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Objectives: To observe the mitochondrial morphology of normal and triple-negative breast cancer cells, extract mitochondria from normal cells, and investigate the effects of mitochondrial transplantation on proliferation, apoptosis, and stemness of triple-negative breast cancer cells. Methods: The morphology of mitochondria was observed by transmission electron microscope. Mitochondria were extracted by mitochondrial extraction kit, mitochondrial protein was identified by western blot, and mitochondrial activity was detected by mitochondrial membrane potential detection kit. MitoTracker Green or MitoTracker Deep Red fluorescent probes were used to label the mitochondria of living cells, and the degree of mitochondria entering LTT cells was observed by confocal laser microscopy at 12, 24, and 96 hours. The effects of mitochondrial transplantation on proliferation, apoptosis, and stemness of breast cancer cells were examined by CCK8, colony formation assay, flow cytometry, and sphere formation assay after 24 hours of mitochondrial transplantation. Results: The mitochondria of normal cells were rod-shaped or elongated, while the mitochondria of triple-negative breast cancer cells were swollen and vacuolated. Western blot results showed that cytochrome c oxidase subunit I (MT-CO1) protein encoded by mitochondria was present in the isolated mitochondria. The content of heat shock protein 60 (HSP60) was higher in mitochondria than that in cytoplasm. The result of the multi-mode microplate reader showed that the content of mitochondrial J-aggregates/monomer was 1.67±0.06, which was significantly higher than 0.35±0.04 of the control group ( P ＜0.001). Exogenous mitochondria were observed in LTT cells at 12, 24, and 96 hours after mitochondrial transplantation. The results of the CCK8 experiment showed that OD450 of LTT cells was 0.27±0.13 after 48 hours transplantation, which was lower than 0.62±0.36 of the control group ( P =0.023). The OD450 of MDA-MB-468 cells was 0.30±0.03, which was lower than 0.65±0.10 of the control group ( P =0.004). After 120 hours of mitochondrial transplantation, OD450 in both groups was still significantly lower than that in the control group (P ＜0.01). The number of clones formed by mitochondrial transplantation of LTT cells was 21.33±7.31, which was lower than 35.22±13.59 of the control group ( P =0.016). Flow cytometry showed that the early apoptosis rate of LTT cells was (30.07±2.15)% after 24 hours of mitochondrial transplantation, which was higher than 2.07±1.58 of the control group ( P ＜0.001). The proportion of early apoptosis in MDA-MB-468 cells was 24.47%±5.22%, which was higher than (7.83±2.06)% in the control group ( P =0.007). In addition, the number of mitochondria transplanted LTT cells into the cell sphere was 46.25±5.40, which was significantly lower than 62.58±6.43 of the control group ( P ＜0.001). Conclusion: Normal mitochondria can enter triple-negative breast cancer cells by co-culture, inhibit the proliferation and stemness of triple-negative breast cancer cells, and promote the apoptosis of triple-negative breast cancer cells.

Background: Antibody-based platforms ( i.e. , ADC) have emerged as one of the most encouraging tools for the cancer resistance caused by cancer stem cells (CSCs) enrichment. Our study might provide a promising therapeutic direction against drug resistance and serve as a potential precursor platform for screening ADC., Methods: The cell migration, invasion, drug resistance, and self-renewal were assessed by the cell invasion and migration assay, wound healing assay, CCK-8 assay, colony formation assay, and sphere formation assay, respectively. The expression profiles of CSCs (ALDH + and CD44 + ) subpopulations were screened by flow cytometry. The western blot and cell immunofluorescence assay were used to evaluate pathway-related protein expression in both anti-ENO1 antibody, MET combined with DPP/CTX-treated CSCs., Results: In the present study, western blot and flow cytometry verified that anti-ENO1 antibody target the CD44 + subpopulation by inhibiting the PI3K/AKT pathway, while metformin might target the ALDH + subpopulation through activation of the AMPK pathway and thus reverse drug resistance to varying degrees. Subsequently, in vitro investigation indicated that anti-ENO1 antibody, metformin combined with cisplatin/cetuximab could simultaneously target ALDH + and CD44 + subpopulations. The combination also inhibited the CSCs proliferation, migration, invasion, and sphere formation; which may result in overcoming the drug resistance. Then, molecular mechanism exploration verified that the anti-ENO1 antibody, metformin combined with cisplatin/cetuximab inhibited the Wnt/β-catenin signaling., Conclusions: The study preliminarily revealed anti-ENO1 antibody combined with metformin could overcome drug resistance against CSCs by inhibiting the Wnt//β-catenin pathway and might serve as a potential precursor platform for screening ADC. More importantly, it is reasonably believed that antibody-based drug combination therapy might function as an encouraging tool for oncotherapy., Competing Interests: The authors declare that they have no competing interests., (© 2024 Shu et al.)

Objective: To investigate the diagnostic efficiency of conventional serum tumor markers and their combination with chest CT for stage ⅠA lung cancer. Methods: A total of 1 155 patients with stage ⅠA lung cancer and 200 patients with benign lung lesions (confirmed by surgery) treated at the Cancer Hospital, Chinese Academy of Medical Sciences from January 2016 to October 2020 were retrospectively enrolled in this study. Six conventional serum tumor markers [carcinoembryonic antigen (CEA), carbohydrate antigen 125 (CA125), squamous cell carcinoma associated antigen (SCCA), cytokeratin 19 fragment (CYFRA21-1), neuron-specific enolase (NSE), and gastrin-releasing peptide precursor (ProGRP)] and chest thin-slice CT were performed on all patients one month before surgery. Pathology was taken as the gold standard to analyze the difference of positivity rates of tumor markers between the lung cancer group and the benign group, the moderate/poor differentiation group and the well differentiation group, the adenocarcinoma group and the squamous cell carcinoma group, the lepidic and non-lepidic predominant adenocarcinoma groups, the solid nodule group and the subsolid nodule group based on thin-slice CT, and subgroups of ⅠA1 to ⅠA3 lung cancers. The diagnostic performance of tumor markers and tumor markers combined with chest CT was analyzed using the receiver operating characteristic curve. Results: The positivity rates of six serum tumor markers in the lung cancer group and the benign group were 2.32%-20.08% and 0-13.64%, respectively; only the SCCA positivity rate in the lung cancer group was higher than that in the benign group (10.81% and 0, P =0.022). There were no significant differences in the positivity rates of other serum tumor markers between the two groups (all P >0.05). The combined detection of six tumor markers showed that the positivity rate of the lung cancer group was higher than that of the benign group (40.93% and 18.18%, P =0.004), and the positivity rate of the adenocarcinoma group was lower than that of the squamous cell carcinoma group (35.66% and 47.41%, P =0.045). The positivity rates in the poorly differentiated group and moderately differentiated group were higher than that in the well differentiated group (46.48%, 43.75% and 22.73%, P =0.025). The positivity rate in the non-lepidic adenocarcinoma group was higher than that in lepidic adenocarcinoma group (39.51% and 21.74%, P =0.001). The positivity rate of subsolid nodules was lower than that of solid nodules (30.01% vs 58.71%, P =0.038), and the positivity rates of stageⅠA1, ⅠA2 and ⅠA3 lung cancers were 33.33%, 48.96% and 69.23%, respectively, showing an increasing trend ( P =0.005). The sensitivity and specificity of the combined detection of six tumor markers in the diagnosis of stage ⅠA lung cancer were 74.00% and 56.30%, respectively, and the area under the curve (AUC) was 0.541. The sensitivity and specificity of the combined detection of six serum tumor markers with CT in the diagnosis of stage ⅠA lung cancer were 83.0% and 78.3%, respectively, and the AUC was 0.721. Conclusions: For stage ⅠA lung cancer, the positivity rates of commonly used clinical tumor markers are generally low. The combined detection of six markers can increase the positivity rate. The positivity rate of markers tends to be higher in poorly differentiated lung cancer, squamous cell carcinoma, or solid nodules. Tumor markers combined with thin-slice CT showed limited improvement in diagnostic efficiency for early lung cancer.

Background: Identification of promising targeted antigens that exhibited cancer-specific expression is a crucial step in the development of novel antibody-targeted therapies. We here aimed to investigate the anti-tumor activity of a novel monoclonal antibody (mAb) 11C9 and identify the antibody tractable target in the hepatocellular cancer stem cells (HCSCs)., Methods: The identification of the targeted antigen was conducted using SDS-PAGE, western blot, mass spectrometry, and co-immunoprecipitation. Silence of HSP90 was induced by siRNA interference. Positive cells were sorted by fluorescence-activated cell sorting. Double-immunofluorescent (IF) staining and two-color flow cytometry detected the co-expression. Self-renewal, invasion, and drug resistance were assessed by sphere formation, matrigel-coated Transwell assay, and CCK-8 assay, respectively. Tumorigenicity was evaluated in mouse xenograft models. RNA-seq and bioinformatics analysis were performed to explore the mechanism of mAb 11C9 and potential targets., Results: MAb 11C9 inhibited invasion and self-renewal abilities of HCC cell lines and reversed the cisplatin resistance. HSP90 (~ 95 kDa) was identified as a targeted antigen of mAb 11C9. Tissue microarrays and online databases revealed that HSP90 was overexpressed in HCC and associated with a poor prognosis. FACS and double-IF staining showed the co-expression of HSP90 and CSCs markers (CD90 and ESA). In vitro and in vivo demonstrated the tumorigenic potentials of HSP90. The inhibition of HSP90 by siRNA interference or 17-AAG inhibitor both decreased the number of invasion, sphere cells, and CD90 + or ESA + cells, as well as reversed the resistance. Bioinformatics analysis and western blot verified that HSP90 activated Wnt/β-catenin signaling., Conclusions: The study preliminarily revealed the anti-tumor activity of mAb 11C9. More importantly, we identified HSP90 as a targeted antigen of mAb 11C9, which functions as an oncogene in phenotype shaping, stemness maintenance, and therapeutic resistance by activating Wnt/β-catenin signaling., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Background: Cancer stem cells (CSCs) are responsible for drug resistance, cancer relapse, and metastasis. Here, we report the first analysis of Palladin expression and its impacts on stem cell-like properties in lung cancer., Methods: Tissue microarrays were used to investigate Palladin expression and its association with prognosis. Immunofluorescence (IF), flow fluorescence assay, and Western blot were performed to detect Palladin expression in 6 NSCLC cell lines. Cell phenotypes and drug resistance were evaluated. Xenograft models were constructed to confirm the role of Palladin in vivo., Results: By using the tissue microarrays, Palladin was identified to be highly expressed in the cytoplasm, specifically in the cytomembrane of NSCLC, and its high expression is associated with poor prognosis. Palladin is widely expressed and enriched in the sphere cells. The in vitro and in vivo studies showed that Palladin promoted stem cell-like properties, including cell viability, invasion, migration, self-renewal abilities, taxol resistance, and tumorigenicity. Western blot revealed that Palladin promoted the accumulation of β-catenin and activated Wnt/β-catenin signaling. Tissue microarrays analysis further confirmed the positive correlation between Palladin and β-catenin. Wnt/β-catenin pathway inhibitor blocked the Palladin-induced enhancement of sphere-forming., Conclusions: Palladin might act as an oncogene by promoting CSCs-like properties and tumorigenicity of NSCLC cells via the Wnt/β-catenin signaling pathway. Besides, Palladin was identified to have the potential as a cell surface marker for LCSCs identification. These findings provide a possible target for developing putative agents targeted to LCSCs., (© 2022 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

The fatality rate of non-small cell lung cancer (NSCLC) has been high due to the existence of cancer stem cells (CSCs). Non-muscle myosin heavy chain 9 (MYH9) can promote the progression of various tumors, but its effect on the stem cell-like characteristics of lung cancer cells (LCCs) has not been clarified. Our research found that the stemness characteristics of LCCs were significantly enhanced by the overexpression of MYH9, and the knockout of MYH9 had the opposite effects. The in vivo with inhibitor blebbistatin further confirmed the effect of MYH9 on the stem cell-like behavior of LCCs. Furthermore, western blotting showed that the expression level of CSCs markers (CD44, SOX2, Nanog, CD133, and OCT4) was also regulated by MYH9. Mechanistic studies have shown that MYH9 regulates stem cell-like features of LCCs by regulating the mTOR signaling pathway, which was supported by sphere formation experiments after LCCs were treated with inhibitors Rapamycin and CHIR-99021. Importantly, high expression of MYH9 in lung cancer is positively correlated with poor clinical prognosis and is an independent risk factor for patients with NSCLC., (© 2021. The Author(s).)

Due to the short-term observation record of the normalized difference vegetation index (NDVI), the research on long-term NDVI changes is scarce, which limits our understanding of the impacts of NDVI changes in the context of global warming. In this study, a regional tree-ring chronology was developed based on the tree-ring samples of Pinus tabuliformis in the middle Qinling Mountains. The results showed that tree-ring width of P. tabuliformis was significantly positively correlated with May-July NDVI ( r =0.624, P <0.01, n =34). The Sig-Free tree-ring width chronology was used to reconstruct May-July NDVI during the period 1825-2018, which explained 38.9% of the total NDVI variance. Results of spatial analysis showed that the reconstructed series could better represent the NDVI changes in the study area. There were six high NDVI periods and five low NDVI periods in the past 194 years. The vegetation grew best in 2006-2018, indicating vegetation cove-rage in the middle of Qinling Mountains had been improved during the warming hiatus. Low NDVI periods in the reconstruction series were consistent with drought over much of study area. Results of wavelet analysis indicated the existence of 2-4 years and 12-16 years cycles in the reconstruction series. SEA analysis showed that the reconstruction series decreased significantly in the El Nino year, while increased significantly in the first to third years after the La Nina event. The growth of P. tabuliformis was predicted to increase slightly under the SSP2-4.5, SSP3-7.0 and SSP5-8.5 scenarios.

Osteosarcoma (OS) is a primary malignant tumor of bone occurring in young adults. OS stem cells (OSCs) play an important role in the occurrence, growth, metastasis, drug resistance and recurrence of OS. CD133 is an integral membrane glycoprotein, which has been identified as an OSC marker. However, the mechanisms of metastasis, chemoresistance, and progression in CD133(+) OSCs need to be further explored. In this study, we aim to explore differences in miRNA levels between CD133(+) and CD133(-) cells from the MG-63 cell line. We found 20 differentially expressed miRNAs (DEmiRNAs) (16 upregulated and 4 downregulated) in CD133(+) cells compared with CD133(-) cells. Hsa-miR-4485-3p, hsa-miR-4284 and hsa-miR-3656 were the top three upregulated DEmiRNAs, while hsa-miR-487b-3p, hsa-miR-493-5p and hsa-miR-431-5p were the top three downregulated DEmiRNAs. In addition, RT-PCR analysis confirmed that the expression levels of hsa-miR-4284, hsa-miR-4485-3p and hsa-miR-3656 were significantly increased, while the expression levels of hsa-miR-487b-3p, hsa-miR-493-5p, and hsa-miR-431-5p were significantly decreased in CD133(+) cells compared with CD133(-) cells. Moreover, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed that predicted or validated target genes for all 20 DEmiRNAs or the selected 6 DEmiRNAs participated in the "PI3K-Akt signaling pathway," "Wnt signaling pathway," "Rap1 signaling pathway," "Cell cycle" and "MAPK signaling pathway". Among the selected six DEmiRNAs, miR-4284 was especially interesting. MiR-4284 knockdown significantly reduced the sphere forming capacity of CD133(+) OS cells. The number of invasive CD133(+) OS cells was markedly decreased after miR-4284 knockdown. In addition, miR-4284 knockdown increased the p-β-catenin levels in CD133(+) OS cells. In conclusion, RNA-seq analysis revealed DEmiRNAs between CD133(+) and CD133(-) cells. MiRNAs might play significant roles in the function of OSCs and could serve as targets for OS treatment. MiR-4284 prompted the self-renewal and invasion of OSCs. The function of miR-4284 might be associated with the Wnt signaling pathway., Competing Interests: The authors declare that they have no competing interests., (© 2021 Shu et al.)

Background: Focusing on antiangiogenesis may provide promising choices for treatment of gastric cancer (GC). This study aimed to investigate the mechanistic role of BCAT1 in the pathogenesis of GC, particularly in angiogenesis., Methods: Bioinformatics and clinical samples analysis were used to investigate the expression and potential mechanism of BCAT1 in GC. BGC823 cells with BCAT1 overexpression or silencing were induced by lentiviral transduction. Cell phenotypes and angiogenesis were evaluated. The relevant proteins were quantized by Western blotting, immunohistochemistry, or immunofluorescence. Xenograft models were constructed to confirm the role of BCAT1 in vivo ., Results: BCAT1 was overexpressed in GC patients and associated with lower survival. BCAT1 expression was correlated with proliferation-, invasion-, or angiogenesis-related markers expression and pathways. Silencing BCAT1 expression suppressed cell viability, colony formation, cycle progression, invasion, and angiogenesis of BGC823 cells, as well as the tumor growth of xenograft models, whereas overexpressing BCAT1 had the opposite results both in vitro and in vivo . Bioinformatics analysis and Western blotting demonstrated that BCAT1 activated the PI3K/AKT/mTOR pathway. The addition of LY294002 reversed the tumor growth induced by BCAT1 overexpression, further verifying this mechanism., Conclusion: BCAT1 might act as an oncogene by facilitating proliferation, invasion, and angiogenesis through activation of the PI3K/AKT/mTOR pathway. This finding could aid the optimization of antiangiogenesis strategies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Shu, Zhan, Sun, Yu, Liu, Sun, Yang, Ran and Sun.)

Background: Tumor-associated antigens (TAAs) can be targeted in cancer therapy. We previously identified a monoclonal antibody (mAb) 12C7, which presented anti-tumor activity in lung cancer stem cells (LCSCs). Here, we aimed to identify the target antigen for 12C7 and confirm its role in LCSCs., Methods: Immunofluorescence was used for antigen localization. After targeted antigen purification by electrophoresis and immunoblot, the antigen was identified by LC-MALDI-TOF/TOF mass spectrometry, immunofluorescence, and immunoprecipitation. The overexpression or silence of ENO1 was induced by lentiviral transduction. Self-renewal, growth, and invasion of LCSCs were evaluated by sphere formation, colony formation, and invasion assay, respectively. High-throughput transcriptome sequencing (RNA-seq) and bioinformatics analysis were performed to analyze downstream targets and pathways of targeted antigen., Results: Targeted antigen showed a surface antigen expression pattern, and the 43-55 kDa protein band was identified as α-enolase (ENO1). Self-renewal, growth, and invasion abilities of LCSCs were remarkably inhibited by ENO1 downregulation, while enhanced by ENO1 upregulation. RNA-seq and bioinformatics analysis eventually screened 4 self-renewal-related and 6 invasion-related differentially expressed genes. GSEA analysis and qRT-PCR verified that ENO1 regulated self-renewal, invasion-related genes, and pathways. KEGG pathway analysis and immunoblot demonstrated that ENO1 inactivated AMPK pathway and activated mTOR pathway in LCSCs., Conclusions: ENO1 is identified as a targeted antigen of mAb 12C7 and plays a pivotal role in facilitating self-renewal, growth, and invasion of LCSCs. These findings provide a potent therapeutic target for the stem cell therapy for lung cancer and have potential to improve the anti-tumor activity of 12C7.

Recent studies have demonstrated that gastric cancer stem cells (CSCs) are a rare sub-group of gastric cancer (GC) cells and have an important role in promoting the tumor growth and progression of GC. In the present study, we demonstrated that the glycolytic enzyme Enolase 1 (ENO1) was involved in the regulation of the stem cell-like characteristics of GC cells, as compared to the parental cell lines PAMC-82 and SNU16, the expression of ENO1 in spheroids markedly increased. We then observed that ENO1 could enhance stem cell-like characteristics, including self-renewal capacity, cell invasion and migration, chemoresistance, and even the tumorigenicity of GC cells. ENO1 is known as an enzyme that is involved in glycolysis, but our results showed that ENO1 could markedly promote the glycolytic activity of cells. Furthermore, inhibiting glycolysis activity using 2-deoxy-D-glucose treatment significantly reduced the stemness of GC cells. Therefore, ENO1 could improve the stemness of CSCs by enhancing the cells' glycolysis. Subsequently, to further confirm our results, we found that the inhibition of ENO1 using AP-III-a4 (ENOblock) could reduce the stemness of GC cells to a similar extent as the knockdown of ENO1 by shRNA. Finally, increased expression of ENO1 was related to poor prognosis in GC patients. Taken together, our results demonstrated that ENO1 is a significant biomarker associated with the stemness of GC cells.

Objective: To investigate the risk factors for concurrent sepsis in neonates with necrotizing enterocolitis (NEC)., Methods: A retrospective analysis was performed for the clinical data of 273 neonates with NEC. The risk factors for concurrent sepsis were analyzed from the aspects of perinatal factors and treatment regimen before the diagnosis of NEC., Results: The incidence rate of concurrent sepsis in NEC was 32.2% (88/273). The neonates with stage III NEC had a significantly higher incidence rate of concurrent sepsis than those with stage II NEC (69.0% vs 15.9%; P<0.05). Of all neonates with sepsis, 62.5% experienced sepsis within 3 days after the diagnosis of NEC, and 37.5% experienced sepsis more than 3 days after the diagnosis. Compared with those without concurrent sepsis, the neonates with concurrent sepsis had significantly lower gestational age and birth weight (P<0.05). The neonates who had scleredema, had stage III NEC, needed gastrointestinal decompression after the diagnosis of NEC, and experienced a long time of gastrointestinal decompression tended to develop sepsis more easily (P<0.05). Scleredema (OR=9.75, 95%CI: 2.84-33.52, P<0.001), stage III NEC (OR=12.94, 95%CI : 6.82-24.55, P<0.001), and gastrointestinal decompression (OR=2.27, 95%CI: 1.14-4.5, P=0.02) were independent risk factors for concurrent sepsis in NEC., Conclusions: Scleredema, stage III NEC, and gastrointestinal decompression are independent risk factors for concurrent sepsis in neonates with NEC.

Objective: To investigate the biological characteristics of monoclonal antibodies against human liver cancer stem cells and its therapeutic effect in combination with cisplatin in the treatment of hepatocellular carcinoma., Methods: Cell culture in serum-free medium and PKH26 staining were used to determine the existence of cancer stem cells in human liver Bel7402-V3 cell line. The co-expression of antigen recognized by monoclonal antibody (McAb) 15D2 and epithelial specific antigen (ESA) and PKH26-positive cells in the Bel7402-V3 cells were detected by immunofluorescence assay. Serum-free suspension culture was used to detect the self-renewal ability of 15D2-positive Bel7402-V3 cells sorted by flow cytometry and the effect of 15D2 on the self-renewal ability of Bel7402-V3 cells. The effect of 15D2 on cisplatin resistance in the cells was examined by CCK8 method. The inhibitory effect of 15D2 combined with cisplatin on the transplanted tumor growth in mice was also observed., Results: Single PKH26-positive cells were observed in the Bel7402-V3 cell spheroids cultured for 11 days. Immunofluorescence assay showed that the 15D2-recognized antigen could be conjugated with PKH26 and ESA and co-localized on Bel7402-V3 cells. The spheroid formation rate of 15D2-positive cells in serum-free medium was significantly higher than that of 15D2-negative cells [(30.4±3.4)% vs. (8.8±1.8)%, P<0.01]. The cisplatin resistance of 15D2-positive cells was obviously higher than that of 15D2-negative cells (IC50: 1.014 μmol/L vs. 0.365 μmol/L). McAb 15D2 significantly suppressed the spheroid formation of Bel7402-V3 cells, with an inhibition rate of 37.5%. McAb 15D2 also notably inhibited the cisplatin resistance of Bel7302-V3 cells. The IC50 was 0.211 μg/ml in the 15D2 group and 0.325 μg/ml in the control group. The mouse experiment showed that the tumor growth rates of 50 mg/kg, 25 mg/kg and 12.5 mg/kg 15D2-treatment groups were 82.6%, 71.4% and 60.0%, respectively; that of the 50 mg/kg 15D2 + cisplatin group was 91.0%, and that of the cisplatin monotherapy was 56.7%., Conclusion: McAb 15D2 is a functional monoclonal antibody targeting liver cancer stem cells, which could be a potential monoclonal antibody drug for the stem cell-targeted therapy of liver cancer.

Ischemic stroke is caused when blood flow to the brain is stopped and is a major cause of death and long term disability across the globe. Excessive release of neurotransmitters is triggered in the brain by ischemia that mediates neuronal damage and causes ischemic injury. In this study, a simple, sensitive, and on-line preconcentration capillary electrophoresis method based on electrokinetic supercharging (EKS) was developed for the determination of the biogenic amines including dopamine (DA), epinephrine (E), and norepinephrine (NE) in C57BL/6 mice brain. Under the optimized conditions, the analytes were concentrated and detected within 10 min. The detection limits for the analytes ranged from 0.42 to 0.57 ng mL(-1) for a mice brain matrix. With the proposed method, the analyses of three neurochemical amines in C57BL/6 mice brain tissue during cerebral ischemic/reperfusion had been performed successfully.

Objective: To evaluate the clinical value of autoantibody spectrum against ovarian cancer associated antigens combine CA(125) in detecting and monitoring ovarian cancer., Methods: Circulating IgG, IgM autoantibodies against ovarian cancer associated antigens which included TM4SF1, C1D, TIZ, OV-142, FXR1 and OV-189 were measured by indirect ELISA in serum from 126 patients with ovarian cancer (prior treatment), 42 patients with benign ovarian masses, 142 healthy women. Cut off value of IgG, IgM autoantibodies were determined by receive operating characteristic (ROC) curve. CA(125) was measured in serum by immunoradiometric assay (IRMA). We evaluated the clinical value of combining multiple autoantibodies (autoantibody spectrum), combining autoantibody spectrum with CA(125) by binary logistic regresion. The positive ratio of autoantibody spectrum in serum (prior and post treatment) of 24 synchronization patients with ovarian cancer was analyzed to evaluate the value in monitoring state of illness., Results: Our data indicated that serum contains IgG, IgM autoantibodies against ovarian cancer associated antigens. The positive ratio of IgG autoantibodies in serum from ovarian cancer patients and cancer-free patients were 34.1% - 47.6% and 13.0% - 19.0%, respectively (P < 0.05). The positive ratio of IgM autoantibodies in serum from ovarian cancer patients and cancer-free patients were 39.7% - 53.2% and 12.0% - 33.2%, respectively (P < 0.05). The positive ratio of IgG autoantibodies against FXR1 and IgM autoantibodies against TIZ, FXR1 and OV-189 in early stage (I-II) ovarian cancer (55.3%, 63.8%, 61.7% and 66.0%) were significantly higher than those in advanced (III-IV) ovarian cancer (34.2%, 39.2%, 26.6%, 45.6%; all P < 0.05). Combining five autoantibodies (TM4SF1 IgG, TM4SF1 IgM, C1D IgG, FXR1 IgG and TIZ IgM) showed significantly improved sensitivity (75.4%, P < 0.05), lower specificity (78.3%, P < 0.05) and similar accuracy (77.1%, P > 0.05) in detecting ovarian cancer compared to those of CA(125) (61.1%, 88.0%, 77.1%). But the autoantibody spectrum showed significantly improved sensitivity in classifying early stage (76.6%), compared to those of CA(125) (51.1%, P < 0.05). Combining autoantibody spectrum with CA(125) showed significantly improved sensitivity (85.7%), specificity (90.8%)and accuracy (88.7%) in detecting ovarian cancer compared to those of autoantibody spectrum alone (all P < 0.05), while CA(125) (61.1%, P < 0.05; 88.0%, P > 0.05; 77.1%, P < 0.05). The positive ratio of combine the autoantibody spectrum with CA(125) was significantly lower in 24 post-treatment serum (42%) compared to the pairing prior treatment serum (88%, P < 0.05)., Conclusion: Combining the autoantibody spectrum against ovarian cancer associated antigens with CA(125) can improve sensitivity, specificity and accuracy in detecting early ovarian cancer and may be used to monitoring state of illness.

The purpose of this study was to investigate invasion- and metastasis-related genes in gastric cancer. To this end, we used the transwell system to select a highly invasive subcell line from minimally invasive parent cells and compared gene expression in paired cell lines with high- and low-invasive potentials. Lysyl oxidase-like 2 (LOXL2) was overexpressed in the highly invasive subcell line. Immunohistochemical analysis revealed that LOXL2 expression was markedly increased in carcinoma relative to normal epithelia, and this overexpression in primary tumor was significantly associated with depth of tumor invasion, lymph node metastasis and poorer overall survival. Moreover, LOXL2 expression was further increased in lymph node metastases compared with primary cancer tissues. RNA interference-mediated knockdown and ectopic expression of LOXL2 showed that LOXL2 promoted tumor cell invasion in vitro and increased gastric carcinoma metastasis in vivo. Subsequent mechanistic studies showed that LOXL2 could activate both the Snail/E-cadherin and Src kinase/Focal adhesion kinase (Src/FAK) pathways. However, secreted LOXL2 induced gastric tumor cell invasion and metastasis exclusively via the Src/FAK pathway. Expression correlation analysis in gastric carcinoma tissues also revealed that LOXL2 promoted invasion via the Src/FAK pathway but not the Snail/E-cadherin pathway. We then evaluated secreted LOXL2 as a target for gastric carcinoma treatment and found that an antibody against LOXL2 significantly inhibited tumor growth and metastasis. Overall, our data revealed that LOXL2 overexpression, a frequent event in gastric carcinoma progression, contributes to tumor cell invasion and metastasis, and LOXL2 may be a therapeutic target for preventing and treating metastases.

Objective: To further explore the effect of annexin I on the tumor growth of human pancreatic cancer in nude mice., Methods: To knock down the expression of annexin I in pancreatic carcinoma cells by RNAi. A nude mouse model of human pancreatic cancer was established by subcutaneous inoculation of human pancreatic cancer cell line Suit-II cells. The effect of annexin I on tumor growth was assessed by tumor growth curve and tumor weight records, and Westen blot and flow cytometry were used to examine the expression of annexin I after annexin I-knocking down., Results: The results of Western blot revealed that the expression of annexin I was significantly decreased in Suit-II cells transfected with pSilencer-annexin I-siRNA1, and almost completely inhibited in the cells transfected with pSilencer-annexin I-siRNA2 and pSilencer-annexin I-siRNA3. The growth of tumors transfected with annexin I-siRNA2 and annexin I-siRNA3 was inhibited by 76.6% and 68.4%, respectively, in comparison with that of tumor from the parent Suit-II cells. At 44 days after tumor cell inoculation, the tumor weight was 0.8987 g (transfected with annexin I-siRNA2) and 0.8992 g (transfected with annexin I-siRNA3), significantly lower (P < 0.001) than that of tumor from parent Suit-II cells (2.5866 g) and transfected with annexin I-siRNAN (2.4070 g)., Conclusion: annexin I promotes the growth and proliferation of pancreatic carcinoma cells in vivo and increases the ability of tumor formation in nude mice. The results of this study support that annexin I may become a potential target in gene therapy for this disease.

Objective: To investigate the value of autoantibody of breast cancer susceptibility 1-associated RING domain (BARD1) splice variant (OV-142) in detection of ovarian cancer., Methods: We cloned OV-142 gene into plasmid pET-30b(+). The recombinant protein of OV-142 was expressed in pET-30b(+) system and purified. The autoantibody of OV-142 was detected by indirect enzyme-linked immunosorbent assay (ELISA)., Results: We successfully constructed the recombinant plasmid of OV-142. The recombinant protein was expressed in pET-30b(+) system and purified. The purification rate of the recombinant protein was up to 90%. The relative amount of autoantibody of OV-142 detected by indirect ELISA was analyzed by receiver operating characteristic curve (ROC) and the cutoff value was determined. Combination of the autoantibody IgG of OV-142 and CA(125) was analyzed by logistic regression. The sensitivity, specificity and accuracy was 71.4%, 89.1%, and 81.9%, respectively, which were higher than IgG (41.3%, 84.2%, 66.8%) and CA(125) (61.1%, 88.0%, 77.1%) when used alone each., Conclusions: OV-142 is a splice variant of BARD1. It may be a potential immunotherapy target of ovarian cancer. Detection of autoantibody of OV-142 is a potent complementary tool of CA(125) in ovarian cancer diagnosis.

Esophageal cancer is characterized by rapid clinical progression and poor prognosis due to adjacent tissue invasion and distant organs metastasis at a very early stage. TM4SF3 (transmembrane 4 superfamily 3), a member of tetraspanin family, has been reported as a metastasis associated gene in many types of tumors. Herein, we described new properties of TM4SF3 in tumor metastasis, which suggested that this gene might be involved in esophageal carcinoma metastasis. Western blotting revealed that TM4SF3 was overexpressed in 57.1% (8/14) of esophageal carcinomas and esophageal carcinoma cell lines with high-invasive potential. Exogenous expression of TM4SF3 in two low-invasive esophageal carcinoma cell lines, KYSE150 and EC9706, significantly promoted cell migration and invasion. Upregulating TM4SF3 expression in EC9706 cells promoted xenograft tumor invading into surrounding tissues, enhanced lung metastasis, and shortened the lifespan of mice (median survival EC9706-TM4SF3 106.5 days versus EC9706-Vector 169.0 days, P < 0.0001) in a spontaneous metastasis model. Further studies demonstrated that ADAM12m was upregulated by TM4SF3 overexpression in vitro and in vivo. Abrogating up-expression of ADAM12m by siRNA significantly suppressed TM4SF3-mediated invasion. Together, these data from our studies indicated that overexpression of TM4SF3 in esophageal cancer conferred advantage to the invasion and metastasis of this destructive disease. Upregulated expression of ADAM12m by TM4SF3 might play a key role in TM4SF3-mediated invasion and metastasis. TM4SF3 and ADAM12m might be potential targets of esophageal carcinoma for anti-metastasis therapy.

Objective: To explore epithelial ovarian cancer (EOC) antigens that are potentially useful for cancer early detection and therapy., Methods: A high quality cDNA library derived from ascites tumor cells of EOC patients (3 cases of serous EOC, 1 case of mucinous EOC, and 1 case of endometrial carcinoma of ovary) was constructed, and the method of combining serological analysis of recombinant cDNA expression libraries (SEREX) and suppression subtractive hybridization (SSH) was used for screening cDNA library. All of the positive clones were sequenced and bioinformatics analysis with BLAST software in GenBank was performed. Serological mini-arrays of recombinant tumor antigens (SMARTA) was used to investigate the prevalence of autoantibodies to these antigens in both 96 ovarian cancer patients and 96 cancer-free controls., Results: Fifty-five positive clones encoding different antigenic genes of EOC recognized by IgG and (or) IgM were obtained. It showed that these 55 clones derived from 45 distinct genes and these genes could be grouped into 6 classes as following according to homology with known expressed sequence tag (EST): (1) known ovarian carcinoma related genes: BARD1, et al; (2) homologous genes with other tumors: TM4SF1, et al; (3) homologous genes with special tissues: ILF3, FXR1, et al; (4) homologous genes with special function: TIZ, C1D, et al; (5) embryo originating genes: PKHD1, et al; (6) novel genes: OV-189, et al. SMARTA results showed that the positive ratio of five EOC antigens TM4SF1 (28% vs. 9%), C1D (21% vs. 6%), BARD1 (23% vs. 5%), FXR1 (23% vs. 8%), OV-189 (31% vs. 13%) which reacting with their IgG autoantibodies, three antigens TIZ (26% vs. 8%), FXR1 (28% vs. 11%), and OV-189 (18% vs. 7%) which reacting with their IgM autoantibodies in patients was higher than in controls (P < 0.05). The positive ratio of EOC antigens FXR1 (34% vs. 16%), and OV-189 (46% vs. 23%) reacting with their IgG autoantibodies and TIZ (40% vs. 18%), FXR1 (46% vs. 18%) which reacting with their IgM autoantibodies in stage I-II patients was higher than that in stage III-IV (P < 0.05). The positive ratio of OV-189 (67% vs. 26%) which reacting with its IgG autoantibodies in well differentiated cases was higher than in moderately-poorly differentiated cases (P < 0.01). Combination of the above antigens showed a 66% sensitivity and 73% accuracy in discriminating EOC. Combination of the autoantibody profile of TM4SF1, C1D, TIZ, BARD1, FXR1, OV-189 with CA125 showed an 83% sensitivity and 80% accuracy in discriminating EOC., Conclusions: The strategy of combination of SEREX and SSH is a potent tool in isolating tumor-associated antigen genes. Autoantibody profile of TM4SF1, C1D, TIZ, BARD1, FXR1, OV-189 are potential tumor markers in EOC detection.

Background & Objective: Tumor growth relies on angiogenesis; angiogenic inhibition can effectively treat tumors. This study was to develop monoclonal antibody library against human hepatic carcinoma vessel endothelial cells (HCVECs), and screen functional monoclonal antibodies with inhibitory effect on the angiogenesis of hepatic carcinoma., Methods: Endothelial cells were separated from human hepatic carcinoma tissue to immunize 10 BALB/c mice. The spleen cells of the immunized mice were fused with SP2/0 cells and cultured on methyl cellulose. Functional monoclonal antibodies of each clone were screened by immunofluorescence, endothelial cell proliferation assay, tube formation assay, and humanized blood vessel in vivo tumor model., Results: A total of 1,442 monoclonal antibody clones were obtained by fusing spleen cells with SP2/0 cells; 119 clones could specifically react with HCVECs. Of the 119 clones, 53 significantly suppressed the proliferation or tube formation of HCVECs. Two of the 53 monoclonal antibody clones suppressed the hepatic tumor growth in vivo, with inhibitory rates of 66.7% and 76.5%., Conclusion: We successfully developed 2 functional monoclonal antibodies which could suppress the hepatic tumor growth in vivo.

Background & Objective: In malignant transformation, mutant gene products and dysregulated proteins can become tumor antigens and activate immunoreactions. Therefore, auto-antibodies exist in sera of cancer patients. Serologic analysis of recombinant cDNA expression libraries (SEREX) using autologous and allogenic patient sera provides a powerful approach to identify tumor antigens. This study was to identify esophageal cancer antigens with SEREX for serologic diagnosis, gene therapy, and immune therapy., Methods: Expression library of cDNA from esophageal squamous cell carcinoma was constructed. SEREX screened out 21 positive clones from the 1.6x10(6) clones in the established library. The 21 positive clones were subcloned to monoclonality and submitted to in vivo excision of pBluescript phagemids. The nucleotide sequences of cDNA inserts were analyzed with DNASIS and BLAST software on EMBL and GenBank. According to the bioinformatics analyses, serologic immunoreactions of 4 colons in 10 samples of esophageal cancer serum and 10 samples of normal control serum were further detected by SADA., Results: Of the 21 positive clones, 4 had no homology to any known genes, 17 were known fragments which were defined as antigens of esophageal cancer for the first time. The serologic immunoreaction rates of 4 selected antigens, including Ribosomal protein S4, and so on, were 40%, 60%, 70%, and 30%, respectively, in cancer sera, and 0%, 10%, 20%, and 20%, respectively, in normal sera., Conclusions: Antigens, such as Ribosomal protein S4, are frequently involved in serologic immunoreactions of esophageal cancer. The 21 antigens identified by the present study can be used as potential targets for gene therapy and serologic biomarkers of esophageal cancer.

Proteins that are unfolded or misfolded in the endoplasmic reticulum (ER) must be targeted for refolding or degradation to maintain the homeostasis of the ER. Derlin-1 was reportedly implicated in the retro-translocation of misfolded proteins from the ER to the cytosol for degradation. In this report, we showed that Derlin-1 was down-regulated in the endothelial cells derived from human hepatic cavernous hemangioma (CHEC) compared with other tested cells. Electron microscopy analysis showed that ER was aberrantly enlarged in CHEC cells, but not in other tested cells. When overexpressed, Derlin-1 induced the dilated ER to return normal size. This ER dynamic was associated with the activation of unfolded protein response (UPR). In CHEC cells where Derlin-1 was down-regulated, increased expression of the immunoglobulin heavy chain-binding protein (Bip) and UPR-specific splicing of X-box DNAbinding protein 1 (XBP1) mRNA were detected, as compared with that in other tested cells, indicating that UPR was activated. After Derlin-1 overexpression, the extent of UPR activation diminished, as evidenced by decreased expression of Bip, reduced amount of the spliced form of XBP1 (XBP1s), and elevated expression of the unspliced form of XBP1 (XBP1u). Taken together, these findings provide another example of a single protein being able to affect ER dynamic in mammalian cells, and an insight into the possible molecular mechanism(s).

Background & Objective: Animal models are indispensable in the studies of tumor endothelial genes and anti-angiogenic therapy. This study was to build a new engrafted tumor model with humanized blood vessels., Methods: Human liver sinusoid endothelial cells (HLSECs) were mixed respectively with human liver cancer cell line BEL7402, human colon cancer cell line LS174T, and human esophageal cancer cell line NEC, and then inoculated into NOD/SCID mice or BALB/c nude mice. The mice inoculated with only tumor cells were used as controls. Tumor growth was observed. Green fluorescent protein (GFP)-labeled HLSECs were observed under fluorescent microscope to detect their survival and tube formation. Microvessel density (MVD) was analyzed by immunohistochemistry. The tumor-bearing mice were treated by anti-HLSEC monoclonal antibody 2B6 to observe its effect on tumor growth., Results: Tumor growth was significantly enhanced by the co-inoculation of HLSECs with BEL7402 cells in NOD/SCID mice; tumor weight was increased by 5.1 folds as compared with that of control. GFP-labeled vessels could easily be observed in the tumors from co-inoculation of HLSECs with BEL7402 cells. Total MVD was increased by 85.7% of control. The humanized MVD was about 10.28-29.28, which was 41%-65% of total MVD. Furthermore, the co-inoculation of HLSECs with NECs, BEL7402, or LS174T cells in nude mice led to 3.3-6.0 folds increase of xenograft weight as compared with control. When treated with 2B6 antibody, humanized MVD was decreased by 65.1% in the engrafted tumors and the tumor weight lost 71.8%., Conclusions: When co-inoculate with human tumor cell lines into mice, HLSECs could survive, proliferate, and contribute to tumor angiogenesis, which may enhance tumor growth. The engrafted tumor in vivo model with humanized vessels can be widely used in the research of functional genes in tumor angiogenesis and anti-angiogenic therapies.

Aim: To gain tumor endothelium associated antigen genes from human liver cancer vascular endothelial cells (HLCVECs) cDNA expression library, so as to find some new possible targets for the diagnosis and therapy of liver tumor., Methods: HLCVECs were isolated and purified from a fresh hepatocellular carcinoma tissue sample, and were cultured and proliferated in vitro. A cDNA expression library was constructed with the mRNA extracted from HLCVECs. Anti-sera were prepared from immunized BALB/c mice through subcutaneous injection with high dose of fixed HLCVECs, and were then tested for their specificity against HLCVECs and angiogenic effects in vitro, such as inhibiting proliferation and inducing apoptosis of tumor endothelial cells, using immunocytochemistry, immunofluorescence, cell cycle analysis and MTT assays, etc. The identified xenogeneic sera from immunized mice were employed to screen the library of HLCVECs by modified serological analyses of recombinant cDNA expression libraries (SEREX). The positive clones were sequenced and analyzed by bio-informatics., Results: The primary cDNA library consisted of 2 x 10(6) recombinants. Thirty-six positive clones were obtained from 6 x 10(5) independent clones by immunoscreening. Bio-informatics analysis of cDNA sequences indicated that 36 positive clones represented 18 different genes. Among them, 3 were new genes previously unreported, 2 of which were hypothetical genes. The other 15 were already known ones. Series analysis of gene expression (SAGE) database showed that ERP70, GRP58, GAPDH, SSB, S100A6, BMP-6, DVS27, HSP70 and NAC alpha in these genes were associated with endothelium and angiogenesis, but their effects on HLCVECs were still unclear. GAPDH, S100A6, BMP-6 and hsp70 were identified by SEREX in other tumor cDNA expression libraries., Conclusion: By screening of HLCVECs cDNA expression library using sera from immunized mice with HLCVECs, the functional genes associated with tumor endothelium or angiogenesis were identified. The modified SEREX, xenogeneic functional serum screening, was demonstrated to be effective for isolation and identification of antigen genes of tumor endothelium, and also for other tumor cell antigen genes. These antigen genes obtained in this study could be a valuable resource for basic and clinical studies of tumor angiogenesis, thus facilitating the development of anti- angiogenesis targeting therapy of tumors.

To demonstrate effects of antisense VEGF(165) to suppress esophageal cancer cells and to improve efficacy of the gene therapy of tumor by using hypoxic environment, hypoxia response element (HRE) was cloned from promoter of VEGF by PCR and employed to construct an eukaryotic expression vector containing luciferase and antisense VEGF(165) by using recombinant DNA techniques. The recombinant vectors were transfered into esophageal cancer cells by lipofectin methods, and hypoxia inducible reporter gene expression was determined by luminometer and the expression of antisense VEGF was evaluated indirectly by ELISA that detected of VEGF. The esophageal cells tansfected by antisense VEGF(165) gene were transplanted into nude mice, in order to evaluate the suppressive effect of antisense VEGF(165). Our results showed that, in vitro, hypoxia increased expression of reporter gene to 3 780 % and enhanced greatly expression of antisense VEGF. In vivo, the growth of esophageal cancer cells transfected by antisense VEGF in the vector containing HRE was suppressed more significantly, with suppression rate being 71.1%, than that by the vector without HRE, whose inhibiting rate 56.1%. It was concluded that antisense VEGF(165) suppressed significantly growth of esophageal cancer, and by using a gene expression vector containing HRE, the expression of target genes could be regulated autonomously by hypoxic environment of tumor and the efficiency of gene therapy could be greatly improved.

Background & Objective: NY-ESO-1 is a tumor antigen from cDNA library of esophageal carcinoma. However, there was no report about its expression in the tissue of esophageal carcinoma. In the current study, the authors examined the frequency of the NY-ESO-1 gene expression in esophageal carcinoma in order to determine whether NY-ESO-1 antigen-based vaccine therapy be available for the patients with esophageal carcinoma., Methods: Reverse transcriptase and polymerase chain reaction amplification techniques were utilized to assay 30 esophageal carcinoma tissues and the matched adjacent normal esophageal tissues for expression of NY-ESO-1 gene. And the cDNA sequence of NY-ESO-1 was cloned from esophageal carcinoma tissues by molecular cloning techniques., Results: Out of 30 esophageal carcinoma tissues, 50% expressed NY-ESO-1 gene while no expressed in the matched adjacent normal esophageal tissues. The sequence of NY-ESO-1 cloned was identical to the sequence of that from GenBank., Conclusions: NY-ESO-1 gene is expressed highly in esophageal carcinoma and NY-ESO-1 antigen can be recognized by cytolytic T lymphocytes when presented by HLA-class-I molecular.

Background & Objective: Carcinoembryonic antigen(CEA), is highly expressed in many kinds of carcinomas, especially in colon carcinoma. Anti-CEA antibodies have great application in diagnosis and therapy of the patients with colon carcinoma. This study was designed to investigate the biodistribution and radioimmunodetection in nude mice bearing human colon carcinoma using 188Re-labeled anti-CEA chimeric antibody., Methods: CEA chimeric antibody and its parent McAb C50 were labeled with 188Re by a stannous chloride reduction method. The radiochemical purity of them were determined by ITLC. The biodistribution and whole body gamma scintigraphy imaging of 188Re-CEA chimeric antibody in nude mice bearing human colon carcinoma were fulfilled. The effect of radioimmunoimage between these two antibodies was compared., Results: 188Re-CEA chimeric antibody showed high radiochemical purity of more than 95%. The specific reactivity of 188Re-CEA chimeric antibody was 356 MBq/mg. Immunoreactivity of 188Re-CEA chimeric antibody was 61% as determined by ELISA. Tumor/kidney ratio (0.75) and tumor/blood ratio(0.99) of 188Re-CEA chimeric antibody were observed in 24 h; compared to the orther organs, the ration is over 1.78. Tumor/kidney ratio (1.02) and tumor/blood ratio (1.12) of 188Re-CEA chimeric antibody were observed in 48 h; compared to the other organs more than 2.08. High uptake of 188Re-CEA chimeric antibody was observed in 20 hours after injection. The radioimmunodetection effect of these two antibodies was approximately same., Conclusions: 188Re-CEA chimeric antibody showed high specific tumor uptake and could fast display clear image in the nude mice bearing human colon carcinoma. Comparing with McAb C50, CEA chimeric antibody has good effect in clinic because of reducing of immunogenicity.

Background & Objective: Human inhibiting angiogenesis factor-1(HIAF-1) expressed in E coli. shown the activity of inhibiting the angiogenesis of tumors. This study was designed to investigate the expression of HIAF-1 in insect cells and to elucidate its biological activity., Methods: Recombinant baculovirus expression vector pAcuW51-HIAF-1 was constructed by recombinant DNA technique and cotransfected with linearized baculovirus DNA into sf9 cells to produce recombinant virus. The expression of recombinant HIAF-1 in insect cells infected by recombinant virus was detected by SDS-PAGE and Western blot. The recombinant protein was purified by affinity chromatography. In vitro endothelial proliferation inhibiting activity of recombinant HIAF-1 was examined by MTT method, its antitumor activity in vivo was studied in human esophageal cancer transplanted in nude mice., Results: HIAF-1 was effectively expressed in insect cells as 26 KD fusing protein and its expression level was about 5-10% of insect cellular total soluble proteins. Recombinant HIAF-1 protein could inhibit endothelial cell proliferation in vitro with IC50 value of 3.1 micrograms/ml and inhibited remarkably growth of human esophageal cancer transplanted in nude mice., Conclusions: The recombinant HIAF-1 with better activity was successfully expressed in insect cells and establish a base for clinical application.

One of the cardinal questions in tumor immunology is the identification of antigenic structures in human tumors that are recognized by host immune system. A powerful new methodology for identifying human tumor antigens eliciting humoral immune response is SEREX (serological identification of antigen by recombinant cDNA expression cloning). Here, by using this method, a recombinant cDNA expression library from lung cancer was analysed and several new tumor antigens were isolated. Using the lambda-ZAP vector, cDNA expression library was constructed from lung cancer tissues of three patients including a moderately differentiated lung adenocarcinoma, a highly differentiated lung squamous cell carcinoma and a moderately differentiated lung adeno-squamous carcinoma. The primary library consisted of 0.8 x 10(6) recombinants. 33 positive clones encoding antigen genes were obtained after immunoscreening, and the nucleotide sequences of cDNA inserts were determined and analysed with DNASIS and BLAST softwares in EMBL and GenBank. These antigen genes included known genes, such as MAGE (melanoma antigen gene), vitiligo-associated protein VIT-1, fibronectin, Na-K-ATPase et al and unknown genes or ESTs. To characterize expression profile of these genes, antibodies in sera from 48 lung cancer patients and 48 health donors were assayed with three antigens (L-8, L-19, L-51) to screen specific and relative serum markers for lung cancer. The results show that positive rates in lung cancer patients are higher than in health donors. Our research indicates that some of these antigens may be related to lung cancer and may be valuable tumor markers in diagnosis of lung cancer.

The McAb C25 against human P185(erbB2) specifically inhibits proliferation of cancer cells overexpressing P185(erbB2). In order to decrease HAMA response in clinical therapy of human cancer using McAb, and to express this antibody efficiently in CHO cells, an anti-P185(erbB2) mouse/human chimeric antibody gene containing variable region of C25 gene was constructed. The expression vectors were constructed using genomic DNA of human IgG1 constant region, and using neo and dhfr genes driven by weaker promoters as selectable marker genes. Variable region genes of C25 were cloned by RT-PCR. VL and VH genes of C25 were sequenced and then inserted, respectively, into the light chain and heavy chain expression vectors. The two expression vectors were cotransfected into CHO-dhfr(-) cells with LipofectAMINE. The specificity of the chimeric antibody was verified using cellular-ELISA and immuno-fluorescence techniques. ELISA and RT-PCR were used toconfirm that the chimeric antibody containing both variable region of C25 and human constant region. At 72 h post-transfection, the chimeric antibody could be detected in supernatant of CHO cells by ELISA assay and the yield was 1 mg/L. After the selection by G418, stepwise MTX pressure (1x10(-8) 2.5x10(-7) mol/L) culture was carried out, and the yield of the chimeric antibody was increased up to 100 mg/L. The chimeric antibody was demonstrated to have the antigen specificity to P185(erbB2) and to carry the human antibody constant region by cellular-ELISA, immuno-fluorescence assay, indirect-ELISA and RT-PCR. The chimeric antibody could inhibit proliferation of SKBR(3) and SKOV(3) cells at the same inhibiting rate asMcAb C25. In conclusion, a mouse/human chimeric antibody against human P185(erbB2) with potential of usage in clinical therapy of human cancer was constructed and highly expressed.

Two hemorrhagic proteases, Lachesis hemorrhagic toxins a and b (LHTa and LHTb), were isolated from the venom of Lachesis muta, which is distributed in Central and South America. One protease showed strong hemorrhagic action, while the other showed weak hemorrhagic activity even though the two enzymes are very similar in their chemical properties. Neither enzyme hydrolyzed arginine esters, but both hydrolyzed casein and reduced fibrinogen. The A alpha chain of fibrinogen was hydrolyzed first, and the B beta chain was hydrolyzed later. The gamma chain of fibrinogen was resistant to hydrolysis. The molecular weights of LHTa and LHTb were very similar, 22,000 and 23,000, respectively. The amino acid composition of LHTa was also similar to that of LHTb. The secondary structure of LHTa as determined by Lippert's equation was 52% alpha helix, 17% beta sheet, and 31% random coil; that of LHTb was 47% alpha helix, 13% beta sheet, and 40% random coil.

By means of CM-Sephadex C-50 column chromatography and gel filtration on Sephadex G-75, a potent platelet function inhibitor was purified from Vipera russelli siamensis venom. It appeared as a single protein band on polyacrylamide gel electrophoresis in the presence or absence of SDS, and consists of 123 amino acid residues. Its NH2-terminal residue is serine. It showed the following characteristics: molecular weight, 13,800; isoelectric point, 10.4; LD50, 0.5 +/- 0.12 mg/kg (i.v.). The platelet inhibitor exhibited phospholipase A2 activity with a specific activity of 35 mumoles/min/mg. From 2 g of the venom, 70 mg of the purified inhibitor was obtained. Inhibition of human platelet aggregation induced by ADP or adrenaline was dose-dependent, with ID50 of 1.14 micrograms/ml or 0.37 microgram/ml, respectively. The platelet aggregation induced by thrombin or collagen was also inhibited and the inhibitory activity on platelet aggregation was heat stable (at 100 degrees C, 20 min) in an acidic medium (pH 5.8), while its phospholipase A2 activity was relatively heat labile under the same condition. The release of 3H-serotonin in platelets stimulated by ADP was also inhibited and this was positively correlated with inhibition of platelet aggregation induced by ADP (r = 0.998, P less than 0.002).