metallothionein and Lung-Neoplasms

Metallothioneins (MTs) are low weight proteins involved in several key cellular processes such as metal ions homeostasis, detoxification and scavenging of free radicals. Four groups of MTs are distinguished: MT-1, MT-2, MT-3 and MT-4. Regardless of the type, MTs are characterized by high content of cysteine, responsible for their biological properties such as binding of relevant zinc and copper ions, as well as toxic ions such as lead and cadmium. MTs were additionally shown to protect cells against oxidative stress damage and participate in differentiation, proliferation and/or apoptosis of normal and cancer cells. Many studies of different neoplasms showed association of elevated MTs levels with occurrence of chemo- and radiotherapy resistance and poor patients' outcome. In this review, we summarize and discuss the potential mechanism of action of metallotioneins in lung physiology and pathology.

Topics: Animals; Biomarkers, Tumor; Carcinoma; Humans; Lung Neoplasms; Metallothionein

Topics: Adenosine Triphosphate; ATP Binding Cassette Transporter, Subfamily B; ATP Binding Cassette Transporter, Subfamily B, Member 1; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Drug Resistance, Neoplasm; Genes, MDR; Humans; Lung Neoplasms; Metallothionein; Neoplasm Proteins

Data obtained from multiple sources indicate that no single mechanism can explain the drug resistance and the poor prognosis of patients with lung cancer. The resistance-related proteins P-glycoprotein, glutathione-dependent enzymes, topoisomerase II, metallothioneins, O-6-alkylguanine-DNA alkyltransferase, thymidylate synthase, dihydrofolate reductase and heat shock proteins have been found in lung carcinomas, but these alone cannot explain the drug-resistant phenotype. Cell cycle-related proteins, angiogenic factors, protooncogenes, and tumor suppressor genes also play a role in the phenotype that is resistant lung cancer. A key future challenge involves determining the relative quantitative contributions of each of these mechanisms to overall resistance.

Topics: Animals; ATP Binding Cassette Transporter, Subfamily B, Member 1; DNA Topoisomerases, Type II; Drug Resistance, Neoplasm; Genes, Tumor Suppressor; Glutathione; Glutathione Transferase; Heat-Shock Proteins; Humans; Lung Neoplasms; Metallothionein; Proto-Oncogenes; Tetrahydrofolate Dehydrogenase; Thymidylate Synthase; Tumor Cells, Cultured

Esterase D (ESD) is a nonspecific esterase widely distributed in various organisms. ESD plays an important role in regulating cholesterol efflux, inhibiting viral replication and lung cancer growth. MT2A (metallothionein 2A) is the most important isoform of metallothionein (MTs) in human and high expression of MT2A in tumors represents poor prognosis and metastatic behavior. However, there are no reports about the molecular mechanism of ESD in the regulation of tumor metastasis. In this study, we found for the first time that activation ESD promoted its interaction with MT2A and decreased the protein level of MT2A, which resulting in the concentration of free zinc ions up-regulated, and inhibited the migration of A549 lung cancer cells in vitro.

Topics: A549 Cells; Carboxylesterase; Cell Line, Tumor; Cell Movement; Humans; Lung Neoplasms; Metallothionein

Osteosarcoma is the most prevalent primary bone malignancy in children and young adults. Resistance to chemotherapy remains a key challenge for effective treatment of patients with osteosarcoma. The aim of the present study was to investigate the preventive role of metallothionein-2A (MT2A) in response to cytotoxic effects of chemotherapy. A panel of human and murine osteosarcoma cell lines, modified for MT2A were evaluated for cell viability, and motility (wound healing assay). Cell-derived xenograft models were established in mice. FFPE tumour samples were assessed by IHC. In vitro experiments indicated a positive correlation between half-maximal inhibitory concentration (IC50) for drugs in clinical practice, and MT2A mRNA level. This reinforced our previously reported correlation between MT2A mRNA level in tumour samples at diagnosis and overall survival in patients with osteosarcoma. In addition, MT2A/MT2 silencing using shRNA strategy led to a marked reduction of IC50 values and to enhanced cytotoxic effect of chemotherapy on primary tumour. Our results show that MT2A level could be used as a predictive biomarker of resistance to chemotherapy, and provide with preclinical rational for MT2A targeting as a therapeutic strategy for enhancing anti-tumour treatment of innate chemo-resistant osteosarcoma cells.

Topics: Animals; Antineoplastic Agents; Biomarkers, Tumor; Cell Death; Cell Line, Tumor; Cell Movement; Gene Expression Regulation, Neoplastic; Gene Silencing; Humans; Inhibitory Concentration 50; Lung Neoplasms; Metallothionein; Mice; Molecular Targeted Therapy; Osteosarcoma; RNA, Messenger; Xenograft Model Antitumor Assays

Lung cancer is the most commonly diagnosed cancer and the most frequent cause of death worldwide. Tesmin (testis‑specific metallothionein‑like protein; MTL‑5) is a 60‑kDa protein which has cysteine‑rich motifs (CXC domain), characteristic of metallothioneins (MTs). Tesmin expression has been observed in germ cells during spermatogenesis, oogenesis and also in various cell nuclei after exposure to heavy metal ions. Yet, the role of tesmin in carcinogenesis is unknown. The aim of the present study was to evaluate the localization and intensity of tesmin expression in non‑small cell lung cancer (NSCLC) and its association with the clinicopathological data of patients. A total of 121 cases of NSCLC and 20 cases of non‑cancerous tissue samples from the surgical margin (control) were used for immunohistochemistry (IHC). In addition, 20 cases of frozen NSCLC tissues and 20 cases of control were used for the in vivo study. Normal lung fibroblasts (IMR‑90) and lung cancer cell lines NCI‑H1703 (lung squamous cell carcinoma), NCI‑H522 and A549 (both adenocarcinomas of the lung) were used for western blot analysis (WB) and RT‑PCR studies. Positive cytoplasmic tesmin expression was observed in 88.42% of the examined cases of NSCLC. Statistical analysis showed increased IHC tesmin expression in cancer cells compared to that noted in the controls. In addition, MTL5 mRNA and WB tesmin protein expression were also higher in cancer cases compared to the controls. A positive correlation between tesmin and Ki‑67 IHC expression was demonstrated (r=0.32; P<0.001). Higher WB tesmin expression was also associated with shorter overall survival (P<0.05, Mantel‑Cox test). The in vitro study revealed higher tesmin protein (WB) and MTL5 (qPCR) in lung cancer cell lines compared to the lung fibroblast control cell line. Higher tesmin expression in cancer cells compared to control cells may suggest a role of tesmin in NSCLC carcinogenesis. A positive correlation between tesmin and Ki‑67 could indicate a possible role of tesmin in the proliferation of NSCLC.

Topics: A549 Cells; Adult; Aged; Aged, 80 and over; Carcinoma, Non-Small-Cell Lung; Case-Control Studies; Cell Line, Tumor; Cell Survival; Cytoplasm; Female; Humans; Lung Neoplasms; Male; Metallothionein; Middle Aged; Prognosis

Metallothioneins (MTs) are cysteine-rich proteins that act as antioxidants. A case-control study was conducted to assess the effects of gene polymorphisms in the MT region on the risk of lung cancer in Japanese subjects: 769 lung cancer cases and 939 non-cancer controls. Associations were evaluated using logistic regression models with adjustment for potential confounders (age, sex, and lifestyle factors including smoking, drinking, and green-yellow vegetable intake). We found five polymorphisms in the MT-1 gene region that showed statistically significant associations with lung cancer. Of these polymorphisms, rs7196890 showed the strongest association (odds ratio: 1.30, P = 0.004, 95% confidence interval: 1.09-1.55). The impact of the polymorphism decreased with the increase of smoking, and virtually no association with lung cancer was observed among heavy smokers whose pack-year values were 30 or more (odds ratio: 1.02, P = 0.93, 95% confidence interval: 0.67-1.55). These results suggest that polymorphisms in the MT gene are moderately associated with the risk of lung cancer and that the associations are modified by lifestyle factors.

Topics: Adult; Aged; Case-Control Studies; Confounding Factors, Epidemiologic; Female; Genetic Predisposition to Disease; Humans; Japan; Lung Neoplasms; Male; Metallothionein; Middle Aged; Polymorphism, Genetic

Inorganic arsenic is a human lung carcinogen. We studied the ability of chronic inorganic arsenic (2 μM; as sodium arsenite) exposure to induce a cancer phenotype in the immortalized, non-tumorigenic human lung peripheral epithelial cell line, HPL-1D. After 38 weeks of continuous arsenic exposure, secreted matrix metalloproteinase-2 (MMP2) activity increased to over 200% of control, levels linked to arsenic-induced cancer phenotypes in other cell lines. The invasive capacity of these chronic arsenic-treated lung epithelial (CATLE) cells increased to 320% of control and colony formation increased to 280% of control. CATLE cells showed enhanced proliferation in serum-free media indicative of autonomous growth. Compared to control cells, CATLE cells showed reduced protein expression of the tumor suppressor gene PTEN (decreased to 26% of control) and the putative tumor suppressor gene SLC38A3 (14% of control). Morphological evidence of epithelial-to-mesenchymal transition (EMT) occurred in CATLE cells together with appropriate changes in expression of the EMT markers vimentin (VIM; increased to 300% of control) and e-cadherin (CDH1; decreased to 16% of control). EMT is common in carcinogenic transformation of epithelial cells. CATLE cells showed increased KRAS (291%), ERK1/2 (274%), phosphorylated ERK (p-ERK; 152%), and phosphorylated AKT1 (p-AKT1; 170%) protein expression. Increased transcript expression of metallothioneins, MT1A and MT2A and the stress response genes HMOX1 (690%) and HIF1A (247%) occurred in CATLE cells possibly in adaptation to chronic arsenic exposure. Thus, arsenic induced multiple cancer cell characteristics in human peripheral lung epithelial cells. This model may be useful to assess mechanisms of arsenic-induced lung cancer.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Arsenic; Carcinogens; Cell Line, Tumor; Cells, Cultured; Epithelial Cells; Epithelial-Mesenchymal Transition; Humans; Lung Neoplasms; Metallothionein; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Phenotype; Proto-Oncogene Proteins; Proto-Oncogene Proteins p21(ras); ras Proteins

Although aberrant DNA methylation has been implicated in the pathophysiology of lung cancer, the role of methylation in multidrug resistance (MDR) of lung cancer has remained unclear. To investigate whether certain distinct DNA methylation pattern is associated with acquired MDR of lung adenocarcinoma, methylated-DNA immunoprecipitation-chromatin immunoprecipitation (MeDIP-ChIP) was utilised to compare the genome-wide promoter methylation of the human lung adenocarcinoma MDR A549/cisplatin (A549/DDP) cells with its progenitor A549 cells. The comparison identified 3617 genes with differentially methylated promoter, of which 1581 were hypermethylated and 2036 were hypomethylated. Then, bisulphite sequencing polymerase chain reaction (PCR) (BSP) and quantitative reverse transcription (RT)-PCR (Q-PCR) were used to validate the promoter methylation of five candidate genes and to determine whether the expression of genes was associated with the promoter methylation. BSP confirmed that the promoter methylation incidence of the hypermethylated genes, G protein-coupled receptor 56 isoform 3 (GPR56), metallothionein 1G (MT1G), and RAS association domain family gene 1 (RASSF1), was significantly higher in A549/DDP cells compared with A549 cells (p<0.001, p=0.0099, and p=0.0165), whereas no significant difference was found in that of the other two genes, CCNL2 and BAD (p=0.0594 and p=0.5546). Additionally, Q-PCR showed that the mRNA expression of the three hypermethylated genes was significantly lower in A549/DDP cells compared with A549 cells (all p<0.001). In conclusion, this study reported for the first time that a distinct promoter methylation pattern is associated with MDR of lung adenocarcinoma A549/DDP cells and suggested that GPR56, MT1G, and RASSF1 might be the potential methylation markers associated with acquired MDR of lung adenocarcinoma.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Cell Line, Tumor; DNA Methylation; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Genome-Wide Association Study; Humans; Lung Neoplasms; Metallothionein; Promoter Regions, Genetic; Receptors, G-Protein-Coupled; Tumor Suppressor Proteins

Nuclear factor (erythroid-derived 2)-like (Nrf)2 and metallothionein have been implicated in carcinogenesis. This study investigated the expression of Nrf2 and of Nrf2-targeted genes (NQO1 and GCLC) and the genes for the metallothionein (MT) isoforms (MT-1A and MT-2A) in human lung cancer and cancer-surrounding tissues.. Surgically removed lung cancer samples (n = 80) and cancer-surrounding tissues (n = 38) were collected from Zunyi Medical College Hospital, China. Total RNA was extracted, purified, and used for real-time reverse transcription-PCR analysis of interested genes.. Expression of the Nrf2-targed genes NQO1 and GCLC tended to be higher (30 to 60%) in lung cancers, but was not significantly different from that in peri-cancer tissues. By contrast, expression of the genes for M)-1A, MT-2A, and the metal transcription factor MTF-1 were three-fold to four-fold lower in lung cancers.. In surgical samples of lung cancer, MT expression was generally downregulated, whereas Nrf2 expression tended to be upregulated. These changes could play an integral role in lung carcinogenesis.

Topics: Biomarkers, Tumor; Humans; Lung Neoplasms; Metallothionein; NAD(P)H Dehydrogenase (Quinone); NF-E2-Related Factor 2; Prognosis; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

Cisplatin-based therapy is a pivotal type of chemotherapy for non-small cell lung cancer (NSCLC) and chemoresistance to cisplatin represents one of the most significant barriers to improving long-term clinical outcomes.. The present study aimed at examining metallothionein (MT) expression in six NSCLC cell lines as well as examining effects of exposure to cisplatin on MT expression in the most cisplatin-resistant (97/97) and the cisplatin-sensitive (DV90) cell lines.. The most cisplatin-resistant NSCLC cell line [97/97; (IC50)=4.659 μM] exposed to the highest concentration of cisplatin (10 μM) exhibited decreased nuclear MT expression (MTn=6) compared to cells cultured in medium with a lower concentration of cisplatin (0, 1 and 5 μM) (MTn=12). A higher cytoplasmic metallothionein expression (MTc=6) was found in the 97/97 cell line exposed to the highest concentration of cisplatin (10 μM), compared to cells cultured in the medium with lower concentrations of cisplatin (0, 1 and 5 μM) (MTc=3). The most cisplatin-sensitive NSCLC cell line (DV90; IC50=0.184 μM) was characterized by a significant decrease of both nuclear and cytoplasmic MT expression with increasing cisplatin concentrations (5 vs. 10 μM).. Nuclear and cytoplasmic expression of MT has no significant impact on the development of cisplatichemoresistance in NSCLC cell lines. The present study suggests that cisplatin resistance in NSCLC is metallothionein-independent.

Topics: Antineoplastic Agents; Carcinoma, Non-Small-Cell Lung; Cisplatin; Humans; In Vitro Techniques; Lung Neoplasms; Metallothionein

Metallothioneins (MT) are intracellular, low molecular weight proteins (6-7 kDa) involved in binding of metal ions, scavenging of free radicals, cell proliferation and apoptosis and resistance to certain chemotherapeutics. Four basic families of MT proteins are distinguished: MT-I, MT-II, MT-III, MT-IV, within each of them different isoforms occur. The study aimed at examining the expression level of nine MT isoforms: MT-1A, -1B, -1E, -1F, -1G, -1H, -1X, MT-2A and MT-IV by using real-time PCR and MT-I/II expression by immunohistochemical (IHC) technique in 69 cases of non-small cell lung cancer (NSCLC) and 12 non-malignant lung tissues (NMLT) and to correlate them with patients clinicopathological data and Ki-67 antigen expression. Out of all the analyzed cases, 62 (89.9%) demonstrated an increased MT-I/II expression. MT-1B, 1F, -1G, -1H and MT-1X were significantly up-regulated, whereas MT-1E was significantly down-regulated in NSCLC as compared to NMLT. Only in two cases MT-IV mRNA expression was noted. Significant positive correlations were observed between each particular MT isoform expressions. Higher MT-1F and MT-1A mRNA expression was associated with larger primary tumor size (P=0.0362 and P<0.0001, respectively). Moreover, up-regulated MT-1F mRNA expression was associated with higher grade of malignancy of NSCLC (P=0.0085). Higher MT-1B mRNA expression was associated with squamocellular and adenocarcinoma subtype of NSCLC (P=0.0358). Univariate analysis showed, that up-regulated MT-1F and MT-2A mRNA predicted poor patients' survival (P=0.0206 and P=0.0097, respectively). The levels of MT-1F and MT-2A mRNA could be considered as new markers of poor prognosis of NSCLC patients.

Topics: Biomarkers, Tumor; Carcinoma, Non-Small-Cell Lung; Down-Regulation; Female; Humans; Ki-67 Antigen; Lung Neoplasms; Male; Metallothionein; Middle Aged; Neoplasm Grading; Prognosis; Protein Isoforms; RNA, Messenger; Up-Regulation

Metallothioneins (MTs) are low molecular weight proteins present both in normal and neoplastic cells. They protect cells from the effects of heavy metals and from damage induced by free radicals. MT bind heavy metals, exert an anti-apoptotic effect and stimulate proliferation of neoplastic cells. The role of MTs in carcinogenesis has not been fully clarified yet. This study aimed at the evaluation of the intensity of metallothionein (MT-I/II) expression in various histological types of non-small cell lung cancer (NSCLC) and correlation of the expression intensity with clinical/pathological parameters and Ki-67 and minichromosome maintaince protein 2 (MCM-2) proliferation markers.. The studies were performed on archival material, originating from 145 patients, 105 men and 40 women (65 adenocarcinomas, 67 squamous cell carcinomas, 13 large cell carcinomas).. A positive correlation was noted between expression of MT-I/II and expressions of Ki-67 (r=0.1863, p=0.0248) and MCM-2 (r=0.1766, p=0.0336) in NSCLC overall. The most pronounced expression of MT-I/II was noted in the large cell carcinomas. The expression of MT-I/II was significantly lower in the adenocarcinomas than in the squamous cell carcinomas (p=0.0028) and large cell carcinomas (p=0.0485). The expression of MT-I/II showed no differences related to individual degrees of NSCLC malignancy. Univariate analysis demonstrated no significant differences in overall survival related to the expression intensity of MT-I/II, Ki-67 or MCM-2, but the survival of the patients with high expression of MT-I/II and Ki-67 in the neoplastic cells, as compared to low expression of MT-I/II and Ki-67, was shorter (the difference approached statistical significance, p=0.067).. MT-I/II expression is evident in proliferating NSCLC neoplastic cells, pointing to the prognostic importance of parallel expression of MT-I/II and Ki-67.

Topics: Biomarkers, Tumor; Carcinoma, Non-Small-Cell Lung; Cell Cycle Proteins; Cell Proliferation; Female; Humans; Ki-67 Antigen; Lung Neoplasms; Male; Metallothionein; Minichromosome Maintenance Complex Component 2; Nuclear Proteins

To identify the DNA methylation biomarkers for the detection of the stage I non-small cell lung cancer (NSCLC).. The methylated state of p16INK4A, ESR1, HOX9, RASSF1A, DAPK1, PTEN, ABCB1, MGMT, APC and MT1G genes that have been reported frequently methylated in lung cancer was determined using methylation-specific PCR in four lung cancer cell lines, 124 cancer tissues of the stage I NSCLC and 26 non-cancerous disease tissues.. The RASSF1A (53/124, 42.74%), APC (49/123, 39.52%), ESR1 (37/124, 29.84%), ABCB1 (31/124, 24.19%, MT1G (25/124, 20.16%) and HOXC9 (17/124, 13.71%) genes were more frequently methylated in the lung tissue from the stage I NSCLC than the non-cancerous lesion patients (2/26, 7.69%, P < 0.01; 2/26, 7.69%, P < 0.01; 2/26, 7.69%, P < 0.05; 1/26, 3.85% P < 0.01; 0/26 0%, P value: <0.01; 0/26, 0%, P < 0.05, respectively). p16INK4A was methylated in 28/124 (22.56%) of cancer tissues and 2/26 (7.69%) of non-cancerous tissues (P value >0.05). No significant association between the methylated state of the genes and the smoking, age or the pathologic types (squamous carcinoma, adenoma and the mixed types) was found. However, p16INK4A methylation was more frequently detected in the male (23/80, 28.75%) than the female (5/44, 11.36%, P > 0.05) patients. MGMT was barely methylated: 1/67, 1.49%), while DAPK1 and PTEN were not at all methylated in the cancer groups.. Methylation analysis in tissue of RASSF1A, APC, ESR1, ABCB1 and HOXC9 genes confirmed 79.8% of the existing diagnosis for the stage I NSCLC at specificity: 73.1%. The insufficiency of predicting disease onset in China, using the previously recommended targets (MGMT, DAPK1 and PTEN) in the United States reflects a potential disease disparity between these two populations. Alternatively, methylated state of this set of genes may be more specific to the late rather than the early stage of NSCLC.

Topics: Adult; Aged; Apoptosis Regulatory Proteins; ATP Binding Cassette Transporter, Subfamily B; ATP Binding Cassette Transporter, Subfamily B, Member 1; Calcium-Calmodulin-Dependent Protein Kinases; Carcinoma, Non-Small-Cell Lung; China; Death-Associated Protein Kinases; DNA Methylation; Estrogen Receptor alpha; Female; Genes, APC; Genes, p16; Homeodomain Proteins; Humans; Lung Neoplasms; Male; Metallothionein; Middle Aged; Neoplasm Staging; PTEN Phosphohydrolase; Tumor Cells, Cultured; Tumor Suppressor Proteins

The Long-Evans Cinnamon (LEC) rat, an animal model of Wilson's disease, is resistant to a variety of chemical carcinogenesis except liver and colon. In the present study, N-ethyl-N-nitrosourea (ENU)-induced transplacental carcinogenesis was examined in male and female LEC, Long-Evans Agouti (LEA), a sibling line of the LEC rat, and F344 rats (n=21). ENU was administered to pregnant rats as a single s.c. injection at a dose of 60 mg/kg body weight on the 17th day after conception. Cerebral/spinal gliomas and trigeminal/spinal nerve schwannomas developed in both LEA and F344 rats at 30 weeks of age, but no nervous system tumors developed in LEC rats, the difference being statistically significant. Lung adenomas also developed in LEA and F344 rats, but not in LEC rats. Semiquantitative RT-PCR demonstrated that metallothionein (MT)1a, MT2 and O(6)-methylguanine-DNA methyltransferase (MGMT) mRNA levels in the liver of LEC rats were higher than those in F344 and LEA rats. In addition, Western blot analysis showed that MT (MT1 plus MT2) in the liver of LEC rats was also higher than that in other strains. Present results suggest that high levels of MT and/or MGMT contribute to the resistance to nitrosamine-induced carcinogenesis in LEC rats.

Topics: Animals; Base Sequence; Carcinogens; Ethylnitrosourea; Female; Liver; Lung Neoplasms; Male; Maternal-Fetal Exchange; Metallothionein; Nervous System Neoplasms; O(6)-Methylguanine-DNA Methyltransferase; Pregnancy; Rats; Rats, Inbred F344; Rats, Inbred LEC; Rats, Long-Evans; RNA, Messenger; Species Specificity

New asymmetric organotellurides exhibiting good antioxidant properties in vitro and in cell culture can be attached to human serum albumin.

Topics: Antioxidants; Cell Survival; Glutathione Peroxidase; Humans; Kinetics; Lung Neoplasms; Metallothionein; Organometallic Compounds; Peroxides; Phenols; Serum Albumin; Sulfhydryl Compounds; Tellurium; Zinc

Minerals are important for cellular functions, such as transcription and enzyme activity, and are also involved in the metabolism of anticancer chemotherapeutic compounds. Profiling of intracellular elements in individual cells could help in understanding the mechanism of drug resistance in tumors and possibly provide a new strategy of anticancer chemotherapy. Using a recently developed technique of scanning X-ray fluorescence microscopy (SXFM), we analyzed intracellular elements after treatment with cis-diamminedichloro-platinum(II) (CDDP), a platinum-based anticancer agent. The images obtained by SXFM (element array) revealed that the average Pt content of CDDP-resistant cells was 2.6 times less than that of sensitive cells, and the zinc content was inversely correlated with the intracellular Pt content. Data suggested that Zn-related detoxification is responsible for resistance to CDDP. Of Zn-related excretion factors, glutathione was highly correlated with the amount of Zn. The combined treatment of CDDP and a Zn(II) chelator resulted in the incorporation of thrice more Pt with the concomitant down-regulation of glutathione. We propose that the generation of an element array by SXFM opens up new avenues in cancer biology and treatment.

Topics: Antineoplastic Agents; Cell Line, Tumor; Chelating Agents; Cisplatin; Drug Interactions; Drug Resistance, Neoplasm; Glutathione; Humans; Inactivation, Metabolic; Lung Neoplasms; Metallothionein; Microscopy, Fluorescence; Platinum; Spectrometry, X-Ray Emission; Zinc

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

The molecular predictor for cisplatin sensitivity in advanced non-small cell lung cancer (NSCLC) is still an open question at present. The purpose of this study was to evaluate the relationship of the cisplatin sensitivity/prognosis with the expression of excision repair cross complement-1 (ERCC-1), metallothionein (MT), and p53 in the paraffin-embedded tissue of advanced non-small cell lung cancer (NSCLC).. From January 1994 to December 2001, 51 pathologically confirmed advanced NSCLC patients were included. All patients received cisplatin contained regimen chemotherapy (Gemzar + DDP or NVB + DDP) as the first line treatment. At the same time, the tumor samples were collected and the expression of ERCC-1, MT, and p53 in the tumor samples were examined by immunohistochemical method. The response rates and survival data were analyzed according to the over-expression or not of these three parameters (negative group/over-expression group). The response rates were compared by Chi(2) test. Kaplan-Meier method and Cox proportional hazards model were used for survival analysis.. In these 51 patients, the expression rates of ERCC-1, MT, and p53 were 42.8%, 57.5%, and 37.8%, respectively. The response rates in negative group/over-expression group of ERCC-1, MT, and p53 were 33.3%/16.7%, 35.3%/27.3%, and 21.4%/35.3%, respectively (1.99, 1.29, and 0.61 times, respectively, P >0.05). The response rate in both ERCC-1 and MT negative group was 37.5%. In both ERCC-1 and MT over-expression (co-expression) group, the response rate was 14.3%. The former was 2.6 times of the latter (P >0.05). The average survival time in negative group/over-expression groups were 621/523 days (for ERCC-1), 556/479 days (for MT), 599/416 days (for p53), respectively. ERCC-1, MT, or p53 over-expression group showed poorer prognosis than those of negative group by Kaplan-Meier analysis. But in multivariate analysis, only p53 over-expression was an independent poor prognostic factor of survival time (Cox regression, P=0.009).. Whether over-expression of ERCC-1 and MT can be used as a molecular marker of cisplatin resistance in advanced NSCLC patients need further study. Over-expression of p53 predicates poor prognosis for patients with advanced NSCLC.

Topics: Adolescent; Adult; Aged; Antineoplastic Agents; Carcinoma, Non-Small-Cell Lung; Cisplatin; DNA-Binding Proteins; Drug Resistance, Neoplasm; Endonucleases; Female; Humans; Lung Neoplasms; Male; Metallothionein; Middle Aged; Neoplasm Staging; Prognosis; Proportional Hazards Models; Survival Rate; Tumor Suppressor Protein p53

Metallothionein (MT) is a low molecular weight protein, which participates in differentiation and proliferation of normal and tumour cells. In some malignant tumours (mammary, renal, ovarian cancers), its increased expression is thought to represent an unfavourable prognostic factor. Non-small-cellular lung cancers (mainly squamocellular cancer and adenocarcinoma) are characterised by ill-defined prognosis, which poses problems in the selection of effective post-surgical therapy. The present study aimed at demonstration of the prognostic significance of MT expression in cells of non-small cell lung cancers, attempting to correlate the intensity of MT expression with G grade and with the intensity of proliferation-associated antigen, Ki-67 expression. The studies were performed on archival paraffin blocks with samples of 25 cases of non-small cell lung cancers (5 squamous cell cancers, 20 adenocarcinomas). In paraffin sections of the studied tumours, immunocytochemical reactions were performed, using mouse monoclonal anti-MT and anti-Ki-67 antibodies. The expressions of MT and Ki-67 were demonstrated in all the studied tumours. An analysis of correlation between the expression of MT, Ki-67 antigen and G grade demonstrated a strong positive relation between the latter two parameters (r=0.70; p<0.05). Less pronounced positive correlations were disclosed between MT expression and G grade (r=0.44; p<0.05) and between MT expression and the expression of Ki-67 antigen (r=0.41; p<0.05). In addition, in 15 cases of examined tumours, survival analysis was performed, which disclosed a shorter survival in patients with high MT expression. The obtained results confirmed the relationship between MT expression and Ki-67 antigen expression, indicating an involvement of the proteins in processes of tumour cell proliferation. In turn, the shorter survival of patients with high expression of MT pointed to prognostic significance of the protein in non-small cell lung cancers.

Topics: Adenocarcinoma; Carcinoma, Non-Small-Cell Lung; Carcinoma, Squamous Cell; Humans; Ki-67 Antigen; Lung Neoplasms; Metallothionein; Survival Analysis

Apoptotic resistance can either be desirable or undesirable, depending on the conditions. In cancer chemotherapy, it is critical that tumor cells are selectively and effectively killed while leaving normal cells undamaged. Since acquisition of apoptotic resistance appears to be a common occurrence during malignant transformation, elucidating the mechanisms underlying apoptotic resistance is an area of intense study. Previous studies have revealed that metallothionein (MT) can protect cells from apoptosis induced by oxidative stress and metals. In the present study, we tested the hypothesis that the presence of MT may somehow modulate apoptosis. Our results revealed a strong linear negative correlation between basal MT levels and etoposide-induced apoptosis in the human tumor cell lines PLC/PRF/5, H460, and HepG2 (r = -0.991). In HepG2 cells, 24 h pretreatment with cadmium resulted in concentration-dependent increases in MT levels and marked decreases in etoposide-induced apoptosis. Zinc pretreatment also resulted in increased MT synthesis and decreased etoposide-induced apoptosis. More importantly, induced MT levels were negatively correlated with sensitivity to etoposide-induced apoptosis (r = -0.965). These suggest that MT may play a role in regulating apoptosis and that modulating MT expression may provide a strategy for altering cellular resistance to chemotherapeutic compounds.

Topics: Apoptosis; Cadmium; Carcinoma, Hepatocellular; Caspase 3; Caspases; Cell Line, Tumor; Cell Survival; DNA Fragmentation; Dose-Response Relationship, Drug; Drug Combinations; Etoposide; Hepatocytes; Humans; Liver Neoplasms; Lung Neoplasms; Metallothionein; Zinc

In 1999, the World Health Organization categorized pulmonary large cell neuroendocrine carcinoma as a variant of large cell carcinoma. However, an optimal treatment for large cell neuroendocrine carcinoma has not been established yet. Recently, multimodality therapy combining both surgery and adjuvant chemotherapy has been reported as a useful treatment for large cell neuroendocrine carcinoma, but the effect of chemotherapy on it has not yet been fully investigated. Thus, we evaluated immunohistochemical data of the expression of drug-resistant proteins in large cell neuroendocrine carcinoma.. We identified 10 large cell neuroendocrine carcinomas (1.2%) out of 850 primary lung cancers that had been surgically resected. We examined the immunohistochemical staining of three drug-resistant proteins, namely, P-glycoprotein, metallothionein and glutathione S-transferase-pi to compare large cell neuroendocrine carcinoma with other histological types of lung cancer.. The mean tumor cell positivity rates for P-glycoprotein, metallothionein and glutathione S-transferase-pi in large cell neuroendocrine carcinoma were 0%, 2.4 +/- 3.6% and 35.0 +/- 37.5%, respectively. The positivity rates for P-glycoprotein and glutathione S-transferase-pi were significantly lower than those in adenocarcinoma (P = 0.0003, P = 0.0009). The positivity rate for glutathione S-transferase-pi was also lower than that in squamous cell carcinoma (P = 0.0387). These drug-resistant proteins showed similar expression pattern in both large cell neuroendocrine carcinoma and small cell carcinoma except glutathione S-transferase-pi.. Immunohistochemical expression of drug-resistant proteins in large cell neuroendocrine carcinoma was lower than that in adenocarcinoma and squamous cell carcinoma, and differences exist in drug-resistance between large cell neuroendocrine carcinoma and small cell carcinoma.

Topics: Aged; Antineoplastic Combined Chemotherapy Protocols; ATP Binding Cassette Transporter, Subfamily B, Member 1; Carcinoma, Large Cell; Carcinoma, Neuroendocrine; Carcinoma, Non-Small-Cell Lung; Female; Follow-Up Studies; Glutathione S-Transferase pi; Glutathione Transferase; Humans; Immunohistochemistry; Isoenzymes; Japan; Lung Neoplasms; Male; Metallothionein; Middle Aged; Neoplasm Staging; Radiotherapy, Adjuvant; Surgical Procedures, Operative

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

Apoptosis involves a series of genetically programmed events associated with endonucleolytic cleavage of DNA. This process is triggered by a variety of agents, including oxidants such as hydrogen peroxide (H(2)O(2)) and it plays a key role in eliminating pre-neoplastic cells from the lung. Failure to do so could favor tumor promotion. The current study demonstrated that alveolar epithelial cells, adapted to cadmium (CdCl(2)) by repeated in vitro exposure, exhibit lower levels of H(2)O(2)-induced apoptosis than similarly challenged non-adapted cells. An immunologic assay, measuring cytoplasmic histone-associated DNA fragments, indicated maximal apoptosis 24 h after exposure to 400 microM H(2)O(2). Non-adapted cells showed a 13-fold increase in oxidant-induced apoptosis while Cd-adapted cells had only a 4-fold elevation. A terminal deoxyribonucleotidyl transferase mediated dUTP nick end labeling (TUNEL) method was used to assess the percentage of cells with DNA breaks consistent with apoptosis. Cd-adapted and non-adapted cells that were not exposed to H(2)O(2) did not differ in TUNEL positivity. However, after H(2)O(2) treatment, the percentage of TUNEL positive cells was 4-fold higher in non-adapted cultures than in adapted ones. Suppression of oxidant-induced apoptosis is due, in part, to up-regulation in the gene expression of several resistance factors including metallothioneins (MT-1 and MT-2), glutathione S-transferases (GST-alpha and GST-pi), and gamma-glutamylcysteine synthetase catalytic subunit (gamma-GCS). These steady-state mRNA changes, determined by Northern blotting, were accompanied by increased levels of MT and gamma-GCS protein, GST activity, and glutathione (GSH). Suppressed oxidant-induced apoptosis, resulting at least in part from these response modifications, could leave pre-neoplastic or neoplastic cells alive, favor clonal expansion, and ultimately lead to cancer development.

Topics: Adaptation, Biological; Apoptosis; Cadmium; Catalysis; Cell Nucleus; Drug Interactions; Enzyme-Linked Immunosorbent Assay; Epithelial Cells; Glutamate-Cysteine Ligase; Glutathione; Glutathione S-Transferase pi; Glutathione Transferase; Homeostasis; Humans; Hydrogen Peroxide; In Situ Nick-End Labeling; Isoenzymes; Lung Neoplasms; Metallothionein; Oxidants; Protein Isoforms; Pulmonary Alveoli; RNA, Messenger; Up-Regulation

Cellular and molecular mechanisms involved in the resistance to cytotoxic heavy metals remain largely to be characterized in mammalian cells. To this end, we have analyzed a metal-resistant variant of the human lung cancer GLC4 cell line that we have selected by a step-wise procedure in potassium antimony tartrate. Antimony-selected cells, termed GLC4/Sb30 cells, poorly accumulated antimony through an enhanced cellular efflux of metal, thus suggesting up-regulation of a membrane export system in these cells. Indeed, GLC4/Sb30 cells were found to display a functional overexpression of the multidrug resistance-associated protein MRP1, a drug export pump, as demonstrated by Western blotting, reverse transcriptase-polymerase chain reaction and calcein accumulation assays. Moreover, MK571, a potent inhibitor of MRP1 activity, was found to markedly down-modulate resistance of GLC4/Sb30 cells to antimony and to decrease cellular export of the metal. Taken together, our data support the conclusion that overexpression of functional MRP1 likely represents one major mechanism by which human cells can escape the cytotoxic effects of heavy metals.

Topics: Antimony; Antineoplastic Agents; Arsenites; ATP Binding Cassette Transporter, Subfamily B, Member 1; ATP-Binding Cassette Transporters; Blotting, Western; Cadmium Chloride; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Fluoresceins; Gene Amplification; Humans; Inhibitory Concentration 50; Lung Neoplasms; Meglumine; Metallothionein; Metals, Heavy; Multidrug Resistance-Associated Proteins; Propionates; Quinolines; RNA, Messenger; Tumor Cells, Cultured; Up-Regulation; Zinc Sulfate

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

Twenty tumoral and peritumoral tissues from patients with lung cancer were analyzed immunohistochemically for the drug resistance-related proteins P-glycoprotein (P-170), topoisomerase II (Topo-II), glutathione S-transferase-pi (GST-pi), metallothionein (MT), heat shock protein-70 (HSP-70) and the putative regulators of resistance (ErbB1, Fos and Jun). Protein expression of Topo-II, GST-pi, MT, HSP-70, ErbB1, Fos and Jun was elevated in tumor tissue in comparison to normal tissue. The different expression of the proteins between tumoral and normal tissues was statistically significant for Topo-II (P = 0.05), MT (P = 0.03), and HSP-70 (P = 0.01), whereas ErbB1 showed a borderline significance. The expression of the proteins was frequently increased in smokers in comparison to non-smokers. In general, the increase of the proteins of smokers corresponded in tumoral and non-tumoral tissue. Different expression was only found with MT and HSP-70 which were higher in tissues of smokers.

Topics: Adult; Aged; ATP Binding Cassette Transporter, Subfamily B, Member 1; DNA Topoisomerases, Type II; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Female; Glutathione Transferase; HSP70 Heat-Shock Proteins; Humans; Lung; Lung Neoplasms; Male; Metallothionein; Middle Aged; Neoplasm Proteins; Oncogene Proteins; Smoking

Microvessel density was investigated by immunostaining endothelial cells for factor VIII antigen in 84 non-small cell lung carcinomas and compared with the expression of several resistance-related proteins. Glutathione S-transferase-pi, thymidylate synthase, metallothionein and, with limitations, P-glycoprotein were overexpressed in tumors with poor vascularization.

Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Carcinoma, Non-Small-Cell Lung; Catalase; Drug Resistance, Neoplasm; Female; Glutathione Transferase; Humans; Isoenzymes; Lung Neoplasms; Male; Metallothionein; Middle Aged; Neoplasm Proteins; Neovascularization, Pathologic; Thymidylate Synthase; Up-Regulation

The aim of the study was to prove whether or not an association exists between the heat shock protein 70 (hsp70) and drug resistance. Tumor samples of 90 patients with previously untreated non-small lung carcinomas were investigated immunohistochemically for expression of resistance related proteins. Additionally, resistance to doxorubicin was determined using a short term test. No association between resistance related proteins. Additionally, resistance to doxorubicin was determined using a short term test. No association between resistance to doxorubicin and hsp70 was found. Of 63 resistant tumors, 33 showed low and 30 high hsp70 expression. Of the 26 sensitive tumors, 11 had low and 16 had high hsp70 expression. No relationship could be found between P-glycoprotein which is related to multidrug resistance and hsp70 expression or between hsp70 expression and expression of topoisomerase II, thymidylate synthase and metallothionein. On the other hand, a trend was noted for tumors with high glutathione S-transferase-pi expression to show high hsp70 expression. In addition, there was a significant relationship between hsp70 and catalase positivity. These data indicate that heat shock and stress promote intracellular oxidative damage and catalase is necessary for protection.

Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Carcinoma, Non-Small-Cell Lung; Catalase; DNA Topoisomerases, Type II; Doxorubicin; Drug Resistance; Glutathione Transferase; Heat-Shock Proteins; Humans; Immunoenzyme Techniques; Lung Neoplasms; Metallothionein; Thymidylate Synthase

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

Metallothionein (MT) shows a protective effect against toxic actions of some antitumor drugs and gamma-irradiation in animals. Preadministration of bismuth subnitrate, an MT inducing drug, significantly reduced side effects of antitumor drugs without affecting antitumor activity of the drugs. This specific effect of bismuth on the toxicity of antitumor drugs appears to be attributable to its specific induction of MT in normal tissues, but not in tumor tissue. Preinduction of MT synthesis in the lung also prevented mice from carcinogenesis caused by cisplatin and melphalan in the lung. On the other hand, zinc compound induced MT synthesis in the tumor, and significantly suppressed the antitumor activity of some antitumor drugs. Propargylglycine (PPG), an inhibitor of cystathionine pathway, significantly inhibited MT induction by zinc in the tumor inoculated in mice, and, consequently, PPG could diminish cisplatin resistance acquired by an increase in the tumor MT levels.

Topics: Alkynes; Animals; Antineoplastic Agents; Bismuth; Cisplatin; Drug Resistance; Female; Glycine; Lung Neoplasms; Melphalan; Metallothionein; Mice; Neoplasms, Experimental; Pargyline

There is sufficient evidence for pulmonary carcinogenicity of inhaled cadmium (Cd) compounds in rats whereas no such evidence was found in mice and hamsters, indicating significant species differences in the pulmonary response to inhaled Cd. We hypothesized that expression of metallothionein (MT) protein in the lung after inhalation of Cd differs between species thereby providing different degrees of sequestration of Cd and protection from its effects. Rats and mice were exposed to 100 micrograms CdCl2 aerosols/m3 for 4 weeks, and the presence of MT was determined in lung and free lung cell homogenates as well as by immunocytochemistry in lung sections up to 28 days post-exposure. Cd exposure significantly increased MT in homogenates of total lung in both species; however, no significant increase of MT in rat lung tissue after removal of free lung cells by lavage was found whereas MT was still significantly increased in lavaged mouse lung tissue throughout the post-exposure time. Histochemical analysis of lung sections revealed that mainly the epithelial cells of the bronchi, bronchioli and alveoli of Cd-exposed mice expressed MT. Baseline MT levels were also greater in the lungs of mice compared to rats. The retained Cd dose per g lung was about 2-fold greater in mice. The greater MT induction upon exposure to the same inhaled Cd concentration and the greater baseline MT levels may offer an explanation for the resistance of mice towards the pulmonary carcinogenic effect of inhaled Cd.

Topics: Administration, Inhalation; Animals; Bromodeoxyuridine; Cadmium; Disease Models, Animal; Humans; Lung; Lung Neoplasms; Male; Metallothionein; Mice; Mice, Inbred BALB C; Rats; Rats, Inbred F344; Species Specificity; Time Factors

Eleven pairs of surgically resected lung cancers and corresponding non-neoplastic lung tissue were evaluated for metallothionein (MT) and metal content (cadmium and copper) by the heme/109Cd binding assay and atomic absorption spectroscopy, respectively. Tissue samples, obtained from patients ranging in age from 51 to 79, included six adenocarcinomas, two small cell carcinomas, one mixed cell carcinoma, one squamous cell carcinoma, and one carcinoma of non-primary origin (i.e., melanoma). Paired t-tests showed that metallothionein and copper concentrations in lung tumor tissue were significantly elevated when compared to non-malignant lung tissue. Cu was the major metal associated with the 10 kDa MT fraction in lung tumors whereas Cd was the primary metal bound to MT from non-neoplastic lung tissue. Since Cu-thionein is also known to be elevated in fetal lung tissue, the possibility exists that MT might represent an oncodevelopmental product which is useful as a biomarker for the early detection of lung carcinoma.

Topics: Adenocarcinoma; Aged; Biomarkers, Tumor; Cadmium; Cadmium Radioisotopes; Carcinoma, Non-Small-Cell Lung; Carcinoma, Squamous Cell; Cell Transformation, Neoplastic; Chromatography, Agarose; Copper; Humans; Lung; Lung Neoplasms; Melanoma; Metallothionein; Middle Aged; Neoplasm Proteins; Protein Binding; Spectrophotometry, Atomic

Previously, we studied the ability of cadmium to initiate or promote tumors in B6C3F1 mice and, contrary to expectation, found that cadmium inhibited development of N-nitrosodiethylamine (NDEA)-initiated and sodium barbital-promoted liver tumors. In this study, the time course of cadmium inhibition of NDEA-initiated tumor formation was studied. A single dose of NDEA (90 mg/kg i.p.) was given at 5 weeks of age (time 0) followed by cadmium (1000 ppm) in drinking water from 2 to 48, 4 to 48, 8 to 48, 16 to 48 and 32 to 48 weeks. The study ended at 48 weeks. NDEA-induced elevations in liver tumor incidence (22 tumor-bearing mice/25 total) over control (5/25) were prevented by cadmium regardless of the period of administration (NDEA + cadmium: 2-48 weeks, 2/25; 4-48 weeks, 1/25; 8-48 weeks, 1/25; 16-48 weeks, 2/25; 32-48 weeks, 6/24). Cadmium alone (2-48 weeks) eliminated (0/25) spontaneously occurring liver tumors (5/25). NDEA-induced lung tumor incidence (25/25) and multiplicity (7.28 tumors/lung) were also reduced by cadmium (maximal decreases 28% and 80%, respectively). Some evidence of a specific deficiency of metallothionein in tumor cells was seen immunohistologically in NDEA-induced hepatic lesions and pulmonary lesions. These results indicate that cadmium prevents or reduces tumor formation in the B6C3F1 mouse liver and lung regardless of the exposure interval and apparently by cell-specific cytotoxicity. Auxiliary studies indicated that in mice bearing multiple liver foci resulting from NDEA treatment there was a marked reduction in basal metallothionein levels and in response to zinc induction.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Anticarcinogenic Agents; Body Weight; Cadmium; Diethylnitrosamine; Liver; Liver Neoplasms, Experimental; Lung; Lung Neoplasms; Male; Metallothionein; Mice; Mice, Inbred C3H; Mice, Inbred Strains; Time Factors

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 examined the efficacy of metallothionein induction in the prevention of the carcinogenic action of cis-platinum and melphalan administered repeatedly to mice over a relatively long period. The increased pulmonary metallothionein induced by bismuth or zinc compounds during the period of chemotherapy with cis-platinum or melphalan protected the mice from carcinogenesis of these drugs in the lung. These results suggested the efficacy of metallothionein inducers in suppression of carcinogenicity considered as a secondary effect of anticancer agents in cancer chemotherapy.

Topics: Animals; Anticarcinogenic Agents; Bismuth; Carcinogens; Chlorides; Cisplatin; Female; Lung; Lung Neoplasms; Melphalan; Metallothionein; Mice; Mice, Inbred A; Nitrates; Zinc Compounds

Forty-eight human squamous cell lung carcinomas of previously untreated patients were analyzed for resistance to doxorubicin and for the presence of topoisomerase II (Topo II), metallothionein (MT), thymidylate synthase (TS) and catalase (Cat). Significant correlations exist between resistance to doxorubicin measured by the in vitro short-term test and overexpression of MT and TS measured by immunohistochemistry. No significant correlation was found between resistance and expression of Topo II or Cat. No significant interrelationship between smoking habits of patients and expression of Topo II, MT, TS or Cat was found.

Topics: Adult; Aged; Carcinoma, Squamous Cell; Catalase; DNA Topoisomerases, Type II; Doxorubicin; Drug Resistance; Enzymes; Female; Humans; Immunohistochemistry; Lung Neoplasms; Male; Metallothionein; Middle Aged; Smoking; Thymidylate Synthase

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

Subpopulations T20 and T27, cloned from the human lung carcinoma line A549, differ significantly in their Cd2+ cytotoxic response. T27 has an LC50 of 31 microM Cd2+ and a cytotoxic response threshold of 5 microM Cd2+, whereas the T20s LC50 is 15 microM Cd2+ and there is no observed threshold for cytotoxicity. Cadmium-induced metallothionein (MT) synthesis, cadmium accumulation, glutathione (GSH) content, and Cd2(+)-induced changes in GSH content were studied in T20 and T27 in an attempt to determine the mechanism(s) causing differential cytotoxic response. MT synthesis measured by following Cd2(+)-induced [35S] incorporation into MT was found not to differ between T20 and T27. There is, however, a difference in Cd2+ accumulation between the two subclones. T20 and T27 cells were exposed to 5 microM Cd2+ for different times or to different concentrations of Cd2+ for 8 h. The T27 subline, which is the more Cd2+ resistant, was found to accumulate significantly more Cd2(+)-both as a function of time exposed to Cd2+ and as a function of Cd2+ concentration. The two subpopulations were found to have comparable initial GSH contents, but showed different Cd2(+)-induced changes in [GSH] when the cells were exposed to 5 microM Cd2+. T27 cells maintained their GSH content following Cd2+ exposure but T20 cells showed a Cd2(+)-induced decrease in GSH content. The results indicate that the difference in Cd2+ cytotoxic response between A549--T20 and A549--T27 cells is not attributable to alterations in MT synthesis nor to a difference in initial GSH content. Relative Cd2+ cytotoxicity also does not in these cells correlate with relative Cd2+ accumulation. The fact that T27 cells accumulate more Cd2+ and yet are more Cd2+ resistant than T20 cells suggests that T27 cells have a much more effective non-MT mechanism to handle intracellular Cd2+. This may involve different GSH metabolism and/or yet undefined molecular factors.

Topics: Cadmium; Cystine; Glutathione; Humans; Lung Neoplasms; Metallothionein; Tumor Cells, Cultured; Tumor Stem Cell Assay

Urokinase-type plasminogen activator, a neutral proteinase, seems to play a central role in the degradation of the extracellular matrix that accompanies a number of biological phenomena including inflammatory reactions and neoplasia. The effect of auranofin and retinoic acid on the plasminogen activator activity expressed by two cell types, i.e. murine macrophages and Lewis lung carcinoma cells, has been investigated. Low concentrations of both drugs (10(-6)-10(-7) M) can inhibit in vitro the induction of plasminogen activator in macrophages stimulated by phorbol 12-myristate 13-acetate. This action occurs rapidly (15 min), is irreversible and is independent of a global cytotoxic effect. Auranofin and retinoic acid remain without effect in macrophages when added after stimulation by the phorbol ester. Both drugs are thus potent inhibitors of the induction of plasminogen activator activity in macrophages, possibly through an interaction with the protein kinase C system. The plasminogen activator activity of Lewis lung carcinoma cells, which is apparently not dependent on a protein kinase C pathway, is not influenced by auranofin or retinoic acid. These observations may contribute to explain: (1) the activity of auranofin and retinoic acid in rheumatoid arthritis, and (2) the antitumor promoting activity of retinoic acid. It would be relevant to assess whether auranofin may exhibit, like retinoic acid, an antitumor-promoting activity.

Topics: Animals; Auranofin; Cell Line; Cells, Cultured; Enzyme Precursors; Kinetics; L-Lactate Dehydrogenase; Lung Neoplasms; Macrophages; Metallothionein; Mice; Plasminogen Activators; Tretinoin; Urokinase-Type Plasminogen Activator

Glutathione (GSH) depletion sensitizes human lung carcinoma (A549-T27) cells to the cytotoxic effects of Cd++. The effects of GSH depletion on Cd++ accumulation and Cd++-induced metallothionein (MT) content were investigated to determine the possible role of these Cd++ responses in the sensitization process. Cellular GSH was depleted to 20% to 25% of control levels with buthionine sulfoximine (BSO), or diethyl maleate (DEM), respectively. Neither treatment significantly affected Cd++-induced accumulation of exogenous 35s-cysteine into intracellular MT in a dose-dependent fashion. The results indicate that neither enhanced Cd++ accumulation nor reduced MT synthesis plays a primary role in affecting enhanced Cd++ cytotoxicity in A549 cells with reduced GSH levels. Although BSO inhibition of GSH synthesis enhanced MT synthesis, it sensitized the cells to Cd++, which suggests an additive effect of GSH and MT in cadmium cytoprotection. This observation also raises the possibility that intracellular cysteine levels limit Cd++-induced MT accumulation rates.

Topics: Buthionine Sulfoximine; Cadmium; Glutathione; Humans; Lung Neoplasms; Maleates; Metallothionein; Methionine Sulfoximine; Sulfur Radioisotopes; Tumor Cells, Cultured

This case report describes the natural history of chronic cadmium intoxication in a woman who was exposed to excessive cadmium in her occupation. We document the clinical, laboratory, and tissue response to the toxicant.

Topics: Adenocarcinoma; Cadmium; Cadmium Poisoning; Environmental Exposure; Female; Humans; Kidney; Liver; Lung Neoplasms; Metallothionein; Microscopy, Electron; Middle Aged; Occupational Diseases