycline showed a good regulation on the expression of P37 protein, which was not detectable without tetracycline induction. When induced with 2 mg/L tetracycline for 60 hours, the P37 protein expression reached maximum level. Cell growth was promoted after being transfected with p37. CONCLUSION: A stable cell line expressing P37 regularly was established, which provides a good cell model for studying p37 function and its molecular mechanism.

Screening and identification of proteins interacting with nucleostemin.

AIM: To identify the proteins interacting with nucleostemin (NS), thereby gaining an insight into the function of NS. METHODS: Yeast two-hybrid assay was performed to screen a human placenta cDNA library with the full length of NS as a bait. X-Gal assay and beta-galactosidase filter assay were subsequently conducted to check the positive clones and the gene was identified by DNA sequencing. To further confirm the interaction of two proteins, the DNA fragment coding NS and the DNA fragment isolated from the positive clone were inserted into the mammalian expression vector pcDNA3 and pcDNA3-myc, respectively. Then, two plasmids were cotransfected into the COS-7 cells by DEAE-dextron. The total protein from the cotransfected cells was extracted and coimmunoprecipitation and Western blot were performed with suitable antibodies sequentially. RESULTS: Two positive clones that interacted with NS were obtained from human placenta cDNA library. One was an alpha isoform of human protein phosphatase 2 regulatory subunit B (B56) (PPP2R5A) and the other was a novel gene being highly homologous to the gene associated with spondylo paralysis. The co-immunoprecipitation also showed that NS specifically interacted with PPP2R5A. CONCLUSION: NS and PPP2R5A interact in yeast and mammalian cells, respectively, which is helpful for addressing the function of NS in cancer development and progression.

[Characterization of the interaction between low density lipoprotein receptor-related protein-associated protein 1 and the cancer and embryo expression protein 65].

OBJECTIVE: To understand the mechanism of cancer and embryo expression protein 65 (CEP65) in cancer development. METHODS: CEP65 was used as a bait protein to isolate the partners of CEP65 from a human placenta cDNA library with yeast two hybrid system. The DNA fragments from positive clone were expressed prokaryotic and mammalian cells respectively, the GST pull-down experiment was performed to ascertain the binding activity. RESULTS: After screening a human placenta cDNA library, the low density lipoprotein receptor-related protein-associated protein 1 (RAP) was isolated and shown to interact with CEP65. GST pull-down assay results indicated that His-CEP65 and GST-RAP expressed by prokaryotic system were immunoprecipitated, and so were Myc-RAP and GST-P65 expressed by mammalian cells. CONCLUSION: RAP can interact with CEP65 and RAP may be a candidate to mediate the function of CEP65.

Preliminary study on proteomics of gastric carcinoma and its clinical significance.

AIM: To explore the preliminary identification of serum protein pattern models that may be novel potential biomarkers in the detection of gastric cancer. METHODS: A total of 130 serum samples, including 70 from patients with gastric cancer and 60 from healthy adults, were detected by surface-enhanced laser desorption and ionization time-of-flight mass spectrometry (SELDI-TOF-MS). The data of spectra were analyzed by Biomarker Patterns Software (BPS). Thirty serum samples of gastric cancer patients and 30 serum samples of healthy adults were grouped into the training group to build models, and the other 70 samples were used to test and evaluate the models. The samples of the test group were judged only with their peaks' height and were separated into cancer group or healthy control group by BPS automatically and the judgments were checked with the histopathologic diagnosis of the samples. RESULTS: Sixteen mass peaks were found to be potential biomarkers with a significant level of P< 0.01. Among them, nine mass peaks showed increased expression in patients with gastric cancer. Analyzed by BPS, two peaks were chosen to build the model for gastric cancer detection. The sensitivity, specificity, and accuracy of the model were 90%, 36/40, 86.7%, 26/30, and 88.6%, 62/70, respectively, which were greatly higher than those of clinically used serum biomarkers CEA (carcinoembryonic antigen), CA19-9 and CA72-4. Stage I/II gastric cancer samples of the test group were all judged correctly. CONCLUSION: The novel biomarkers in serum and the established model could be potentially used in the detection of gastric cancer. However, large-scale studies should be carried on to further explore the clinical impact on the model.

[Eukaryotic expression and biological activities of anti-p185 erbB2 mouse/human chimeric antibody].

OBJECTIVE: Overexpression of the HER2/neu oncogene is a frequent molecular event in multiple human cancers. Being a cancer antigen, p185(erbB2) is an ideal target for immunotherapy. In order to decrease the immunogenicity of mouse anti-p185(erbB2) monoclonal antibody in human cancer therapy, we constructed the eukaryotic expression vector of anti-p185(erbB2) chimeric monoclonal antibody and verified expression of the chimeric antibody in CHO-dhfr(-) cell. METHODS: The variable regions of light chain and heavy chain were amplified with RT-PCR and inserted into the chimeric antibody vector pWSD2. After CHO-dhfr(-) cells were transfected with recombination plasmid by lipofectAMINE, the chimeric antibody expressing level was identified with RT-PCR, indirect-ELISA, and Western blot. The specificity of the anti-p185(erbB2) chimeric antibody was testified with ELISA assay and immunoprecipitation. Moreover, the effects of chimeric antibody on the proliferation of breast cancer cell line SKBR3, which is overexpressing p185(erbB2), were measured with MTT assay in vitro. RESULTS: The anti-p185(erbB2) chimeric antibody eukaryotic expression vector was constructed successfully and the expression of the chimeric antibody in CHO-dhfr(-) was verified by RT-PCR, indirect-ELISA, and Western blot. ELISA assay showed that chimeric antibody reacted with cells overexpressing p185(erbB2) specifically, but did not react with that non-overexpressing p185(erbB2). Immunoprecipitation test confirmed that the chimeric antibody could bind to p185(erbB2) specifically. The MTT assay demonstrated that the chimeric antibody could inhibit the growth of SKBR3 cells overexpressing p185(erbB2) . CONCLUSION: The anti-p185(erbB2) mouse/human chimeric antibody that was expressed in CHO-dhfr(-) cells can bind to p185(erbB2) specifically and inhibit proliferation of SKBR3 cells overexpressing p185(erbB2) . It has a potential application in biotherapy of cancer.