ABSTRACT
The mitotic spindle is assembled by the coordinated action of centrosomes and kinetochore microtubules. An evolutionally conserved protein family, transforming acidic coiled-coil (TACC), has been shown to be involved in this process. In humans, TACC3 is aberrantly expressed in a variety of human cancers, but its biological significance remains to be elucidated. Here, using a novel compound targeting TACC3, spindlactone (SPL), we show that the perturbation of TACC3 selectively inhibited the nucleation of centrosome microtubules in ovarian cancer cells. In contrast to centrosome microtubules, the kinetochore microtubules were robustly assembled, forming ectopic spindle poles that resulted in multipolar spindles. Interestingly, the extensive inhibition of TACC3 partially suppressed the nucleation of kinetochore microtubules. These dose-dependent effects of SPL were consistent with the results observed by the depletion of TACC3 and its binding partner, colonic and hepatic tumor overexpressed gene protein (TOGp). Although these proteins both have roles in the assembly of centrosome and kinetochore microtubules, their contributions were spatiotemporally different. Notably, SPL did not affect spindle assembly in normal cells. Furthermore, the oral administration of SPL significantly suppressed tumor growth in vivo. The unique mechanism of action of SPL not only enables it to be used as a tool to dissect the molecular basis of spindle assembly but also to provide a rationale for the use of TACC3 as a molecular target for cancer treatment. This rationale offers an opportunity to develop new strategies for cancer chemotherapy that overcome the limitations of microtubule toxins and expand their scope and clinical efficacy.
Subject(s)
Antineoplastic Agents/pharmacology , Coumarins/pharmacology , Microtubule-Associated Proteins/metabolism , Spindle Apparatus/metabolism , Animals , Cell Line, Tumor , Cell Proliferation/drug effects , Centrosome/metabolism , Heterografts , Humans , Mice , Mice, Inbred NOD , Mice, SCID , Microtubule-Associated Proteins/antagonists & inhibitors , Microtubules/metabolism , Time-Lapse Imaging , Tumor Burden/drug effectsABSTRACT
The formation of the bipolar spindle is responsible for accurate chromosomal segregation during mitosis. The dynamic instability of microtubules has an important role in this process, and has been shown to be an effective target for cancer chemotherapy. Several agents that target non-microtubule mitotic proteins, including the motor protein Eg5, Aurora kinases and Polo-like kinases, are currently being developed as chemotherapeutic drugs. However, because the efficacies of these drugs remain elusive, new molecular targets that have essential roles in tumor cells are desired. Here, we provide in vivo evidence that transforming acidic coiled-coil-3 (Tacc3) is a potential target for cancer chemotherapy. Using MRI, we showed that Tacc3 loss led to the regression of mouse thymic lymphoma in vivo, which was accompanied by massive apoptosis. By contrast, normal tissues, including the thymus, showed no overt abnormalities, despite high Tacc3 expression. in vitro analysis indicated that Tacc3 depletion induced multi-polar spindle formation, which led to mitotic arrest, followed by apoptosis. Similar responses have been observed in Burkitt's lymphoma and T-ALL. These results show that Tacc3 is a vulnerable component of the spindle assembly in lymphoma cells and is a promising cancer chemotherapy target.
Subject(s)
Burkitt Lymphoma/pathology , Carrier Proteins/physiology , Fetal Proteins/physiology , Lymphoma/pathology , Neoplasm Regression, Spontaneous/genetics , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/pathology , Thymus Neoplasms/pathology , Animals , Burkitt Lymphoma/genetics , Carrier Proteins/genetics , Fetal Proteins/genetics , Genes, p53 , Humans , Lymphoma/genetics , Mice , Microtubule-Associated Proteins , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/genetics , Thymus Neoplasms/geneticsABSTRACT
BACKGROUND: We investigated, in the panel of 60 human tumour cell lines of the National Cancer Institute (NCI-60), whether the R72P polymorphism of TP53 and the T309G polymorphism of MDM2 were associated to the in vitro cytotoxicity of anticancer agents, extracted from the NCI database. For validation, the same study was performed independently on a second panel of tumour cell lines, JFCR-45. METHODS: Both SNPs were identified in cell DNA using PCR-RFLP techniques confirmed by direct sequencing and by pyrosequencing. For the analysis of the results, the mutational status of p53 was taken into account. RESULTS: In the NCI-60 panel, the TP53 rare-allele frequency was 32% and the MDM2 rare-allele frequency 39%. The MDM2 alleles were distributed according to Hardy-Weinberg equilibrium whereas this was only found, for the TP53 alleles, in p53 non-mutated cell lines. Comparable results were obtained in the JFCR-45 validation set. The TP53 SNP had low impact on anticancer drug cytotoxicity in either panel. In contrast, the MDM2 gene polymorphism had a major impact on anticancer drug cytotoxicity, essentially in p53 non-mutated cell lines. Presence of the rare allele was associated to significantly higher MDM2 protein expression and to increased sensitivity to DNA-interfering drugs. In the JFCR-45 panel, a similar effect of the MDM2 gene polymorphism was observed, but was less dependent on the p53 mutational status. CONCLUSIONS: We hypothesised that cell lines harbouring the MDM2 G allele presented a lower availability of p53 for DNA repair, translating into higher sensitivity to DNA-damaging agents.
Subject(s)
Genes, p53 , Neoplasms/drug therapy , Neoplasms/genetics , Proto-Oncogene Proteins c-mdm2/genetics , Alleles , Cell Line, Tumor , Drug Screening Assays, Antitumor , Gene Expression , Genotype , Humans , Mutation , Polymerase Chain Reaction , Polymorphism, Restriction Fragment Length , Polymorphism, Single Nucleotide , Proto-Oncogene Proteins c-mdm2/biosynthesis , Tumor Suppressor Protein p53/biosynthesis , Tumor Suppressor Protein p53/geneticsABSTRACT
We found that a lectin, Datura stramonium agglutinin, induced irreversible differentiation in C6 glioma cells. The differentiated cells had long processes, a low rate of proliferation and a high content of glial fibrillary acidic protein. When the medium was replaced with Datura stramonium agglutinin-free medium after 1 h, cell proliferation continued to be inhibited. Experiments with several other lectins indicated that both recognition of linear N-acetyllactosamine repeats and recognition of multiantennary units of cell-surface glycans were required for the inhibition of C6 proliferation. Proliferation of four human glial tumour cells was also inhibited by Datura stramonium agglutinin. Further, these differentiated human glial tumour cells had long processes and a high content of glial fibrillary acidic protein similar to differentiated C6 glioma cells. Taken together, these observations suggest that Datura stramonium agglutinin may be useful as a new therapy for treating glioma without side effects.
Subject(s)
Agglutinins/pharmacology , Brain Neoplasms/pathology , Cell Differentiation/drug effects , Cell Division/drug effects , Datura stramonium/chemistry , Glioma/pathology , Lectins/pharmacology , Astrocytes/cytology , Astrocytes/metabolism , Brain Neoplasms/metabolism , Cell Line , Glial Fibrillary Acidic Protein/metabolism , Glioma/metabolism , Humans , Tumor Cells, CulturedABSTRACT
Estrogen plays an important role in many physiological events including carcinogenesis and the development of human breast cancer. However, the molecular mechanisms of estrogen signaling in cancers have not been clarified hitherto and accurate therapeutic prediction of breast cancer is earnestly desired. We first carried out estrogen-responsive expression profiling of approximately 9000 genes in estrogen receptor-positive human MCF-7 breast cancer cells. Based on the results, estrogen-responsive genes were selected for production of a custom-made cDNA microarray. Using a microarray consisting of the narrowed-down gene subset, we first analyzed the time course of the estrogen-responsive gene expression profiles in MCF-7 cells, resulting in subdivision of the genes up-regulated by estrogen into early-responsive and late-responsive genes. The expression patterns of several genes were confirmed by Northern blot analysis. We also analyzed the effects of the estrogen antagonists ICI 182780 and 4-hydroxytamoxifen (OHT) on the estrogen-responsive gene expression profiles in MCF-7 cells. While the regulation of most of the genes by estrogen was completely abolished by ICI 182780, some genes were partially regulated by estrogen even in the presence of OHT. Furthermore, the estrogen-responsive gene expression profiles of twelve cancer cell lines derived from the breast, ovary, stomach and other tissues were obtained and analyzed by hierarchical clustering including the profiles in MCF-7 cells. Several genes also showed up-regulation or down-regulation by estrogen in cell lines other than MCF-7 cells. The significance of the estrogen-responsive genes identified in these analyses concerning the nature of cancer is discussed.
Subject(s)
Estrogens/physiology , Gene Expression Profiling/methods , Oligonucleotide Array Sequence Analysis/methods , Blotting, Northern , Breast Neoplasms/diagnosis , Breast Neoplasms/genetics , Estradiol/pharmacology , Estrogen Antagonists/pharmacology , Estrogen Receptor alpha , Female , Humans , Neoplasms, Hormone-Dependent/diagnosis , Neoplasms, Hormone-Dependent/genetics , Receptors, Estrogen/analysis , Tumor Cells, CulturedABSTRACT
BACKGROUND: Macrophages often infiltrate into solid tumor tissues. Tumor-associated macrophages (TAMs) are known to play a crucial role in tumor progression. Monocyte chemoattractant protein-1 (MCP-1) is one of the major chemokines capable of inducing chemotactic migration of monocytes. METHODS: With the objective of investigating the clinical significance of MCP-1, the authors analyzed the expression of MCP-1 and of some other molecules by immunohistochemistry in 230 samples of primary breast carcinoma tissue. MCP-1 staining was performed using an anti-MCP-1 monoclonal antibody, and it was assessed by grading the percentage of stained cells. RESULTS: It was found that 117 breast tumor specimens (51%) had intensive staining in tumor cells. The expression of MCP-1 in tumor cells had a significant correlation with the expression of thymidine phosphorylase and membrane type 1-matrix metalloproteinase. In addition, MCP-1 expression tended to be associated with the accumulation of TAMs, which were counted by CD68 staining, and with microvessel density. MCP-1 expression in TAMs was correlated significantly with the histologic vessel invasion of tumor cells. CONCLUSIONS: The results of this study suggest that MCP-1 may play key roles in macrophage recruitment, in the expression of angiogenic factors, and in the activation of matrix metalloproteinases in patients with breast carcinoma.
Subject(s)
Breast Neoplasms/metabolism , Carcinoma, Ductal, Breast/metabolism , Chemokine CCL2/metabolism , Neovascularization, Pathologic , Angiogenesis Inducing Agents , Breast Neoplasms/blood supply , Breast Neoplasms/pathology , Carcinoma/metabolism , Carcinoma/pathology , Carcinoma, Ductal, Breast/blood supply , Carcinoma, Ductal, Breast/pathology , Humans , Immunohistochemistry , Neovascularization, Pathologic/metabolism , Receptors, Estrogen/metabolism , Receptors, Progesterone/metabolism , Tumor Cells, CulturedABSTRACT
PURPOSE: Glyoxalase I (GLO1) is an enzyme that plays a role in the detoxification of methylglyoxal, a side-product of glycolysis. We previously reported that GLO1 was a resistant factor to antitumor agent-induced apoptosis, and that S-p-bromobenzylglutathione cyclopentyl diester (BBGC), an effective inhibitor of GLO1, selectively sensitized to etoposide the drug-resistant human leukemia cells that overexpressed GLO1. In this study, we quantitatively measured GLO1 enzyme activity in various human solid tumor cells, and the antiproliferative effect of the GLO1 inhibitor was examined. EXPERIMENTAL DESIGN: BBGC-induced apoptosis was assessed by flow cytometry. To evaluate antitumor activity of BBGC in vivo, we developed human cancer xenografts in nude mice. RESULTS: We found that GLO1 enzyme activity was higher in all of the 38 human cancer cell lines that we examined than in the normal tissue samples. Moreover, GLO1 activity was frequently elevated in human lung carcinoma cells. Positive correlation between cellular GLO1 activity and BBGC sensitivity was observed in the lung cancer cell lines. Human lung cancer NCI-H522 and DMS114 cells, expressing higher GLO1 activity, underwent apoptosis when treated with BBGC, whereas A549 cells, expressing lower activity, did not. BBGC induced the activation of the stress-activated protein kinases c-Jun NH(2)-terminal kinase 1 (JNK1) and p38 mitogen-activated protein kinase (MAPK), which led to caspase activation in GLO1-overexpressing tumor cells. BBGC significantly inhibited the growth of xenografted DMS114 and human prostate cancer DU-145. CONCLUSIONS: Our present results indicate that GLO1 is a tumor-specific target enzyme especially in human lung carcinoma cells and that the GLO1 inhibitor is a potent chemotherapeutic agent to repress GLO1-overexpressing human tumors.
Subject(s)
Apoptosis/drug effects , Enzyme Inhibitors/pharmacology , Glutathione/pharmacology , Lactoylglutathione Lyase/antagonists & inhibitors , Lung Neoplasms/prevention & control , Animals , Caspases/drug effects , Caspases/metabolism , Cell Division/drug effects , Dose-Response Relationship, Drug , Enzyme Activation/drug effects , Glutathione/analogs & derivatives , Humans , Lactoylglutathione Lyase/metabolism , Lung Neoplasms/enzymology , Lung Neoplasms/pathology , Mice , Mice, Nude , Mitogen-Activated Protein Kinase 8 , Mitogen-Activated Protein Kinases/drug effects , Mitogen-Activated Protein Kinases/metabolism , Time Factors , Xenograft Model Antitumor AssaysABSTRACT
The higher frequency of human lung adenocarcinoma in females than in males, strongly suggests the involvement of gender dependent factors in the etiology of this disease. This is the first investigation of estrogen receptor (ER) beta in human lung. Immunohistochemical staining revealed ERbeta expression in normal lung and in atypical adenomatous hyperplasia (AAH), considered as a precancerous lesion for adenocarcinomas. Adenocarcinomas showed significantly higher expression of ERbeta than squamous cell carcinomas. On the contrary, ERalpha expression was not detected in all cases. The functional integrity of ERbeta such as the binding ability to estrogen responsive element (ERE) and transcriptional activity was confirmed using a human lung cancer cell line, RERF-LC-OK. Colony formation of this cell was significantly reduced in the presence of pure antiestrogen. We conclude that ERbeta, but not ERalpha, is present in lung tissues with an important physiological function in normal lung. Furthermore, ERbeta may play a role in growth and development of adenocarcinomas.
Subject(s)
Lung Neoplasms/pathology , Receptors, Estrogen/physiology , Adenocarcinoma/genetics , Adenocarcinoma/pathology , Adenocarcinoma/physiopathology , Aged , Bronchi/cytology , Bronchi/physiology , Carcinoma, Squamous Cell/genetics , Carcinoma, Squamous Cell/pathology , Carcinoma, Squamous Cell/physiopathology , Cell Adhesion , Estrogen Receptor beta , Female , Genes, Reporter , Humans , Hyperplasia , Lung/cytology , Lung/pathology , Lung/physiology , Lung Neoplasms/genetics , Lung Neoplasms/physiopathology , Male , Middle Aged , Receptors, Estrogen/analysis , Receptors, Estrogen/genetics , Reference Values , Respiratory Mucosa/cytology , Respiratory Mucosa/physiology , Transfection , Tumor Cells, CulturedABSTRACT
In vitro cytotoxic activities of 24 quaternary protoberberine alkaloids related to berberine have been evaluated using a human cancer cell line panel coupled with a drug sensitivity database. Extending the alkyl chain at position 8 or 13 strongly influenced the cytotoxic activity, that is, relative lipophilicity as well as the size of the substituent affects cytotoxicity. The highest level of activity was observed in 8- or 13-hexyl-substituted derivatives of berberine. Structure-activity relationships are described.
Subject(s)
Antineoplastic Agents, Phytogenic/chemical synthesis , Berberine Alkaloids/chemical synthesis , Berberine/analogs & derivatives , Berberine/chemical synthesis , Animals , Antineoplastic Agents, Phytogenic/chemistry , Antineoplastic Agents, Phytogenic/pharmacology , Berberine/chemistry , Berberine/pharmacology , Berberine Alkaloids/chemistry , Berberine Alkaloids/pharmacology , Brain Neoplasms/metabolism , Breast Neoplasms/metabolism , Colonic Neoplasms/metabolism , Crystallography, X-Ray , Databases, Factual , Doxorubicin/pharmacology , Female , Humans , Lung Neoplasms/metabolism , Magnetic Resonance Spectroscopy , Mice , Mitomycin/pharmacology , Molecular Structure , Ovarian Neoplasms/metabolism , Papaver/chemistry , Plants, Medicinal , Stomach Neoplasms/metabolism , Structure-Activity Relationship , Tumor Cells, Cultured/drug effectsABSTRACT
Cyclic hydroxamic-acid-containing peptide 1 (CHAP1), designed as a hybrid of trichostatin A and trapoxin, is a lead compound for the development of potent inhibitors of histone deacetylase (HDAC). In this study, we synthesized a series of CHAP derivatives and evaluated their biological activities by monitoring the potency of their inhibition of HDAC activity, their ability to augment the expression of MHC class-I molecules in B16/BL6 cells, and their effect on cell proliferation. A structure-activity relationship study using these three assay systems revealed several requirements of their structure for the strong inhibition of HDAC not only in the cell-free situation, but also in cells. When the structures of CHAP derivatives are represented as cyclo(-Asu(NHOH)-AA(2)-AA(3)-Pro or Pip-)(n), where Asu(NHOH) and Pip are zeta-hydroxamide-alpha-aminosuberic acid and pipecolic acid, respectively, (a) the tetrapeptide structure (n = 1) was better than the octapeptide one (n = 2); (b) AA(2) and AA(3) should be hydrophobic; and (c) the combination of amino acid chirality should be LDLD for the strongest inhibition of HDAC in cells (LDLD > LLLD, LDLL > LLDL). cyclo(-L-Asu(NHOH)-D-Tyr(Me)-L-Ile-D-Pro-) or CHAP31 was selected as one of the strongest CHAPs, and its biological activity was characterized further. CHAP31 was much more stable in the presence of cultured cells (t(1/2) > 3000 h) than trichostatin A (t(1/2) = 14.7 h) or trapoxin A (t(1/2) = 2.10 h). CHAP31 exhibited antitumor activity in C57BL x DBA/2 F(1) (BD2F(1)) mice bearing B16/BL6 tumor cells. Furthermore, CHAP31 inhibited the growth in four of five human tumor lines implanted into nude mice. These results suggest CHAP31 to be promising as a novel therapeutic agent for cancer treatment.
Subject(s)
Antineoplastic Agents/pharmacology , Enzyme Inhibitors/pharmacology , Histone Deacetylase Inhibitors , Peptides, Cyclic/pharmacology , Animals , Antineoplastic Agents/chemistry , Drug Screening Assays, Antitumor , Enzyme Inhibitors/chemistry , Humans , Hydroxamic Acids/chemistry , Melanoma, Experimental/drug therapy , Melanoma, Experimental/enzymology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Inbred DBA , Mice, Nude , Peptides, Cyclic/chemistry , Proline/chemistry , Stereoisomerism , Structure-Activity Relationship , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
13,14-Dihydro-15-deoxy-Delta7-prostaglandin A1 methyl ester (TEI-9826), an antitumor prostaglandin analog, is a candidate for clinical trial. In the present study, we examined its biological stability in vitro, antitumor activity in vitro and in vivo, and pharmacokinetics. Although TEI-9826 was rapidly hydrolyzed to the carboxylic acid form (TOK-4528), TOK-4528 as well as Delta12-prostaglandin J2 (PGJ2) were found to be stable in rat, mouse and human serum in vitro. TEI-9826 exhibited nearly identical or greater potential antitumor activity compared to Delta12-PGJ2 and Delta7-PGA1 in vitro against Colon26 tumor cells. Further evaluation of TEI-9826 using the 38 human cancer cell lines panel and COMPARE analysis suggested that its mode of action is quite different from other anticancer agents that are currently used. TEI-9826 was integrated into lipid microspheres (Lipo TEI-9826) for dosing. Growth inhibition by Lipo TEI-9826 against Colon26 tumor inoculated s.c. in mice depended on administration route, i.e. at 80 mg/kg, no growth suppressive effect was observed for daily bolus i.v., but significant growth suppressive effect was observed for daily i.p., daily s.c. every other day s.c. and 4 times a day continuous (5 min) i.v. These tumor growth-suppressive effects were cytostatic and the tumor started to regrow at the end or a few days after the end of administration. The pharmacokinetic study suggested that maintaining the blood level of TEI-9826 and/or TOK-4528 was essential for their antitumor effects. These results show that continuous i.v. infusion might be the most suitable administration method of Lipo TEI-9826 for clinical trial.
Subject(s)
Antineoplastic Agents/administration & dosage , Colonic Neoplasms/drug therapy , Prostaglandins A/administration & dosage , Tumor Cells, Cultured/drug effects , Animals , Antineoplastic Agents/pharmacokinetics , Colonic Neoplasms/metabolism , Colonic Neoplasms/pathology , Dose-Response Relationship, Drug , Drug Carriers , Drug Delivery Systems , Drug Screening Assays, Antitumor , Drug Stability , Humans , Infusions, Intravenous , Liposomes , Male , Mice , Microspheres , Prostaglandins A/pharmacokinetics , Rats , Rats, Wistar , Tumor Stem Cell AssayABSTRACT
IL-12 directly acts on T cells and NK cells to induce IFN-gamma production. IFN-gamma plays an important role in anti-tumor effect of IL-12. In spite of various functions of IL-12 on immunocytes, the direct effect of IL-12 on tumor cells has not been fully clarified. The present study investigated the direct effect of IL-12 on eight murine tumor cell lines in vitro. IL-12 did not directly up-regulate expression of MHC class I on tumor cells, but enhanced IFN-gamma-induced up-regulation of MHC class I expression in MC-38, MCA102, MCA205 and MCA207 cells. IL-12 alone did not activate STAT1, but IL-12 enhanced IFN-gamma-mediated STAT1 phosphorylation in MC-38, MCA102, MCA205, MCA207 and Colon-26-NL-17 cells, which expressed IL-12 receptor beta1 mRNA. In the other side, Panc-02, B16-BL6 and 266-6 cells were not affected by IL-12, in which expression of IL-12 receptor beta1 mRNA was not detected. Anti-IL-12 mAb inhibited the direct effect of IL-12 on MC-38 cells. Moreover, nuclear localization of NF-kappaB was observed after stimulation of IL-12 or IL-12 p40 in MC-38 and Colon-26-NL-17 cells, but not in Panc-02 cells. These findings suggest that IL-12 directly acts on tumor cells through IL-12 receptor beta1 to activate NF-kappaB and enhance IFN-gamma-mediated STAT1 phosphorylation.
Subject(s)
DNA-Binding Proteins/metabolism , Interferon-gamma/pharmacology , Interleukin-12/pharmacology , NF-kappa B/metabolism , Trans-Activators/metabolism , Active Transport, Cell Nucleus/drug effects , Animals , Antibodies, Monoclonal/immunology , DNA/metabolism , Flow Cytometry , Histocompatibility Antigens Class I/metabolism , Interferon-gamma/metabolism , Interleukin-12/agonists , Interleukin-12/antagonists & inhibitors , Interleukin-12/immunology , Mice , Phosphorylation/drug effects , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptors, Interleukin/metabolism , Receptors, Interleukin-12 , STAT1 Transcription Factor , STAT4 Transcription Factor , Tumor Cells, Cultured , Up-Regulation/drug effectsABSTRACT
Most anticancer drugs cause DNA strand breaks and finally induce cell cycle arrest or cell death. To identify genes involved in these effects, we examined gene expression profiles in human lung cancer A549 cells before and after adriamycin treatment, using a cDNA array technique. In this manner, we identified several up- or down-regulated genes in cells undergoing G2 arrest following adriamycin treatment; among them, cyclin B1 expression was dramatically reduced. The reduction in cyclin B1 expression and G2 arrest were also seen after treatment with etoposide and irinotecan. Previous reports have shown that overexpression of p53 represses cyclin B1 transcription. However, cisplatin neither reduced cyclin B1 expression nor induced G2 phase arrest, while it induced a comparable amount of p53 protein. These results suggest that a reduction in cyclin B1 expression plays a role in the mechanism of action of certain anticancer drugs, including adriamycin, which induce G2 arrest in cancer cells.
Subject(s)
Cisplatin/pharmacology , Cyclin B/genetics , Doxorubicin/pharmacology , Antineoplastic Agents/pharmacology , Base Sequence , CDC2 Protein Kinase/metabolism , Cyclin B/metabolism , Cyclin B1 , DNA Primers/genetics , Down-Regulation/drug effects , Gene Expression Profiling , Gene Expression Regulation, Neoplastic/drug effects , Humans , Lung Neoplasms/drug therapy , Lung Neoplasms/genetics , Oligonucleotide Array Sequence Analysis , Phosphorylation , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Neoplasm/genetics , RNA, Neoplasm/metabolism , Tumor Cells, CulturedABSTRACT
Deoxynybomycin was identified as an inducer of p21the/WAF1 gene following screening using a reporter, p21/luciferase. The present study examined its anti-proliferative effect on human tumor cell lines. Deoxynybomycin selectively inhibited growth of human osteoblastic sarcoma Saos-2, gastric cancer TMK-1, and monocytic leukemia THP-1 cells, but did not affect survival of normal human fibroblasts at doses up to 5 microg/ml. Results from an assay system using a panel of 39 human cancer cell lines indicated that deoxynybomycin has selective cytotoxic activity against lung carcinoma cell lines. Deoxynybomycin induced apoptosis in Saos-2, TMK-1, and THP-1 cells as revealed by DNA fragmentation and TUNEL assays. It inhibited topoisomerase I but not topoisomerase II. These results suggest that deoxynybomycin may be useful in cancer chemotherapy.
Subject(s)
Antibiotics, Antineoplastic/pharmacology , Apoptosis/drug effects , Enzyme Inhibitors/pharmacology , Topoisomerase I Inhibitors , Cyclin-Dependent Kinase Inhibitor p21 , Cyclins/genetics , Humans , Promoter Regions, Genetic , Quinolones/pharmacology , Tumor Cells, CulturedABSTRACT
Metastasis, a major factor contributing to poor prognosis of cancer patients, is caused by a complex series of events that involve many genes. To investigate this process, we analyzed by differential display three cell lines that had been established from a murine colon adenocarcinoma (colon 26), NL4, NL17, and NL22, each of which possessed a different potential for metastasis in mice. We report here the identification of a novel gene, ream (reduced expression associated with metastasis), which showed significantly lower expression in NL17 and NL22 with a high potential for metastasis than in NL4 without a metastatic potential. The human counterpart of murine ream expressed two sizes of transcript, 4.4 kb and 1.8 kb, both encoding the same 367-amino acid peptide, which appeared to contain four membrane-spanning regions. The cDNA showed no significant homology to any known genes in the public database. Human REAM was found to lie within an 800-kb segment of 8p21.3-22, where we had previously identified a commonly deleted region in colorectal and hepatocellular carcinomas. Its expression was reduced in more than half of the human colorectal cancers we examined, particularly in advanced stages with liver metastasis. Furthermore, we identified somatic mutations of this gene in a colorectal cancer, a hepatocellular carcinoma, and a nonsmall lung cancer among 111 human tumors of various stages examined.
Subject(s)
Chromosomes, Human, Pair 8/genetics , Colorectal Neoplasms/genetics , Colorectal Neoplasms/pathology , Gene Expression Regulation, Neoplastic , Liver Neoplasms/genetics , Liver Neoplasms/secondary , Adenocarcinoma/genetics , Adenocarcinoma/pathology , Amino Acid Sequence , Animals , Cloning, Molecular , DNA Mutational Analysis , DNA, Complementary/genetics , Humans , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Mice , Molecular Sequence Data , Tumor Cells, CulturedSubject(s)
Antibiotics, Antineoplastic/pharmacology , Cell Division/drug effects , Neoplasms, Experimental/drug therapy , Animals , Graft Survival/drug effects , Humans , Mice , Mice, Nude , Neoplasm Transplantation , Neoplasms, Experimental/pathology , Random Allocation , Thiazines/pharmacology , Transplantation, Heterologous , Tumor Cells, Cultured/drug effectsABSTRACT
PURPOSE: We examined the anti-tumor effect of 5-S-GAD, a novel potent inhibitor of protein tyrosine kinases, isolated from the flesh fly in order to investigate the potential use of this compound as an anti-tumor agent. METHODS: In vitro growth inhibition was evaluated using the alamarBlue assay kit. In vivo anti-tumor activity was evaluated by i.p. treatment of 5-S-GAD against xenografted melanoma (LOX-IMV1) and breast carcinoma (MDA-MB-435S) in nude mice. RESULTS: Of 38 human cancer cell lines examined, this compound showed significant cytotoxicity toward two estrogen-negative breast carcinomas (MDA-MB-231 and MDA-MB-435S) and one malignant melanoma (LOX-IMV1) in vitro, indicating that it exhibits selective cytotoxicity to certain tumor cell lines. In accordance with its in vitro anti-tumor effect, 5-S-GAD was shown to significantly repress the growth of sensitive tumor cells in nude mice. CONCLUSION: These results indicate that 5-S-GAD is potentially useful to treat certain human cancer.
Subject(s)
Antineoplastic Agents/therapeutic use , Breast Neoplasms/pathology , Dihydroxyphenylalanine/analogs & derivatives , Diptera/chemistry , Glutathione/analogs & derivatives , Mammary Neoplasms, Experimental/drug therapy , Melanoma, Experimental/drug therapy , Melanoma/pathology , Animals , Antineoplastic Agents/pharmacology , Benzoquinones , Dihydroxyphenylalanine/pharmacology , Dihydroxyphenylalanine/therapeutic use , Drug Screening Assays, Antitumor , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/therapeutic use , Female , Glutathione/pharmacology , Glutathione/therapeutic use , Humans , Lactams, Macrocyclic , Mice , Mice, Nude , Molecular Structure , Neoplasm Proteins/antagonists & inhibitors , Neoplasm Transplantation , Protein-Tyrosine Kinases/antagonists & inhibitors , Quinones/pharmacology , Rifabutin/analogs & derivatives , Transplantation, Heterologous , Tumor Cells, Cultured/drug effects , Tumor Cells, Cultured/transplantationABSTRACT
IAP is a family of protein that has baculovirus IAP repeat (BIR) domains and inhibits apoptosis. We found a human IAP family gene, which we named Apollon, encoding a huge protein (530 kDa) that contains a single BIR domain and a ubiquitin-conjugating enzyme domain, that is a human homolog of BRUCE. Apollon protein was expressed in four of six brain cancers (gliomas), and one of five ovarian cancers in 38 human cancer cell lines that we examined. Among the brain cancer cell lines, SNB-78 expressed a high level of Apollon, and this cell line shows resistance against various anticancer drugs. Treating SNB-78 cells with antisense oligonucleotide against Apollon reduced the expression of Apollon protein, and significantly sensitized the cells to apoptosis induced by cisplatin and camptothecin. These results suggest that Apollon protects SNB-78 cells from undergoing apoptosis and, at least in part, plays a role in tumorigenesis and drug resistance of this cell line.