Generation of free radicals and induction of DNA adducts by activation of heterocyclic aromatic amines via different metabolic pathways in vitro

Food-derived heterocyclic aromatic amines (HCAs) have proved to be carcinogenic in both rodents and nonhuman primates. Two different metabolic pathways are suggested for the metabolic activation of HCA. The hepatic pathway proceeds via a two-step process involving N-hydroxylation by cytochrome P4501A2 and subsequent O-acetylation by N-acetyltransferase-2. An alternative pathway may be of particular interest in extrahepatic tissues and proceeds via one-electron oxidation catalyzed by prostaglandin H synthase (PHS), rendering free-radical metabolites. In this study, we investigated the metabolic activation of two HCAs, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), by two different enzyme systems in vitro, generating different primary and secondary reactive metabolites. Rat liver S9 mix and PHS were used as the activating system and represent the hepatic and extrahepatic pathways, respectively. Electron-spin resonance spectroscopy showed that both IQ and PhIP exerted inhibiting effects on PHS-mediated formation of hydroxyl radicals during the conversion of arachidonic acid to prostaglandins. Evidence for the formation of HCA free radicals was presented in an indirect way by the formation of glutathione-derived thiyl radicals, with purified PHS as the activating system. Activation by S9 mix did not result in the formation of detectable radical metabolites, showing that the two metabolic routes primarily led to the formation of different metabolites. In all electron-spin resonance experiments, IQ appeared to be more effective than PhIP. In contrasts, DNA adduct analysis by means of (32)P-postlabeling showed similar adduct patterns for S9 and PHS in single-stranded and double-stranded salmon testes DNA after incubation with PhIP, indicating the ultimate formation of a common reactive intermediate. For IQ, activation by PHS led to an additional adduct spot that was not present after S9 activation. Furthermore, activation of IQ resulted in higher adduct levels compared with PhIP for both activation pathways. Overall, adduct levels were higher in single-stranded DNA than double-stranded DNA. Our results showed that the hepatic and extrahepatic pathways resulted in different primary metabolites, while the ultimate formation of a similar reactive intermediate for PhIP, possibly an arylnitrenium ion, suggested that both pathways could play an important role in the onset of carcinogenesis.     

Low dose 2-CdA schedule activity in splenic marginal zone lymphomas

Splenic Marginal Zone Lymphoma (SMZL) is a rare clinicopathological entity among marginal zone lymphomas. SMZL is an indolent lymphoma usually treated by splenectomy. A subset of patients is characterized by a more aggressive clinical course and poor prognosis. Treatment of these cases and second-line therapy for relapsed patients have not been yet identified. We report 10 cases treated with cladribrine (5 mg/m(2)/week) for six courses. Six patients (60%) achieved partial response, two patients (20%) achieved a complete response and the two remaining patients did not respond and died as a result of progression of the disease. The treatment was well tolerated. A total of 60% of the patients had an overall survival rate of 48 months and 24 months progression-free-survival was achieved by 37% with a median time of progression-free-survival of 17 months. Interestingly, in addition to a relevant percentage of hematological remission, some patients also experienced a molecular remission. We conclude that this treatment is safe and well tolerated and is able to induce a substantial number of responses. Our results suggest that this schedule is well tolerated and could be an useful alternative to splenectomy.     

Non‐invasive estimation of 10B‐4‐borono‐L‐phenylalanine‐derived boron concentration in tumors by PET using 4‐borono‐2‐18F‐fluoro‐phenylalanine

In boron neutron capture therapy (BNCT), ¹⁰B‐4‐borono‐L‐phenylalanine (BPA) is commonly used as a ¹⁰B carrier. PET using 4‐borono‐2‐¹⁸F‐fluoro‐phenylalanine (¹⁸F‐FBPA PET) has been performed to estimate boron concentration and predict the therapeutic effects of BNCT; however, the association between tumor uptake of ¹⁸F‐FBPA and boron concentration in tumors remains unclear. The present study investigated the transport mechanism of ¹⁸F‐FBPA and BPA, and evaluated the utility of ¹⁸F‐FBPA PET in predicting boron concentration in tumors. The transporter assay revealed that 2‐aminobicyclo‐(2.2.1)‐heptane‐2‐carboxylic acid, an inhibitor of the L‐type amino acid transporter, significantly inhibited ¹⁸F‐FBPA and ¹⁴C‐4‐borono‐L‐phenylalanine (¹⁴C‐BPA) uptake in FaDu and LN‐229 human cancer cells. ¹⁸F‐FBPA uptake strongly correlated with ¹⁴C‐BPA uptake in 7 human tumor cell lines (r = .93; P < .01). PET experiments demonstrated that tumor uptake of ¹⁸F‐FBPA was independent of the administration method, and uptake of ¹⁸F‐FBPA by bolus injection correlated well with BPA uptake by continuous intravenous infusion. The results of this study revealed that evaluating tumor uptake of ¹⁸F‐FBPA by PET was useful for estimating ¹⁰B concentration in tumors.     

Regulation of the differentiation-related gene Drg-1 during mouse skin carcinogenesis.

Differentiation-related gene-1 (Drg-1) has been identified as a gene whose expression is increased in several processes related to differentiation, but its function is currently unknown. In this report, we show that Drg-1 was expressed in keratinocytes, this expression being rapidly increased as a result of induction by 12-O-tetradecanoylphorbol-13-acetate (TPA) or the presence of an activating form of Ha-ras. Induction by TPA occurred both in cultured cell lines and primary keratinocytes as well as in mouse skin after a single TPA application. Overexpression of Drg-1 was also observed in TPA-induced hyperplastic skin. In agreement, mouse skin papillomas and carcinomas also overexpressed Drg-1. In addition, Drg-1 was induced when keratinocytes were forced to differentiate by calcium switch or serum starvation. Analysis of the expression of Drg-1 during the keratinocyte cell cycle demonstrated relatively high levels of Drg-1 mRNA in G(0), which increased in early G(1) and decreased afterwards in late G(1)/S. In situ analysis showed an accumulation of Drg-1 in the suprabasal layers of the skin, as well as in the more differentiated areas of mouse skin papillomas. These results suggest that, in addition to being upregulated during keratinocyte differentiation, the Drg-1 gene might have a complex function in skin tumorigenesis.     

Oxidative DNA damage and 8-hydroxy-2-deoxyguanosine DNA glycosylase/apurinic lyase in human breast cancer

To test the hypothesis that oxidative stress is involved in breast cancer, we compared the levels of 8-hydroxy-2-deoxyguanosine (8-oxo-dG), an oxidized DNA base common in cells undergoing oxidative stress, in normal breast tissues from women with or without breast cancer. We found that breast cancer patients (N = 76) had a significantly higher level of 8-oxo-dG than control subjects (N = 49). The mean ( +/- SD) values of 8-oxo-dG/10(5) dG, as measured by high-performance liquid chromatography electrochemical detection, were 10.7 +/- 15.5 and 6.3 +/- 6.8 for cases and controls, respectively (P = 0.035). This difference also was found by immunohistochemistry with double-fluorescence labeling and laser-scanning cytometry. The average ratios (x10(6)) of the signal intensity of antibody staining to that of DNA content were 3.9 +/- 7.2 and 1.1 +/- 1.4 for cases (N = 57) and controls (N = 34), respectively (P = 0.008). There was no correlation between the ages of the study subjects and the levels of 8-oxo-dG. Cases also had a significantly higher level of 8-hydroxy-2-deoxyguanosine DNA glycosylase/apurinic lyase (hOGG1) protein expression in normal breast tissues than controls (P = 0.008). There was no significant correlation between hOGG1 expression and 8-oxo-dG. Polymorphism of the hOGG1 gene was very rare in this study population. The previously reported exon 1 polymorphism and two novel mutations of the hOGG1 gene were found in three of 168 cases and two of 55 controls. In conclusion, normal breast tissues from cancer patients had a significantly higher level of oxidative DNA damage. The elevated level of 8-oxo-dG in cancer patients was not related to age or to deficiency of the hOGG1 repair gene.     

Intestinal carcinogenesis of two food processing contaminants, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and 5-hydroxymethylfurfural, in transgenic FVB min mice expressing human sulfotransferases

Humans express sulfotransferases (SULTs) of the SULT1A subfamily in many tissues, whilst the single SULT1A gene present in rodents is mainly expressed in liver. The food processing contaminants, 5‐hydroxymethylfurfural (HMF) and 2‐amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]pyridine (PhIP), are bioactivated by human SULT1A1 and SULT1A2. FVB multiple intestinal neoplasia (Min) mice, which spontaneously develop tumors and flat aberrant crypt foci (ACF) in intestine, were crossed with transgenic FVB mice expressing human SULT1A1 and 1A2 (hSULT) in several tissues, giving rise to wild‐type and Min mice with and without hSULT. One‐week‐old Min mice with or without hSULT were given HMF (375 or 750 mg/kg bw) or saline by gavage three times a week for 11 wk. In another experiment, the F1 generation received subcutaneous injections of 50 mg/kg bw PhIP or saline 1 wk before birth, and 1, 2, and 3 wk after birth. HMF did not affect the formation of tumors, but may have induced some flat ACF (incidence 15–20%) in Min mice with and without hSULT. No control mouse developed any flat ACF. With the limitation that these putative effects were weak, they were unaffected by hSULT expression. The carcinogenic effect of PhIP increased in the presence of hSULT, with a significant increase in both incidence (31–80%) and number of colonic tumors (0.4–1.3 per animal). Thus, intestinal expression of human SULT1A1 and 1A2 might increase the susceptibility to compounds bioactivated via this pathway implying that humans might be more susceptible than conventional rodent models. © 2011 Wiley Periodicals, Inc.     

Effect of serum deprivation on constitutive production of granulocyte-colony stimulating factor and granulocyte macrophage-colony stimulating factor in lung cancer cells

We previously established 2 lung cancer cell lines, OKa-C-1 and MI-4, which constitutively produce an abundant dose of granulocyte-colony stimulating factor (G-CSF) and granulocyte macrophage-colony stimulating factor (GM-CSF). Many other cases with G-CSF or GM-CSF producing tumors have been reported up to the present. However, the biological properties of the overproduction of G-CSF and GM-CSF by tumor cells have not been well known. Several reports demonstrated the presence of an autocrine growth loop for G-CSF and GM-CSF in nonhematopoietic tumor cells. We showed that exogenous G-CSF and GM-CSF stimulated cell growth in a dose-dependent manner in OKa-C-1 and MI-4 cells. We could detect the presence of G-CSF and GM-CSF receptors in both cell lines by RT-PCR analysis. We have previously shown that inflammatory cytokines, tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta enhance the expression of G-CSF and GM-CSF in the cell lines. However, the factors that regulate constitutive production of G-CSF or GM-CSF by tumor cells are still unknown well. In our study, we first reported that serum deprivation stimulated constitutive production of G-CSF and GM-CSF by lung tumor cells through activation of nuclear factor (NF)-kappaB and p44/42 mitogen-activated protein kinase (MAPK) pathway signaling. We suggest that G-CSF and GM-CSF constitutively produced by tumor cells could grow tumor itself and rescue tumor cells from the cytotoxicity of serum deprivation.     

DICKKOPF-4 activates the noncanonical c-Jun-NH2 kinase signaling pathway while inhibiting the Wnt-canonical pathway in human renal cell carcinoma

BACKGROUND:

To the authors' knowledge, the functional significance of the Wnt antagonist dickkopf homolog 4 (DKK4) has not been investigated previously in renal cancer.

METHODS:

The authors initially observed that the expression of DKK4 was significantly higher in renal cancer tissues compared with adjacent normal kidney tissues. To assess the function of DKK4, stable DKK4‐transfected cells were established, and functional analyses were performed, including a T‐cell factor/lymphoid enhancer factor (TCF/LEF) reporter assay and tests for cell viability, colony formation, apoptosis, cell cycle, invasive capability, wound‐healing capability, and in vivo tumor growth.

RESULTS:

The relative TCF/LEF activity was significantly lower in DKK4‐transfected cells compared with empty vector, and nuclear β‐catenin expression was decreased in DKK4 transfectants. In addition, expression levels of the β‐catenin downstream effector proteins cyclin D1 and c‐Myc were decreased in DKK4 transfectants. However, greater invasiveness and migration were observed in stably transfected DKK4 cells. Increased growth of DKK4‐transfected tumors also was observed in nude mice. Members of the Wnt noncanonical/c‐Jun‐NH2 kinase (JNK) signaling pathway also were effected, such as c‐Jun, which had significantly increased expression and phosphorylation in DKK4‐stable transfectants, and matrix metalloproteinase‐2, which had significantly increased expression in DKK4‐stable transfectants.

CONCLUSIONS:

This is the first study to indicate that DKK4 expression is increased in renal cancer tissues and that DKK4 activates the noncanonical JNK signaling pathway while inhibiting the Wnt‐canonical pathway. Cancer 2011. © 2010 American Cancer Society.     

Anti-tumor efficacy of the nucleoside analog 1-(2-deoxy-2-fluoro-4-thio-beta-D-arabinofuranosyl) cytosine (4'-thio-FAC) in human pancreatic and ovarian tumor xenograft models

1-(2-deoxy-2-fluoro-4-thio-beta-D-arabinofuranosyl) cytosine (4'-thio-FAC) is a deoxycytidine analog that has been shown previously to have impressive anti-proliferative and cytotoxic effects in vitro and in vivo toward colorectal and gastric tumors. In our present studies, the pharmacokinetic behavior in nude mice and the effectiveness of 4'-thio-FAC against human pancreatic and ovarian tumor growth were assessed in comparison with standard chemotherapeutic agents. Potent in vitro anti-proliferative effects were observed against pancreatic (Capan-1, MIA-PaCa-2, BxPC-3) and ovarian (SK-OV-3, OVCAR-3, ES-2) cancer cell lines with IC(50) of 0.01-0.2 microM. In vivo anti-tumor activity was evaluated in nude mice bearing subcutaneously (s.c.) implanted human pancreatic tumor xenografts or intraperitoneally (i.p.) disseminated human ovarian xenografted tumors. Oral daily administration of 4'-thio-FAC for 8-10 days significantly inhibited the growth of gemcitabine-resistant BxPC-3 pancreatic tumors and induced regression of gemcitabine-refractory Capan-1 tumors. 4'-Thio-FAC was also a highly effective inhibitor of ovarian peritoneal carcinomatosis. In the SK-OV-3 and ES-2 ovarian cancer models, 4'-thio-FAC prolonged survival to a greater extent than that observed with gemcitabine. Furthermore, the superiority of 4'-thio-FAC to carboplatin and paclitaxel was demonstrated in the ES-2 clear cell ovarian carcinoma model. Studies provide evidence that 4'-thio-FAC is a promising new alternative to gemcitabine and other chemotherapeutic drugs in the treatment of a variety of tumor indications, including pancreatic and ovarian carcinoma.     

Inhibition of tumor growth by biodegradable microspheres containing all-trans-retinoic acid in a human head-and-neck cancer xenograft

Retinoids play essential roles in the regulation of cell differentiation and in the proliferation of various epithelial tissues, and atRA is one such active metabolite of retinoids. However, despite the known functions of atRA, its clinical applications are limited due to the induced metabolism by the specific cytochrome P-450s in the liver. To overcome the limitation, parenteral administration of atRA-loaded biodegradable microspheres, the PDLLA/PLE microspheres containing atRA, was suggested previously. We evaluated chemotherapeutic efficacy of atRA-loaded microspheres in a human head-and-neck xenograft/nude mouse model. When atRA-loaded microspheres were administered s.c. at 200 mg/kg body weight to athymic nude mice, plasma concentration of atRA could be maintained in a range of 1.2 to 3.7 x 10(-8) M for 4 weeks. As a result, the tumor volume of human head-and-neck cancer was reduced compared to the control group by 51.3% (p < 0.01) at 14 days and by 49.2% (p < 0.05) at 28 days.     

A synthetic compound, 1,5-bis(2-methoxyphenyl)penta-1,4-dien-3-one (B63), induces apoptosis and activates endoplasmic reticulum stress in non-small cell lung cancer cells

Endoplasmic reticulum (ER) stress-induced cancer cell apoptosis has become a novel signaling target for the development of therapeutic drugs for cancer treatment. Curcumin, a dietary phytochemical, exhibits growth-suppressive activity against cancer cells via multitarget mechanisms. However, the low stability and poor pharmacokinetics significantly limit its clinical applications. Thus, we designed and synthesized a novel monocarbonyl analog of curcumin, 1,5-bis(2-methoxyphenyl) penta-1,4-dien-3-one (B63). This compound exhibited a higher chemical stability in cultural medium and a better intracellular profile than curcumin. Treatment with B63 potently induced apoptosis of human non-small cell lung cancer (NSCLC) cells in a dose-responsive manner, while exhibiting no cytotoxicity in normal lung fibroblast cells. Its antitumor effect was associated with the ER stress-mediated apoptotic pathway and, ultimately, the activation of the caspase cascades. However, curcumin at the same concentrations did not cause ER stress in H460 cells. Further, C/EBP homologous protein knockdown by siRNA attenuated B63-induced cell apoptosis, indicating that the apoptotic pathway is ER stress-dependent. In vivo, the volume and weight of the tumor were reduced significantly by pretreating the H460 tumor cells with B63 before implantation. Taken together, these insights on the novel compound B63, from both chemical and biological perspectives, may provide a novel anticancer candidate for the treatment of NSCLC.     

2-Deoxy-L-ribose inhibits the invasion of thymidine phosphorylase-overexpressing tumors by suppressing matrix metalloproteinase-9

Thymidine phosphorylase (TP), an enzyme involved in pyrimidine metabolism, is identical with an angiogenic factor, platelet-derived endothelial cell growth factor. 2-Deoxy-D-ribose (D-dRib), the degradation product of thymidine generated by TP activity, has been suggested to be a downstream mediator of TP function. 2-Deoxy-L-ribose (L-dRib), a stereoisomer of D-dRib, inhibited the promotion of angiogenesis, tumor growth and metastasis by TP. In our study, we have shown that nude mice inoculated with TP-overexpressing KB/TP cells had shorter survival times than those injected with control KB/CV cells. KB/TP tumors were also more highly invasive than KB/CV tumors in mice. The expression levels of matrix metalloproteinase (MMP)-9 in KB/TP tumors were significantly higher than those in KB/CV tumors. L-dRib and a TP inhibitior, TPI, extended the survival period of KB/TP tumor-bearing mice. L-dRib also reduced MMP-9 mRNA levels in KB/TP tumors. Furthermore, L-dRib suppressed the mRNA level of MMP-9 in cultured KB/TP cells, and the invasive activity of the cells. L-dRib may be useful for the suppression of invasion of TP-expressing tumor cells.     

Betulinic acid decreases ER‐negative breast cancer cell growth in vitro and in vivo: Role of Sp transcription factors and microRNA‐27a:ZBTB10

Betulinic acid (BA), a pentacyclic triterpenoid isolated from tree bark is cytotoxic to cancer cells. There is evidence that specificity proteins (Sps), such as Sp1, Sp3, and Sp4, are overexpressed in tumors and contribute to the proliferative and angiogenic phenotype associated with cancer cells. The objective of this study was to determine the efficacy of BA in decreasing the Sps expression and underlying mechanisms. Results show that BA decreased proliferation and induced apoptosis of estrogen‐receptor‐negative breast cancer MDA‐MB‐231 cells. The BA‐induced Sp1, Sp3, and Sp4 downregulation was accompanied by increased zinc finger ZBTB10 expression, a putative Sp‐repressor and decreased microRNA‐27a levels, a microRNA involved in the regulation of ZBTB10. Similar results were observed in MDA‐MB‐231 cells transfected with ZBTB10 expression plasmid. BA induced cell cycle arrest in the G2/M phase and increased Myt‐1 mRNA (a microRNA‐27a target gene), which causes inhibition in G2/M by phosphorylation of cdc2. The effects of BA were reversed by transient transfection with a mimic of microRNA‐27a. In nude mice with xenografted MDA‐MB‐231 cells, tumor size and weight were significantly decreased by BA treatment. In tumor tissue, ZBTB10 mRNA was increased while mRNA and protein of Sp1, Sp3 and Sp4, as well as mRNA of vascular endothelial growth factor receptor (VEGFR), survivin and microRNA‐27a were decreased by BA. In lungs of xenografted mice, human β2‐microglobulin mRNA was decreased in BA‐treated animals. These results show that the anticancer effects of BA are at least in part based on interactions with the microRNA‐27a‐ZBTB10‐Sp‐axis causing increased cell death. © 2012 Wiley Periodicals, Inc.     

High frequency of neurolymphomatosis as a relapse disease of intravascular large B-cell lymphoma

BACKGROUND:

Intravascular large B‐cell lymphoma (IVL) is characterized by lymphoma cell proliferation in the lumina of small vessels in various organs. A high incidence of neurologic symptoms associated with the central nervous system has been reported, but peripheral nerve involvement (neurolymphomatosis [NL]) rarely has been described.

METHODS:

The medical records from patients who were diagnosed with IVL over the past 4 years were reviewed. A diagnosis of NL was made based on the combination of neurologic symptoms and their correspondence with imaging studies, such as magnetic resonance imaging (MRI), ¹⁸F‐fluoro‐2‐deoxy‐D‐glucose (FDG)‐positron emission tomography/computed tomography (PET/CT), and/or the histologic confirmation of lymphoma cells within the peripheral nerves, nerve root/plexuses, or cranial nerves.

RESULTS:

Four patients with NL were identified among 11 patients who had IVL. All cases of NL occurred as relapsed disease during or shortly after the completion of chemotherapy. Although MRI studies of the brains and whole spines revealed nerve infiltration by gadolinium enhancement in 2 patients, the technology was not sensitive enough to detect such infiltration in the remaining 2 patients. In contrast, FDG‐PET/CT studies successfully revealed cranial or peripheral nerve lesions in all 4 patients and was useful for evaluating therapeutic response. Patients received treatment with high‐dose methotrexate with or without other systemic chemotherapy, which achieved varied success. Further studies will be needed to determine the optimal treatment.

CONCLUSIONS:

Considering the rarity of IVL and NL, the current observations suggested that IVL may have a predilection not only for the vessels but also for both the central and peripheral nervous systems. Cancer 2011. © 2011 American Cancer Society     

Induction of glandular stomach cancers in Helicobacter pylori-infected Mongolian gerbils by 1-nitrosoindole-3-acetonitrile

Helicobacter pylori (H. pylori) infection and high intake of various traditional salt-preserved foods are regarded as risk factors for human gastric cancer. We previously reported that Chinese cabbage contains indole compounds, such as indole-3-acetonitrile, a mutagen precursor. 1-Nitrosoindole-3-acetonitrile (NIAN), formed by the treatment of indole-3-acetonitrile with nitrite under acidic conditions, shows direct-acting mutagenicity. In the present study, NIAN administration by gavage to Mongolian gerbils (MGs) at the dose of 100 mg/kg two times a week resulted in three adduct spots (1.6 adducts/10(8) nucleotides in total), detected in DNA samples from the glandular stomach by (32) P-postlabeling methods. Treatment with six consecutive doses of 100 mg/kg of NIAN, two times a week for 3 weeks, induced well-and moderately-differentiated glandular stomach adenocarcinomas in the MGs at the incidence of 31% under H. pylori infection at 54-104 weeks. Such lesions were not induced in MGs given broth alone, broth + NIAN or infection with H. pylori alone. Thus, endogenous carcinogens formed from nitrosation of indole compounds could be critical risk factors for human gastric cancer development under the influence of H. pylori infection.     

Reliability of serum 1,3‐beta‐d‐glucan assay in patients undergoing renal replacement therapy: a review of the literature

The serum 1,3‐beta‐d ‐glucan (BDG) test is a pan‐fungal serum marker considered to detect the majority of pathogenic fungi, including Aspergillus spp. and Candida spp. For this review we searched for publications dealing with serum BDG levels in patients undergoing renal replacement therapy (RRT). The influence of various different membrane materials used for RRTs in these publications on serum BDG has been reviewed. We found that unmodified cellulose containing membranes increased the serum BDG levels highly, whereas conflicting results have been observed for modified cellulose containing materials. Synthetic materials (e.g. polysuflone) had no influence on serum BDG levels in the majority of the reviewed publications.