metallothionein and Carcinoma--Small-Cell

To search genes sensitivity to the anti-cancer drugs navelbine (NVB) and docetaxel (DOC) in small-cell lung cancer (SCLC) and non-small-cell lung cancer (NSCLC) cell strains.. The sensitivity of 4 strains of SCLC and 6 strains of NSCLC to NVB and DOC was evaluated using the MTT assay. The expression of 1291 sensitive-related genes to the anti-cancer drugs in 10 lung cancer cell strains was measured using cDNA macroarrays and the relationship was analyzed.. In total, there were 56 (r > or = 0.4) genes sensitive to NVB and DOC. For NVB: 36 genes were sensitive to NVB, 20 co-expressed genes between the SCLC+NSCLC set and the NSCLC set; 27 expressed genes and 7 specially expressed genes in the SCLC+NSCLC set; and 29 expressed genes and 9 specially expressed genes in the NSCLC set. For DOC, 50 genes were sensitive to DOC, 12 co-expressed genes between the SCLC+NSCLC set and the NSCLC set; 24 expressed genes and 12 specially expressed genes in the SCLC+NSCLC set; and 38 expressed genes and 26 specially expressed genes in the NSCLC set. The genes sensitive to NVB and DOC in lung-cancer cell stains were mainly divided into the following 4 categories: signal transduction molecules, cell factors, transcription factors and metabolism-related enzymes and inhibitors.. There were obvious differences in genes related to NVB and DOC between SCLC and NSCLC cell strains, but the same as categories of function.

Topics: Antineoplastic Agents, Phytogenic; Carcinoma, Non-Small-Cell Lung; Carcinoma, Small Cell; Cell Line, Tumor; Cell Survival; Cluster Analysis; Clusterin; Docetaxel; Dose-Response Relationship, Drug; Galectin 1; Gene Expression Regulation, Neoplastic; Humans; Inhibitory Concentration 50; Lung Neoplasms; Metallothionein; Oligonucleotide Array Sequence Analysis; Taxoids; Tissue Inhibitor of Metalloproteinase-1; Vinblastine; Vinorelbine

Over-expression of cellular metallothionein occurs frequently in human tumours but the underlying mechanism remains unknown. The aim of this study was to assess metallothionein expression in cases of lung carcinoma and to correlate it with histopathological parameters.. Tumour tissue samples from 89 patients with lung carcinoma were immunostained by the streptavidin-biotin-peroxidase technique, using a monoclonal antibody against both metallothionein-1 and -2 isoforms. Positive matallothionein immunostaining was prominent in 44 out of 89 (49%) and negative in 45 out of 89 (51%) cases of lung carcinoma examined. Metallothionein positivity was prominent in 32 out of 43 (74%) cases of squamous cell lung carcinoma, and in 12 out of 35 (34%) cases of adenocarcinoma, while it was negative in all 11 cases of small-cell lung carcinoma examined, presenting a statistically significant difference between the different histological types. The intensity of metallothionein staining revealed a statistically significant difference between the squamous cell and adenocarcinoma cases examined. The pattern and extent of metallothionein staining in tumour cells and the expression of metallothionein in stromal cells were not correlated with histopathological parameters (type and grade) in metallothionein-positive cases of lung carcinoma examined. No association was found between metallothionein expression and lymph node status in the examined cases of lung carcinoma.. Our findings indicate that expression of metallothionein was evident in squamous cell lung carcinoma and adenocarcinoma, but absent in small-cell lung carcinoma, supporting evidence for participation of this protein in the biological mechanisms underlying the carcinogenic evolution in the lung.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Carcinoma, Small Cell; Carcinoma, Squamous Cell; Female; Humans; Immunoenzyme Techniques; Lung Neoplasms; Male; Metallothionein; Middle Aged

Patients with small cell carcinoma of the lung (SCLC) are known to have an extremely poor prognosis, with a 5-year survivor rate of only 5%. Chemotherapeutic drug resistance is a major obstacle to curative therapy in patients with SCLC.. The authors evaluated retrospectively the expression of metallothionen (MT), proliferating cell nuclear antigen (PCNA), p53, and retinoblastoma gene product (RBGP) in biopsy samples from 58 patients with SCLC prior to standard chemotherapy. The objective was to study the correlation between MT and other molecular markers in SCLC and correlate these data with the clinical outcome of patients. The authors studied 28 short-term survivors (STS; survival < 24 months) and 30 long-term survivors (LTS; survival > 24 months).. In line with expectations, the authors found a strong inverse association between stage and survival. Of 58 patients with SCLC, 26 patients (45%; 17 STS and 9 LTS) showed MT expression, 55 patients (94%; 28 STS and 27 LTS) were positive for PCNA, 28 patients (48%; 16 STS and 12 LTS) were positive for p53, and only 6 patients (10%; 1 STS and 5 LTS) showed positivity for RBGP. On comparing the percent positivity of various markers in the two survivor groups, there was greater frequency of expression of MT, PCNA, and p53 and lower RBGP expression in the STS group compared with the LTS group. However, only the difference in expression of MT between the two survivor groups was statistically significant (Fisher exact test; P = 0.034). Multivariable analysis using a logistic regression model showed a significant association between MT expression and patient survival after adjusting for disease stage (chi-square test; P = 0.022). There was also a statistically significant association between MT expression and p53 expression (chi-square test; P = 0.001).. In this study, of the molecular markers studied, the authors demonstrated that only MT overexpression was independently predictive of short-term survival in patients with SCLC undergoing chemotherapy.

Topics: Adult; Aged; Biomarkers; Carcinoma, Small Cell; Female; Humans; Immunoenzyme Techniques; Logistic Models; Lung Neoplasms; Male; Metallothionein; Middle Aged; Neoplasm Staging; Prognosis; Proliferating Cell Nuclear Antigen; Retinoblastoma Protein; Retrospective Studies; Survival Analysis; Tumor Suppressor Protein p53

The combination of VP-16 and cisplatin is one of the most active regimens available for the treatment of small cell lung cancer (SCLC), however, most tumors eventually become resistant to these drugs.. To investigate the problem of resistance to VP-16 and cisplatin in patients with SCLC, we established two resistant sublines from the drug sensitive human SCLC line, NCI-H209, by in vitro selection in VP-16 and cisplatin.. The VP-16-selected cell line, H209/VP, was more than 100-fold resistant to VP-16, and displayed cross-resistance to VM-26 and other topoisomerase II interactive drugs, but not to vinca alkaloids. There was no difference in accumulation of VP-16 in H209/VP compared with its parent cell line. The level of topoisomerase II-alpha was reduced to 8% of that in the parent cell line, and there was an altered form of this enzyme with a molecular weight of 160 kilodaltons (kDa), in addition to the normal 170 kDa protein. The cisplatin-selected cell line, H209/CP, was 11.5-fold resistant to cisplatin, with only a low level of cross-resistance to other platinum compounds including carboplatin, tetraplatin, iproplatin, and lobaplatin. This line was highly cross-resistant to vinca alkaloids, but not to anthracyclines or epipodophyllotoxins. The H209/CP cell line was not resistant to cadium chloride, suggesting that alterations in metallothionein are unlikely to be a cause of resistance. Although glutathione (GSH) levels were increased nearly 2-fold in H209/CP, there was no difference in levels of the GSH-related enzymes glutathione-S-transferase, glutathione peroxidase, and glutathione reductase, compared with the parent line. The H209/CP line had a 1.4-fold elevation of topoisomerase II-alpha. The accumulation of cisplatin was reduced in this cell line, and there were fewer DNA-interstrand cross links formed in the presence of cisplatin in H209/CP, compared with the parent line. Neither H209/VP nor H209/CP expressed MDR1, the gene for P-glycoprotein. The MRP gene was expressed at a slightly higher level in the H209/VP cell line, but there was no significant increase in expression of this gene in the H209/CP cell line.. The resistance of the H209/VP cell line is associated with an alteration of topoisomerase II-alpha, whereas the resistance in the H209/CP line is associated with reduced drug accumulation.

Topics: Antibiotics, Antineoplastic; Antineoplastic Agents; Antineoplastic Agents, Phytogenic; ATP Binding Cassette Transporter, Subfamily B, Member 1; Cadmium; Cadmium Chloride; Carcinoma, Small Cell; Cell Line; Chlorides; Cisplatin; Cross Reactions; DNA; DNA Topoisomerases, Type II; Drug Resistance, Neoplasm; Etoposide; Gene Expression Regulation, Neoplastic; Glutathione; Glutathione Peroxidase; Glutathione Reductase; Glutathione Transferase; Humans; Lung Neoplasms; Metallothionein; Platinum Compounds; Podophyllotoxin; Teniposide; Tumor Cells, Cultured; Vinca Alkaloids

Expression of v-ras(H) in NCI-H82 human small cell lung cancer (SCLC) cells results in a line (NCI-H82ras(H)) with a non-small cell phenotype (Mabry et al., Proc Natl Acad Sci USA 85: 6523-6527, 1988). This v-ras(H) -associated phenotypic change is prevented by treatment with trans-retinoic acid (tRA) (Kalemkarian et al., Cell Growth Differ 5: 55-60, 1994). The present studies were performed to examine changes in drug sensitivity that accompanied these phenotypic changes. v-ras(H) expression was associated with increased metallothionein-IIa (MT-IIa) mRNA and decreased levels of nonprotein sulfhydryls in NCI-H82ras(H) cells compared with -H82 cells. These changes were accompanied by the development of CdCl2 resistance without any change in cisplatin sensitivity. In contrast, growth of parental NCI-H82 cells in 1 microM tRa resulted in increased MT-IIa mRNA without any change in nonprotein sulfhydryls. In these cells, a 3.3-fold increase in cisplatin IC50 was observed. Examination of the action of topoisomerase (topo) poisons revealed that NCI-H82 and -H82ras(H) cells had indistinguishable levels of topo II polypeptides and indistinguishable sensitivities to etoposide, an agent that is often combined with cisplatin clinically. On the other hand, v-ras(H) expression was accompanied by a 2-fold increase in topo I activity and a 1.7-fold decrease in IC50 for the topo I-directed agent camptothecin. These changes resulted in 30-fold lower survival of NCI-H82ras(H) cells compared with -H82 cells at camptothecin concentrations as low as 10 nM. In summary, these studies demonstrate that chronic tRA treatment is accompanied by decreased cisplatin sensitivity in NCI-H82 human SCLC cells. In contrast, v-ras(H) expression is not associated with any change in cisplatin or etoposide sensitivity, but is accompanied by increased camptothecin sensitivity.

Topics: Antineoplastic Agents; Antineoplastic Agents, Phytogenic; Camptothecin; Carcinoma, Small Cell; Cell Survival; Cisplatin; Drug Resistance; Etoposide; Genes, ras; Humans; Lung Neoplasms; Metallothionein; Phenotype; RNA, Messenger; RNA, Transfer; Sulfhydryl Compounds; Transfection; Tumor Cells, Cultured

The expression of intrinsic resistance to cisplatin in two lung cancer cell lines, one derived from a small cell carcinoma (SW1271) and the other from an adenocarcinoma (A549), relative to a drug-sensitive small cell line SW900, was characterized by: (i) expression of cross-resistance to mitomycin C and cadmium chloride, but increased sensitivity to adriamycin and etoposide; (ii) significantly decreased cisplatin uptake; (iii) elevated levels of glutathione which could be reduced by buthionine L-sulfoximine resulting in significant sensitization of the cells to cisplatin; (iv) a lack of consistent modification of metallothionein content and expression of levels of glutathione S-transferase, glutathione reductase and glutathione peroxidase or of activities of DT-diaphorase or catalase; (v) significantly reduced total DNA-platination levels immediately following a 1 h cisplatin treatment with 10 micrograms/ml (33.3 microM); (vi) increased removal of Pt-GG and Pt-AG adducts by the A549 cells, consistent with increased repair capacity, but a lack of removal of these major adducts by the SW1271 cells indicative of tolerance of this drug-induced DNA damage. These data therefore provide evidence of differential formation, repair and tolerance of DNA damage following exposure of three human lung carcinoma cell lines to cisplatin.

Topics: Adenocarcinoma; Carcinoma, Small Cell; Catalase; Cisplatin; DNA; DNA Adducts; DNA Damage; DNA Repair; DNA, Neoplasm; Drug Resistance; Drug Screening Assays, Antitumor; Gene Expression; Glutathione; Glutathione Peroxidase; Glutathione Reductase; Glutathione Transferase; Humans; Kinetics; Lung Neoplasms; Metallothionein; NAD(P)H Dehydrogenase (Quinone); Tumor Cells, Cultured

We have established cis-diamminedichloroplatinum(II) (cisplatin) resistant human small cell lung cancer cell lines, H69/CDDP0.2 and H69/CDDP, to investigate the mechanism of acquired resistance to cisplatin. H69/CDDP0.2 and H69/CDDP were 6- and 11-fold resistant to cisplatin compared with the H69 parental cell line. H69/CDDP was also resistant to cadmium chloride (2-fold), cis-diammine(glycolato)platinum (4-fold), 4-hydroperoxycyclophosphamide (3-fold) and 3-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-1-(2-chloroethyl)-1-nitrosour ea (4-fold) if the drug concentrations that inhibit cell growth by 50% from growth inhibition assay were compared. There was no significant difference in the cisplatin accumulation among these cell lines. Although DNA interstrand cross-link formations, determined by filter elution assay in H69/CDDP0.2 and H69/CDDP, was decreased to 20 to 30% of that in H69 parental cells, the repair capacity of DNA interstrand cross-links was equivalent in all three cell lines. Intracellular glutathione content was equal in all cell lines. H69/CDDP had the highest glutathione S-transferase activity (H69, 11 nmol/min/mg protein, H69/CDDP0.2, 12 nmol/min/mg protein; H69/CDDP, 74 nmol/min/mg protein, respectively) and an overexpression of glutathione S-transferase pi mRNA. The drug concentrations that inhibit cell growth by 50% for cisplatin in all cell lines were decreased by treatment with ethacrynic acid, an inhibitor of glutathione S-transferase pi, but this did not alter the relative degree of resistance. Intracellular metallothionein content (H69, 14 pmol/mg protein, H69/CDDP0.2, 22 pmol/mg protein; H69/CDDP, 33 pmol/mg protein, respectively) and expression of metallothionein mRNA were correlated with the drug concentrations that inhibit cell growth by 50% of the three cell lines for cisplatin and cadmium chloride. The present study suggested the importance of metallothionein in the mechanisms of cisplatin resistance.

Topics: Antineoplastic Agents; Carcinoma, Small Cell; Cell Line; Cell Survival; Cisplatin; Drug Resistance; Glutathione; Glutathione Transferase; Humans; Kinetics; Lung Neoplasms; Metallothionein