Inhibition of metastasis to lung of a human nasopharyngeal carcinoma cell line CNE-2L2 transfected with pRc/CMV-antisense 6A8 cDNA in nude mice

The growth of CNE-2L2 cell, a cloned line of human nasopharyngeal carcinoma with a high potentiality of metastasis to lung was inhibited to a certain extent after transfection with a recombinant antisense expression vector of a cDNA encoding a human a-mannosidase (pRc/CMV-antisense 6A8 cDNA) (the Genbank accession number of 6A8 cDNA is U37248) in comparison with that of the cell transfected with the Mock and of the wild cell. Two months after a subcutaneous inoculation of CNE-2L2 cell into the axilla of nude mice metastatic lesions in the lung were observed in 9/10 mice (90%) with grade III in 8 mice and grade II in one mouse in the wild cell group, in 6/8 mice (75%) with grade III in one mouse, grade II in 2 mice and grade I in 3 mice in the Mock-transfection group, in only 3/10 mice (30%) with all grade I in pRc/CMV-antisense 6A8 cDNA-transfection group.

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.

Effect of heparin on apoptosis in human nasopharyngeal carcinoma CNE2 cells

In order to study the mechanism of the effect of heparin on apoptosis in carcinoma cells, the nasopharyngeal carcinoma cell line CNE2 was used to identify the effect of heparin on apoptosis associated with the expression of c-myc, bax, bcl-2 proteins by use of Hoechst 33258 staining, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL), agarose gel electrophoresis, and flow cytometry, as well as Western blot analysis. The results showed that heparin induced apoptosis of CNE2 cells including the morphologic changes such as reduction in the volume, and the nuclear chromatin condensation, as well as the 'ladder pattern' revealed by agarose gel electrophoresis of DNA in a concentration-dependent manner. The number of TUNEL-positive cells was dramatically increased to 33.6+/-1.2% from 2.8+/-0.3% by treatment with heparin in different concentrations (10 to approximately 40 kU/L). The apoptotic index was increased to 32.5% from 3.5% by detecting SubG1 peaks on flow cytometry. Western blot analysis showed that levels of bcl-2, bax and c-myc were significantly overexpressed by treatment with the increase of heparin concentrations. These results suggest that heparin induces apoptosis of CNE2 cells, which may be regulated by differential expression of apoptosis-related genes.

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.

ESTABLISHMENT OF A NOVEL CELL LINE (HNE_(1)) DERIVED FROM A NASOPHARYNGEAL CARCINOMA

A novel epithelial cell line, designated HNE1 was established from a biopsy specimen of a naso-pharyngeal carcinoma (NPC). Electron microscopic examination of the HNE1 cells demonstrated bi-directional differentiation, with some cells displaying features of poorly differentiated squamous cell carcinoma, while other cells appeared to have the morphology of poorly differentiated adenocarcinoma. The HNE1 cell line has been passaged more than 100 times over a period of one year. We recently reported that the Epstein-Barr virus (EBV) nuclear antigen (EBNA) was detected in a low pe rcentage of the HNE1 cells examined at subcultures 5-81; the cells were also shown to be EBV DNA positive. Tumorigenicity of the HNE1 cells was demonstrated by xentransplanta tion in athymic nude mice. The developed tumors were characterized as well-differentiated squamous cell carcinomas upon histological examination. Kar yotypic analysis of the HNE1 cells demonstrated an aneuploidy with a modal chromosomal number of 74 at passage 5 and 101 at passage 20; 15 marker chromosomes were identified. The frequency of spontaneous sister chromatid exchange was found ot be very high (87.6±0.4/cell).

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.

Establishment and characterization of a novel nasopharyngeal carcinoma cell line (SUNE2) from a Cantonese patient

The undifferentiated form of nasopharyngeal carcinoma (NPC) is the most common malignant head and neck cancer in South China, especially in Cantonese populations. However, few NPC cell lines have been established from the patients in this region. In this study, we established a new NPC cell line, termed SUNE2, from a Cantonese patient with undifferentiated NPC. This cell line had extremely low concentrations of Epstein-Barr virus (EBV) DNA in long-term culture and expressed low levels of latent membrane protein 1 (LMP1), latent membrane protein 2A (LMP2A), BamH1-A right frame 1 (BARF1), EBV-encoded RNA-1 (EBER1), and EBV-encoded RNA-2 (EBER2) in early passages. SUNE2 cells also showed much stronger transforming ability than 5-8F cells in colony formation assays and anchorage- independent growth assays in soft agar, and they only need 2 weeks to form tumors in nude mice. In summary, the SUNE2 cell line is a new in vitro model that can be used for further research on the mechanisms underlying the occurrence and development of NPC.

Modulating effects of survivin antisense oligonucleotide on changes of apoptosis and cell cycle of human hepatocellular carcinoma cell line SMMC-7721

To investigate the modulating effects of survivn antisense oligonucletode (ASODN) on the cell cycle and apoptosis of human hepatocellular carcinoma (HCC) cell line SMMC-7721 and explore its mechanism.Methods Survivin ASODN was transfected into SMMC-7721 cells mediated by DOTAP liposomal reagent.Electron microscopy,flow cytometry and RT-PCR were used to detect the changes in cell ultrastructure,apoptosis,cell cycle and the expression of cyclinB1 mRNA,respectively.Results After transfection of survivin ASODN,the expression of cyclinB1 mRNA in the cells significantly increased and increase in G2-M arrest and apoptosis appeared.Meanwhile,the cell ultrastructure had apoptotic changes such as chromatin condensation and apoptotic body formation.Conclusion Survivin ASODN can induce the expression of cyclinB1 that may result in G2-M arrest.Consequently,apoptosis is triggered.Survivin ASODN transfection might be an improtant new treatment for HCC.14 refs,2 figs,1 tab.

Orthotopical transplantation of human renal carcinoma tissue into nude mice and the establishment of a high metastatic cell line MRCC

Objective To establish a SOI model of human renal carcinoma and a high metastatic cell subline. Methods A human renal cell line RCC-9863 has been established by inoculating a human renal tumor tissue into nude mice s. c.. When RCC-9863 passaged for 20 times, the tissue from the same xemotransplant tumor were used to construct SOI model. Cultured the metastatic tissue in vitro, the tumor cell suspension was then injected orthotopically, The metastatic tissue obtained underwent the same procedure again. At last, the metastatic tumor was cultured in vitro and cloned. Results 15 days later, a tumor mass sized 1. 7 cm × 0. 6 cm in the nude mouse’s renal parenchyma was grown which lobulated, rude, and with multiply blood vessels and 55 days later later the mouse became moribund and metastases in the lungs were formed. The transplanted renal tumor in the SOI model grew fast and invasively and metastasized to lungs, lymphatic node and liver. A subline, MRCC, with metastatic ability to the lung was selected.

Inhibitory effect of endostatin expressed by human liver carcinoma SMMC7721 on endothelial cell proliferation in vitro

AIM: To construct a stable transfectant of human liver carcinoma cell line SMMC7721 that could secret human endostatin and to explore the effect of human endostatin expressed by the transfectant on endothelial cell proliferation. METHODS: Recombinant retroviral plasmid pLncx-Endo containing the cDNA for human endostatin gene together with rat albumin signal peptide was engineered and transferred into SMMC7721 cell by lipofectamine. After selection with G418, endostatin-transfected SMMC7721 cells were chosen and expanded. Immunohistochemical staining and Western blot were used to detect the expression of human endostatin in transfected SMMC7721 cells and its medium. The conditioned medium of endostatin-transfected and control SMMC7721 cells were collected to cultivate with human umbilical vein endothelial cells for 72 hours. The inhibitory effect of endostatin, expressed by transfected SMMC7721 cells, on endothelial proliferation in vitro was observed by using MTT assay. RESULTS: A 550 bp specific fragment of endostatin gene was detected from the PCR product of endostatin-transfected SMMC7721 cells. Immunohistochemistry and Western blot analysis confirmed the expression and secretion of foreign human endostatin protein by endostatin-transfected SMMC7721 cells. In vitro endothelial proliferation assay showed that 72 hours after cultivation with human umbilical vein endothelial cells, the optical density (OD) in group using the medium from endostatin-transfected SMMC7721 cells was 0.51 +/- 0.06, lower than that from RPMI 1640 group (0.98 +/- 0.09) or that from control plasmid pLncx-transfected SMMC7721 cells (0.88 +/- 0.11). The inhibitory rate for medium from endostatin-transfected SMMC7721 cells was 48%, significantly higher than that from empty plasmid pLncx-transfected SMMC7721 cells (10.2%, P【0.01). CONCLUSION: Human endostatin can be stably expressed by SMMC7721 cell transferred with human endostatin gene and its product can significantly inhibit the proliferation of human umbilical vein endothelial cell in vitro.

Inhibitory effect of a new gossypol derivative apogossypolone (ApoG2) on xenograft of human prostate cancer cell line PC-3

Objective: To investigate the inhibitory effect of apogossypolone (ApoG2) on prostate cancer cell line PC-3 in vivo, and explore its mechanism. Methods: The models of transplantation tumors in Balb/c nu/nu mice were established via subcutaneous injection of PC-3 cells and the tumor-transplanted mice were divided into 4 groups: control group and three ApoG2 treatment groups, with 10 mice in each group. Volumes of the tumor were estimated every 2 d and the morphology of tumor tissues was observed. Immunohistochemistry was employed to observe the expression of Bcl-2, PCNA, CD31, caspase-3 and caspase-8 in tumor tissues. Results: ApoG2 (2.5 mg/kg-10 mg/kg) given intraperitoneally once a day can obviously inhibit the growth of subcutaneous prostatic carcinoma implant. The tumor volume decreased obviously when the treatment dosage was bigger than 5.0 mg/kg (P<0.01). Meanwhile, ApoG2 decreased the expression of PCNA and CD31, and enhanced the expression of caspases-3, caspase-8 in tumor tissues. Conclusion: ApoG2 exert an inhibitory effect on prostatic carcinoma possibly by inducing apoptosis and inhibiting tumor angiogenesis.

Inhibitory effects of apogossypolone on subcutaneous implants of human LNCaP prostatic carcinoma cells

Objective:The aim of this study was to investigate the inhibitory effect of apogossypolone (ApoG2) on subcutaneous implants of human LNCaP prostatic carcinoma cells, and explore its mechanism. Methods:To establish human LNCaP prostatic carcinoma cell line subcutaneous xenograft models and observe the inhibitory effect of ApoG2 on the tumor model. Immunohistochemistry was employed to observe the expression of Bcl-2, PCNA, CD31, caspase-3 and-8 in tumor tissues. The microvessel density was calculated. Results:ApoG2 could obviously inhibit the growth of subcutaneous prostatic carcinoma implant. ApoG2 decreased the expression of PCNA and CD31, and increased the expression of caspases-3,-8 in tumor tissues. Conclusion:ApoG2 has an inhibitory effect on prostatic carcinoma implants.

Relation between the Expression of K-ras in Hep-2 Cells and Development of Laryngeal Carcinoma~*

Objective： To investigate the expression of K-ras in human laryngeal squamous cell carcinoma cell lines （Hep-2） and its significance for establishing a solid foundation for further study of the relationship between human laryngeal squamous cell carcinoma and K-ras gene point mutations. Methods： The expression of K-ras in human laryngeal squamous cell carcinoma cell lines （Hep-2） and human pancreatic carcinoma cell lines （MIAPaCa-2） was detected by using RT-PCR. Results： The expression of K-ras mRNA in Hep-2 and MIAPaCa-2 was strong and positive. Conclusion： The expression of K-ras mRNA in human laryngeal squamous cell carcinoma cell lines （Hep-2） is positive. Development of laryngeal carcinoma might be related to the activation of K-ras gene point mutation.

Role of JNK signaling pathway in sensitivity to radiotherapy of nasopharyngeal carcinoma

Objective:The aim of the study was to investigate the effect of c-Jun N-terminal protein kinase(JNK) signaling pathway on influencing the sensitivity to radiotherapy of human nasopharyngeal carcinoma CNE cells.Methods:Human nasopharyngeal carcinoma CNE multicellular spheroids(MCS) were constructed with three dimensional cell culture methods.Western blot was employed to analyze the activity of JNK signaling pathway in MCS after X-ray irradiation,and the expression of caspase-3 protein before and after using SP600125(a special inhibitor of JNK).X-ray induced cell apoptosis in MCS before and after treated with SP600125 were detected by TUNEL.Results:The level of JNK phosphorylation in MCS was a dynamic course after radiation,and there was a phosphorylation peaks at 2 h later,the apoptotic rate of MCS(P < 0.05) and the expression of caspase-3 protein(P < 0.05) were significantly increased after treated with SP600125.Conclusion:The transient activation of JNK played a important role in sensitivity to radiotherapy of CNE MCS via mediating survival signals,blocking this pathway accelerate cell apoptosis,which may be related to the increased expression of caspase-3.

探讨姜黄素通过NF-κB对鼻咽癌CNE-2Z细胞的生长抑制作用、诱导凋亡作用及其机制研究

目的:探讨姜黄素通过NF-κB对鼻咽癌CNE-2Z细胞增殖的影响。方法:MTT法测定姜黄素对鼻咽癌CNE-2Z细胞增殖的影响,采用流式细胞仪测定姜黄素不同浓度下的CNE-2Z细胞凋亡情况,Western Blot测定NF-κB蛋白的表达。结果:姜黄素能影响CNE-2Z细胞的NF-κB蛋白表达,使NF-κB蛋白表达水平降低,说明姜黄素对CNE-2Z的增殖和转移有一定的影响。姜黄素对CNE-2Z的作用效果受作用时间和浓度影响,其对CNE-2Z细胞24 h的IC50数值为(43.514~45.435)μmol/L,48 h的IC50数值为(8.366~13.11)μmol/L,72 h的IC50数值为(5.658~8.469)μmol/L。结论:姜黄素能够通过NF-κB蛋白的表达,来抑制鼻咽癌CNE-2Z细胞增殖与侵袭。姜黄素浓度越高,作用时间越长,CNE-2Z细胞增殖抑制效果越好。

青天葵总黄酮对人鼻咽癌CNE-2荷瘤裸鼠皮下肿瘤的抑制作用

目的:研究青天葵总黄酮(TFNF)对BALB/c裸鼠人鼻咽癌细胞皮下种植模型中TGF-β1/Erk/MAPK通路的调控作用。方法:BALB/c裸鼠皮下接种CNE-2细胞(1.0×107细胞/ml)建立鼻咽癌荷瘤模型,苏木精-伊红染色(HE)行病理判断,免疫组化法(IHC)观察细胞周期调节蛋白-67(Ki67)、增殖细胞核抗原(PCNA)的表达水平。免疫印迹法检测转化生长因子-β1(TGF-β1)、细胞外调节蛋白激酶(Erk)、p-Erk、丝裂原活化蛋白激酶(MAPK)、p-MAPK的蛋白表达水平。试剂盒法评估肾脏、肝脏、脾脏中超氧化物歧化酶(SOD)及丙二醛(MDA)水平。结果:与模型组比较,高剂量TFNF显著抑制荷瘤小鼠肿瘤体积与质量,显著降低Ki67(55.19%)、PCNA(46.65%)表达水平,提高肾脏、肝脏、脾脏中SOD水平至3.88、2.98、3.71U/mgprot,降低肾脏、肝脏、脾脏的MDA水平至2.19、1.05、2.85nmol/mgprot。TFNF可降低肿瘤组织中TGF-β1(47.36%)、p-Erk/Erk(81.13%)、p-MAPK/MAPK(19.80%)的蛋白表达水平。结论:TFNF可调控TGF-β1/Erk/MAPK通路抑制肿瘤生长,并减轻荷瘤裸鼠主要脏器的氧化损伤发生。

Inhibitory effect of metformin on the proliferation of human hepatoma HepG2 cells and its potential mechanism

Objective： This work aimed to study the inhibitory effect and the related mechanism of metformin （MET） on the proliferation of human hepatoma HepG2 cells. Methods： Human hepatoma HepG2 cells were treated with MET （0, 2, 10, and 50 mM）. The inhibitory effect of MET on the proliferation of HepG2 cells was determined by MTT method. The apoptosis of HepG2 cells was detected by flow cytornetry. The expression of cyclin D1 in HepG2 cells was examined by Western blot. ROS-DHE fluorescence probe was used to stain the reactive oxygen species （ROS） generated by HepG2 cells after treat- ment. Results： MET could inhibit the proliferation of HepG2 cells in a dose and time dependent manner. MET promoted the apoptosis of HepG2 cells. In addition, MET suppressed the expression of cell cycle protein cyclin D1 and induced the produc- tion of ROS in HepG2 cells. Conclusion： MET can inhibit the proliferation of human hepatoma HepG2 cells and induce cell apoptosis. Meanwhile, MET has the ability to decrease the expression of cyclin D1 and induce ROS generation, which may be involved in the mechanism of inhibiting hepatoma cells proliferation.