naphthoquinones and Cell-Transformation--Neoplastic

Defective or abortive repair of DNA lesions has been associated with carcinogenesis. Therefore it is imperative for a cell to accurately repair its DNA after damage if it is to return to a normal cellular phenotype. In certain circumstances, if DNA damage cannot be repaired completely and with high fidelity, it is more advantageous for an organism to have some of its more severely damaged cells die rather than survive as neoplastic transformants. A number of DNA repair inhibitors have the potential to act as anticarcinogenic compounds. These drugs are capable of modulating DNA repair, thus promoting cell death rather than repair of potentially carcinogenic DNA damage mediated by error-prone DNA repair processes. In theory, exposure to a DNA repair inhibitor during, or immediately after, carcinogenic exposure should decrease or prevent tumorigenesis. However, the ability of DNA repair inhibitors to prevent cancer development is difficult to interpret depending upon the system used and the type of genotoxic stress. Inhibitors may act on multiple aspects of DNA repair as well as the cellular signaling pathways activated in response to the initial damage. In this review, we summarize basic DNA repair mechanisms and explore the effects of a number of DNA repair inhibitors that not only potentiate DNA-damaging agents but also decrease carcinogenicity. In particular, we focus on a novel anti-tumor agent, beta-lapachone, and its potential to block transformation by modulating poly(ADP-ribose) polymerase-1.

Topics: Anticarcinogenic Agents; Cell Transformation, Neoplastic; DNA Breaks, Double-Stranded; DNA Breaks, Single-Stranded; DNA Repair; Humans; Models, Biological; Naphthoquinones; Neoplasms; Poly(ADP-ribose) Polymerases

Effects of compounds that inhibit repair of DNA lesions in cells have been reported frequently. The consequences include altered incidence of carcinogenicity in vivo, tumorigenic transformation of cultured cells, mutations, and increased lethality as well as sister-chromatid exchanges and chromosome aberrations. This literature is reviewed here, with major emphasis on methylxanthines (caffeine in particular) and nicotinamide analogs. Existing information is also summarized on a novel potent repair inhibitor, beta-lapachone. Compounds that inhibit both DNA replication and repair are not discussed in detail since they have been reviewed often, but miscellaneous inhibitors of repair are summarized in a table. The relatively small number of experiments performed on the anticarcinogenic effects of methyl-xanthines and nicotinamide analogs gave very conflicting results. Some investigators report decreased carcinogenicity of DNA-damaging agents when caffeine was provided, but others obtained the opposite effect. The three studies with nicotinamide analogs all reported enhanced tumorigenicity of carcinogens. The data are too few to enable firm conclusions to be drawn regarding the possibility of using repair inhibitors to prevent cancer in humans. Variations of experimental conditions, carcinogens, cells, etc. have provided conflicting results. The possibility of cancer prevention is, nevertheless, so important that further investigations with DNA-repair inhibitors, particularly with human cells, seem very well justified.

Topics: Animals; Antibiotics, Antineoplastic; Carcinogens; Cell Transformation, Neoplastic; DNA Damage; DNA Repair; Naphthoquinones; Niacinamide; Xanthines

Metabolic reprogramming is an important feature of host-pathogen interactions and a hallmark of tumorigenesis. The intracellular apicomplexa parasite

Topics: Animals; Antiprotozoal Agents; Biological Transport; Carrier Proteins; Cattle; Cell Line, Transformed; Cell Transformation, Neoplastic; Enzyme Inhibitors; Gene Expression Regulation; Glucose; Host-Pathogen Interactions; Hypoxia-Inducible Factor 1, alpha Subunit; Lymphocytes; Membrane Proteins; Metabolic Networks and Pathways; Naphthoquinones; NIMA-Interacting Peptidylprolyl Isomerase; Protozoan Proteins; RNA, Small Interfering; Signal Transduction; Theileria; Thyroid Hormone-Binding Proteins; Thyroid Hormones

Topics: Animals; Antineoplastic Agents; Carcinoma, Pancreatic Ductal; Cell Transformation, Neoplastic; Ceruletide; Disease Models, Animal; Disease Progression; Gene Knockdown Techniques; Heterografts; Humans; Metaplasia; Mice; Mice, Inbred C57BL; Mice, Nude; Mice, Transgenic; Naphthoquinones; Pancreas; Pancreatic Neoplasms; Precancerous Conditions; Snail Family Transcription Factors; Tumor Cells, Cultured

SIRT2 is involved in the development of a variety of cancers. Shikonin is a natural compound that is known to have antitumor effects. This study aims to assess the effects of shikonin on the development and metastatic progression of colorectal cancer (CRC) through regulation of SIRT2 expression and whether this effect is related to the phosphorylation of extracellular signal-regulated kinases (ERKs). The results demonstrated that SIRT2 is downregulated in CRC biopsy samples (n=31) compared with the adjacent non-cancerous tissues (ANCT, n=26). Furthermore, CRC metastases were positive for SIRT2 despite a lack of expression in the primary tumor. In addition, data from an in vitro assay revealed that overexpression of SIRT2 inhibited the proliferation and metastatic progression of SW480 cells while blocking of SIRT2 expression induced the proliferation and metastatic progression of HT29 cells. Shikonin inhibited the viability, migration and invasion of SW480 cells and it also inhibited the tumor growth in the nude mice model; while AGK2 (a specific inhibitor of SIRT2) reversed these effects. Epidermal growth factor (EGF, an activator of ERK) and ERK-overexpression inhibited the effects of shikonin on SIRT2 expression, proliferation and metastasis in SW480 cells. However, this proliferative effect of EGF was reversed by SIRT2 overexpression. In conclusion, these results suggest that SIRT2 is a new therapeutic target for the treatment of CRC. The antitumor effects of shikonin on CRC seem to be mediated by SIRT2 upregulation via phospho-ERK inhibition.

Topics: Animals; Antineoplastic Agents; Carcinogenesis; Cell Line, Tumor; Cell Movement; Cell Transformation, Neoplastic; Colorectal Neoplasms; Down-Regulation; Extracellular Signal-Regulated MAP Kinases; Female; Gene Expression Regulation, Neoplastic; Humans; Male; Mice; Middle Aged; Naphthoquinones; Neoplasm Invasiveness; Neoplasm Metastasis; Sirtuin 2

Infectious agents develop intricate mechanisms to interact with host cell pathways and hijack their genetic and epigenetic machinery to change host cell phenotypic states. Among the Apicomplexa phylum of obligate intracellular parasites, which cause veterinary and human diseases, Theileria is the only genus that transforms its mammalian host cells. Theileria infection of bovine leukocytes induces proliferative and invasive phenotypes associated with activated signalling pathways, notably JNK and AP-1 (ref. 2). The transformed phenotypes are reversed by treatment with the theilericidal drug buparvaquone. We used comparative genomics to identify a homologue of the peptidyl-prolyl isomerase PIN1 in T. annulata (TaPIN1) that is secreted into the host cell and modulates oncogenic signalling pathways. Here we show that TaPIN1 is a bona fide prolyl isomerase and that it interacts with the host ubiquitin ligase FBW7, leading to its degradation and subsequent stabilization of c-JUN, which promotes transformation. We performed in vitro and in silico analysis and in vivo zebrafish xenograft experiments to demonstrate that TaPIN1 is directly inhibited by the anti-parasite drug buparvaquone (and other known PIN1 inhibitors) and is mutated in a drug-resistant strain. Prolyl isomerization is thus a conserved mechanism that is important in cancer and is used by Theileria parasites to manipulate host oncogenic signalling.

Topics: Animals; Cattle; Cell Line; Cell Transformation, Neoplastic; Drug Resistance; Host-Parasite Interactions; Humans; Leukocytes; Naphthoquinones; NIMA-Interacting Peptidylprolyl Isomerase; Parasites; Peptidylprolyl Isomerase; Protein Stability; Proto-Oncogene Proteins c-jun; Signal Transduction; SKP Cullin F-Box Protein Ligases; Theileria; Transcription Factor AP-1; Ubiquitination; Xenograft Model Antitumor Assays; Zebrafish

The M2 isoform of pyruvate kinase M2 (PKM2) has been shown to be up-regulated in human skin cancers. To test whether PKM2 may be a target for chemoprevention, shikonin, a natural product from the root of Lithospermum erythrorhizon and a specific inhibitor of PKM2, was used in a chemically-induced mouse skin carcinogenesis study. The results revealed that shikonin treatment suppressed skin tumor formation. Morphological examinations and immunohistochemical staining of the skin epidermal tissues suggested that shikonin inhibited cell proliferation without inducing apoptosis. Although shikonin alone suppressed PKM2 activity, it did not suppress tumor promoter-induced PKM2 activation in the skin epidermal tissues at the end of the skin carcinogenesis study. To reveal the potential chemopreventive mechanism of shikonin, an antibody microarray analysis was performed, and the results showed that the transcription factor ATF2 and its downstream target Cdk4 were up-regulated by chemical carcinogen treatment; whereas these up-regulations were suppressed by shikonin. In a promotable skin cell model, the nuclear levels of ATF2 were increased during tumor promotion, whereas this increase was inhibited by shikonin. Furthermore, knockdown of ATF2 decreased the expression levels of Cdk4 and Fra-1 (a key subunit of the activator protein 1. In summary, these results suggest that shikonin, rather than inhibiting PKM2 in vivo, suppresses the ATF2 pathway in skin carcinogenesis.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Activating Transcription Factor 2; Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Carcinogens; Carrier Proteins; Cell Line; Cell Proliferation; Cell Transformation, Neoplastic; Cyclin-Dependent Kinase 4; Epidermis; Epithelial Cells; Female; Gene Expression Regulation, Neoplastic; Humans; Membrane Proteins; Mice; Mice, Inbred DBA; Naphthoquinones; Proto-Oncogene Proteins c-fos; Pyridines; Signal Transduction; Skin Neoplasms; Thyroid Hormone-Binding Proteins; Thyroid Hormones; Transcriptional Activation

Chemoprevention has been a pivotal and effective strategy during the skin cancer treatment. Using human skin normal and tumor samples, we demonstrated that both the expression and activity levels of pyruvate kinase M2 (PKM2) were higher in skin tumor tissues than normal tissues, suggesting that PKM2, one of important metabolic enzyme, might serve as a target for skin cancer prevention and/or therapy. Shikonin, a small-molecule active chemical, has been studied as an anti-cancer drug candidate in human cancer models. However, the mechanism of action and the chemopreventive potential of shikonin are unclear. Herein, we used the skin epidermal JB6 P+ cells and demonstrated that shikonin suppressed the tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) induced neoplastic cell transformation and PKM2 activation in the early stage of carcinogenesis. Mitochondrial functions were inhibited by TPA treatment, as indicated by reduced mitochondrial membrane potential and mitochondrial respiration, which were restored by shikonin. We also examined the levels of lactate as a glycolysis marker, and shikonin suppressed its increase caused by tumor promoter treatment. Modulation of cell metabolism by shikonin was associated with G2-M phase accumulation, and Fra-1 (a major subunit of activator protein 1 in skin tumorigenesis) downregulation. In addition, we demonstrated that AMP-activated protein kinase (AMPK), an energy sensor, which is inactivated by TPA, shikonin could reverse AMPK activity. These results suggest that shikonin bears chemopreventive potential for human skin cancers in which PKM2 is upregulated, which might be mediated by inhibiting oncogenic activation, PKM2 activation, and mitochondrial dysfunction.

Topics: AMP-Activated Protein Kinases; Animals; Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; Blotting, Western; Carrier Proteins; Cell Adhesion; Cell Proliferation; Cell Respiration; Cell Transformation, Neoplastic; Cells, Cultured; Epidermis; Humans; Membrane Potential, Mitochondrial; Membrane Proteins; Mice; Mitochondria; Naphthoquinones; Oxygen Consumption; Tetradecanoylphorbol Acetate; Thyroid Hormone-Binding Proteins; Thyroid Hormones

Angiogenesis is a hallmark of tumor development and metastatic progression, and anti-angiogenic drugs targeting the VEGF pathway have shown to decrease the disease progression in cancer patients. In this study, we have analyzed the anti-proliferative and anti-angiogenic property of plumbagin in cisplatin sensitive, BRCA2 deficient, PEO-1 and cisplatin resistant, BRCA2 proficient PEO-4 ovarian cancer cells. Both PEO-1 and PEO-4 ovarian cancer cells are sensitive to plumbagin irrespective of BRCA2 status in both normoxia and hypoxia. Importantly, plumbagin treatment effectively inhibits VEGF-A and Glut-1 in PEO-1 and PEO-4 ovarian cancer cells. We have also analyzed the p53 mutant, cisplatin resistant, and BRCA2 proficient OVCAR-5 cells. Plumbagin challenge also restricts the VEGF induced pro-angiogenic signaling in HUVECs and subsequently endothelial cell proliferation. In addition, we observe a significant effect on tumor regression among OVCAR-5 tumor-bearing mice treated with plumbagin, which is associated with significant inhibition of Ki67 and vWF expressions. Plumbagin also significantly reduces CD31 expression in an ear angiogenesis assay. Collectively, our studies indicate that plumbagin, as an anti-cancer agent disrupts growth of ovarian cancer cells through the inhibition of proliferation as well as angiogenesis.

Topics: Angiogenesis Inhibitors; Animals; Antineoplastic Agents; Antineoplastic Agents, Phytogenic; BRCA2 Protein; Calcium; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Cisplatin; Endothelial Cells; Female; Glucose Transporter Type 1; Humans; Ki-67 Antigen; Mice; Mice, SCID; Naphthoquinones; Neovascularization, Pathologic; Ovarian Neoplasms; Platelet Endothelial Cell Adhesion Molecule-1; Random Allocation; Tumor Suppressor Protein p53; Vascular Endothelial Growth Factor A; Xenograft Model Antitumor Assays

Our efforts are concerned with identifying features of incomplete malignant transformation caused by non viral pathogens. Theileria parva (T. parva) is a tick-transmitted protozoan parasite that can cause a fatal lymphoproliferative disease in cattle. The T. parva-infected lymphocytes display a transformed phenotype and proliferate in culture media like the other tumor cells, however those cells will return to normal after antiprotozoal treatment reflecting the incomplete nature of transformation. To identify signaling pathways involved in this form of transformation of T. parva-infected cells, we screened a library of anticancer compounds. Among these, TIBC, a specific inhibitor of MDM2, markedly inhibited proliferation of T. parva-infected lymphocytes and promoted apoptosis. Therefore we analyzed MDM2 function in T. parva-infected cells. Several T. parva-infected cell lines showed increased expression level of MDM2 with alternatively spliced isoforms compared to the lymphoma cells or ConA blasts. In addition, buparvaquone affected MDM2 expression in T. parva transformed cells. Moreover, p53 protein accumulation and function were impaired in T. parva-infected cells after cisplatin induced DNA damage despite the increased p53 transcription level. Finally, the treatment of T. parva-infected cells with boronic-chalcone derivatives TIBC restored p53 protein accumulation and induced Bax expression. These results suggest that the overexpression of MDM2 is closely linked to the inhibition of p53-dependent apoptosis of T. parva-infected lymphocytes. Aberrant expression of host lymphocyte MDM2 induced by cytoplasmic existence of T. parva, directly and/or indirectly, is associated with aspects of this type of transformation of T. parva-infected lymphocytes. This form of transformation shares features of oncogene induced malignant phenotype acquisition.

Topics: Amino Acid Sequence; Animals; Apoptosis; bcl-2-Associated X Protein; Cattle; Cell Line; Cell Transformation, Neoplastic; Cisplatin; DNA Damage; Enzyme Activation; Lymphocyte Activation; Molecular Sequence Data; Naphthoquinones; NF-kappa B; Protein Isoforms; Proto-Oncogene Proteins c-mdm2; Signal Transduction; T-Lymphocytes; Theileria parva; Tumor Suppressor Protein p53

Sepantronium bromide (YM155) is an antitumor drug in development and is a first-in-class chemical entity, which is a survivin suppressant. We developed a radiosynthesis of [(11)C]YM155 to non-invasively evaluate its tissue and tumor distribution in mice bearing human prostate tumor xenografts.. Methods utilizing [(11)C]acetyl chloride and [(11)C]methyl triflate, both accessible with automated radiosynthesis boxes, were evaluated. The O-methylation of ethanolamine-alkolate with [(11)C]methyl triflate proved to be the key development toward a rapid and efficient process. The whole-body distribution of [(11)C]YM155 in PC-3 xenografted mice was examined using a planar positron imaging system (PPIS).. Sufficient quantities of radiopharmaceutical grade [(11)C]YM155 were produced for our PET imaging and distribution studies. The decay corrected (EOB) radiochemical yield was 16-22%, within a synthesis time of 47 min. The radiochemical purity was higher than 99%, and the specific activity was 29-60 GBq/μmol (EOS). High uptake levels of radioactivity (%ID/g, mean±SE) were observed in tumor (0.0613±0.0056), kidneys (0.0513±0.0092), liver (0.0368±0.0043) and cecum (0.0623±0.0070). The highest tumor uptake was observed at an early time point (from 10 min after) following injection. Tumor-to-blood and tumor-to-muscle uptake ratios of [(11)C]YM155, at 40 min after injection, were 26.5 (±2.9) and 25.6 (±3.6), respectively.. A rapid method for producing a radiopharmaceutical grade [(11)C]YM155 was developed. An in vivo distribution study using PPIS showed high uptake of [(11)C]YM155 in tumor tissue. Our methodology may facilitate the evaluation and prediction of response to YM155, when given as an anti-cancer agent.

Topics: Animals; Antineoplastic Agents; Carbon Radioisotopes; Cell Line, Tumor; Cell Transformation, Neoplastic; Humans; Imidazoles; Inhibitor of Apoptosis Proteins; Male; Mice; Naphthoquinones; Positron-Emission Tomography; Prostatic Neoplasms; Radiochemistry; Survivin; Tissue Distribution; Whole Body Imaging

Ultraviolet A (UVA) radiation (320-400 nm) is considered a major cause of human skin photoaging and skin cancer. Overexpression of cyclooxygenase-2 (COX-2) leads to prostanoid formation in skin tissue, disturbs the balance between proliferation and apoptosis, and subsequently promotes tumorigenesis. The peptidyl-prolyl isomerase Pin1 is known to be overexpressed in most cancer cell types and plays an important role in oncogenesis. Here, we studied whether exposure of JB6 Cl41 mouse epidermal cells to UVA affects COX-2 expression and the possible involvement of Pin1 activation. UVA increased COX-2 protein expression and prostaglandin E2 production in an energy-dependent manner. Pre-exposure of JB6 Cl41 cells to UVA potentiated epidermal growth factor-induced anchorage-independent growth; this effect was significantly suppressed by inhibition of COX-2. UVA-stimulated COX-2 expression was significantly decreased by inhibition of Pin1. The increased COX-2 gene transcription in response to UVA was preceded by activation of the transcription factors nuclear factor-κB (NF-κB), cAMP response element-binding protein (CREB), CCAAT/enhancer-binding proteins α and β (C/EBPα and C/EBPβ) and c-Jun/activator protein-1 (AP-1). Pin1 inhibitor treatment suppressed the activation of NF-κB, CREB, and C/EBP by UVA irradiation. Conversely, JB6 C141 cells overexpressing Pin1 showed increased basal COX-2 expression and NF-κB, CREB, C/EBP, and AP-1 activities. These results suggest that UVA-induced COX-2 expression is mediated by Pin1 activation and this is associated with malignant transformation of epidermal cells.

Topics: Animals; CCAAT-Enhancer-Binding Proteins; Cell Line; Cell Transformation, Neoplastic; Cyclic AMP Response Element-Binding Protein; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Dinoprostone; Enzyme Activation; Enzyme Induction; Epidermal Growth Factor; Epidermis; Epithelial Cells; Meloxicam; Mice; Naphthoquinones; NF-kappa B; NIMA-Interacting Peptidylprolyl Isomerase; Peptidylprolyl Isomerase; Signal Transduction; Thiazines; Thiazoles; Transcription Factor AP-1; Ultraviolet Rays

Juglone (5-hydroxy-1,4-naphthalenedione) from black walnut trees induces apoptosis and inhibits proliferation of various malignant cells. Here, we investigated whether juglone affects 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced cell transformation through the phosphoinositol 3-kinase (PI3K) pathway. The results showed that TPA- and endothelial growth factor (EGF)-induced anchorage-independent colony formation were suppressed in a dose-dependent manner by treatment of JB6 CI41 mouse skin epidermal cells with juglone (2.5 and 5 µM). We demonstrated that juglone suppressed PI3K activity via direct binding to PI3K by sepharose 4B pull-down assay and western blot analysis. Juglone significantly suppressed TPA-induced protein kinase B (AKT) and c-Jun phosphorylation and c-fos activation, but not mitogen-activated protein-kinase kinase (MEK), extracellular signaling-regulated kinase (ERK) or 90 kDa ribosomal protein S6 kinase (RSK) phosphorylation. Juglone significantly blocked activator protein-1 (AP-1) and cyclooxygenase-2 (COX-2) activation more than the PI3K inhibitors LY294002 and wortmannin. Overall, these results showed the anticancer efficacy of juglone targeting PI3K to prevent TPA-induced tumorigenesis.

Topics: Androstadienes; Animals; Cell Line; Cell Transformation, Neoplastic; Chromones; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Endothelial Growth Factors; Extracellular Signal-Regulated MAP Kinases; JNK Mitogen-Activated Protein Kinases; MAP Kinase Signaling System; Mice; Morpholines; Naphthoquinones; Phosphatidylinositol 3-Kinase; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-fos; Ribosomal Protein S6 Kinases, 90-kDa; Skin; Tetradecanoylphorbol Acetate; Transcription Factor AP-1; Wortmannin

Constitutively activated STAT3 plays a pivotal role in oncogenesis and metastasis in many human cancers, and STAT3 has been validated as a novel anticancer drug target. Thus, the identification of small molecules that modulate STAT3 activity could be of great therapeutic importance. The aim of this study was to isolate novel modulators of the STAT3 signaling pathway from the roots of Polygonum cuspidatum by bioassay-guided fractionation using a STAT3 reporter gene assay. 2-Methoxystypandrone (1), as well as three anthraquinones (2-4), were identified as major active components of P. cuspidatum. Compound 1 demonstrated a potent inhibitory effect on STAT3 activation and significantly inhibited cell proliferation of human breast cancer cells, especially those with constitutively activated STAT3 (IC₅₀ = 2.7-3.1 µM). The SAR analysis of quinone analogues suggested that the phenolic and carbonyl groups are the key structures contributing to their inhibitory activities against the STAT3 signaling.

Topics: Anthraquinones; Apoptosis; Biological Assay; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Dose-Response Relationship, Drug; Fallopia japonica; Female; Humans; Medicine, Chinese Traditional; Naphthoquinones; Phosphorylation; Plant Extracts; Plant Roots; Signal Transduction; STAT3 Transcription Factor

p53 is frequently mutated by genetic alternation or suppressed by various kinds of cellular signaling pathways in human cancers. Recently, we have revealed that p53 is suppressed and eliminated from cells by direct binding with oncogenic K-Ras-induced Snail. On the basis of the fact, we generated specific inhibitors against p53-Snail binding (GN25 and GN29). These chemicals can induce p53 expression and functions in K-Ras-mutated cells. However, it does not show cytotoxic effect on normal cells or K-Ras-wild-type cells. Moreover, GN25 can selectively activate wild-type p53 in p53(WT/MT) cancer cells. But single allelic mt p53 containing cell line, Panc-1, does not respond to our chemical. In vivo xenograft test also supports the antitumor effect of GN25 in K-Ras-mutated cell lines. These results suggest that our compounds are strong candidate for anticancer drug against K-Ras-initiated human cancers including pancreatic and lung cancers.

Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; Cell Transformation, Neoplastic; Child; Drug Evaluation, Preclinical; Female; Genes, ras; Humans; Mice; Mutation; Naphthoquinones; Neoplasms; Protein Binding; Small Molecule Libraries; Snail Family Transcription Factors; Transcription Factors; Tumor Suppressor Protein p53; Xenograft Model Antitumor Assays

Phosphorylation of proteins on serine or threonine residues that immediately precede proline (pSer/Thr-Pro) is specifically catalyzed by the peptidyl-prolyl cis-trans isomerase Pin1 and is a central signaling mechanism in cell proliferation and transformation. Although Pin1 is frequently overexpressed in hepatocellular carcinoma (HCC), the molecular mechanism of Pin1 in HCC has not been completely elucidated. Here, we show that Pin1 interacts with p70S6K in vitro and ex vivo. Overexpression of Pin1 resulted in enhanced p70S6K phosphorylation induced by insulin in SK-HEP-1 cells. In contrast, Pin1(-/-) mouse embryonic fibroblasts (MEFs) exhibited significantly decreased insulin-induced p70S6K phosphorylation compared with Pin1(+/+) MEFs. Furthermore, Pin1 enhanced the insulin-induced extracellular signal-regulated protein kinase (ERK)1/2 phosphorylation through its interaction with p70S6K, whereas the inhibition of p70S6K activity by rapamycin suppressed insulin-induced ERK1/2 phosphorylation in SK-HEP-1 cells. Hence, Pin1 affected activator protein-1 activity through p70S6K-ERK1/2 signaling in SK-HEP-1 cells. Most importantly, Pin1-overexpressing JB6 Cl41 cells enhanced neoplastic cell transformation promoted by insulin much more than green fluorescent protein-overexpressing JB6 Cl41 control cells. These results imply that Pin1 amplifies insulin signaling in hepatocarcinoma cells through its interaction with p70S6K, suggesting that Pin1 plays an important role in insulin-induced tumorigenesis and is a potential therapeutic target in hepatocarcinoma.

Topics: Animals; Antineoplastic Agents; Carcinoma, Hepatocellular; Cell Transformation, Neoplastic; Cells, Cultured; Drug Synergism; Embryo, Mammalian; Fibroblasts; Gene Expression Regulation, Neoplastic; Green Fluorescent Proteins; Humans; Hypoglycemic Agents; Immunoblotting; Immunosuppressive Agents; Insulin; Liver Neoplasms, Experimental; Mice; Mice, Knockout; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Naphthoquinones; NIMA-Interacting Peptidylprolyl Isomerase; Peptidylprolyl Isomerase; Phosphorylation; Ribosomal Protein S6 Kinases, 70-kDa; RNA, Small Interfering; Signal Transduction; Sirolimus; Transcription Factor AP-1; Two-Hybrid System Techniques

The peptidyl-prolyl isomerase Pin1 is frequently up-regulated in human cancers in which Rel/nuclear factor-kappaB (NF-kappaB) is constitutively activated, but its role in these cancers remains to be determined, and evidence is still lacking to show that Pin1 contributes to cell transformation by Rel/NF-kappaB. Rel/NF-kappaB transcriptional and oncogenic activities are modulated by several posttranslational modifications and coregulatory proteins, and previous studies showed that cytokine treatment induces binding of Pin1 to the RelA subunit of NF-kappaB, thereby enhancing RelA nuclear localization and stability. Here we show that Pin1 associates with the Rel subunits of NF-kappaB that are implicated in leukemia/lymphomagenesis and modulates their transcriptional and oncogenic activities. Pin1 markedly enhanced transformation of primary lymphocytes by the human c-Rel protein and also increased cell transformation by the potent viral Rel/NF-kappaB oncoprotein v-Rel, in contrast to a Pin1 mutant in the WW domain involved in interaction with NF-kappaB. Pin1 promoted nuclear accumulation of Rel proteins in the absence of activating stimuli. Importantly, inhibition of Pin1 function with the pharmacologic inhibitor juglone or with Pin1-specific shRNA led to cytoplasmic relocalization of endogenous c-Rel in human lymphoma-derived cell lines, markedly interfered with lymphoma cell proliferation, and suppressed endogenous Rel/NF-kappaB-dependent gene expression. Together, these results show that Pin1 is an important regulator of Rel/NF-kappaB transforming activity and suggest that Pin1 may be a potential therapeutic target in Rel/NF-kappaB-dependent leukemia/lymphomas.

Topics: Amino Acid Sequence; Animals; Cell Nucleus; Cell Proliferation; Cell Transformation, Neoplastic; Cells, Cultured; Chickens; Humans; Lymphoma; Molecular Sequence Data; Multigene Family; Naphthoquinones; NF-kappa B; NIMA-Interacting Peptidylprolyl Isomerase; Oncogene Proteins v-rel; Peptidylprolyl Isomerase; Protein Binding; Protein Transport; Sequence Homology, Amino Acid; Up-Regulation

Infection of bovine T cells and B cells with the intracellular protozoan parasite Theileria parva induces a transformed phenotype with characteristics comparable to leukemic cells. The transformed phenotype reverts on drug-induced parasite death, and the cured lymphocytes acquire a resting phenotype and eventually die by apoptosis if not further stimulated. Here, we show that both lymphocyte proliferation and activation of the transcription factor AP-1 are mediated by Src-family protein tyrosine kinases (PTKs) in a parasite-dependent fashion. Src-family PTKs are known to be present in glycolipid-enriched microdomains (GEMs), also called lipid rafts, and to be negatively regulated by PTK Csk complexed to tyrosine-phosphorylated transmembrane adapter protein PAG (phosphoprotein associated with GEMs) also called Cbp (Csk-binding protein). We, therefore, purified GEMs from proliferating infected B cells and from growth-arrested cells that had been drug-cured of parasites. Proliferation arrest led to a striking increase of PAG/Cbp expression; correspondingly, the amount of Csk associated with PAG/Cbp in GEMs increased markedly, whereas PTK Hck accumulation in GEM fractions did not alter on growth arrest. We propose that Theileria-induced lymphocyte proliferation and permanent activation of Hck stems from down-regulation of PAG/Cbp and the concomitant constitutive loss of the negative regulator Csk from the GEMs of transformed B cells.

Topics: Animals; Antiprotozoal Agents; B-Lymphocytes; Cattle; Cell Division; Cell Transformation, Neoplastic; CSK Tyrosine-Protein Kinase; Enzyme Activation; Enzyme Inhibitors; Lymphocyte Activation; Membrane Microdomains; Membrane Proteins; Naphthoquinones; Phenotype; Phosphatidylinositol 3-Kinases; Phosphoproteins; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-hck; Pyrimidines; Signal Transduction; src-Family Kinases; Theileria parva; Transcription Factor AP-1

Most chemotherapeutic drugs kill cancer cells by indirectly activating checkpoint-mediated apoptosis after creating nonselective damage to DNA or microtubules, which accounts for their toxicity toward normal cells. We seek to target cancer cells by directly activating checkpoint regulators without creating such damage. Here, we show that beta-lapachone selectively induces apoptosis in cancer cells without causing the death of nontransformed cells in culture. This unusual selectivity against cancer cells is preceded by activation of S-phase checkpoint and selective induction of E2F1, a regulator of checkpoint-mediated apoptosis. This study suggests direct checkpoint activation as a strategy against cancer.

Topics: Antibiotics, Antineoplastic; Antineoplastic Agents; Apoptosis; Blotting, Western; Cell Cycle; Cell Cycle Proteins; Cell Death; Cell Division; Cell Line; Cell Transformation, Neoplastic; Coloring Agents; DNA-Binding Proteins; Dose-Response Relationship, Drug; E2F Transcription Factors; E2F1 Transcription Factor; Flow Cytometry; Humans; Models, Biological; Naphthoquinones; Neoplasms; Retinoblastoma Protein; S Phase; Tetrazolium Salts; Thiazoles; Transcription Factors; Tumor Cells, Cultured

2-Hydroxy-1,4-naphthoquinone (HNQ; Lawsone; CAS 83-72-7) is the principal natural dye ingredient contained in the leaves of Henna (Lawsonia inermis). Published genotoxicity studies on HNQ suggested it was a weak bacterial mutagen for Salmonella typhimurium strain TA98 or was more clearly mutagenic for strain TA 2637, both in the presence of metabolic activation. HNQ was unable to induce sex-linked recessive lethal mutations in Drosophila melanogaster. However, a small increase in micronucleus frequency was reported in the bone marrow of mice at a single mid-range dose level, 24h after intraperitoneal injection. In view of the wide use of Henna hair dyes it was deemed necessary to conduct a thorough investigation, under Good Laboratory Practice conditions, of the genotoxicity of HNQ. HNQ was non-mutagenic in bacterial (Ames test) or mammalian (V79 hprt) assays. It was borderline positive in a mouse lymphoma tk mutation assay and a chromosome aberration test (CHO cells), results that may reflect a similar clastogenic mechanism. Negative in vivo genotoxicity results were noted in the rat hepatocyte in vivo/in vitro UDS test, in peripheral lymphocytes (chromosome aberrations) of rats receiving repeated oral doses of HNQ at the MTD for 28 days, and in mouse and hamster bone marrow chromosome aberration tests. However small, but statistically significant increases in the incidence of bone marrow micronuclei were observed in two out of five tests at 72 h after dosing, but not at 24 or 48 h. There was evidence of haematotoxicity at 72 h, which may have been enhanced by the vehicle (DMSO) used in the positive tests. As erythropoiesis and administration of haematotoxic agents are known to induce small increases in the frequency of bone marrow micronuclei, typically at delayed sampling times, the data suggest that the positive 72 h response produced by HNQ is consistent with stimulation of haematopoiesis subsequent to haematological toxicity of HNQ, and not due to a DNA-reactive mechanism. Overall, the weight of evidence suggests that Henna and HNQ pose no genotoxic risk to the consumer.

Topics: Animals; Bone Marrow Cells; Cell Transformation, Neoplastic; Coloring Agents; Cricetinae; DNA; Dose-Response Relationship, Drug; Female; Hepatocytes; Leukemia L5178; Male; Mesocricetus; Mice; Micronuclei, Chromosome-Defective; Mutagenicity Tests; Mutagens; Naphthoquinones; Rats; Rats, Inbred Strains; Salmonella typhimurium

Transformed Chinese hamster embryo fibroblasts (CHEF), which gradually increase in tumor-forming ability in nude mice, were isolated from normal diploid CHEF/18 cells. Transformed CHEF cells (i.e. T30-4 > 21-2M3 > 21-2 > normal CHEF/18) showed gradual increases in potentially lethal damage (PLD) survival recovery. beta-Lapachone and camptothecin, modulators of topoisomerase I (Topo I) activity, not only prevented survival recovery in normal as well as in tumor cells, but enhanced unscheduled DNA synthesis. These seemingly conflicting results are due to the fact that Topo I activity can be modulated by inhibitors to convert single-stranded DNA lesions into double-stranded breaks. Increases in unscheduled DNA synthesis may result from a continual supply of free ends, on which DNA repair processes may act. Altering Topo I activity with modulators appears to increase X-ray lethality via a DNA lesion modification suicide pathway. Cells down-regulate Topo I immediately after ionizing radiation to prevent Topo I-mediated lesion modification and to enhance survival recovery.

Topics: Animals; Camptothecin; Cell Line, Transformed; Cell Survival; Cell Transformation, Neoplastic; Cells, Cultured; Cricetinae; Cricetulus; DNA Repair; DNA Topoisomerases, Type I; Embryo, Mammalian; Fibroblasts; Mice; Mice, Nude; Naphthoquinones; Radiation Tolerance; Topoisomerase I Inhibitors

The transforming activity of luteoskyrin (LS), a bis-anthraquinoid mycotoxin produced by Penicillium islandicum Sopp., and a hepatocarcinogen in rodents, was examined by an in vitro transformation assay using mouse embryonal Balb/3T3 A31-1-1 cells. The results revealed that LS induced type III foci at 0.5 micrograms/ml, and that the cells selected from these foci by soft-agar cloning grew with a high saturation density. Thus, it was confirmed that LS not only induces hepatic tumours in laboratory animals, but also transforms in vitro cultured mammalian cells. The tumorigenicity of the transformants obtained was confirmed by transplantation into nude mice and by image analysis with IIIIn. A transfection assay, using calcium phosphate co-precipitation, demonstrated that the DNA of the cloned cells transformed NIH3T3 cells. Northern blot also revealed transcriptional activation of c-myc and c-Ha-ras oncogenes. The possible participation of LS-derived hydroxy radicals in the formation of genetic lesions was discussed.

Topics: Animals; Cell Transformation, Neoplastic; Cells, Cultured; DNA; Mice; Mice, Nude; Mycotoxins; Naphthoquinones; Neoplasms, Experimental; Oncogenes

Beta-lapachone is a potent inhibitor of DNA repair in mammalian cells and activates topoisomerase I. We show that beta-lapachone can prevent the oncogenic transformation of CHEF/18A cells by ionizing radiation. Potentially lethal DNA damage repair (PLDR) occurs while x-irradiated cells are held in medium containing low serum prior to replating. PLDR processes permitted survival recovery but also drastically increased the number of foci per plate (i.e., transformation) of CHEF/18A cells. By blocking PLDR with beta-lapachone, both survival recovery and enhanced transformation were prevented. At equivalent survival levels, exposure of x-irradiated cells to beta-lapachone resulted in an 8-fold decrease in the number of foci per dish as compared to the number of transformants produced after PLDR. Early PLDR-derived increases in transformation may be the result of error-prone genetic rearrangements dependent on topoisomerase I, which are thereby prevented by beta-lapachone. Beta-lapachone exposure decreased the rejoining of DNA strand breaks and produced additional double-strand breaks in x-irradiated cells during PLDR. The activation of topoisomerase I by beta-lapachone may convert repairable single-strand DNA breaks into the more repair-resistant double-strand breaks, thereby preventing PLDR and neoplastic transformation. These results suggest a new direction for the development of anticarcinogenic agents.

Topics: Animals; Antibiotics, Antineoplastic; Cell Line; Cell Survival; Cell Transformation, Neoplastic; DNA Damage; DNA Repair; Dose-Response Relationship, Radiation; Naphthoquinones; X-Rays

Topics: Cell Transformation, Neoplastic; Cybernetics; Diethylstilbestrol; Hot Temperature; Hydrogen Peroxide; Hydrogen-Ion Concentration; Membranes; Naphthoquinones; Neoplasms; Vitamin K