Effect of alkyl chain length on inhibition of N-nitrosomethylbenzylamine-induced esophageal tumorigenesis and DNA methylation by isothiocyanates

This study was undertaken to evaluate the inhibitory effectsof benzyl isothiocyanate (BITC), phenethyl isothiocyanate (PEITC),3-phenylpropyl isothiocyanate (PPITC) or 4- phenylbutyl isothiocyanate(PBITC) on N-nitrosomethylbenzylamine (NMBA)-induced esophagealtumorigenesis in male Fisher 344 rats. Groups of 15 male ratswere fed modified AIN-76A diet or diet containing the four isothiocyanatesat concentrations of 2.5, 1.0 and 0.4 µmol/g diet for25 weeks. After two weeks, rats were administered 0.5 mg/kgNMBA S.C. once weekly for 15 weeks. Additional controls receivedmodified AIN-76A diet only or diet containing the high concentrationof isothiocyanates (2.5 µmol/g) only. No tumors were foundin any of the groups that were not administered NMBA. Rats treatedwith NMBA only developed 6.7±0.8 tumors/animal. Tumorincidences in rats treated with 2.5 and 1.0 µmol PEITC/gdiet, and with all three dietary concentrations of PPITC wereinhibited by 60/100% compared to controls. Tumor multiplicitieswere inhibited by 83–100% by PEITC or PPITC at all dietaryconcentrations tested. PPITC clearly had a stronger inhibitoryeffect on NMBA tumorigenesis than did PEITC. Compared to PEITCand PPITC, BITC and PBITC had little inhibitory effect on tumormultiplicity and no effect on NMBA tumor incidence. In general,the occurrence of preneoplastic lesions (acanthoses, hyperkeratose,leukoplakias and leukokeratoses) was inhibited in a similarmanner as tumor incidence and multiplicity, except that no experimentaldiet resulted in a significant reduction of the incidence ofacanthoses and hyperkeratoses. As with their effects on tumorigenicityand formation of premalignant lesions, the inhibitory effectsof the isothiocyanates on NMBA-induced DNA methylation 24 hafter administration followed the order: PPITC > PEITC >PBITC > BITC.     

Chemopreventive activity of thiol conjugates of isothiocyanates for lung tumorigenesis

A series of L-cysteine (L-Cys), glutathione (GSH), and N-acetyl-L- cysteine (NAC) conjugates of phenethyl (PEITC), benzyl (BITC), and 6- phenylhexyl isothiocyanate (PHITC) were studied for their inhibitory activity toward metabolic activation of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in mouse lung microsomes. Selected compounds, PEITC, PEITC-GSH, PEITC-NAC and PHITC-NAC, were also assayed for the potential chemopreventive activity toward NNK-induced lung tumorigenesis in A/J mice. Results showed that PEITC and its conjugates inhibited NNK metabolism with decreasing potency: PEITC < PEITC-GSH > PEITC-Cys > PEITC-NAC. PHITC and its GSH and NAC conjugates exhibited nearly 10 times higher inhibitory activity toward NNK metabolism than the PEITC counterparts. In the tumor bioassay, as expected, the conjugates exhibited inhibitory activity against lung tumorigenesis induced by NNK. PEITC-GSH was not inhibitory at 4 micromol/mouse, but it inhibited approximately 32% of lung tumor multiplicity at 8 micromol/mouse. PEITC-NAC at 5 and 20 micromol/mouse both inhibited approximately 30% tumor multiplicity. Among all the conjugates examined, PHITC-NAC was the most potent. At a 5-micromol dose, it completely inhibited tumor multiplicity and incidence to the background level observed in the control group. These results revealed that the structure-activity relationships of the conjugates are similar to those found with their parent isothiocyanates (ITCs), i.e., the potency increased with the increasing alkyl chain length from two to six carbons in arylalkyl ITCs, suggesting that a common active species is involved. The inhibitory activity of ITC conjugates and the expected low toxicity make thiol conjugates of ITC a promising new series of chemopreventive agents.      

Effects of alkyl chain length on the inhibition of NNK-induced lung neoplasia in A/J mice by arylalkyl isothiocyanates

Six homologous arylalkyl isothiocyanates were evaluated fortheir abilities to inhibit pulmonary adenomas induced by thetobacco-specific nitrosamine 4-(methylnitrosamino)-l-(3-pyridyl)-1-butanone(NNK) in A/J mice. Four consecutive daily doses (5 µmol/mouse)of phenyl isothiocyanate (PITC), benzyl isothiocyanate (BITC),phenethyl isothiocyanate (PETTC), 3-phenylpropyl isothiocyanate(PPITC), 4-phenylbutyl isothiocyanate (PBITC), 4-oxo-4-(3-pyridyl)-butylisothiocyanate (OPBITC) and corn ofl were administered to miceby gavage. Two hours following the final dosing, mice were administeredsaline or 10 µmol of NNK in saline i.p. Pulmonary adenomaswere counted at 16 weeks after NNK administration. The miceadministered only corn oil prior to NNK developed an averagemultiplicity of 9.2 tumors/ mouse. Pretreatment with PITC, BITCand OPBITC had no significant effects on NNK-induced lung neoplasia.However, PEITC pretreatment resulted in a 64% reduction of lungtumor multiplicity, but did not affect the percentage of micethat developed tumors. Both PPITC and PBITC decreased tumormultiplicity by 96% and the percentage of tumor-bearing animalsby >60%. These results, in conjunction with our previouswork, demonstrate a general trend of increasing inhibition ofNNK-induced lung neoplasia by arylalkyl isothiocyanates withincreasing alkyl chain length. This study also demonstratesthe remarkable inhibitory activities of PPITC and PBITC, twoisothiocyanates that had not previously been tested as chemopreventiveagents.     

High incidence of nitrosamine-induced tumorigenesis in mice lacking DNA repair methyltransferase

The enzyme O6-methylguanine-DNA methyltransferase repairs alkylation- induced DNA damage, O6-methylguanine and O4-methylthymine, the former being formed more frequently. Previously, by means of gene targeting, we generated mice in which alleles for methyltransferase were disrupted. We now use these mouse lines, which are totally deficient in methyltransferase activity, to examine protective effects of the enzyme against tumor formation. In gene-targeted female mice given an i.p. injection of 5 mg/kg of dimethylnitrosamine, a larger number of liver and lung tumors occurred, as compared with normal female mice treated in the same manner. In male mice given a lower dose of carcinogen, the difference between normal and gene-targeted mice was statistically insignificant although more tumors did form in the gene-targeted mice. Methyltransferase apparently afforded protection from nitrosamine- induced tumorigenesis.      

Structure-activity relationships for inhibition of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone lung tumorigenesis by arylalkyl isothiocyanates in A/J mice

Phenethyl isothiocyanate (PEITC), 3-phenylpropyl isothiocyanate (PPITC), 4-phenylbutyl isothiocyanate (PBITC), and the newly synthesized 5-phenylpentyl isothiocyanate (PPeITC), 6-phenylhexyl isothiocyanate (PHITC), and 4-(3-pyridyl)butyl isothiocyanate (PyBITC) were tested for their abilities to inhibit tumorigenicity and DNA methylation induced by the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in the lungs of A/J mice. Mice were administered isothiocyanates by gavage for 4 consecutive days at doses of 5, 1, or 0.2 mumol/day prior to administration of 10 mumol of NNK by i.p. injection. Mice were sacrificed 16 weeks after NNK administration and pulmonary adenomas were quantitated, PEITC effectively inhibited NNK-induced lung tumors at a dose of 5 mumol/day but was not inhibitory at doses of 1 or 0.2 mumol/day. PPITC, PBITC, PPeITC, and PHITC were all considerably more potent inhibitors of NNK lung tumorigenesis than PEITC. While virtually no differences in inhibitory activity could be ascertained for PPITC, PBITC, and PPeITC, PHITC appeared to be the most potent tumor inhibitor of all of the compounds. At a dose of 0.2 mumol/day, PHITC pretreatment reduced tumor multiplicity by 85%. PyBITC, an analogue of both NNK and PBITC, was ineffective as an inhibitor. Using the same protocol, the compounds were found to have qualitatively similar inhibitory effects on NNK-induced DNA methylation when administered at 1 mumol/day. These results extend our previous findings that increased alkyl chain length enhances the inhibitory activity of an arylalkyl isothiocyanate toward NNK lung tumorigenesis and demonstrate the exceptional chemopreventive potentials of two new isothiocyanates, PPeITC and PHITC.     

The dual role of fibulins in tumorigenesis

The innate immune molecule Lipocalin 2 (LCN2) was initially shown to combat bacterial infection by binding bacterial siderophores, hence impairing microbial iron sequestration. In recent years, it has become apparent that LCN2 is over-expressed in cancers of diverse histological origin and that it facilitates tumorigenesis by promoting survival, growth, and metastasis. Herein, we discuss emerging evidence that substantiates two functional roles for LCN2 in cancer: promotion of the epithelial-to-mesenchymal transition (EMT) that facilitates an invasive phenotype and metastasis, and sequestration of iron that results in cell survival and tumorigenesis. Further, we present evidence that upregulated LCN2 expression in solid tumors is induced by hypoxia and pro-inflammation, microenvironmental noxae that converge to cause an endoplasmic reticulum (ER) stress response. Taken together, it appears that tumor cells exploit the beneficial innate immune function of LCN2 to support uncontrolled growth. This duplicity in function highlights LCN2 and its upstream driver, the ER stress response, as key targets for cancer therapy.     

The role of hedgehog signalling in tumorigenesis

Well differentiated thyroid carcinomas provide a unique model of human, epithelial cell carcinogenesis. Their molecular characterization has allowed to associate specific genetic alterations to the two papillary and follicular histotypes which, despite their common origin, display different biological and clinical behaviors. A common mechanism of oncogenic activation has been observed in these tumors, based on the peculiar characteristic of thyroid epithelium to generate fusion transforming genes by chromosomal rearrangements. The reasons for this peculiar uniqueness of thyrocytes are not known, but a structural explanation, based on the spatial contiguity in the interphase nuclei of thyrocytes of the two fused genes and enzymatic features of these cells which render them apoptosis resistant to DNA damage, have been proposed to account for this behavior.     

The dual role of fibulins in tumorigenesis

The human fibulin family consists of seven complex extracellular glycoproteins originally characterized as components of elastic fibers in connective tissue. However, beyond its structural role, fibulins are involved in complex biological processes such as cell adhesion, migration or proliferation. Indeed, they have proved to be essential elements in normal physiology, as shown by mouse models lacking these proteins, that evidence several developmental abnormalities and pathological features. Their relevance is also apparent in tumorigenesis, an aspect that has started to be intensely studied. Distinct fibulins are expressed in both tumor and stromal cells and are subjected to multiple expression regulations with either anti or pro-tumor effects. The mechanistic insights that underlie these observations are now commencing to emerge, portraying these proteins as very versatile and active constituents of connective tissue. The aim of this review is to highlight the most relevant connections between fibulins and cancer.     

The function of IGF-IR in NNK-mediated lung tumorigenesis

The type I insulin-like growth factor receptor (IGF-IR) is associated with many different types of cancer and it has been found to be involved in many aspects of the malignant phenotype, such as mitogenesis, survival, transformation and metastasis. This receptor has been observed to be overexpressed in the majority of lung cancer cell lines and human lung tumor biopsies. Two doxycycline-inducible transgenic mouse models in which the human IGF-IR was overexpressed in either the Clara cells or the type II alveolar cells of the lung were used in this study to examine the interaction between the nicotine derivative, nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and the IGF-IR. NNK was injected into both of the transgenic mouse models that overexpress human IGF-IR in the lung tissue in order to determine whether IGF-IR overexpression would affect NNK-induced tumorigenesis. No significant differences in the overall tumor burden were found between mice overexpressing the IGF-IR transgene that were treated with NNK and those that were not, however NNK-treated mice expressing high levels of IGF-IR transgene developed larger tumors than mice expressing high levels of IGF-IR transgene that did not receive NNK injections. In addition, endogenous murine IGF-IR was found to be expressed at high levels in the tumors that developed in the wild type, NNK injected mice suggesting that NNK induces lung tumors through inducing endogenous IGF-IR expression.     

The insulin receptor is essential for virus-induced tumorigenesis of Kaposi's sarcoma

Kaposi sarcoma (KS), a multifocal neoplasm of the skin that can spread to visceral organs, is the most prevalent malignant tumor in acquired immuno deficiency syndrome (AIDS) patients. KS-associated herpesvirus (KSHV or HHV8) is considered the primary etiological factor of this malignancy, as well as of primary effusion lymphoma and multicentric Castleman's disease. KS lesions are characterized by proliferating spindle cells of endothelial cell (EC) origin. The action of the insulin-like growth factor (IGF) system has been implicated in many malignancies, and recent data have demonstrated that the IGF-I receptor (IGF-IR) is required for in vitro growth of the KS-derived KSIMM cell line. To examine whether the IGF pathway is also involved in KSHV-mediated transformation of ECs, we examined the expression and function of the IGF system in KSHV-infected, immortalized dermal microvascular EC (E-DMVEC). The expression of the insulin receptor (IR) was strongly induced in latently infected E-DMVEC, whereas the expression levels of the IGF-IR remained unchanged. Gene knockdown of IR, but not IGF-IR, prevented the characteristic focus formation seen in KSHV-infected E-DMVEC. Similarly, treatment with the IR-specific small-molecule inhibitor HNMPA-(AM(3)) inhibited postconfluent growth. These data suggest a role for the IR, but not the IGF-IR, in KSHV-induced transformation of vascular ECs.     

Requirement of essential fatty acid for mammary tumorigenesis in the rat

In an attempt to determine the requirement of essential fatty acid for dimethylbenz(a)anthracene-induced mammary tumorigenesis, rats were fed diets containing different levels of linoleate: 0.5, 1.1, 1.7, 2.2, 3.5, 4.4, 8.5, or 11.5%. Each diet contained 20% of fat by weight, with varying amounts of coconut oil and corn oil added to achieve the desired levels of linoleate. Mammary tumorigenesis was very sensitive to linoleate intake and increased proportionately in the range of 0.5 to 4.4% of dietary linoleate. Regression analysis indicated that a breakpoint occurred at 4.4%, beyond which there was a very poor linear relationship, suggesting the possibility of a plateau. From the intersection of the regression lines in both the upper and lower ranges, the level of linoleate required to elicit the maximal tumorigenic response was estimated to be around 4%. The differences in tumor yield could not be correlated with changes in prostaglandin E concentration in the mammary fat pads of normal animals maintained on similar diets, suggesting that linoleate may act by some other mechanism to stimulate mammary tumorigenesis.     

Angiotensin II type 2 receptor gene deficiency attenuates susceptibility to tobacco-specific nitrosamine-induced lung tumorigenesis: involvement of transforming growth factor-beta-dependent cell growth attenuation

To clarify an involvement of angiotensin II signaling in lung neoplasia, we have examined the effect of angiotensin II receptor deficiency on 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung tumorigenesis. Male angiotensin II type 2 receptor (AT2)-null mice with an SWR/J genetic background and control wild-type mice were treated with NNK (100 mg/kg, i.p.) or saline vehicle. NNK treatment caused the development of lung tumors in all wild-type control mice (100 % tumor prevalence), but only 85% of AT2-null mice developed tumors. The tumor multiplicity in AT2-null mice (1.9 +/- 0.3) was significantly smaller than that in wild-type mice (4.1 +/- 0.9). Primary cultured lung fibroblasts prepared from both AT2-null and wild-type mice markedly increased the colony counts of A549 lung cancer cells in soft agar, but a consistently higher colony count was observed with the wild-type fibroblasts (fold increase in colony number, 5.6 +/- 0.5) than with the AT2-null fibroblasts (3.5 +/- 0.8). The underlying mechanism by which angiotensin II regulates cancer cell growth is due to the regulation of active transforming growth factor-beta (TGF-beta) production. Although the total level of TGF-beta was significantly stimulated when A549 cells were cocultured with either type of fibroblasts, the level of active TGF-beta in the conditioned medium was consistently higher with AT2-null fibroblasts than with wild-type fibroblasts. These results imply that the AT2 receptor negatively regulates the level of active TGF-beta and thus increases NNK-induced lung tumorigenesis. The AT2 receptor function in lung stromal fibroblasts may be a potential modulator of tumor susceptibility in chemical carcinogen-induced lung tumorigenesis.     

DNA羟甲基化酶基因TET1在肿瘤发生中的作用

TET1 （ten-eleven-translocation 1）是一种羟甲基化酶基因,该酶能够催化5甲基胞嘧啶（5-methyl-cytosine,5mC）形成5 羟甲基胞嘧啶（5-hydroxymethyl-cytosine,5hmC）,在DNA甲基化调控中发挥重要作用。最近研究表明, TET1 的低表达与肿瘤发 生有关,可作为癌症治疗潜在标志物,表明 TET1 可作为肿瘤抑制基因。此外,除了它的双氧酶活性外, TET1 还可以诱导上皮细 胞间质转变,并充当调节基因转录的辅助活化因子,如癌症的低氧应答基因的调节因子。这表明 TET1 也可做为癌基因促进肿瘤 的发生。因此,在癌症和发育生物学中,TET1的调控机制是十分复杂的, TET1 基因突变也已有报道。本文就TET1在肿瘤发生中 的不同作用进行了综述,深入阐述TET1的作用对拓展DNA去甲基化机制的认识及发现肿瘤治疗新靶标具有重要价值。     

A role for Ubc9 in tumorigenesis

The post-translational modifications ubiquitination and sumoylation have been implicated in regulating many critical cellular pathways. Like ubiquitination, sumoylation is a multistep process involving maturation, activation, conjugation and deconjugation. Ubc9 is a sole E2-conjugating enzyme essential for sumoylation. We have previously shown that alterations of Ubc9 expression affect tumor drug responsiveness. However, it is not clear whether there is any link between sumoylation and tumorigenesis, even though alterations of the ubiquitination pathway can lead to the development of cancer. In this study, we found that Ubc9 expression levels were elevated in ovarian tumors compared to the matched normal ovarian specimens, suggesting that Ubc9 may play a role in tumorigenesis. To test this, we overexpressed a dominant-negative mutant of Ubc9 (Ubc9-DN) and wild-type Ubc9 (Ubc9-WT) in the MCF-7 human breast tumor cells. Inoculating these cells as xenografts in mice revealed that tumors expressing Ubc9-WT grew better than the vector control, while tumors expressing Ubc9-DN exhibited reduced growth. This pattern was also seen in these cells when grown in culture. To better understand the mechanism behind this observation, we profiled gene expressions in these cells by microarray analysis and found alterations in expression of the pro-oncogene bcl-2 in these Ubc9-DN- and Ubc9-WT-expressing cells. Consistent with the bcl-2 results, subsequent studies revealed a higher rate of apoptosis and poor survival for the MCF-7 cells expressing Ubc9-DN, which are associated with downregulation of bcl-2. Together, these results suggest a role for Ubc9 in tumorigenesis at least partially through regulation of bcl-2 expression.     

A role for cholinesterases in tumorigenesis?

Hydrolysis of the neurotransmitter acetylcholine by acetylcholinesterase (ACHE) and butyrylcholinesterase (BCHE) is the rate-limiting step in the termination of cholinergic signaling at neuromuscular junctions. A growing body of evidence suggests that these enzymes also play a role in tumorigenesis. The ACHE and BCHE genes are amplified, mutated, and/or aberrantly expressed in a variety of human tumor types. These changes could be the result of chromosome breakage, since there is an unusually high frequency of chromosomal abnormalities near the map positions of these genes (3q26-ter and 11p-ter, respectively) in such tumors, particularly hemopoietic malignancies. Both ACHE and BCHE contain the consensus peptide motif S/T-P-X-Z, which is found in many substrates of cdc2-related protein kinases. Here we consider the intriguing possibility that phosphorylation by cdc2-related kinases may be the molecular mechanism linking cholinesterases with tumor cell proliferation. We also discuss the notion that inhibition of these enzymes by commonly used organophosphorous poisons may be tumorigenic in humans.     

Transplacental lung tumorigenesis in the athymic mouse

Female BALB/c nu/+ mice, pregnant by nu/+ males (nu: gene for hairlessness-athymia) were given injections of urethan, a transplacental tumorigen, on Day 17 or 19 of gestation. After an average of 16 weeks under clean conventional conditions, the incidence of primary lung tumors was similar in nude and normal offspring treated with carcinogen on either gestational day, with a higher incidence after treatment of Day 19. Thus, the absence of thymus did not affect the occurrence of transplacentally induced primary lung tumors or alter the well-known perinatal increase in sensitivity. Histologically, the nu/nu tumors differed from normal in the appearance of many atypical basophilic cells and in a tendency to invade both the parenchyma and the pleural surface. These results suggested progression of the lung adenomas to a more atypical, invasive form, a progression that may have occurred prematurely in the absence of thymus-dependent immune response.