[Show abstract][Hide abstract] ABSTRACT: Drug resistance of lung cancer cells is one of main factors which affect the outcome of chemotherapy. It has been reported that abnormal p53 gene is well assosiated with chemotherapy resistance of tumor cells. The aim of this study is to evaluate the effects of p53 gene on drug resistance in human lung cancer cell lines, so as to provide foundation of choosing individual chemotherapy drugs in clinical treatment.
The expression vectors which contain p53cDNA and p53 antisense cDNA respectively were constructed and were confirmed by sequencing. Transfected the 801D, a human lung cancer cell line with recombined plasmids by lipofectin mediating. Several kinds of monoclone cell lines, pEGFP-801D,pEGFP-sense p53-801D(including sense p53,pEGFP-p53(RS)-801D),pEGFP-antisense p53-801D(including antisense p53,pEGFP-p53(AS)-801D),which contained p53 of different status were obtained. Green fluorescence was observed through fluorescence microscopy. The extraneous gene was detected by PCR. MTT assay was taken to determine the drug resistance of each cell line to chemotherapy agents. Cell cycle and apoptosis induced by antitumor drugs were examined by flow cytometer.
Extraneous sense p53 and antisense p53 were proved to be linked to plasmid respectively by sequencing.Green fluorescence was found in transfected cell lines. The IC50 of pEGFP-p53(AS)-801D cell line(0.26+/-0.09 mug/mL) to Cisplatin(DDP) decreased markedly compared with 801D(0.55+/-0.19 mug/mL,P<0.05)and pEGFP-801D(0.77+/-0.13mug/mL,P<0.05). The IC50 value of pEGFP-p53(RS)-801D to DDP is 0.43+/-0.25 mug/mL,which is significantly lower than that of pEGFP-801D(P =0.000)but higher than that of pEGFP-p53(AS)-801D(P <0.05). pEGFP-p53(RS)-801D cell line showed a notably smaller value of IC50(2.34+/-0.43 ng/mL) to Paclitaxel(TAX) than 801D(8.40+/-1.50 ng/mL, P <0.05)did. The IC50 value of pEGFPp53(RS)-801D is lower than that of pEGFP-801D(6.41+/-1.98 ng/mL),but not markedly. The IC50 of pEGFP-p53(RS)-801D cell line to 5-Fluorouracil(5FU) is 2.08+/-0.18 mug/mL, which is obviously lower than that of 801D(4.90+/-1.12 mug/mL,P <0.05) and pEGFP-801D(3.41+/-0.86 mug/mL,P <0.05). By the assay of flow cytometer, G2 phase arrest was observed when pEGFP-p53(AS)-801D was treated with DDP. TAX induced G2 phase arrest in pEGFP-p53(RS)-801D. A increased S phase proportion was induced by 5FU in pEGFP-p53(RS)-801D. The cell lines experienced apoptosis and necrosis when they were treated with either DDP or TAX.
p53 gene of different status have different effects on resistance of chemotherapy agents in lung cancer cell lines. p53 mutation and deletion are related to drug resistance of DDP. p53 deletion connects with chemoresistance of TAX and 5FU. Neither p53 mutation nor p53 deletion is associated with drug resistance of Navelbine(NVB) and Gemcitabine(GEM). It is helpful to choose sensitive drugs according to the p53 status of patients for enhancing the efficiency of chemotherapy.
Zhongguo fei ai za zhi = Chinese journal of lung cancer 04/2008; 11(2):157-64. DOI:10.3779/j.issn.1009-3419.2008.02.001
[Show abstract][Hide abstract] ABSTRACT: Matrix metalloproteinase-9 (MMP-9), endostatin (ES) and vascular endothelial growth factor (VEGF) are important angiogenic regulators for many neoplasms. The aim of this study is to judge clinical and prognostic values of detection of serum MMP-9, ES and VEGF in patients with non-small cell lung cancer (NSCLC).
Serum levels of MMP-9, ES and VEGF were detected in 92 patients with NSCLC, 50 patients with pulmonary benign disease and 52 healthy controls by ELISA method.
The serum levels of MMP-9, ES and VEGF in NSCLC patients were significantly higher than those in patients with pulmonary benign disease and healthy controls (P=0.000, P=0.000, P=0.000). The sensitivity and specificity of serum MMP-9 was 92.51% and 79.10% with a cutoff value of 117.17 μg/L, 88.32% and 74.25% for ES with a cutoff value of 100.31 μg/L, and 83.40% and 75.63% for VEGF with a cutoff value of 380.32 ng/L. Serum MMP-9 and ES levels were significant prognostic factors for lung cancer patients (P=0.0145, P=0.008). The change of serum MMP-9 level after chemotherapy was a useful indicator of prognosis for NSCLC patients (P=0.0322).
The serum levels of MMP-9, ES and VEGF are significantly increased in patients with NSCLC. They might be used as prognostic parameters in patients with NSCLC.
Zhongguo fei ai za zhi = Chinese journal of lung cancer 04/2007; 10(2):138-40. DOI:10.3779/j.issn.1009-3419.2007.02.13
[Show abstract][Hide abstract] ABSTRACT: With the development of antibody technology, more and more immunoconjugates are used in clinical treatment for different cancers. The aim of this study is to investigate the inhibitive effects of 5F11-DOX immunoconjugate on human lung adenocarcinoma cell line LTEP-A2 in vitro and in vivo and to explore the potential mechanism.
The 5F11-DOX immunoconjugate was produced by diluted glutaraldehyde crosslinking. The killing efficiency of 5F11-DOX was detected by clonogenic assay. The distribution of DOX was observed under fluorescence microscope and the 5F11 location was determined by immunohistochemistry. The therapeutic efficacy of 5F11-DOX and free DOX was detected on subcutaneous or intraperitoneal exnogenic transplanted tumors of human lung adenocarcinoma A2 cells in nude mice.
5F11-DOX of 0.04mg/L could kill all the A2 cells in vitro and the killing efficiency was 10 times as that of the free DOX. Fluorescence microscopy showed that fluorescence of DOX in 3mg/L 5F11-DOX group was much stronger than that in 3mg/L free DOX group after treating A2 cells with 3mg/L 5F11-DOX or DOX for 3h, then incubating the cells with fresh medium for another 24 hours. Immunohistochemistry showed that 5F11 located in cell membrane and cytoplasm and fluorescence microscopy proved that DOX located inside the cells. The average sizes of subcutaneous or intraperitoneal exnogenic transplanted tumors in 5F11-DOX group were obviously smaller than those of the control group and free DOX group at the same dosage (P < 0.05), and the anti-tumorogenicity efficacy of 5F11-DOX was 4-8 times as that of free DOX. The HE staining showed that extensive necrosis occurred in the center of tumors and around cancer nests in 5F11-DOX group.
The killing efficacy of 5F11-DOX on human lung adenocarcinoma cell line A2 is obviously higher than that of the free DOX.
Zhongguo fei ai za zhi = Chinese journal of lung cancer 12/2005; 8(6):495-500. DOI:10.3779/j.issn.1009-3419.2005.06.02
[Show abstract][Hide abstract] ABSTRACT: Cytochrome P450 2A6 (CYP2A6) plays an important role in oxidation of nicotine and in activation of tobacco-related carcinogens. It has been suggested that individuals with defective CYP2A6 allele are at a lower risk of developing lung cancer. This study is to investigate the frequency of CYP2A6 gene deletion and the relationship of CYP2A6 genetic polymorphism with lung cancer risk in Chinese.
A case-control study which detected CYP2A6 genotype of 180 patients with lung cancer and 224 controls by PCR-based genotype assay was conducted.
No relationship was found between the frequency of CYP2A6 gene deletion and lung cancer risk. There was only one case of CYP2A6 del/del genotype in the controls. The frequency of CYP2A6 del allele was 13.8% in the controls, and 12.8% in lung cancer cases. The CYP2A6 del/del genotype was not found in lung cancer cases.
There is no difference in frequency of CYP2A6 gene deletion between lung cancer cases and controls.
Zhongguo fei ai za zhi = Chinese journal of lung cancer 08/2005; 8(4):297-9. DOI:10.3779/j.issn.1009-3419.2005.04.09
[Show abstract][Hide abstract] ABSTRACT: To investigate the relations between metabolizing enzymes' genetic polymorphism and lung cancer risk in Chinese, especially in heavy smokers.
CYP1A1, 2D6, 2E1 and GSTM1 genotypes were detected in 180 patients with lung cancer and 224 controls by PCR-based genotype assays.
CYP1A1 variant allele, CYP2D6 wild allele, CYP2E1 A genotype, GSTM1-null genotype were found to be associated with lung cancer. The individuals who carried GSTM1-null genotype and one of the CYP1A1, CYP2D6, CYP2E1 'in risk' genotypes had a 2.24-2.69 fold increased risk of lung cancer. The heavy smokers had a significantly increased risk of lung cancer than the non-smokers who carried the same genotype of metabolizing enzymes. The heavy smoker who carried all the four 'in risk' genotypes of metabolizing enzymes had an obviously increased risk of lung cancer (OR=9.85, 95%CI=2.30-45.71).
The individuals who carry the 'in risk' genotype of metabolizing enzymes have an increased risk of lung cancer. It is positively associated with tobacco carcinogen dose.
Zhongguo fei ai za zhi = Chinese journal of lung cancer 04/2004; 7(2):112-7. DOI:10.3779/j.issn.1009-3419.2004.02.08
[Show abstract][Hide abstract] ABSTRACT: To study the inhibition effects of both extraneous right sense and antisense p53 on malignant phenotype of human lung cancer cell line.
The named 801D cell line with p53 deletion and mutation at 248 code was selected as a model in vitro. The recombined plasmid pEGFP-p53(RS) and pEGFP-p53(AS) were constructed. The extraneous gene was detected by PCR. The p53 mutation protein was examined by immunohistochemical stain of p53 antibody. The inhibition effect of extraneous p53 on tumor growth in vitro were determined by clonogenic survival assay. FCM analysis was carried out in cells. The inhibition effect on malignant growth of extraneous p53 in vivo was observed by heteroplastic transplant on nude mouse.
The transfected cell lines, pEGFP-p53(AS)-801D, pEGFP-p53(RS)-801D and pEGFP-801D were established. Presence of extraneous p53 and neo genes in pEGFP-p53(AS)-801D and pEGFP-p53(RS)-801D was proved by PCR and green fluorescence was found out in those cells under the microscope. Mutant protein in pEGFP-p53(AS)-801D was negative by immunohistochemical stain. The malignant growth of these transfected cell lines was inhibited comparing with parents in vivo and in vitro. Inhibition rate of colony formation was 62.0% for pEGFP-p53(AS)-801D and 80.8% for pEGFP-p53(RS)-801D. The tumorigenicity in nude mice was suppressed. Inhibition effects of extraneous right sense p53 on malignant growth of 801D was more distinct. FCM analysis showed that pEGFP-p53(AS)-801D cells were arrested at G1 phase.
The transfected cell lines with extraneous right sense and antisense p53 appear that malignant growth can be inhibited in vivo and in vitro.
Zhongguo fei ai za zhi = Chinese journal of lung cancer 08/2002; 5(4):245-9. DOI:10.3779/j.issn.1009-3419.2002.04.02
[Show abstract][Hide abstract] ABSTRACT: To construct and express a phage display library of anti human lung cancer monoclonal antibody 5F-11.
Immunoglobulin variable regions (VH,VL) were amplified from 5F-11 hybridrom by RT-PCR. ScFv genes consisting of VH DNA and VL DNA joined together by a linker DNA were cloned into a phage vector pCANTAB5E. After 4 rounds of screening with lung adenocarcinoma cell line A2 as antigen, an enriched secondary phage display library was obtained.
A recombinant phage display library with total of 8×10⁷ pfu/ml was established. Randomized clones from unselected library digested with BstNⅠ showed different patterns, however, those from selected library showed that phages with special pattern were enriched. Twenty-three out of 30 clones were found to respond strongly to A2 cell lines.
The ScFv of anti-lung adenocarcinoma monoclonal antibody 5F-11 can be successfully produced, which may be useful to widen the application of the antibody.
Zhongguo fei ai za zhi = Chinese journal of lung cancer 04/2002; 5(2):119-22. DOI:10.3779/j.issn.1009-3419.2002.02.12
[Show abstract][Hide abstract] ABSTRACT: To study the effects of extraneous p53 antisense RNA on malignant growth and sensitivity to cisplatin of human lung cancer cell line.
801D cell line with p53 deletion and mutation at 248 codon was selected as a parent cell line. An 1.8 kb human p53 full length cDNA was inserted into a mammalian expression vector PEGFP to construct a p53 antisense RNA recombined plasmid PEGFP-p53(AS) and GFP gene at plasmid was a report gene to monitor extraneous gene expression. The extraneous gene was detected by PCR. The p53 mutation protein was examined by immunohitochemical stain of p53 monoclonal antibody. The inhibition growth efficacy of extraneous p53 in vitro was determined by clonogenic survival assay. Sensitivity of cells to cisplatin was examined with MTT assay. FCM analysis was performed to measure the effect of p53 antisense RNA on cell cycle.
Two cell lines, PEGFP-p53(AS)-801D and PEGFP-801D, were established after transfection of 801-D cells by lipofection and selection. Presence of extraneous p53 gene in PEGFP-p53(AS)-801D was proved by PCR and expression of extraneous p53 was estimated when green fluorescence in those cells was found out under the fluorescent microscopy. Mutated p53 protein in parent cell line 801D was positive and in PEGFP-p53(AS)-801D was negative with immunochemical stain. The inhibition rate of colony formation was 61% for PEGFP-p53(AS)-801D (P < 0.001). The sensitivity of PEGFP-p53(AS)-801D cells to cisplatin was increased. FCM analysis showed that the cell line was arrested at G1 phase.
p53 mutation at 248 code plays an important role on malignant growth and resistance to cisplatin of human lung cancer cell line 801D. Malignant growth of cells with p53 deletion and mutation at 248 codon can be inhibited by extraneous p53 antisense RNA, and simultaneously the sensitivity to cisplatin is also increased.
Zhongguo fei ai za zhi = Chinese journal of lung cancer 02/2002; 5(1):1-5. DOI:10.3779/j.issn.1009-3419.2002.01.01