Sorting and gene mutation verification of circulating tumor cells of lung cancer with epidermal growth factor receptor peptide lipid magnetic spheres.

BACKGROUND: This study aimed to identify an efficient, simple, and specific method of detecting mutations in the epidermal growth factor receptor (EGFR) gene in isolated lung cancer circulating tumor cells (CTCs) and to improve the ability to obtain tumor tissue clinically. METHODS: EGFR peptide lipid magnetic spheres (EG-P-LMB) were prepared by reverse evaporation, and characterization and cell capture efficiency assessed. The peripheral blood samples of 30 lung cancer patients were isolated and identified with the EG-P-LMB using 20 healthy volunteers as controls. Finally, the isolated CTCs were tested for EGFR gene mutations, and the tissue samples selected for comparison. RESULTS: The prepared magnetic spheres had a smaller particle size and higher stability according to the particle size potential test. Their morphology was homogeneous by atomic force observation, and the UV test showed that there were peptides on the surface. The separation efficiency of EG-P-LMB was greater than 90% in PBS and greater than 80% in the blood simulation system. Compared with the tissue sample results, the positive rate of EGFR gene mutations was 94%. The CTC test results of 27 patients were consistent with the tissue test results of the corresponding patients, and the consistency with the tissue comparison test results was 90% (27/30). CONCLUSIONS: EG-P-LMB can effectively capture CTCs in the peripheral blood of patients with lung cancer. CTC detection can accurately identify mutations in the EGFR gene and improve the ability to obtain tumor tissue in clinical practice. KEY POINTS: SIGNIFICANT FINDINGS OF THE STUDY: EG-P-LMB can effectively capture CTCs in the peripheral blood of patients with lung cancer. CTC detection can accurately identify mutations in the EGFR gene and improve the ability to obtain tumor tissue in clinical practice. WHAT THIS STUDY ADDS: This study added EGFR peptide lipid magnetic spheres to capture CTCs in the blood. Genetic testing was performed and compared with tissues. It solves the problem of clinically difficult tumor tissue sampling.

Molecular cloning and characterization of α-amylase/subtilisin inhibitor from rhizome of Ligusticum chuanxiong.

OBJECTIVES: To clone and characterize a novel bi-functional α-amylase/subtilisin inhibitor (LASI) from the rhizome of Ligusticum chuanxiong, a traditional Chinese medicine. RESULTS: The LASI showed strong homology with members of the Kunitz trypsin inhibitor family. Its putative amino acid sequence has a 40 % identity with that of the α-amylase/subtilisin inhibitor from rice. LASI gene without signal peptide was expressed in E. coli Rosetta. After purification, the recombinant LASI protein was inhibitory against not only α-amylase from porcine pancreas, Helicoverpa armigera, Spodoptera litura and Plutella xylostella, but also subtilisin A, but not against trypsin or chymotrypsin. In addition, the expression level of LASI in rhizome was higher than that in leaf and LASI expression was enhanced by salt, chilling and drought treatment. CONCLUSIONS: This is the first member of the Kunitz-protease inhibitor family identified in traditional Chinese medicine and it might be involved in the plant defense responses against lepidopterous pests, microorganisms and abiotic stresses.

Everolimus synergizes with gefitinib in non-small-cell lung cancer cell lines resistant to epidermal growth factor receptor tyrosine kinase inhibitors.

PURPOSE: Epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) therapy is considered as one of the most important treatments for patients with advanced non-small-cell lung cancer (NSCLC). However, not all patients benefit from this therapy because of primary or acquired resistance, both of which are usually caused by the activation of alternative signaling pathways. Thus, a combination of different signaling pathway inhibitors is a promising strategy. We used the mammalian target of rapamycin (mTOR) inhibitor everolimus in combination with gefitinib in NSCLC cell lines to analyze the efficacy of this combination regimen and the underlying molecular mechanism. METHODS: Acquired gefitinib-resistant cell lines, together with EGFR wild-type and mutant primary gefitinib-resistant NSCLC cell lines, were treated with everolimus alone, gefitinib alone, or the combination of the two drugs, and the effects were evaluated using cell proliferation assays. The effects of everolimus and gefitinib on the EGFR pathway in NSCLC cell lines were determined by Western blot analysis. RESULTS: Combined treatment resulted in synergistic antitumor effects in gefitinib-resistant cells A549 and H1975. The combination index (CI) of cells increased with increasing dose of everolimus. Everolimus demonstrated no apparent inhibition of phosphorylated Akt (p-Akt) and phosphorylated p44/42 MAPK (p-MAPK) in H1650 cells. Additionally, in gefitinib-resistant cell lines, the combination of gefitinib and everolimus not only showed stronger inhibition of phosphorylated mTOR and phosphorylated p70S6K expression than either drug alone but also reduced the levels of p-Akt and p-MAPK in both cell lines. CONCLUSIONS: Our data showed that the combination of everolimus and gefitinib exhibits dose-dependent synergism in primary and acquired gefitinib-resistant NSCLC cells. Thus, a preclinical rationale exists for the use of everolimus to enhance the efficacy of gefitinib in EGFR-TKI-resistant patients with NSCLC.

[Epithelial growth factor receptor mutation status to the effective of survival in non-small cell lung cancer after surgery].

OBJECTIVES: To investigate the relationship between the epithelial growth factor receptor (EGFR) mutation status and clinicopathological factors, and to analyze the mutation on the effect in non-small cell lung cancer (NSCLC) after surgery. METHODS: The NSCLC patients who were resected and detected EGFR gene from March 2009 to March 2011 were retrospectively reviewed. The relationship between EGFR mutation status and clinicopathological factors, tumor markers, prognostic was analyzed. RESULTS: The mutation and the wild group had 169 and 214 patients respectively. EGFR mutation in female, non-smoking, adenocarcinoma and less than 60 years old accounted for 63.91%, 61.54%, 88.76% and 62.13% with statistical significance compared with male (χ(2) = 53.490, P = 0.000), smoking (χ(2) = 48.568, P = 0.000), non-adenocarcinoma (χ(2) = 105.560, P = 0.000) and more than 60 years old (χ(2) = 6.057, P = 0.017). Disease free survival (DFS) of the wild group was better than mutation group (χ(2) = 11.329, P = 0.001). In addition, there were some relations between mutation status and excision repair cross complementing (ERCC1) protein, carcinoembryonic antigen (CEA), squamous cell carcinoma (SCC) and Cyfra21-1. ERCC1(+) (χ(2) = 6.739, P = 0.012), SCC(χ(2) = 16.839, P = 0.000) and Cyfra21-1(χ(2) = 6.638, P = 0.013) more than normal value was common in wild group. Increased CEA was common in mutation group (χ(2) = 5.436, P = 0.023). CONCLUSIONS: EGFR mutation is commonly found in female, non-smoking, adenocarcinoma and less than 60 years old NSCLC patients. The wild group obtains better DFS than mutation group. Tumor markers may predict the mutation status, which need further research.

[A clinical and prognostic retrospective analysis of IIIA-N2 non-small cell lung cancer].

OBJECTIVES: To analyze the clinical conditions of postoperative patients with IIIA-N2 non-small cell lung cancer (NSCLC) and the prognostic factors related with survival of NSCLC, and to investigate the influence of operation and therapy on prognosis. METHODS: Clinical data of 657 inpatient cases with IIIA-N2 NSCLC admitted from January 2000 to December 2005 was retrospectively reviewed. The Kaplan-Meier method was used for survival analysis. The Log-rank law was applied to analyze the relationship between the variables and the prognosis in monovariate analysis, while Cox proportional hazard regression model was used to make multivariate analysis. RESULTS: The 1-, 3-and 5-year accumulative survival rates of the operative patience were 64.4%, 26.0% and 17.9%, respectively. The median survival time was 18 months. In monovariate analysis, the main unfavorable factors that affect life span involve were the diameter of tumor, T stage, skip metastasis of N2 lymph node, the number of metastatic lymph nodes, the metastasis of subcarinal lymph nodes, adjuvant chemotherapy, the cycle of adjuvant chemotherapy, postoperative radiotherapy, and the modality of therapy (the effect of naive surgery was disappointed, while the prognosis of the patients with adjuvant chemoradiotherapy was better than those with chemotherapy alone). A multivariate analysis using Cox regression identified 5 factors of prognosis: the diameter of tumor (P = 0.001), the metastasis of subcarinal lymph nodes (P = 0.019), the number of metastatic lymph nodes (P = 0.006), the cycle of adjuvant chemotherapy (P = 0.007), postoperative radiotherapy (P = 0.055), and adjuvant chemoradiotherapy (P = 0.026). CONCLUSIONS: The 5-year survival rate of the patients with IIIA-N2 Non-small cell lung cancer is poor. Tumor size, the metastasis of subcarinal lymph nodes, the number of metastatic LNs, the cycle of adjuvant chemotherapy, and postoperative radiotherapy have an effect on the prognosis. The prognosis of postoperative patients with single-level N2 and multi-level N2 disease is similar, and the key point of survival is the number of nodes involved. The therapeutic effect of patience given adjuvant chemoradiotherapy is superior to those treated with adjuvant chemotherapy.

Role of urinary biomarkers of N,N-dimethylformamide in the early detection of hepatic injury among occupational exposed workers.

AIM: To determine the sensitive and convenient biomarkers for the early detection of hepatic injury in N,N-dimethylformamide (DMF) exposed workers. METHODS: Seventy-nine individuals in a synthetic leather factory were investigated with questionnaire survey. The air samples, urine samples, and blood samples were collected at the specific time point. Airborne DMF and the urine metabolites of DMF were measured by gas chromatography (GC), high-performance liquid chromatography (HPLC), and gas chromatography-mass spectrometry (GC-MS). Traditional liver function tests and hepatic fibrosis parameters were performed by auto-chemistry analyzer and ELISA methods. RESULTS: The urine concentration of N-acetyl-S-(N-methylcarbamoyl)-cysteine (AMCC), one of the metabolites of DMF, was positively correlated with activities of liver function enzymes. About 60% subjects with urine AMCC concentration above 40 mg/g creatinine showed raised liver enzymes activities. In terms of hepatic fibrosis parameters, we found 4 of 5 abnormal total serum bile acid (SBA) and 4 of 4 abnormal serum hyaluronidase (HA) among workers with higher amount of urine AMCC. CONCLUSION: Workers exposed to DMF with higher urine AMCC levels were more likely to develop liver diseases. In addition, SBA and HA have the potential to act as early indicators of toxic hepatic fibrosis activities for occupational health surveillance.

[Correlation of DNA-dependent protein kinase catalytic subunit expression to radiosensitivity of non-small cell lung cancer cell lines].

BACKGROUND AND OBJECTIVE: DNA-dependent protein kinase catalytic subunit (DNA-PKcs) plays an important role in repairing irradiation-induced DNA double-strand break (DSB), and affects the radiosensitivity of tissue cells. This study was to detect the expression of DNA-PKcs in different non-small cell lung cancer (NSCLC) cell lines and evaluate its correlation to radiosensitivity. METHODS: The content and activity of DNA-PKcs in five NSCLC cell lines A549, H1299, L78, PGCL3 and H460 were measured by Western blot and the DNA-PK activity assay. Cell survival was analyzed using clonogenic formation assay. RESULTS: The radiosensitivities of five NSCLC cell lines were different. The values of survival fraction at 2 Gy (SF2) were 0.74 in A549 cells, 0.25 in H1299 cells, 0.21 in H460 cells, 0.48 in PGCL3 cells, and 0.58 in L78 cells. The protein levels of DNA-PKcs were 3.26+/-0.98 in A549 cells, 0.51+/-0.07 in L78 cells, 0.51+/-0.11 in H1299 cells, 0.86+/-0.23 in H460 cells, and 2.60+/-0.76 in PGCL3 cells. The activity values of DNA-PKcs were 8.30+/-1.03 in A549 cells, 2.45+/-0.52 in H1299 cells, 0.11+/-0.02 in H460 cells, 4.13+/-0.87 in PGCL3 cells, and 0.42+/-0.07 in L78 cells. In adenocarcinoma and large cell carcinoma cell lines, SF2 were correlated to DNA-PKcs content (P<0.05, r=0.95) and activity (P=0.03, r=0.98). CONCLUSION: DNA-PKcs is an important factor to predict the radiosensitivity in adenocarcinoma and large cell lung cancer cell lines.

Expression of cyclin D1 splice variants is differentially associated with outcome in non-small cell lung cancer patients.

Real-time reverse transcription polymerase chain reaction and immunohistochemistry were used to evaluate the messenger RNA (mRNA) and protein expression levels of total cyclin D1 and its splice variants (cyclin D1a and cyclin D1b) in 102 paired malignant and nonmalignant tissues from patients with non-small cell lung cancer, respectively. The expression levels of total cyclin D1 and its splice variants were significantly up-regulated in malignant tissues than in nonmalignant tissues at both mRNA and protein levels. Although the expression levels of cyclin D1a were higher than those of cyclin D1b, the relative expression ratios of cyclin D1b mRNA between malignant and nonmalignant lung tissues were obviously higher than those of cyclin D1a mRNA. Analysis of variance showed that cyclin D1b mRNA expression was significantly associated with the histologic grade, lymph node metastasis, distant metastasis, and tumor stage of patients, whereas cyclin D1a mRNA expression was not related to clinicopathologic characteristics except sex. Patients with cyclin D1b mRNA expression above the median value had shorter survival than those below the median value (P = .033). Similarly, cyclin D1b immunopositivity was also associated with histologic grade, and patients with immunostaining positivity for cyclin D1b showed poor survival (P = .005). Multivariate analysis demonstrated that cyclin D1b immunopositivity was an independent risk factor in survival of patients with non-small cell lung cancer (P = .018). Our data show that cyclin D1b, rather than canonical cyclin D1a, might contribute to the development of non-small cell lung cancer. Cyclin D1b would be a better prognostic indicator for non-small cell lung cancer as compared to total cyclin D1 or cyclin D1a.

Better survival with EGFR exon 19 than exon 21 mutations in gefitinib-treated non-small cell lung cancer patients is due to differential inhibition of downstream signals.

Somatic mutations in the epidermal growth factor receptor (EGFR) kinase domain are associated with sensitivity to tyrosine kinase inhibitors (TKIs) in patients with non-small cell lung cancer (NSCLC). Our clinical data showed NSCLC patients with exon 19 deletions survived longer following gefitinib treatment than those with exon 21 point mutations. We aimed to investigate whether these two mutations produced differences in phosphorylation of EGFR and downstream signals. Two stable cell lines expressing these mutations were obtained by transfection. Inhibition of phosphorylation of EGFR, Akt, and Erk by gefitinib was detected using Western blotting, and cell inhibition tests were conducted to evaluate the bio-behavior. Gefitinib inhibited the phosphorylation of EGFR, Akt, and Erk to a greater degree in exon 19 deletion cells than in L858R cells. Gefitinib produced G1 arrest in more of the cells with exon 19 deletion than with L858R. This might be attributable to patient selection in TKIs therapy.