The Effects of Nimesulide Combined with Cisplatin on Lung Cancer

To study the effects of cyclooxygenase 2 selective inhibitor Nimesulide (NIM) combined with Cisplatin (DDP) on human lung cancer and the possible mechanisms, the proliferation and ap-optosis of human lung cancer cell line A549 were evaluated by MTT reduction assay and flow cy-tometry respectively. The inhibitory effect on neoplasia in vivo was tested on nude mice subcutane-ously implanted tumor. Our results showed that NIM and DDP could inhibit A549 cell proliferation in a concentration-dependent pattern; this action was enhanced when NIM (25 /μmol/L) was given in combination with DDP and they worked in a synergistic or additive pattern as DDP concentration≥1 μg/ml. NIM and DDP could induce A549 cells apoptosis and the action was augmented when used in combination (P＜0.01). NIM and DDP could inhibit the growth of subcutaneously implan-ted tumors on nude mice (P＜0.05,P＜0.01) and the inhibitory rate of NIM combined with DDP was significantly higher than that of NIM or DDP group (P＜0.01, P＜0.01). It is concluded that combined use of NIM and DDP has significant synergistic antitumor effects on lung cancer cell line A549 and in animals in vivo. The synergy may be achieved by growth inhibition and apoptosis in-duction.     

Effect of lumiracoxib on proliferation and apoptosis of human nonsmall cell lung cancer cells in vitro

Background Lumiracoxib is a highly selective cyclooxygenase-2 （COX-2） inhibitor with antiinflammatory, analgesic and antipyretic activities comparable with class specific drugs, but with much improved gastrointestinal safety. No studies have examined lumiracoxib for antitumorigenic activity on human nonsmall cell lung cancer cell lines in vitro or its possible molecular mechanisms. Methods The antiproliferative effect of lumiracoxib alone or combined with docetaxol on A549 and NCI-H460 lines was assessed by 3-（4, 5-dimethylthiazol-2-yl）-2, 5-diphenyltetrazolium bromide （MTT） assay. Drug-drug interactions were analyzed using the coefficient of drug interaction （CDI） to characterize the interactions as synergism, additivity or antagonism. Morphological changes were observed by acridine orange fluorescent staining. Extent of apoptosis was determined by flow cytometry. Results Lumiracoxib （15-240 pmol/L） has an inhibitory effect on the proliferation of A549 and NCI-H460 cell lines in concentration- and time-dependent manners with the ICso values of 2597 pmol/L and 833 pmol/L, respectively. The synergistic effect was prominent when lumiracoxib （15-240 pmoVL） was combined with docetaxol （0.2-2 pmol/L） （CDI 〈1）. Fluorescent staining showed that lumiracoxib could induce apoptosis in A549 and NCI-H460 cells. Lumiracoxib treatment also caused an increase of the sub-G1 fraction in each cell line and resulted in an increase of G0/Gl-phase cells and a decrease of S-phase cells. Conclusions Lumiracoxib had antiproliferative effect on the human nonsmall cell lung cancer cell lines A549 and NCI-H460 and had a significant synergy with docetaxol, which may be related to apoptotic induction and cell cycle arrest.     

Inhibitory activity of nuclear factor-κB potentiates cisplatin-induced apoptosis in A549 cells

Whether inhibiting the activity of nuclear factor （NF）-κB potentiates cisplatin-induced apoptosis in non-small cell lung cell line A549 cells was investigated. The recombinant plasmid pcDNA3.1（＋）/IκBα expressing IκBα was constructed. The in vitro cultured A549 cells were transfected with pcDNA3.1 （＋）/IκBα alone, or pcDNA3.1（＋）/IκBα combined with cisplatin. The mitochondrial membrane potential （△ψm） was determined by rhodamine 123, the activity of caspase-3 was tested by colorimetric assay, and cell apoptosis was detected by flow cytometry with the annexin V/propidium iodide assay. The results showed that the activity of NF-κB in A549 cells was inhibited by transfecting pcDNA3.1（＋）/IκBα. Transfection of pcDNA3.1（＋）/IκBα alone did not promote apoptosis. Treatment of cisplatin alone had a little effect on cell apoptosis. Transfection of pcDNA3.1（＋）/IκBα combined with cisplatin treatment significantly induced apoptosis of A549 ceils. It was concluded that inhibiting the activity of NF-κB potentiated cisplatin-induced apoptosis of A549 cells.     

Inhibitory Activity of Nuclear Factor-κB Potentiates Cisplatin-induced Apoptosis in A549 Cells

Whether inhibiting the activity of nuclear factor(NF)-κB potentiates cisplatin-induced apoptosis in non-small cell lung cell line A549 cells was investigated.The recombinant plasmid pcDNA3.1( )/IκBα expressing IκBα was constructed.The in vitro cultured A549 cells were transfected with pcDNA3.1( )/IκBα alone,or pcDNA3.1( )/IκBα combined with cisplatin.The mito-chondrial membrane potential(?ψm) was determined by rhodamine 123,the activity of caspase-3 was tested by colorimetric assay,and cell apoptosis was detected by flow cytometry with the annexin Ⅴ/propidium iodide assay.The results showed that the activity of NF-κΒ in A549 cells was inhibited by transfecting pcDNA3.1( )/IκΒα.Transfection of pcDNA3.1( )/IκΒα alone did not promote apoptosis.Treatment of cisplatin alone had a little effect on cell apoptosis.Transfection of pcDNA3.1( )/IκΒα combined with cisplatin treatment significantly induced apoptosis of A549 cells.It was concluded that inhibiting the activity of NF-κB potentiated cisplatin-induced apoptosis of A549 cells.     

Effects of inotodiol extracts from inonotus obliquus on proliferation cycle and apoptotic gene of human lung adenocarcinoma cell line A549

Objective:To observe the proliferation inhibition,apoptosis,and cell proliferation cycle of human lung carcinoma cell line A549 treated with Inotodiol extracts from Inonotus obliquus and explore the possibility of Inotodiol extracts from Inonotus obliquus as a new tumor chemopreventive drug.Methods:Human lung cancer cell line A549 was treated with different concentrations of Inotodiol,the effects of Inotodiol on cell apoptosis,the expression of Ki-67,Bcl-2,Bax,and p53 and cell cycle were detected by TUNEL assay,immunohistochemistry, and flow cytometry assay respectively.Results:Inotodiol extracts had antiproliferation effect on human lung carcinoma cell line A549.The expression of Ki-67 decreased with the increase of Inotodiol concentration and exposure time(P<0.05),in a dose-dependent and time-dependent manner.The typical characteristics of the apoptosis of A549 cells treated with Inotodiol were observed,and the apoptotic rate of A549 cell at 48 h was the highest by TUNEL assay.Inotodiol arrested A549 cells in the S phase,and apoptotic peak was observed by flow cytometry.Immunocytochemistry indicated that the expression of Bcl-2 protein decreased,while the expression of p53 and Bax proteins increased in A549 cells treated with Inotodiol,compared with the control cells(P<0.05).Conclusion:Inotodiol can inhibit proliferation and induce the apoptosis of A549 cells,and its molecular mechanism may be associated with the up-regulating expression of p53 and bax proteins and down-regulating expression of Bcl-2 protein,which arrested A549 cells in S phase.     

Inhibitory Activity of Nuclear Factor-κB Potentiates Cisplatin-induced Apoptosis in A549 Cells

Whether inhibiting the activity of nuclear factor (NF)-κB potentiates cisplatin-induced apoptosis in non-small cell lung cell line A549 cells was investigated. The recombinant plasmid pcDNA3.1( )/IκBα expressing IκBα was constructed. The in vitro cultured A549 cells were trans-fected with pcDNA3.1( )/IκBα alone, or pcDNA3.1( )/IκBα combined with cisplatin. The mitochondrial membrane potential (△ψm) was determined by rhodamine 123, the activity of caspase-3 was tested by colorimetric assay, and cell apoptosis was detected by flow cytometry with the annexin V/propidium iodide assay. The results showed that the activity of NF-κB in A549 cells was inhibited by transfecting pcDNA3.1( )/IκBα. Transfection of pcDNA3.1( )/IκBα alone did not promote apoptosis. Treatment of cisplatin alone had a little effect on cell apoptosis. Transfection of pcDNA3.1( )/IκBα combined with cisplatin treatment significantly induced apoptosis of A549 cells. It was concluded that inhibiting the activity of NF-κB potentiated cisplatin-induced apoptosis of A549 cells.     

Variation of Protein''''s Expression Correlated to the Drug Resistance after Sequential Anti-cancer Treatment in Human Lung Cancer Cell Line

Multi-drug resistance is one of the leading causes for fai- lure to treat patients with cancer. This study is to explore the expression of the proteins correlated with chemoresistance in a human lung cancer cell line (LPET-a-1) repeatedly treated by anti-cancer drugs. LPET-a-1 was cultivated in the plates with 96 wells, where anti-cancer drugs-doxorubicin, etoposide, and cisplatin were diluted in different concentrations. MTT assay was used to an- alyze the effect of these drugs on cell gr…     

EXPRESSION DIFFERENCES OF VASCULAR GROWTH FACTORS IN HUMAN LUNG ADENOCARCINOMA CELL LINE A549 AND CISPLATIN-RESISTANT HUMAN LUNG ADENOCARCINOMA CELL LINE A_(549)~(DDP)

Objective To elucidate the expression differences of vascular growth factors in human lungadenocarcinoma cell line A549 and cisplatin-resistant human lung adenocarcinoma cell line A549DDP . Methods RT-PCR and immunohistochemistry was used to detect the mRNA and protein expressions of vascular endothelial growth factor ( VEGF) and basic fibroblast growth factor ( bFGF) in A549 and A549DDP . Results VEGF and bFGF mRNA were expressed in A549 and in A549DDP VEGF and bFGF mRNA expression levels in A549DDP were significant higher than those in A549 (P< 0 .025 ). VEGF and bFGF protein expressions were all strong positive in A549 and A549DDP. Conclusion There are certain differences between VEGF and bFGF expressions in A549 and A549DDP . Drug-resistance of lung cancer is associated with those above genes over-expressions. Over-expression of vascular growth factors are related to drug resistance of lung cancer.     

Co-transfection of MRP and bcl-2 antisense S-oligodeoxynucleotides reduces drug resistance in cisplatin-resistant lung cancer cells

Objective To detect the influence of antisense s-oligodeoxynucleotides](S-ODNs) of bd-2 and multidrug resistamce-associated protein (MRP) genes multidrug resistance-associated protein gene and bcl-2 antisense S-oligodeoxynucleotides on cisplatin-resistant lung adenocarcinoma cell line A(549)(DDP)which overexpresses both bcl-2 and MRP.Methods A(549)(DDP)cells were treated with sense and antisense S-ODN mediated by lipofection. Expression of MRP and bcl-2 mRNA and protein in the treated cells was measured by RT-PCR and flow cytometry (FCM), respectively. Apoptosis was identified by DNA electrophoresis and terminal deoxynucleotidyl transferase (TdT)-mediated biotin dUTP nick end-labeling(TUNEL). The degree of drug resistance of the treated cells was detected by a cell viability 3’-［4,5-dimethylthiazol-2-yl］-2,5-diphenyl-tefrazolium bromide thiazolylblue (MTT) assay. Results Expression of bcl-2 and MRP significantly decreased in the cells treated with bcl-2 or/and MRP antisense S-ODN for 48h as compared to the cells untreated and sense-treated (P&lt;0.05). Resistance to cisplatin in the cells treated with bcl-2 or/and MRP antisense S-ODN decreased by 60.6% (6.5 times), 56.4% (7.2 times) and 71.0% (4.8 times), respectively, which paralleled the decrease of bcl-2 and MRP expression. Similarly, the resistance to etoposide and epirubicin in antisense-treated cells also reduced in parallel to decreases of the two gene expressions. The drug resistance in sense-treated cells was similar to that in untreated cells. Statistically significant dose-and concentration-dependent increases of apoptotic cells were observed in the groups exposed to 100 μmol/L cisplatin for 48 h after treatment by bcl-2 or/and MRP antisense.Conclusion Bcl-2 and MRP were at least additive and possibly synergistic in conferring drug resistance in a cisplatin-resistant lung adenocarcinoma cell line. Antisense S-ODN could attenuate drug resistance by promoting cells apoptosis, which might lead to a new treatment for patients with non-small cell lung cancers (NSCLCs) who are refractory to conventional chemotherapy.     

Effects of Banxia Xiexin Decoction (半夏泻心汤) on Cisplatin-Induced Apoptosis of Human A549 Lung Cancer Cells

Objective: To examinie the synergistic effects of Banxia Xiexin Decoction(半夏泻心汤,Known as Banhasasim-tang in Korean) extract(BXDE) on cisplatin-induced cytotoxicity in the A549 human lung cancer cell lines. Methods: A549 cells were treated with varying concentrations(50–200 μg/m L) of cisplatin and BXDE alone or in combination for 96 h. We used 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan assay and flow cytometry to analyze cell viability and apoptosis, respectively. Results: The exposure of cells to cisplatin and BXDE alone or in combination decreased cell viability dose-and time-dependently(P0.05), which was found to be mediated by the apoptotic pathway as confirmed by the increase in the annexin V+/propidium iodide-stained cell population and a ladder pattern of discontinuous DNA fragments. Furthermore, the apoptosis was inhibited by the pan-caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp(OMe) fluoromethylketone(z-VAD-FMK). Conclusions: BXDE significantly potentiated apoptotic effects of cisplatin in A549 cells. Moreover, apoptosis induced by BXDE might be the pivotal mechanism mediating its chemopreventative action against cancer.     

Anticancer activity of total flavonoids isolated from Xianhe Yanling Recipe (仙鹤延龄方)

Objective:To investigate the anticancer activity of the total flavonoids isolated from a herbal formula,Xianhe Yanling Recipe（仙鹤延龄方）,a recipe commonly used in cancer patients in China.Methods: The in vitro anticancer activity of the total flavonoids was determined using the 3-（4,5-dimethylthiazole-2-yl）-2,5- diphenyltetrazolium bromide（MTT） assay on three cancer cell lines:MCF-7（a human breast adenocarcinoma cell line）,HepG-2（a human hepatocellular carcinoma cell line） and ES-2（a human ovarian cancer cell line）. The in vivo anticancer effect of the total flavonoids was assessed in a mouse tumor model bearing H22-induced hepatocellular carcinoma,and cisplatin was used as a positive control.Results:The total flavonoids exerted a powerful inhibitory effect on the three cell lines,with 50%inhibiting concentrations(IC₅₀) of 24.948,31.569 and 6.923μg/mL,respectively.In vivo studies showed that the total flavonoids had dose-dependent inhibitory effects on hepatocellular carcinoma in mice.Conclusion:The total flavonoids from Xianhe Yanling Recipe have potential anticancer activity,and further researches and development are warranted.     

Effects of Exogenous p16^ink4a Gene on Biological Behaviors of Human Lung Cancer Cells

The effects of exogenous p16ink4a gene on biological behaviors of human lung cancer cell line with homozygous deletion of p16ink4a gene were investigated. Exogenous p16ink4a gene was transfected by lipofectin into human lung cell line A549, in which p16ink4a gene was homozygously deleted. The expression of p16ink4a mRNA and protein was detected by RT-PCR and immunocyto-chemistry, respectively. The changes in the behaviors of the transfected cell lines in vitro and in vivo were observed. In the transfected cell line A549, the exogenous p16ink4a gene could be stably ex-pressed. The growth of A549 cells transfected with p16ink4a gene was obviously slowed down. Flow cytometry revealed that transfection of the exogenous p16ink4a gene resulted in A549 cell lines arrest in G1 phase of cell cycle. The tumorigenicity of these transfected cells in nude mice could be inhib-ited, and the tumor growth of nude mice was significantly suppressed. It was concluded that exoge-nous p16ink4a gene may be stably expressed in human lung cancer cell line A549. The expression of the introduced p16ink4a could block lung cancer cells to entry into S phase of cell cycle and inhibit tumor malignant growth both in vitro and in vivo.     

Heparanase antisense oligodeoxynucleotide inhibits angiogenesis and metastasis of human mammary carcinoma cell xenografts in nude mice

Objective： To evaluate the inhibitory effect of heparanase antisense oligodeoxynucleotide （AS-ODN） on the angiogenesis and metastasis of human mammary carcinoma cell xenografts in nude mice. Methods： The AS-ODN complementary to the start codon region of heparanase mRNA and its control, scrambled nonsense oligodeoxynucleotide （NS-ODN） were designed and synthesized. A subcutaneous growth model and an acute hematogenous metastasis model of human mammary carcinoma were established in nude mice and were treated with ODNs. The heparanase expression in tumor was evaluated by RT-PCR and Western blot. The microvessel density （MVD） was measured by immunohistochemistry for factor VS. The tumor volume was calculated and lung metastatic nodules were counted. Results ： The heparanase expression, MVD, tumor volume and lung metastatic nodules in AS-ODN treated group were significantly decreased compared with that in NS-ODN treated group and that in PBS group （P〈0.01）. Conclusion ： Heparanase AS-ODN has significant inhibitory effect on the angiogenesis and metastasis of human mammary carcinoma cell xenografts in nude mice.     

Genistein sensitizes ovarian carcinoma cells to chemotherapy by switching the cell cycle progression in vitro

Objective： To address how genistein sensitizes the chemotherapy-resistant ovarian carcinoma cells and promotes apoptosis in the respect of cell cycle and the regulation of survivin expression in the process. Methods： Ovarian SKOV-3 carcinoma cell line was treated with genistein or cisplatin either alone or in combination. Cell viability was showed by MTT method. Cell cycle and apoptosis were detected by flow cytometry. Survivin mRNA and protein were revealed by RT-PCR and immunocytochemistry, respectively. Results： Genistein could reduce the cell viability in a dose-dependent manner, while cisplatin did so at a much higher level. In contrast, if the two agents were treated in combination, half growth inhibition （IC50） value for cisplatin was reduced remarkably and the effect was synergistic as analyzed by isobologram. In particular, the reduced cell viability was exhibited by a switch in cell cycle progression, as the cells were arrested in G2/M phase and the G0/G1 phase- fraction was significantly decreased. The reduced cell viability appeared to involve apoptosis, based on our results from flow cytometry and Hoechst 33258 staining. In the meanwhile, genistein performed the inhibitory effect on cisplatin-induced survivin expression. Conclusion： Genistein can sensitize ovarian carcinoma cells to cisplatin therapy with the inhibition of survivin expression as the potential mechanism.     

Inhibition Mechanism of Novel Pyrazolo[1,5-a]pyrazin-4(5H)-one Derivatives Against Proliferation of A549 and H322 Cancer Cells

Objective To explore the inhibition mechanism and safety of pyrazolo[1,5-a]pyrazin-4(5H)-one derivatives against proliferation of human lung cancer A549 cells, H322 cells, and human umbilical vein endothelial cell (HUVEC). Methods Cells were treated with 40μmol/L of the ppo3a, ppo3b, ppo3i, and 0.1% DMSO (control) for 48 hours, respectively. Apoptosis was determined by Hoechst 33258 staining assay in H322 and A549 cells. Cell cycle distribution was determined by flow cytometry analysis in A549 cell. LC3-II, p53, and heat shock protein (HSP) 70 protein levels were detected by Western blotting in A549 cells treated with ppo3b for 48 hours. The morphology and viability of HUVEC were observed by inverted microscope and sulforhodamine B (SRB) assay. Results Ppo3a, ppo3b, and ppo3i significantly induced apoptosis in H322 and A549 cells. A strong G1-phase arrest was concomitant with the growth inhibitory effect on A549 cells. Ppo3b effectively elevated the p53 protein level, but significantly reduced the HSP70 protein level. There were no significantly inhibitory effect on the morphology and viability of HUVEC when treated with ppo3a, ppo3b, and ppo3i. Conclusions ppo3a, ppo3b, and ppo3i could inhibit H322 proliferation through apoptosis and inhibit A549 through apoptosis and G1-phase arrest. The protein p53 and HSP70 might involve in the inhibition effects. These derivatives might be a clue to find effective and safe drug for lung cancers.     

In Vitro Cytotoxicity of Conjugate of Mitomycin C with Murine Monoclonal Antibody against Gastric Cancer Cell Line

In the present study, the conjugate of mitomycin C (MMC) with a murine monoclonalantibody against gastric cancer, MG11, was prepared and its cytotoxicity on gastric cancer cell lineKATOⅢ was determined. MMC was modified with glutaric anhydride to introduce a carboxylicgroup at N-lα position. The MMC derivative was then treated with N-hydroxysuccinimide anddicyclohexylcarbodimide to from MMC active ester, which was combined with monoclonal antibodyMG11 for preparation of MG11-MMC conjugate, which showed highly selective cytotoxicity uponhuman gastric cancer cell line KATOⅢ in culture. In the 48h exposure test, the cytotoxicity ofMG11-MMC conjugate on target cells was found to be similar to that of free MMC. The conjugatehad only a little effect upon nontarget cells (human normal embryonic lung cell line SL_7). The re-sults suggest that the selective cytotoxicity on target cells of MG11-MMC is mediated by theantibody.     

ROLE OF ERK1／2 KINASE IN CISPLATIN-INDUCED APOPTOSIS IN HUMAN OVARIAN CARCINOMA CELLS

Objective To investigate the role of extracellular regulated kinase (ERK1/2) pathway in cisplatin-induced apoptosis in human ovarian carcinoma cells.Methods Cisplatin-induced apoptosis were stained with DAPI and was assessed microscopically in human epithelial adenocarcinoma ovarian cell line SKOV3 cells. ERK activation was determined by Western blotting using an anti-phosphoERK antibody to detect ERK activity. The effect of PD98059 on ERK activity induced by cisplatin was detected by MTT assay.Results Marked apoptosis of SKOV3 cells resulted from 48 hours treatment with 20 μg/mL cisplatin. Strong activation of ERK was led to by 15 μg/mL cisplatin. Dose response and time course of cisplatin induced apoptosis in SKOV3 cells.Cisplatin-induced ERK activation occurred at 12 hours and increased to highest induction at 24 hours by Western blotting.The effect of PD 98059 on ERK activity induced by cisplatin at the concentration of 100 μmol/L PD 98059. Statistically significant decreased in cell survival were observed with 100 μmol/L PD 98059 at 15 and 20 μg/mL cisplatin (P＜ 0.05).Conclusions Cisplatin activates the ERK signaling pathway in ovarian cancer cell line SKOV3. Inhibition of ERK activity enhances sensitivity to cisplatin cytotoxity in ovarian cancer cell line SKOV3. Evaluation of ERK activity could be useful in predicting which ovarian cancer will response most favorably to cisplatin therapy.     

Inhibitory Effect of Pulmonary Carcinoma by Adenovirus-Mediated CD/UPRT Gene

The cell killing effects and bystander effects of double suicide gene on pulmonary carcinoma cells were explored. Lung adenocarcinoma cells (A549) were transfected with different liters of adenovirus vector and followed with different concentrations of 5-FC after a recombinant adenovirus vector carrying CD/UPRT gene (Ad-CD/UPRT) was constructed. The cell viability was measured by MTT assay 4 days later. The cell viability was dropped to 30.57 %-8.62 % after 10 MOI of Ad-CD/UPRT transfected and 5-FC (10-1000μg/mL) administration. Furthermore, Ad-CD/UPRT-infected A549 cells showed a profound neighbor cell killing effect in the same methods. These results suggested that Ad-CD/UPRT/5-FC system can effectively suppress growth of lung adenocarcinoma cells, which may provide a novel and powerful candidate for lung cancer gene therapy strategies.