Empirical studies about quercetin increasing chemosensitivity on human lung adenocarcinoma cell line A549

Objective： The present study was designed to investigate whether quercetin exerts increasing chemosensitivity on human lung adenocarcinoma cells when quercetin combined with cisplatin （DDP） and vincristine （VCR） in vitro respectively and its possible antitumor mechanism. To provide experimental proof for clinical combination application. Methods： Using intermittent administration of high dose VCR, human lung adenocarcinoma sensitive cell line （A549/S） was induced to VCR- resistant human lung adenocarcinoma cell line （A549NCR）. MTT assay was adapted for examing the 50% inhibition （IC50） value of DDP and VCR on A549/S and A549/VCR when quercetin combined with DDP and VCR respectively. Results： IC50 of DDP on A549/S and A549/VCR was 10.18 and 12.35 mg/L, and the IC50 of VCR on the two cell lines was 1.21 and 12.77 rag/L, respectively. The resistance fold of A549/VCR on VCR and DDP was 10.55 and 121, respectively. When quercetin at concentration of 50, 100 and 200 pmol/L in combination with DDP and VCR respectively, the IC50 of DDP and VCR on A549/S and A549/VCR were obvious decreased （P 〈 0.05 - P 〈 0.01）. Conclusion： The experiment results suggested that quercetin could increase the chemosensitivity and partly revise the resistance of A549NCR.

Protein Profile of Human Lung Squamous Carcinoma Cell Line NCI-H226

Objective To construct a database of human lung squamous carcinoma cell line NCI-H226 and to facilitate discovery of novel subtypes markers of lung cancer. Method Proteomic technique was used to analyze human lung squamous carcinoma cell line NCI-H226. The proteins of the NCI-H226 cells were separated by two-dimensional gel electrophoresis and identified by mass spectrometry. Results The results showed that a good reproducibility of the 2-D gel pattern was attained. The position deviation of matched spots among three 2-D gels was 1.95±0.53 mm in the isoelectric focusing direction, and 1.73±0.45 mm in the sodium dodecyl sulfate-polyacrylamide gel electrophoresis direction. One hundred and twenty-seven proteins, including enzymes, signal transduction proteins, structure proteins, transport proteins, etc. were characterized, of which, 29 identified proteins in NCI-H226 cells were reported for the first time to be involved in lung cancer carcinogenesis. Conclusion The information obtained from this study could provide some valuable clues for further study on the carcinogenetic mechanism of different types of lung cancer, and may help us to discover some potential subtype-specific biomarkers of lung cancer.

Up-Regulation of the Gap Junction Intercellular Communication by Tea Polyphenol in the Human Metastatie Lung Carcinoma Cell Line

Our previous study has proven that tea polyphenol has a role in lung neoplasms. The present communication was to investage the anti-proliferation effect of tea polyphenol on the PG cells, which was a high metastatic human lung carcinoma cell line, by 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-diphenytetrazoliumromide (MTT) cell viability assay, and to study the change of intracellular calcium concentration, connexin43 (Cx43) expression, gap junctional intercellular communication (GJIC) and cell cycle distribution after the tea polyphenol treatment by laser scanning confocal microscopy and flow cytometry. The results showed that 1) tea polyphenol could kill the PG cells in a dose-depent manner via inhibiting the PG cell proliferation and blocking the PG cell cycle progression staying in G0/G1 phase and not transfering in S and G2/M phases to reduce the PG cell proliferation index;2) the increases of intracellular calcium concentration, GJIC and Cx43 expression were related with the tea polyphenol doses. The data suggested that tea polyphenol could inhibit the growth of PG cells, which mechanism was associated with the up-regulation of GJIC.

The effect of antisense oligodeoxynucleotides targeting Aurora A kinase on cell proliferation and chemosensitivity to paclitaxel in human lung cancer cell line A549

Objective： Aurora A kinase representing a family of evolutionadly conserved mitotic serine/threonine kinases has been found elevated in human lung adenocarcinoma cell line A549. It is suggested that the overexpression of Aurora A contributes to the carcinogenesis, chromosomal instability （CIN）, and de-differentiation of lung cancers. To address its possibility as a therapeutic target for lung cancer, we employed the antisense oligodeoxynucleotide （ASODN） technique to inhibit Aurora A expression and investigate its effects on tumor growth and cell cycle of A549, as well as the chemosensitivity to paclitaxel. Methods： Aurora AASODN was synthesized and transfected into A549 cells by lipofectAMINE 2000. Aurora A mRNA and protein expression were examined by reverse transcription-polymerase chain reaction （RT-PCR） and Western blot respectively. Cell cycle distribution was observed by flow cytometer. MTT assay was used to evaluate cell inhibition ratio before and after transfection. Results： The proliferation of the A549 cells was inhibited by Aurora A ASODN dose and time dependently. It was also observed that the IC50 of A549 cells after 48 hours＇ treatment of ASODN was about 300 nmol/L and under such circumstances, the Aurora A mRNA and protein expression significantly decreased （P 〈 0.05）, along with the induction of accumulation of cells in S phase and the G2-M transition. Furthermore, cell inhibition ratio of the combination of Aurora AASODN and paclitaxel was higher significantly than paclitaxel （P 〈 0.05） or Aurora AASODN alone （P 〈 0.05）. Conclusion： Inhibition of Aurora A expression can result in the suppression of cell growth and chemosensitizing activity to paclitaxel in human lung cancer cell line A549.

Reversal effect of recombinant human Endostatin on cisplatin resistance in A549/DDP human lung adenocarcinoma cells in vitro

Objective： Recombinant human Endostatin （rh-Endostatin, YH-16） can reverse cisplatin resistance in A549/DDP cells. However, the possible effect of rh-Endostatin in reversing DDP-resistance in A549/DDP cells and the mechanism are needed to be investigated. Methods： Lung adenocarcinoma cell line A549 and its DDP-resistant cell line A549/DDP were treated with DDP and/or recombinant human Endostatin. Difference in drug resistance was analyzed between different regi- mens and between different cell lines after a 72 h-treatment in vitro. And below the non-cytotoxic concentration of rh-End- ostatin, the possibility of rh-Endostatin in reversing DDP-resistance in A549/DDP was evaluated. The resistance protein which was detected in the study included P glycoprotein （P-gp） and topoisomerase II （Topo-II）. Results： Rh-Endostatin below 400 IJg/mL showed no cytotoxicity in either A549 or A549/DDP after 72 h-treatment with it. The inhibited concentration of 50% （IC50） observed for DDP was （0.79 _＋ 0.05） IJg/mL in A549 and （13.2 ＋ 1.1） in A549/DDP respectively. IC50 was reduced to 2.57 ＋ 0.05 #g/mL in A549/DDP treated by rh-Endostatin below the non-cytotoxic concentrations in combination with DDP, with a reversal fold （RF） of 5.14 and a relative reversal rate of 85.6%. Apoptotic rates were 2.01%, 13.47% and 29.26% re- spectively for cells treated with rh-Endostain, DDP, and the combination. The rate of the A549/DDP control group was 0.99%. The expression level of P-gp or Topo-II was higher in A549/DDP cells than in A549 cells. Rh-Endostatin may partially reverse DDP-resistance in A549/DDP cells in vitro, with a probable mechanism related to lowering expression of P-gp and Topo-II. Conclusien： Rh-Endostatin of non-cytotoxic dose partially reversed cisptatin resistance in cisplatin-resistant human lung adenocarcinoma cell line A549/DDP. Rh-Endostatin reversed the resistance of A549/DDP cells to DDP, which may be related to decreased protein expression of P-gp and Topo-II in A549/DDP cells.

Effects of paclitaxel on cell proliferation and apoptosis and its mechanism in human lung adenocarcinoma A549 cells

Objective： To investigate the effect of paclitaxel on cell proliferation and apoptosis of human lung adenocarcinoma A549 cells line and its mechanism in vitro. Methods ： Cell growth inhibition of paclitaxel on A549 cells was analyzed by MTT assay. Cell apoptosis was detected by DNA cytofluorometry, Hoechst33258 staining when treated with paclitaxel for 48 hours. Meanwhile, Cell cycle and apoptotic rate were analyzed by flow cytometry. The protein expressions of Bax and Bcl-2 were studied by Western Blot. Results： Paclitaxel inhibited the proliferation of A549 cells in a time-and dose-dependant manner. Hoechst33258 staining indicated that apoptosis was induced by paclitaxel. After treated for 48 hours, cell apoptosis rates of 25 nmo1/L, 50 nmol/L and 100 nmol/L paclitaxel groups were 11.52 ± 1.94% ,17.73 ±2.53%, and 29.32 ±5.51% respectively, which were significantly higher than those of control group 5.88 ±1.07%（all P 〈 0.01 ）, and apoptosis rate increased in dose-dependant manner. Meanwhile, G2/M stage cell percentage of 25 nmol/L, 50 nmol/L and 100 nmol/L paclitaxel groups were 42.52 ± 6.25%, 40.46 ± 5.81%, and 35.34 ±6.17% respectively,which were significantly higher than that of control group 22.32 ± 3.30%（all P 〈 0.01 ）; Western blot showed that paclitaxel increased the expression of Bax and decreased the expression of Bcl-2 in dose-dependant manner. Conclusion： Paclitaxel can inhibit A549 cell proliferation in a time-and dose-dependant manner. Its mechanism may be related to arresting cell cycle in G2/M stage and induce cell apoptosis by up-modulating Bax expression and down-modulating Bcl-2 expression.

INFLUENCE OF IMMUNE STATUS OF THE IMMUNE DEFICIENT MICE ON THE METASTATIC PHENOTYPES OF THE HETEROGENEOUS CLONAL SUBLINES OF HUMAN LUNG GIANT CELL CARCINOMA

By using cell cloning technique, 4 sublines (A,C,D,E) were isolated from a cell line of human lung giant cell carcinoma (PLA-801). After subcutaneous inoculation in T-cell deficient BALB/c nude mice, the incidence of tumor growth and spontaneous metastasis were the highest in subline D, moderate in sublines A and E, and lowest in subline C. Tumor cells of subline C also showed similar low tumorigenicity in another T-cell deficient 615/ PB1 nude mice.However, in 615/PB1 beige nude mice with con-genitally combined immune-deficiency in both T and NK cell activity, tumor cells of the rarely metastatic subline C do produce significantly high frequency of tumor growth and spontaneous metastasis.Morphological studies (light microscope, electron microscope and immunohistochemistry) showed rich microfilaments and Vimentin positive in the cytoplasm of metastatic tumor cells. This may imply a possibility that tumor cells differentiate towards the direction favourable to spreading and metastasis.

Antineoplastic effects of deoxyelephantopin,a sesquiterpene lactone from Elephantopusscaber, on lung adenocarcinoma (A549) cells

OBJECTIVE： Deoxyelephantopin, a sesquiterpene lactone from Elephantopus scaber, showed inhibition of the growth of various tumor cells in vitro. In the present study, we investigated the cytotoxicity and apoptosis-inducing capacity of deoxyelephantopin on lung adenocarcinoma （A549） cells. METHODS： The cytotoxic effect of deoxyelephantopin on A549 cells and normal lymphocytes was evaluated using 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide （MTT） assay and 50% inhibitory concentration （IC50） value was determined. The self-renewal and proliferating potential of A549 cells after treatment with deoxyelephantopin were examined by colony formation assay. Cellular morphology of deoxyelephantopin-treated cells was observed using phase- contrast microscopy. The induction of apoptosis was evaluated using acddine orange and ethidium bromide staining, Hoechst 33342 staining, terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end-labeling （TUNEL） assay, DNA fragmentation analysis and Annexin V-fluorescein isothiocyanate staining by flow cytometry. Activation of caspases was detected using fluorogenic substrate specific to caspases 2, 3, 8 and 9 and flow cytometric analysis. The total cellular DNA content and expression of cleaved poly （ADP-ribose） polymerase was also analyzed. RESULTS： Deoxyelephantopin exhibited cytotoxicity to A549 cells （IC50 = 12.287 μg/mL）, however, there was no toxicity towards normal human lymphocytes. Deoxyelephantopin suppressed the colony-forming ability of A549 cells in a dose-dependent manner. Acridine orange, ethidium bromide and Hoechst 33342 staining showed cell shrinkage, chromosomal condensation and nuclear fragmentation, indicating induction of apoptosis. Deoxyelephantopin increased apoptosis of A549 cells, as evidenced by more TUNEL-positive cells. DNA fragmentation and Annexin V staining revealed late-stage apoptotic cell population. Deoxyelephantopin inhibited A549 cell growth by cell cycle arrest at G2/M phase and induced apoptosis through both extrinsic and intrinsic pathways. CONCLUSION： These results suggest that deoxyelephantopin has great potential as a new chemotherapeutic agent to be developed further for the treatment of lung cancer.

REVERSION OF MALIGNANT PHENOTYPES OF HUMAN LUNG SQUAMOUS CARCINOMA CELLS BY ORNITHINE DECARBOXYLASE ANTISENSE RNA

Abnormally elevated activity of ornithine decarboxylase (ODC), and subsequent polyamine accumulation are intimately associated with the genesis.development and metastasis of cancer. In the present study, to control the growth of tumor cells, ODC antisense RNA was used to transfect human lung squamous carcinoma cell line LTEP-78. Compared with the parental cells, growth of the antisense transfected LTEP-78 cells arrested in G0/Gl phase and colony formation in soft agarose and tumorigenicity in nude mice were significantly reduced. Nucleic acid hybridization demonstrated that the transfectants expressed a high level of ODC antisense RNA and a significantly reduced level of endogenous ODC mRNA.The results suggest that the reversion of malignant phenotypes of human lung squamous carcinoma cells transfected with ODC antisense RNA is associated with the inhibition of polyamine biosynthesis.

EXPRESSION AND REVERSION OF DRUG RESISTANCE-AND APOPTOSIS-RELATED GENES OF A DDP-RESISTANT LUNG ADENOCARCINOMA CELL LINE

Objective: To investigate the co-expression of drug resistance- and apoptosis-related genes of cisplatin (CDDP)-selected lung adenocarcinoma cell line A 549 DDP for compared to the parental cell line A549, and reverse of drug resistance by antisense s-oligodeoxynucleotides (S-ODNs) of differentially expressed genes. Methods: Sense and antisense S-ODN were transferred into A 549 DDP cells by lipofectin. The expression of drug resistance and apoptosis related genes was examined by RT-PCR, immunocytochemistry and flow cytometry, respectively. Apoptostic cells were identified by DNA electrophoresis and terminal deoxynucleotidyl transferase (TdT)-mediated biotin dUTP nick end-labeling(TUNEL). Drug resistance of tumor cells was detected by a cell viability (MTT) assay. Results: The expression of bcl-2 was positive and that of multidrug resistance-associated protein (MRP) at mRNA and protein level was increased in A 549 DDP compared to A549 cells. MDR1, c-myc and topoisomeras II (TOPO II) were similarly co-expressed in two cell lines. Both cell lines were negative for c-erbB-2 expression. In A 549 DDP cells, the expression of bcl-2 and MRP was significantly inhibited by their respective antisense S-ODNs. Antisense S-ODNs could also decrease significantly drug resistance of A 549 DDP cells to CDDP by promoting cell apoptosis. Conclusion: Both intrinsic and acquired drug resistance were involved in co-expression of multiple MDR-related genes in lung adenocarcinoma. Cooperation of bcl-2 and MRP genes appeared to play an important action to confer the resistance of A 549 DDP cells to CDDP. Their antisense S-ODNs are responsible for the decrease of drug resistance of this cell line by promoting apoptosis.

Quantitative analysis of butyric acid-induced nuclear ultrastructural alterations in cells of human lung giant cell carcinoma in vitro

The effects of butyric acid(BA)on the nuclear ultrastructure of humanlung giant cell carcinoma(Strain PLA-801 D)were observed with digital imageprocessing.It was found that the length of the nuclear circumference of the tu-mor cells incubated with 2mmol of BA was approximately equal to that of thecontrol whereas the nuclear area was increased by 1.4times,which implies thatthe nuclear profile tends to become more regular after BA treatment.In addition,the optical density of the nuclei of the experimental group decreased significantlyas compared with that of the control,which indicates that the chromatin in thenuclei was decreased by BA.It was concluded on the basis of the findings thatBA may have a biological effect of reverse-transformation on the malignant cells.

Alterations and Its Mechanisms of Wnt Signal Pathway in Human High-matastatatic Large Cell Lung Cancer Cell Line L9981 by Transfecting with Nm23-H1 Gene

Backgroud and Objective Tumor metastasis is not only the malignant marker and characteristics of lung cancer, but also the main cause of failure to cure and lose their life of the

Extraction of Phenolic Compounds from Olive Leaf Extracts and Their Effect on Proliferation of Human Carcinoma Cell Lines

The aim of this work was to evaluate the effect of different olive leaf extracts (OLE) from different leaf growing stages on human carcinoma cell lines. OLE were tested in human carcinoma cell lines in vitro and cells were plated in 96-microtiter culture plates for each OLE concentration. Fresh (F) and freeze-dried (FD) leaves exhibited phenolic compounds in the range of 2.09 ± 0.10 to 8.44 ± 0.64 and 7.72 ± 0.56 to 24.65 ± 1.9 mg gallic acid equivalents/g leaves, respectively. OLE from several Portuguese olive tree cultivars were found to inhibit the growth of human carcinoma cell lines in a range of 2.09 - 8.44 μg phenolic compound/well (209 - 844 μg/ml) and 0.07 - 2.40 μg phenolic compounds/well (7 - 240 μg/ml) for fresh and freeze-dried leaves, respectively. Young (Y) leaves have revealed the highest cell growth inhibition ranging from about 95% for Cobran?osa, followed by 90% for Cobran?osa, 90% for Arbequina and 75% for Arbequina for cell lines A549, HeLa, A431 and OE21, respectively. The lowest cell growth inhibition (35%) was observed for Galega (Y) leaf extract on cell line A549. However, FD samples exhibited a distinctive pattern since cell growth inhibition was highest at highest extract dilution tested, for A431 (Galega Y) followed by A549 (Cobran?osa Y) with cell inhibition of 75% and 70%, respectively. The data presented in this work strongly suggest that OLEs inhibit the growth of human carcinoma cell lines.

Effect of antisense human telomerase RNA on malignant behaviors of gastric carcinoma cell line SGC-7901

Objective:To studytheeffectsof antisensehumantelomeraseRNA(ahTR)transfectionon themalignantbeha-viorsof gastriccarcinomacelllineSGC-7901anditspotentialroleingenetherapyfortumor.Methods:AnantisensehTR eukaryoticexpressionvectorcontainingthesequenceof templateregionof telomererepeatswas transfectedintogastric carcinomacelllineSGC-7901withliposomeDOTAP.Theexpressionsof hTRRNAandantisensehTRRNAwereob-servedwithRT-PCR,telomeraseactivitywithPCR-ELISA.Telomerelengthwas measuredwithSouthernblot.Cellmor-phologyandcellularproliferationcapacitywerestudiedwithMTTassay.Cellcycledistributionandapoptoticstatewere observedwithflowcytometry.Efficiencyof cloneformationin softagarandtumorigencityin nudemicewereexamined andevaluatedinahTR-transfected7901cells,andplasmidpCL-neotransfected7901cellsandparental7901cellsserved as control.Results:AnantisensehTReukaryoticexpressionvectorwastransfectedinto7901cellssuccessfully.Thetelom-eraseactivityin ahTR-transfected7901cellswas decreasedfrom100%to about25%,andtelomerelengthin thecells shortenedfrom4.08kb to3.35kb at60populationdoublings(PDs).Comparedwithparental7901andpCL-neotransfect-ed7901cells,ahTR-transfected7901cellsdisplayedsomemorphologicalchanges,includingdecreasedcellatypiaandnu-cleus/cytoplasmratiounderlightmicroscope.Furthermore,ahTR-transfected7901cellsdisplayedgrowthinhibition,de-creasedinvasivecapacityin Borden’schamberinvasivemodel,increasedG 0 /G 1 phaserateandapoptoticrate,andrestored contactinhibitionanddensityinhibition.Surprisingly,ahTR-transfected7901cellslosttheircapacityof cloneformationin softagarandcarcinogensisinnudemice.Conclusion:AntisensehTRtransfectioncaninduce7901celldifferentiationand reverseitsmalignantphenotype.Thisstudyprovidesan excitingapproachfor cancertherapythroughtheinhibitionof telomeraseactivitywithantisensegeneandothertelomeraseinhibitors.

CONDITIONED MEDIUM OF HUMAN NASOPHARYNGEAL CARCINOMA EPITHELIOID CELL LINE CNE_(1) CONTAINED THE ACTIVITIES OF TRANSFORMED GROWTH-INHIBITING FACTORS

The hormone defined serum free conditioned medium (SFCM) of human nasopharyngeal carcinoma epithelioid cell line (CNE1) was assayed by both the 3H-thymidine incorporation test and the soft agar test. It was found that the SFCM stimulated the growth of long-term serum-free cultured CNE4 cells in ac-cordence with the fact that the growth rate of long-term serum-free cultured CNE1 cells was directly proportional to the plating density. Alternatively 5% SFCM inhibited the growth of short-term serum-free cultured CNE4 cells by 51% in which the indicator cell remained the responsiveness state of growing in the serum-supplemented medium to the effector of interest. Furthermore, SFCM resulted in the inhibition of anchorage-independent growth of CNE4 cells and A431 cells. Also in soft agar test. SFCM reduced the colony formation of NRK(?),9F cells in the presence of EGF or EGF plus TGF-β. These finding suggested that CNE4 secreted autocrine growth stimulating factor(s) and growth inhibiting factor(s) in the serum-free medium, the latter strongly reverse malignant phenotypes of CNE4 and A431 cells in serum-supplemented surrounding.

Study of Pravastatin on Intervention of the Apoptosis in Human Lung Adenocarcinoma A549

Lung cancer is one of the serious threats to human health and life of malignant diseases, on a global scale; it has become one of the major lung cancer deaths. Due to the growth of the tumor and the main reason is that apoptosis is inhibited, therefore, it can induce apoptosis in lung cancer cells that is an important measure for the treatment of lung cancer, which is one of the effective means to reduce lung cancer mortality. In this paper, A549 human lung adenocarcinoma cell line, for example, has the use of chemical genetics of these emerging technological platforms, research pravastatin on apoptosis in human lung adenocarcinoma A549 intervention, while providing a theoretical basis for the development of new lung cancer therapy.

The Difference of the Copy Number Variation and Loss of Heterozygosity of Human Lung Large Cell Cancer Cell Line with Different Metastatic Potential

Background and Objective It has been proven that copy number gain/or loss (copy number variation CNV) in uences gene expression and result in phenotypic variation by

Effects of Methylseleninic Acid on the Proliferation and Cell Cycle in Human High Metastatic Large Cell Lung Cancer Cell Line L9981 and Its Molecular Mechanism

Background and Objective Lung cancer is the rst killer of human being in the whole world. Recently, although many treatment strategies have been developed, the anti-cancer effects

The Inhibiting Effects and It's Molecular Mecanisms of the Fungi of Huai Er's on Human Large Cell Lung Cancer Cell Line L9981

Background and Objective Lung cancer, which threatens human’s health and life, is the malignant tumor with the most rapid increase of morbidity. Although recent years the basic