Xuehui Yang

Cell Cycle Protein Cyclin Y Is Associated With Human Non-Small-Cell Lung Cancer Proliferation and Tumorigenesis

BACKGROUND The role of cell cycle protein cyclin Y (CCNY) in non-small-cell lung cancer (NSCLC) is not clear. Hence, the aim of this study was to explore the potential role of CCNY in lung cancer. PATIENTS AND METHODS Real-time quantitative polymerase chain reaction was used for detecting the expression of CCNY mRNA in 60 samples from patients with NSCLC. The functional role of CCNY in NSCLC cells was evaluated by small interfering RNA-mediated depletion of the protein followed by analysis of cell proliferation, anchorage-independent growth, and xenograft growth. RESULTS CCNY mRNA is overexpressed (N = 60) in samples from patients with NSCLC. Furthermore, CCNY mRNA expression positively correlated with histologic types (squamous cell carcinoma vs. adenocarcinomas; P = .048) and with the tumor size (size > 3 cm vs. size ≤ 3 cm; P = .010) in NSCLC. Functionally, CCNY depletion was shown to inhibit cell proliferation and anchorage-independent growth in lung cancer cells. Moreover, the proliferation effects were increased when CCNY was overexpressed in lung cancer cells. Finally, CCNY was shown to support H1299 and 95D xenograft growth in nude mice. CONCLUSION We reported for the first time (to the best of our knowledge) that CCNY was overexpressed in samples of NSCLC. CCNY mRNA expression associated with histologic types of NSCLC and promoted the malignant growth of lung cancer cell line in vivo and in vitro. Thus, these results validated the role of CCNY as a clinically relevant human oncoprotein and warrant further investigation of CCNY as a biomarker and a therapeutic target in NSCLC.

Clinical Significance and Diagnostic Value of Survivin Autoantibody in Non-small Cell Lung Cancer Patients

BACKGROUND AND OBJECTIVE Lung cancer is one of the most common cancers worldwide and causes more deaths per year than other cancers. Autoantibody was one hotspot in tumor marker research. But the clinical value of Survivin autoantibody is not clear in lung cancer patients at present. The aim of this study is to determine whether Survivin autoantibody could be used as a diagnostic factor of lung cancer and what the clinical value of Survivin autoantibody was in nonsmall cell lung cancer (NSCLC). METHODS Survivin cDNA sequence was obtained by RT-PCR and inserted into prokaryotic expression vector pET30a(+). Fusion protein of Survivin was expressed in E. coil BL21(DE3). SDS-PAGE and Western blot were used to confirm the fusion protein. The Survivin protein was purified by Ni-NTA agarose affinity chromatography. At last, indirect ELISA was used to detect Survivin autoantibody level in the serums of 215 samples from NSCLC patients and serums from 20 samples of nonmalignant lung disease patients as well as 89 samples of healthy persons were also detected as control. RESULTS The recombinant vector pET30a(+)/Survivin was contructed and the Survivin protein is successfully expressed in E.coil BL21(DE3) as inclusion body. Indirect ELISA was done to detect Survivin autoantibody in these serums using purified Survivin protein. It was shown that the positive rate of Survivin autoantibody was 19.5%, with a specificity of 88.9% in NSCLC patients. The expression of Survivin autoantibody has correlation with the volume of tumor and the metastasis of tumor in NSCLC patients (P < 0.05). In our research, positive detection rate of NSCLC was much improved by detecting Survivin autoantibody combined with CEA compared to other tumor markers combined with CEA. CONCLUSIONS In this study, purified fusion protein of Survivin was successfully obtained. Indirect ELISA for detecting Survivin autoantibody in serum of NSCLC patients using purified Survivin protein was established. Our results indicated that Survivin autoantibody as a latent value of tumor marker could be used for clinical diagnosis in NSCLC patients.

Overexpression of cyclin Y in non-small cell lung cancer is associated with cancer cell proliferation.

Cyclin Y (CCNY) is a key cell cycle regulator that acts as a growth factor sensor to integrate extracellular signals with the cell cycle machinery. The expression status of CCNY in lung cancer and its clinical significance remain unknown. The data indicates that CCNY may be deregulated in non-small cell lung cancer, where it may act to promote cell proliferation. These studies suggest that CCNY may be a candidate biomarker of NSCLC and a possible therapeutic target for lung cancer treatment.

Cyclooxygenase-2 is associated with malignant phenotypes in human lung cancer

The objective of the present study was to investigate whether cyclooxygenase-2 (COX-2) is associated with malignancy, and to investigate its molecular mechanisms in human lung cancer tumor malignancy. The present study used RNA interference (RNAi) methodology and celecoxib, a COX-2 inhibitor, to investigate the effect of COX-2 knockdown on the proliferation and invasion abilities of lung cancer cells and the molecular mechanisms involved. Human lung adenocarcinoma A549-si10 and LTEP-A2 cells transfected with a specific small interfering RNA (A549-si10 and LTEP-A2-si10, respectively) grew more slowly compared with parental cell lines and cells transfected with pU6. The colony formation of A549-si10 and LTEP-A2-si10 cells was also reduced. In addition, A549-si10 and LTEP-A2-si10 cells were characterized by decreased metastatic and invasive abilities. The proliferation and invasive potential of parental A549 and LTEP-A2 cells was inhibited following treatment with celecoxib. In vivo, a COX-2 knockdown resulted in a decrease of proliferation and reduction of vascular endothelial growth factor (VEGF), matrix metalloproteinase-2 (MMP-2) and endothelial growth factor receptor (EGFR) expression in A549 xenografts. In conclusion, the present study revealed that COX-2 plays a extremely important role in tumor growth, infiltration and metastasis via the regulation of VEGF, MMP-2 and EGRF expression. Therefore, COX-2 is a potential therapeutic target for lung cancer.

A truncated p53 in human lung cancer cells as a critical determinant of proliferation and invasiveness

As a transcription factor, p53 must accumulate in the nucleus to be effective. Signals related to nuclear localization are distributed mainly in the C-terminal of p53. So these nuclear location domains were reserved and the other part of the C-terminal was removed in this study. We investigated whether the truncated p53 (p53(DEL)) may affect proliferation and invasive potential of human lung cancer cells. H1299 and 801D cells expressing full-length p53 and the p53(DEL) were obtained by screening. Cell proliferation assay, cell apoptotic analysis, cell migration assay, and invasion assay were performed. Protein expression was examined by Western blotting. The data showed H1299-p53(DEL) and 801D-p53(DEL) cells grew more slowly than H1299-p53 and 801D-p53 cells, respectively. The colony formation of H1299-p53(DEL) and 801D-p53(DEL) cells reduced. The truncated p53 induced cell apoptosis. The expression levels of Bax and p53 upregulated modulator of apoptosis were increased in H1299-p53(DEL) and 801D-p53(DEL) cells. H1299-p53(DEL) and 801D-p53(DEL) cells were also characterized by decreased migration and invasion. The expression of the truncated p53 resulted in upregulation of E-cadherin and downregulation of Vimentin, Slug, Twist1, and zinc finger E-box-binding homeobox 1, which suggested the truncated p53 inhibited epithelial–mesenchymal transition occurrence. The above-mentioned characteristics were reverted by treatment of with pifithrin-a, a p53 inhibitor. These findings support the existence of a direct link between the p53(DEL), proliferation, epithelial–mesenchymal transition, and invasiveness in human lung cancer cells. So the p53(DEL) is a promising target for prevention and treatment of lung cancer.

[The Effects of Interfering COX-2 Gene Expression on Malignant Proliferation of Human Lung Adenocarcinoma A2 Cell in vitro.].

BACKGROUND COX-2 was highly expressed in many tumor tissues and was involved in the initiation and development of tumors. The RNAi technique is a method to inhibit gene expression economically, quickly, efficiently and specifically. This study used RNAi technique to explore the interfering effect of COX-2 gene expression and the influence on the malignant proliferation of A2 cells after quenching COX-2 in vitro . METHODS Three COX-2 siRNA expression vectors with human U6 promoter were constructed. The COX-2 siRNA vectors and the vacant vector (pEGFP) were transfected into A2 cells with lipofectamine respectively. The cell strains transfected were selected. The change of COX-2 expression levels was examined by Western blot and RT-PCR. The effects on the proliferation of A2 cells after silencing COX-2 were detected by cell growth curve and clonogenic assay in vitro . RESULTS The three siRNA and U6 promoter cloned into pEGFP plasmid were validated by PCR, restriction endonucleases identification, DNA sequencing and BLAST alignment. The cell strains transfected were coded as A2-3, A2-7, A2-10 and A2-p respectively. Green fluorescence was seen in A2-p cells and not in A2-3, A2-7 and A2-10 cells in 24 h, 48 h and 72 h after transfected. The results of RT-PCR and Western blot showed the three siRNA expression vectors acted effectively and the expression of COX-2 was inhibited in different extent. In contrast to A2 cells, COX-2 mRNA levels of A2-3, A2-7 and A2-10 cells were reduced 15.6%, 20.4% and 64.2% respectively, and COX-2 protein expressions of them were reduced 23.7%, 36.7% and 60.2% respectively. The results of cell growth curve and clonogenic assay showed the growth of A2-10 cell slowed and the clonal formation rate was reduced. However the growth of A2-3 and A2-7 cells had no obvious changes vs controls (A2 and A2-p). CONCLUSIONS Silencing COX-2 gene in vitro by RNAi technique can significantly inhibit the malignant proliferation of A2 cells.

A better experimental method to detect the sensitivity of cancer cells to anticancer drugs after adenovirus-mediated introduction of two kinds of p53 in vivo.

p53 plays an important role in drug responses by regulating cell cycle progression and inducing programmed cell death. The C-terminal of p53 self-regulates the protein negatively; however, whether it affects the sensitivity of cancer cells to anticancer drugs is unclear. In this study, two experimental methods were used to compare the sensitivity to anticancer drugs of human lung 801D cancer cells transfected with adenovirus bearing either full-length p53 or the deleted-C-terminal p53 in vivo. Adenovirus-mediated deliveries of full-length or deleted-C-terminal p53 were performed after development of tumors (the first method) or by infection into cells before xenotransplantation (the second method). The results showed that infection with the deleted-C-terminal p53 increased 801D cell sensitivity to anticancer drugs in the second, but not in the first method, as indicated by greater tumor-inhibition rates. In addition, compared with the first method, the second method resulted in viruses with more uniformly infected cells and the infection rates between groups were similar. This yielded smaller within-group variations and greater uniformity among transplanted tumors. The second method could circumvent the difficulties associated with intratumoral injection.

Effects of p53 gene on drug resistance in human lung cancer cell lines

BACKGROUND 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. METHODS 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. RESULTS 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. CONCLUSIONS 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.

Effects of p53 antisense RNA on malignant phenotype and sensitivity to cisplatin of human lung cancer cell line

BACKGROUND To study the effects of extraneous p53 antisense RNA on malignant growth and sensitivity to cisplatin of human lung cancer cell line. METHODS 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. RESULTS 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. CONCLUSIONS 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.

[The construction and expression of phage display library of anti human lung adenocarcinoma monoclonal antibody 5F-11].

BACKGROUND To construct and express a phage display library of anti human lung cancer monoclonal antibody 5F-11. METHODS 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. RESULTS 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. CONCLUSIONS 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.