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<p><b>BACKGROUND</b>As a new member of inhibitor of apoptosis protein(IAP) family,Livin,especially Livin α,is known to be involved in occurrence and development of lung cancer.Livin is an important mechanism of chemotherapy resistance of lung cancer cell.The aim of this study is to set up Livin isoform(α & β)-specific gene silencing system in SPC-A1 cells by gene transfection and RNA interference(RNAi),and to explore the different functions and value of the isoforms in enhancing chemosensitivity of SPC-A1 cells.</p><p><b>METHODS</b>Livinα+β,Livinα and Livinβ specific siRNA were expressed stably in SPC-A1 cells,respectively.MTT was performed to study sensitivity of the cells to chemotherapy drugs.In vivo experiment was performed to test sensitivity of mouse bearing tumor to cisplatin after gene silencing of Livin.</p><p><b>RESULTS</b>After silencing of Livinα+β,Livinα and Livinβ genes,sensitivity of SPC-A1 cells to many chemotherapy drugs(including cisplatin,carboplatin,cyclophosphamide and adriblastine) was markedly increased(P < 0.05).Among them,gene silencing of Livinα+β showed the strongest enhancement effect on chemosensitivity of SPC-A1 cells(P < 0.01).Animal experiment showed that tumor inhibition rate of pSilencer-Livinα+β,pSilencer-Livinα and pSilencer-Livinβ groups was 146.1%,130.7% and 110.5%,respectively.</p><p><b>CONCLUSIONS</b>The results suggest that Livin isoform,especially Livinα+β is hopeful to be a molecular target for increasing sensitivity of lung cancer cell to chemotherapy.Gene silencing may be a new means of gene therapy for non-small cell lung cancer.</p>
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<p><b>BACKGROUND</b>Smoking plays an important role in the development of carcinoma of human lung,but the mechanism is unknown.The objective of this study is to investigate the mutation in the coding region of mitochondrial DNA(mtDNA) in patients with squamous cell carcinoma of the lung and to explore its significance in carcinogenesis.</p><p><b>METHODS</b>White blood cells,pericarcinomatous tissues and cancer tissues were obtained from 15 cases of lung squamous cell carcinoma and mtDNA was extracted by one step method.The part of coding region fragments was amplified by PCR.Mutations were determined by DNA sequencing.</p><p><b>RESULTS</b>Fifteen pairs of matched pericarcinomatous tissues and cancer tissues were screened for mutation in coding regions,18 mutations were detected in the coding region of mtDNA in 11 patients,of which 10 cases were smokers.</p><p><b>CONCLUSIONS</b>The mutations of the coding region of mtDNA in squamous cell carcinoma of the lung may be correlative with smoking.</p>
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<p><b>BACKGROUND</b>It has been proven that mitochondrial DNA (mtDNA) noncoding region plays a very important role in process of transcription. The mutation in mtDNA noncoding region can influence the transcription and translation process of mtDNA. The aim of this study is to investigate the variation status in noncoding region of mtDNA in lung cancer cell lines, and discuss its meaning in carcinogenesis.</p><p><b>METHODS</b>The 7 lung cancer cell lines, A549, SPC-A-1, Calu-3, LTEP-a-2, QG-56, 95-D and NCI-H446, were cultured. Then the mtDNA of them was extracted with one-step method and variation status of noncoding region was analyzed, compared to the Cambridge sequence of mtDNA.</p><p><b>RESULTS</b>Different variations existed in the mtDNA noncoding regions of 7 lung cancer cell lines. The variation rate of LTEP-a-2 (1.82%) was the highest one, and A549 (0.21%) was the lowest one.</p><p><b>CONCLUSIONS</b>The mtDNA noncoding region sequencing is accomplished firstly in 7 lung cancer cell lines. It may provide a reference and background for related researches.</p>
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<p><b>BACKGROUND</b>Mitochondrial DNA (mtDNA) is the only hereditary substance besides nucleus, which is composed of a code region and a non-code D-loop region. The aim of this study is to investigate the hypervariable region II (HVRII) mutation of mtDNA in peripheral blood leucocyte, pericancerous tissues and cancer tissues of lung squamous cell carcinoma patients, and to explore its significance.</p><p><b>METHODS</b>White blood cells, pericancerous tissues and cancer tissues were obtained from 15 cases of lung squamous cell carcinoma patients and mtDNA were extracted by one step method. HVRII fragments were amplified by PCR. Mutations were determined by DNA sequencing and the mutations of HVRII were analysed.</p><p><b>RESULTS</b>In 15 lung squamous cell carcinoma patients, 14 patients showed mutation in HVRII(93.33%), 88 mutations were found totally. Eighty-seven mutations located in H-strand origin region, especially in the conserved sequence blocks and the mtTF1, 2 binding site (TFX and TFY).</p><p><b>CONCLUSIONS</b>The results suggest that the mutation frequency of HVRII in cancer tissues of lung squamous cell carcinoma patients is very high and it might play an important role in carcinogenesis of the lung.</p>
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<p><b>BACKGROUND</b>p16INK4a and p14ARF, encoded by gene INK4a/ARF located at chromosome 9p21, are cyclin dependent kinase (CDK) inhibitors. Both p16INK4a and p14ARF are cell cycle regulatory proteins and play an important role in Rb and p53 passways respectively. In this study, wild-type INK4a/ARF gene was transfected into human lung adenocarcinoma cell line A549, in which this gene site was lost, and the effects on the cell's biological behavior were investigated.</p><p><b>METHODS</b>The recombinant eukaryotic expression plasmids pcDNA3-p16INK4a and pcDNA3-p14ARF were transfected into A549 by cationic liposome method. By RT-PCR, immunocytochemistry and Western blot after G418 selection, A549 cells that could stably express p16INK4a and p14ARF were obtained. As a control, the parental cell and negative control cell with plasmid pcDNA3-LacZ were used. Inhibition of proliferation was measured by MTT assay. The cell growth curve was drawn according to cell counts. Cell cycle distribution was measured by flow cytometry (FCM), the apoptosis indexes were observed at the same time. The colony formation rate was counted by staining the cells with Coomassie brilliant blue.</p><p><b>RESULTS</b>The introduction of exogenous INK4a and ARF caused significantly growth inhibition of A549. By FCM, more percentage of A549-p16INK4a-p14ARF cells couldn't pass through the checkpoint G1. The percentage of A549-p16INK4a-p14ARF cells inhibited at G0/G1 was 59.9%, 50.3% for A549-vector and 51.2% for A549. The statistical differences were significant between A549-p16INK4a-p14ARF cell and A549-vector cell (P=0.025) and between A549-p16INK4a-p14ARF cell and A549 cell (P=0.043). The apoptosis index of A549-p16INK4a-p14ARF cell was 8.0% and 2.7% for both A549-vector and A549 cell (P < 0.01). The colony formation ability of A549-p16INK4a-p14ARF was weaker than that of A549-vector and A549, they were 63%, 87% and 85% respectively.</p><p><b>CONCLUSIONS</b>The wild-type INK4a/ARF gene can be co-introduced effectively into A549 cell by cationic liposome method. The reexpression of p16INK4a and p14ARF in A549 can inhibit the growth and enhance the apoptosis. This trial will be helpful in using gene therapy of lung cancer in the future.</p>
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<p><b>BACKGROUND</b>The relationship between mitochondrial DNA (mtDNA) mutation and tumor is the hot point in cancer research field by now. The existent methods for extracting mtDNA are time-consuming and complicated. In order to further research the relationship of the mutation of mtDNA and development of lung cancer, it is necessary to establish a rapid and simple method for extracting mtDNA from lung cancer tissue.</p><p><b>METHODS</b>Lung cancer tissues were lysed in base-denaturalization liquid and mtDNA was directly extracted from them by chloroform:isoamyl alcohol (24:1). The mtDNA was confirmed by PCR.</p><p><b>RESULTS</b>The extracted mtDNA samples were not combinated with protein and 1528bp PCR products of (mtDNA) were detected from 40 samples of analyzing patients. About (7.97±0.12)μg of mtDNA could be extracted from 1 gram of cancer tissue.</p><p><b>CONCLUSIONS</b>The method is simple, rapid and efficient to extract (mtDNA) from human lung cancer tissues and it could be used to handle small amount tissues or large number of samples in clinical and scientific research.</p>
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Objective To evaluate the therapeutic efficacy and side effects of cyclopnosphamide, adriamycin, cisplatin (CAP) or CAP combined with verapamil(VPL) in chemotherapy of patients with advanced lung adenocarcinoma. Methods A total of 56 patients(male: 27, female: 29, average age: 48 years old, age range: 32-78 years old) with stage Ⅲb/Ⅳ lung adenocarcinoma confirmed pathologically from April 1998 to December 2001 were divided into two groups: group A(30 patients), treated with cyclophosphamide (CTX, 600 mg?m -2 ?d -1 ) on days 1 and 8, adriamycin (ADM, 40 mg/m 2) on day 1, and cisplatin (CDDP, 30 mg?m -2 ?d -1 ) on days 1, 2 and 3 and group B(26 patients), treated with the same dose of CTX, ADM and CDDP and an additional oral VPL treatment (60 mg t.i.d . on days 1-7). Each of the 3 cycles was repeated every 21 days. Results The patients in group A had a lower response rate (26.7% vs 38.5%, P
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0.05).The major adverse effects were myelo-suppression,nausea,vomiting,phlebitis and alopecie.There were highest incidence of thrombocytopenia(51.1%) in GP regimen group compared with other groups,especially in the stage of Ⅲ? and Ⅳ?(totally accounting for 24.4%)(P
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Objective To explore the silence of B-cell specific Moloney murine leukaemia virus insertion site 1(Bmi-1)by RNA interference on the proliferation of human leukemia cell line K562 and its mechanisms.Methods Small interfering RNA(siRNA)targeting Bmi-1 gene was designed and double-stranded siRNA was chemically synthesized.After double-stranded siRNA was transfected into K562 cells with Lipofectamine 2000,the proliferation of K562 cells was detected by MTT colorimetry,cell cycle was determined by flow cytometry,and the expression of Bmi-1 and P16 were analyzed by Western blotting.Results The siRNA targeting human Bmi-1 gene effectively prolonged the double time of K562 cells,increased the percentage of cells at G1 phase,and the expression of Bmi-1 was significantly down-regulated but the expression of P16 was up-regulated.Conclusion The siRNA targeting human Bmi-1 gene inhibits the proliferation of K562 cells,and up-regulates the expression of P16 in the cells.
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Objective To observe the changes of retinal sensitive cellular ultrastructure of rats in critical period of visual development.Methods Ten normal healthy Wistar rats were chosen,eight of which were neonatal rats and two were mature rats.The eight neonatal rats were randomized into four groups(n=2 in each group) and respectively sacrificed on postborn day 0,15,20,25.The eyeballs of the neonatal rats and the mature rats were resected and the retinas were observed by electron microscope.Results The rat retinal sensitive cellular ultrastructure on postborn day 0 was immature and the cellular arrangement was not clear.The organelle of sensitive cell in critical period developed mature gradually and the arrangement of them was very clear.Conclusion The retinal sensitive cell of rats develops and matures gradually in critical period.
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Objective To investigate the relationship between hypervariable regionsⅠ(HVRⅠ) mutation of mitochondrial DNA (mtDNA) and lung squamous carcinoma and to explore its significance in carcinogenesis. Methods White blood cells and carcinoma tissues were obtained from 13 cases of lung squamous carcinoma patients and mtDNA were extracted by one step method. HVRⅠfragments were amplified by PCR. Mutations were determined by DNA sequencing. Results In 13 lung squamous carcinoma patients, 8 cases showed mutation in HVR Ⅰ, and 30 mutations were found in 25 different nucleotide sites, in which 17 were point mutations and 13 were insertions and deletions, including a 10-bp deletion in one patient. Conclusion These results suggest that the mutation rate of HVRⅠsequence in lung squamous carcinoma tissue was relatively high, and point mutation might play an important role in lung carcinogenesis.
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<p><b>BACKGROUND</b>To elucidate the pattern and mechanism of cisplatin-induced apoptosis and its role in tumor chemotherapy.</p><p><b>METHODS</b>Apoptosis induced by cisplatin in human lung adenocarcinoma cell line A549 was detected by cell morphology, agarose gel electrophoresis, DNA-end-labeling and flow cytometry analysis techniques.</p><p><b>RESULTS</b>Cisplatin-induced apoptosis of A549 cells persisted and augmented gradually from 12 to 72 hours after treated with 3 mg/L cisplatin. All of A549 cells treated respectively with 1, 3, 5 and 7 mg/L cisplatin showed apoptosis. Apoptotic effects increased in a time-dependent pattern and a concentration-dependent pattern. A549 cells were blocked in G1 phase after treated with cisplatin.</p><p><b>CONCLUSIONS</b>Induction of cell apoptosis may be an important mechanism of anti-tumor efficacy of cisplatin.</p>
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Objective To develop a new method to induce the gene transfection in high efficiency for eukaryotic cells in vitro. Methods Four kinds of p14ARF gene primarily deleted human carcinoma cell line including H460,A549,U251,and PC-3 were transfected with the human p14ARF expression vector (pCI-neo-p14ARF) by using the new nonliposomal transfection reagent Fugene 6. The efficiency of gene transfer was determined by screening the cells in G418. Results After 21 days' selection, G418-resistant clones were shown in all the transfected plate. PCR product of p14ARF gene was positive in all the G418-resistant clones. Cytotoxicity of Fugene 6 was detected. The cell proliferation activity was not affected when it was cultured in a high dose of Fugene 6. Conclusion These results demonstrate that Fugene 6 is a rapid, feasible, reproducible, and noncytotoxic gene transfection approach for eukaryotic expression vector in vitro.
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The major problem in lung cancer chemotherapy is the emergence of inherent and acquired drug resistance of the cancer cells. The cancer becomes resistant not only to the drugs used initially, but also to those to which it has not yet been exposed, This condition is known as multidrug resistance (MDR). MDR has been found to be related to three members of the ABC-superfamily of membrane transport proteins (transporters) and one member of the non-ABC-superfamily of transporter. The formers are P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP), and breast cancer resistance pro-tein (BCRP). The latter is lung resistance-related protein (LRP). These findings provided new molecular targets to predict drug resistance, and an atraumatic detection method by using Tc-99m methoxyisobutyl isonitrile ( 99m Tc-MIBI ) SPECT has been developed for predicting MDR in lung cancer. Based on these transporters, different strategies are tried attempting to reverse drug resistance in lung cancer. Encouraging results have been acquired, but further research is necessary.
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Objective To investigate the influence of co-expression state of p14 ARF and p16 INK4a protein on radiochemotherapy and length of survival period in patients with non-small cell lung cancer(NSCLC)after resection. Methods Thirty-five patients with negative p14 ARF and p16 INK4a co-expression and 20 patients with positive p14 ARF and p16 INK4a co-expression were enrolled for the study. The co-expression of the said proteins were previously determined by immunohistochemistry (S-P). Clinical pathological characteristics were compared between two groups, and the survival time and the results of radiochemotherapy of patients were respectively recorded and analysed. Results No significant differences were found in age, TNM stages, degree of differentiation, recurrence/metastasis and radiochemotherapy between two groups. However, there was significant differences in sex, smoking index, and pathological classification. It was found that 2-year, 3-year and total survival rate were significantly lower in patients with p14 ARF and p16 INK4a co-expression than those with positive co-expression (P
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Objective To investigate the effects of co-transfection of the plasmid containing the wildtype (wt) INK4a/ARF gene on the chemosensitivity of human lung adenocarcinoma cell line A549. Methods Recombinant eukaryotic expression vectors pcDNA3-p16 INK4a and pcDNA3-p14 ARF were co-transfected into A549 cells by cationic liposome, in which the site of both genes was lost. By RT-PCR, immunocytochemistry and Western blotting analysis, G418 positive clone was obtained. The parental cell and negative control cell with plasmid pcDNA3-LacZ were used as controls. The 50% inhibition concentration (IC 50 ) of five commonly-used chemotherapy drugs and the apoptosis index (AI) of IC 50 were analysed respectively. Results Compared with the parental cells and negative control cells, the influence of the five drugs on IC50 was different. The values of IC 50 of doxorubicin and cisplatin decreased significantly but that of taxol, topotecan and vinorelbine remained unchanged. The apoptosis index of doxorubicin and cisplatin was much higher than that of other three drugs. Conclusion The chemosensitivity of a part of anti-cancer drugs could be changed by transfection of exogenous INK4a/ARF gene into lung adenocarcinoma cell A549.
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Objective To investigate the differential expression of mitochondrial genome and apoptosis-related genes in human lung adenocarcinoma A549 cell line induced by cisplatin. Methods A549 cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS; Hyclone) in 150-mm dishes, and the test cells were exposed to 0.5?g/ml cisplatin continuously for 20h. Same volume PBS solution was added to culture of control cells instead of cisplatin. Human mitochondrial genome oligonucletide micoarray was used to detect the differential expression of 26 target genes. The apoptosis status was analyzed by the flow cytometry technique. Results Compared with the control cells, the expression levels of tRNA-Cys, tRNA-Asn, 12S rRNA and cytochrome oxidase subunit I were remarkably elevated in test cells. The expression levels of bax, NADH dehydrogenase subunit I, NADH dehydrogenase subunit Ⅱ and tRNA-Ser, tRNA-Asp, tRNA-Arg and tRNA-Ala also were elevated to some extent. Apoptosis rate of test cells was 8.5%?0.78%(n=5), which was remarkably higher than that of control cells 1.3%?0.56%(n=5,P
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Objective To study the curative effect of percutaneous radiofrequency ablation (PRFA) on liver tumors. Methods A total of 66 patients underwent PRFA under ultrasound guidance for the total frequencies of 146. AFP, B-US, and CT examinations were conducted before and after the operation. A follow-up for 6 months was carried out. Results PRFA was successfully performed on all patients. Serious complications and death were found in 2 cases. The follow-up revealed complete disappearance of tumor masses in 6 cases. The diameter of tumor reduced by 1/2 were found in 24 cases, and