Objective: To observe the biological difference of multi-drug resistant nasopharyngeal carcinoma (NPC) cell line CNE2/DDP and its parental cell line CNE2, as well as analyze the characteristics of multi-drug resistant cells. Methods: The difference in morphological features, growth characteristics and colony formation rate in vitro were examined and compared under light microscope. Flow cytometry was used to evaluate the cell cycle distribution and the expression of P-170 and ABCG2 proteins. Drug sensitivity was measured by MTT assay. Tumorigenicity was evaluated after subcutaneous injection of 1 × 106 CNE2/DDP or CNE2 cells into BALB/c nude mice. Results: Under light microscope CNE2/DDP cells were smaller and showed a round shape and the CNE2 cells appeared polygonal cell bodies. CNE2/DDP cells had relatively longer colony doubling time than CNE2 cells (29.46 h vs 21.03 h) indicating that the growth of CNE2/DDP cells was slower. There was significant difference in the cell cycle distribution between CNE2/DDP and CNE2 cells [ (65.12 ± 1.67) % vs (44.9 ± 2.2) % in G0/G1 phase, (23.63 ± 0.42)% vs (39.67 ± 1.27) % in S phase, and (10.93 ± 0.25) % vs (13.23 ± 0.31)% in G2/M phase, P < 0.01]. Expression of P-170 and ABCG2 proteins in CNE2/DDP cells were significantly higher than that in CNE2 cells [(75.93 ± 0.86)% vs (2.83 ± 0.40)%, (43.37 ± 0.42) % vs (4.07 ± 0.59)% P < 0.01]. There was no significant difference in the resistance indexes of CNE2/DDP cells to DDP,5-FU and VCR before and after transplantation. The tumorigenicity time was (17.17 ± 1.17) d and (10.00 ± 2.68) d respectively for CNE2/DDP and CNE2 cells after being transplanted into BALB/c nude mice. Conclusion: CNE2/DDP has typical multi-drug resistance features and could be used for the research of drug-resistant NPC.

Objective To investigate the inhibitory effect of adriamycin in combination with hyperthermia on apoptosis and bcl-2 expression in the chronic leukemic cell line K562/AO2 in vitro.Methods The working con- centration of adriamycin against K562/AO2 determined by using methyl thiazolyl tetrazolium(MTT) assay was used to treat the chronic leukemic cell line K562/AO2 in vitro alone or in combination with hyperthermia induced using a hot water bath at 40,41 or 42℃.The inhibitory effect was evaluated by MTT assay.The apoptosis rates and bcl-2 ex- pression of K562/AO2 were determined by flow cytometry.Results The working concentration of adriamycin in the study was defined as its 50% inhibition concentration (IC50).A 60 min session of hyperthermia at 40℃,41℃or 42℃was associated with significant growth inhibition of the cell line K562/AO2.Adriamycin chemotherapy alone and with hyperthermia significantly inhibited the growth of K562/AO2.All treatments significantly increased apoptosis rates and down-regulated bcl-2 expression of the K562/AO2 cell line.Conclusion Adriamycin chemotherapy com- bined with 60 min sessions of hyperthermia showed significant suppression effect on K562/AO2 cell proliferation.The treatment can increase apoptosis rates and down-regulate bcl-2 expression.

OBJECTIVETo analyze the changes of inhibitory killer cell immunoglobulin-like receptors (KIRs), NKG2D receptor and the cytotoxicity of natural killer (NK) cells induced by persistent exposure to CNE2 cells.

METHODSThe HLA-class I genotypes of CNE2 cells and KIR genotypes were determined by PCR with sequence-specific primers (PCR-SSP). The expressions of KIR2DL1, KIR2DL3, KIR3DL1, and NKG2D by the NK cells (freshly isolated NK cells, NK cells cocultured with 100 U/ml IL2 or with 100 U/ml IL2 and CNE2 cells as the control, IL2 and CNE2 groups, respectively) were analyzed by flow cytometry. Cytotoxicity of NK cells against CNE2 cells were detected by LDH releasing assay.

RESULTSThe HLA genotypes of CNE2 cells were A2, 24, B18, 35, Cw4, 7. NK cells isolated from 3 healthy donors expressed KIR2DL1, KIR2DL3, and KIR3DL1. After 4, 24 and 48 h of culture, NK cells in CNE2 group displayed higher KIR2DL1, KIR2DL3 but lower NKG2D expression than those in the control and IL2 groups (P<0.01), whereas the latter two groups showed no significant difference in KIR2DL1, KIR2DL3, and NKG2D expressions (P>0.05), and no difference in KIR3DL1 expression was found between the 3 groups (P>0.05). After 24 h of culture, the cytotoxicity against CNE2 cells mediated by the NK cells in IL2 and CNE2 groups were (26.96-/+1.47) % and (2.74-/+1.64) % at E:T ratios of 10:1, and (35.74-/+3.59)% and (4.57-/+2.41) % at E:T ratio of 20:1, respectively. NK cells in CNE2 group displayed lower cytotoxicity than those in IL2 group (P<0.01).

CONCLUSIONSPersistent exposure to tumor cells expressing NKG2D ligands can lead to downregulated expression of NKG2D receptor, increased expression of KIRs and reduction of NK-mediated cytolysis. These results elucidate the molecular mechanism of reduced cytotoxicity mediated by the edited NK cells and indicate that blocking HLA-class I-bound KIRs or enhancing the expression of NKG2D may promote NK cell-mediated cytolysis.

Cell Line, Tumor ; Cell Survival ; immunology ; Cytotoxicity, Immunologic ; immunology ; Flow Cytometry ; Humans ; Killer Cells, Natural ; cytology ; immunology ; metabolism ; NK Cell Lectin-Like Receptor Subfamily K ; Nasopharyngeal Neoplasms ; immunology ; metabolism ; pathology ; Receptors, Immunologic ; metabolism ; Receptors, KIR ; metabolism ; Receptors, KIR2DL1 ; metabolism ; Receptors, KIR2DL3 ; metabolism ; Receptors, Natural Killer Cell

OBJECTIVETo evaluate the growth-inhibiting and pro-apoptotic effect of kaempferol in human esophageal squamous carcinoma Eca-109 cells and explore the mechanism.

METHODSThe effect of kaempferol on Eca-109 cell proliferation in vitro was measured by MTT assay. TUNEL staining was used to detect the cell apoptosis following kaempferol treatment. The changes in Bax and Bcl-2 mRNA expressions in response to kaempferol treatment were determined by RT-PCR, and the caspase-3 and caspase-9 activities were evaluated using colorimetric assay.

RESULTSKaempferol significantly inhibited Eca-109 cell proliferation (P<0.05) in a concentration-dependent manner and induced obvious cell apoptosis. RT-PCR showed that after kaempferol treatment caused up-regulated Bax and down-regulated Bcl-2 mRNA expression. The colorimetric assay revealed significantly increased caspase-3 and caspase-9 activities in Eca-109 cells following kaempferol treatment (P<0.01).

CONCLUSIONKaempferol can induce apoptosis of Eca-109 cells via a mitochondria-dependent pathway.

Antineoplastic Agents, Phytogenic ; pharmacology ; Apoptosis ; drug effects ; Carcinoma, Squamous Cell ; pathology ; Caspase 3 ; metabolism ; Caspase 9 ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Esophageal Neoplasms ; pathology ; Humans ; Kaempferols ; pharmacology ; Mitochondria ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; bcl-2-Associated X Protein ; metabolism

The study was aimed to investigate the expression of HLA class I molecules and MHC class I chain-related molecules A/B (MICA/MICB) in K562 and adriamycin (ADM)-resistant K562 cell lines (K562/AO2) and their effect on cytotoxicity of NK cells. Expression of HLA class I molecules and MICA/MICB on the surface of K562 and K562/AO2 cell lines were analyzed by flow cytometry. Cytotoxicity of NK cells (isolated from 3 healthy persons) against K562 and K562/AO2 cells were detected by LDH releasing assay at different effect-to-target cell ratios (E:T). In blocking experiments, anti-MHC class I monoclonal antibody (McAb) (W6/32, a pan anti-HLA class I antibody) and anti-MHC class I chain-related molecules McAb (BAMO-1, specifically against MICA and MICB) were added to the target cells at E:T of 10:1. The results showed that the expression of MHC class I chain-related molecules on K562 was higher than that on K562/AO2 (P=0.000), and HLA class I molecules were not detectable on both cells. Cytotoxicities of NK cells against K562 and K562/AO2 cells were (29.32 +/- 0.12)%, (45.33 +/- 0.78)%, (58.37 +/- 0.87)%, (72.37 +/- 0.96)% and (12.47 +/- 0.91)%, (24.36 +/- 1.11)%, (33.29 +/- 1.03)%, (53.87 +/- 1.27)% at E:T ratios of 5:1, 10:1, 20:1 and 30:1 respectively (P=0.000), the cytotoxicity of NK cells on K562 cells was significantly higher than that on K562/A02 cells at different E:T ratios. Blocking experiments confirmed that at E:T of 10:1 killing of NK cells against K562 and K562/AO2 cells was efficiently inhibited by BAMO-1, whereas W6/32 had no effect on K562 and K562/AO2 cells. It is concluded that the expression of MHC class I chain-related molecules on K562 and K562/AO2 cells is correlated with NK cell-mediated lysis. NK cells display higher cytotoxicity against parental K562 cells than multi-drug resistant K562/AO2 cells. Down-regulation of MICA/B in multi-drug resistant tumor cell lines leads to reduction of susceptibility to NK lysis.
Cytotoxicity, Immunologic ; immunology ; Doxorubicin ; pharmacology ; Drug Resistance, Neoplasm ; immunology ; Genes, MHC Class I ; genetics ; Histocompatibility Antigens Class I ; immunology ; Humans ; K562 Cells ; Killer Cells, Natural ; immunology

OBJECTIVETo analyze the expression of NKG2D ligands on human nasopharyngeal carcinoma cell line CNE2 and the multidrug-resistant lin CNE2/DDP and investigate its impact on cytotoxicity of natural killer (NK) cells.

METHODSExpression of NKG2D ligands on the surface of CNE2 and CNE2/DDP cells was analyzed by flow cytometry, and their HLA genotypes, along with inhibitory killer cell immunoglobulin-like receptors (KIRs) expressed on NK cells from 5 healthy donors, were determined by PCR with sequence specific primers. Cytotoxicity of NK cells against CNE2 and CNE2/DDP cells was evaluated by LDH-releasing assay at different effector-to-target ratios (E:T). In blocking experiments, different monoclonal antibodies (mAb) were added to the target cells at the E:T of 20:1 ratio.

RESULTSThe HLA genotypes of CNE2 and CNE2/DDP cells were A2, 24, B18, 35, Cw4, 7, and the inhibitory KIR genotypes of the 5 healthy donors were KIR2DL1, KIR2DL3, KIR3DL1, and KIR3DL2, mismatched with the HLA -class I molecules expressed by the CNE2 and CNE2/DDP cells. The expression of MHC class I chain-related proteins A and B (MICA and MICB) on CNE2 cells was higher than that on CNE2/DDP cells (P<0.01), and ULBP1 and ULBP3 were not detectable. NK cells displayed highly in vitro cytotoxicity against CNE2 and CNE2/DDP cells with a mean cell lysis rate of (10.50-/+2.17)%, (4.98-/+0.95)%; (27.68-/+1.47) %, (15.48-/+2.10) %; (36.99-/+3.13) %, (28.46-/+4.30) %; (55.00-/+2.20) %, (40.95-/+2.21) %, respectively, corresponding to the E:T ratios of 5:1, 10:1, 20:1, and 30:1 (P<0.01). Blocking experiments confirmed that killing of CNE2 and CNE2/DDP cells by NK cells was efficiently inhibited by anti-MICA, anti-MICB, and anti-ULBP2 mAbs, whereas anti-ULBP1 and anti-ULBP3 mAbs had no effects on the cytotoxicity of the NK cells.

CONCLUSIONExpression of NKG2D ligands on CNE2 and CNE2/DDP cells is correlated with NK cell-mediated lysis, and NK cells display higher cytotoxicity against parental CNE2 cells than the multidrug-resistant CNE2/DDP cells.

Antibodies ; pharmacology ; Cell Line, Tumor ; Cytotoxicity, Immunologic ; drug effects ; immunology ; Drug Resistance, Multiple ; Flow Cytometry ; Genotype ; HLA Antigens ; genetics ; Histocompatibility Antigens Class I ; immunology ; metabolism ; Humans ; Killer Cells, Natural ; immunology ; NK Cell Lectin-Like Receptor Subfamily K ; immunology ; metabolism ; Nasopharyngeal Neoplasms ; immunology ; metabolism ; pathology ; Polymerase Chain Reaction ; Receptors, KIR ; genetics

OBJECTIVETo explore the expression of MICA/B in human esophageal cancer, and to analyze its correlation with clinicopathological features.

METHODSThe expression of MICA/B in 40 cases of esophagus carcinoma and corresponding normal esophageal mucosa tissues were examined by immunohistochemistry.

RESULTSThe positive rate of expression of MICA/B protein in the esophageal carcinoma was 75.0% (30/40), and that in the corresponding normal esophageal mucosa was 0 (0/40). Up-regulation of MICA/B expression was found in the esophageal carcinomas. The expression of MICA/B was related with histological grade of the esophageal carcinoma (P = 0.012).

CONCLUSIONMICA/B protein plays an important role in the esophageal carcinogenesis, and my become a useful molecular marker for the diagnosis of esophageal carcinoma.

Adult ; Aged ; Aged, 80 and over ; Biomarkers, Tumor ; metabolism ; Esophageal Neoplasms ; diagnosis ; metabolism ; pathology ; Female ; Histocompatibility Antigens Class I ; metabolism ; Humans ; Immunohistochemistry ; Male ; Middle Aged ; Neoplasm Grading ; Up-Regulation

OBJECTIVETo observe the effect of thermotherapy on the intracellular adriamycin concentration and apoptosis of Raji cells in vitro.

METHODSThe working concentration of adriamycin against Raji cells was determined with MTT assay. Raji cells were subjected to thermotherapy (at 40 degrees Celsius;, 41 degrees Celsius; or 42 degrees Celsius;) and chemotherapy with adriamycin alone or in conjunction, and the cell survival rates were determined 48 h after the treatment. The cell growth inhibition effect of the treatment was evaluated with MTT assay, and the apoptotic rates of Raji cells and alteration of intracellular adriamycin concentration were determined by flow cytometry.

RESULTSThe IC(50) of adriamycin was defined as the working concentration in the experiment. Thermotherapy at 40, 41 and 42 degrees Celsius; for 60 min in conjuction with chemotherapy significantly inhibited the growth of Raji cells (P<0.01). The results of flow cytometry showed that thermotherapy and adriamycin chemotherapy, used either alone or in combination, significantly increased the apoptotic rates of Raji cells (P<0.01), and thermotherapy remarkably increased the intracellular concentration of adriamycin.

CONCLUSIONAdriamycin chemotherapy combined thermotherapy for 60 min can increase the intracellular concentration of adriamycin and the apoptosis rates of Raji cells.

Antibiotics, Antineoplastic ; pharmacology ; Apoptosis ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cell Survival ; drug effects ; Combined Modality Therapy ; Doxorubicin ; metabolism ; pharmacology ; Flow Cytometry ; Hot Temperature ; Humans ; Hyperthermia, Induced ; Inhibitory Concentration 50 ; Intracellular Space ; metabolism ; Lymphoma ; drug therapy ; metabolism ; pathology

This study was purposed to investigate the inhibitory effect, apoptosis, Bcl-2 and P-gp expression of K562/AO2 cells by hyperthermia combined with adriamycin. The working concentration of adriamycin against K562/AO2 was determined by MTT assay. The hyperthermia and chemotherapy were used alone or in combination, then the cell survival rate was detected at 48 hours. The inhibitory effect was evaluated by MTT assay. The apoptosis rate, Bcl-2 and P-gp expression of K562/AO2 were determined by flow cytometry. The concentration of adriamycin in the experiment was defined as its IC(50) at 48 hours action. The results indicated that the hyperthermia at 40, 41 and 42 degrees C for 60 minutes showed obvious inhibitory effect on K562/AO2 cells (p < 0.01). Adriamycin chemotherapy combined with hyperthermia showed more obvious inhibitory effect on K562/AO2. According to flow cytometric analysis, the hyperthermia and adriamycin used alone or in combination could obviously increase the apoptosis rate and down-regulate Bcl-2 and P-gp expression of K562/AO2 cells (p < 0.01). It is concluded that the adriamycin chemotherapy combined with hyperthermia for 60 minutes shows obvious inhibitory effect on K562/AO2 cells, which increases the apoptosis rate and down-regulates expression of Bcl-2 and P-gp.
ATP-Binding Cassette, Sub-Family B, Member 1 ; metabolism ; Antibiotics, Antineoplastic ; pharmacology ; Apoptosis ; drug effects ; Doxorubicin ; pharmacology ; Drug Resistance, Neoplasm ; Gene Expression Regulation, Neoplastic ; Humans ; Hyperthermia, Induced ; K562 Cells ; Proto-Oncogene Proteins c-bcl-2 ; metabolism

OBJECTIVETo study the expenses of hospitalization among the population in the flood disaster areas of Dongting Lake in Hunan province in 1998.

METHODSDescriptive epidemiologic study were conducted to analyze hospitalization expenses of the residents of 55 villages in flood disaster areas in 1998; single factors analysis and logarithmic linear regression analysis were carried out to explore influencing factors about hospitalization expenses of the residents.

RESULTSThe hospitalization rate was 4.59% with an average hospitalization expenses of 667.42 Yuan in the flood disaster areas' residents of Dongting Lake in 1998. Compared with populations without suffering from flood, hospitalization rate and the average hospitalization expenses of flood disaster Areas' residents of Dongting Lake in 1998 were higher and had significant difference. The average hospitalization expenses in 1998 was affected by flood types, family income, gender, age, literacy, occupation, outcome after leaving the hospital and hospital ranks.

CONCLUSIONThese results implied that the flood disease aggravated inhabitants' burden of disease in Dongting Lake areas; the factors influencing the average hospitalization expenses were multiple, and synthetic measures should be taken in the prevention and control of flood disaster.

Adolescent ; Adult ; Aged ; Child ; Child, Preschool ; China ; Cost of Illness ; Disasters ; Female ; Hospital Charges ; statistics & numerical data ; Hospitalization ; economics ; Humans ; Infant ; Linear Models ; Male ; Middle Aged ; Regression Analysis ; Rural Population