Marco A De Velasco

Abstract: Resveratrol (3, 4', 5-trihydroxy-trans-stilbene), a natural phytoalexin found in grapes and wine, has anti-proliferative activity on human-derived cancer cells. In our study, we used a conventional condensation reaction between aldehydes and amines to provide a number of aza-resveratrol (3, 4', 5-trihydroxy-trans- aza-stilbene) derivatives in an attempt to screen for compounds with resveratrol's action but with increased potency. Aza-resveratrol and its hydroxylated derivative (3, 4, 4', 5-tetrahydroxy-trans- aza-stilbene) showed a more enhanced anti-proliferative effect than resveratrol in an MCF-7 breast carcinoma cell line. To identify the cellular targets of the aza derivatives of resveratrol, we conjugated the latter aza-stilbene compound with epoxy-activated agarose and performed affinity purification. Macrophage migration inhibitory factor (MIF), a proinflammatory cytokine, was identified as a major target protein in MCF-7 cell lysates using a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF MS). The aza-resveratrol and its hydroxylated derivative, but not resveratrol, were also found to be potent inhibitors of MIF tautomerase activity, which may be associated with their inhibitory effects on MIF bioactivity for cell growth.

Abstract: Knowledge gained from the identification of genetic and epigenetic alterations that contribute to the progression of prostate cancer in humans is now being implemented in the development of functionally relevant translational models. GEM (genetically modified mouse) models are being developed to incorporate the same molecular defects associated with human prostate cancer. Haploinsufficiency is common in prostate cancer and homozygous loss of PTEN is strongly correlated with advanced disease. In this paper, we discuss the evolution of the PTEN knockout mouse and the cooperation between PTEN and other genetic alterations in tumor development and progression. Additionally, we will outline key points that make these models key players in the development of personalized medicine, as potential tools for target and biomarker development and validation as well as models for drug discovery.

Abstract: Survival rate for patients presenting muscle invasive bladder cancer is very low, and useful therapeutic target has not been identified yet. In the present study, new COX2 downstream signals involved in urothelial carcinoma cell survival were investigated in vitro and in vivo.

Abstract: Molecular targeting agents have become formidable anticancer weapons showing much promise against refractory tumors and functional peptides and are among the more desirable of these nanobio-tools. Intracellular delivery of multiple functional peptides forms the basis for a potent, non-invasive mode of delivery, providing distinctive therapeutic advantages. We examine the growth suppression efficiency of human renal cell carcinoma (RCC) by single-peptide targeting. We simultaneously introduced p16INK4a tumor suppressor peptides by Wr-T-mediated peptide delivery. Wr-T-mediated transport of p16INK4a functional peptide into 10 RCC lines, lacking expression of the p16INK4a molecule, reversed the specific loss of p16 function, thereby drastically inhibiting tumor growth in all but 3 lines by >95% within the first 96 h. In vivo analysis using SK-RC-7 RCC xenografts in nude mice demonstrated tumor growth inhibition by the p16INK4a peptide alone, however, inoculation of Wr-T and the p16INK4a functional peptide mixture, via the heart resulted in complete tumor regression. Thus, restoration of tumor suppressor function with Wr-T peptide delivery represents a powerful approach, with mechanistic implications for the development of efficacious molecular targeting therapeutics against intractable RCC.

Abstract: Activin A is a multi-functional cytokine belonging to the transforming growth factor-β (TGF-β) superfamily; however, the effect of activin A on angiogenesis remains largely unclear. We found that inhibin β A subunit (INHBA) mRNA is overexpressed in gastric cancer (GC) specimens and investigated the effect of activin A, a homodimer of INHBA, on angiogenesis in GC.

Abstract: BIBF 1120 is a potent, orally available triple angiokinase inhibitor that inhibits VEGF receptors (VEGFR) 1, 2, and 3, fibroblast growth factor receptors, and platelet-derived growth factor receptors. This study examined the antitumor effects of BIBF 1120 on hepatocellular carcinoma (HCC) and attempted to identify a pharmacodynamic biomarker for use in early clinical trials.

Abstract: Forkhead box Q1 (FOXQ1) is a member of the forkhead transcription factor family, and it has recently been proposed to participate in gastric acid secretion and mucin gene expression in mice. However, the role of FOXQ1 in humans and especially in cancer cells remains unknown. We found that FOXQ1 mRNA is overexpressed in clinical specimens of colorectal cancer (CRC; 28-fold/colonic mucosa). A microarray analysis revealed that the knockdown of FOXQ1 using small interfering RNA resulted in a decrease in p21(CIP1/WAF1) expression, and a reporter assay and a chromatin immunoprecipitation assay showed that p21 was one of the target genes of FOXQ1. Stable FOXQ1-overexpressing cells (H1299/FOXQ1) exhibited elevated levels of p21 expression and inhibition of apoptosis induced by doxorubicin or camptothecin. Although cellular proliferation was decreased in H1299/FOXQ1 cells in vitro, H1299/FOXQ1 cells significantly increased tumorigenicity [enhanced green fluorescent protein (EGFP): 2/15, FOXQ1: 7/15] and enhanced tumor growth (437 +/- 301 versus 1735 +/- 769 mm3, P < 0.001) in vivo. Meanwhile, stable p21 knockdown of H1299/FOXQ1 cells increased tumor growth, suggesting that FOXQ1 promotes tumor growth independent of p21. Microarray analysis of H1299/EGFP and H1299/FOXQ1 revealed that FOXQ1 overexpression upregulated several genes that have positive roles for tumor growth, including VEGFA, WNT3A, RSPO2, and BCL11A. CD31 and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling staining of the tumor specimens showed that FOXQ1 overexpression mediated the angiogenic and antiapoptotic effect in vivo. In conclusion, FOXQ1 is overexpressed in CRC and enhances tumorigenicity and tumor growth presumably through its angiogenic and antiapoptotic effects. Our findings show that FOXQ1 is a new member of the cancer-related FOX family.

Abstract: Heparan sulfate proteoglycan syndecan-1, CD138, is well known to be associated with cell proliferation, adhesion, and migration in various types of malignancies. In the present study, we focused on the role of syndecan-1 in human urothelial carcinoma of the urinary bladder. Silencing of syndecan-1 by siRNA transfection down-regulated transcriptional factor junB and the long isoform of FLICE-inhibitory protein (FLIP long), resulting in the induction of apoptosis in the urothelial carcinoma cell lines UMUC2 and UMUC3. Knockdown of junB and FLIP long as well as syndecan-1 silencing mediated apoptosis that was inhibited by pan-caspase inhibitors. Transurethral injection of syndecan-1 siRNA into the urinary bladder significantly reduced syndecan-1 gene expression and growth of red fluorescent-labeled KU-7/RFP bladder cancer cells in the mouse orthotopic bladder cancer model. Immunohistochemical examination showed high syndecan-1 protein expression in high-grade, superficial, and deep invasive carcinomas (pT1 and >or=pT2) as well as carcinoma in situ, but not in low-grade and noninvasive phenotypes (pTa). In addition, the percentage of cancer cells positive for syndecan-1 at initial diagnosis was statistically associated with the frequency of bladder cancer recurrence after transurethral resection. In conclusion, syndecan-1 might contribute to urothelial carcinoma cell survival and progression; therefore, this molecule could be a new therapeutic target in human urinary bladder cancer.

Abstract: Renal ischemia-reperfusion injury (IRI), leading to acute kidney injury, is a frequent complication with renal transplantation and it is associated with graft function. Its pathogenesis involves ischemia, vascular congestion and reactive oxygen metabolites. Carvedilol is an antihypertensive drug with potent anti-oxidant properties. In this study we investigated the protective effects of carvedilol in a rat renal IRI model.

Abstract: We previously reported that personalized peptide vaccine (PPV) therapy in combination with leutenizing hormone-releasing hormone (LH-RH) analog and estramustine phosphate in certain cases is safe and capable of inducing both immune responses and clinical responses for metastatic castration-resistant prostate cancer (CRPC) patients. In the present study, PPV monotherapy was given to CRPC patients. Twenty-three patients with metastatic CRPC were treated with PPV without any additional treatment modalities, including LH-RH analogs. Samples were analyzed for peptide-specific cytotoxic T-lymphocyte (CTL) precursor analysis and peptide-reactive IgG. Toxicity and immunological and clinical responses were assessed on a three-monthly basis. Seventeen patients were available for immunological and clinical evaluation. The vaccines were well tolerated, with grade 3 erythema at injection sites in only one patient. Augmentation of CTL or IgG responses to at least one of the peptides was observed in six of 17 (35%) and 15 of 17 (88%) patients tested, respectively. Among 57 peptides used, 9 and 36 peptides induced CTL and IgG responses, respectively. Delayed-type hypersensitivity reaction was observed in eight of 17 patients. More than 30% prostate-specific antigen (PSA) decline was observed in four of 17 patients. Of these, one patient achieved a complete PSA response and another patient showed a partial PSA response with profound shrinking of lymph node metastases and prostate. The overall median survival time was 24 months (range, 5-37 months). These results suggest that PPV monotherapy appears to be safe and capable of inducing peptide-specific immune responses and clinical responses in CRPC patients. This trial was registered with University Hospital Medical Information Network (UMIN) number R000003339.

Abstract: We recently identified a novel human AlkB homologue, ALKBH8, which is expressed in various types of human cancers including human urothelial carcinomas. In examining the role and function of ALKBH8 in human bladder cancer development in vitro, we found that silencing of ALKBH8 through small interfering RNA transfection reduced reactive oxygen species (ROS) production via down-regulation of NAD(P)H oxidase-1 (NOX-1) and induced apoptosis through subsequent activation of c-jun NH(2)-terminal kinase (JNK) and p38. However, we also found that JNK and p38 activation resulted in phosphorylation of H2AX (gammaH2AX), a variant of mammalian histone H2A, which contributes to the apoptosis induced by silencing ALKBH8 and NOX-1. Silencing of ALKBH8 significantly suppressed invasion, angiogenesis, and growth of bladder cancers in vivo as assessed both in the chorioallantoic membrane assay and in an orthotopic mouse model using green fluorescent protein-labeled KU7 human urothelial carcinoma cells. Immunohistochemical examination showed high expression of ALKBH8 and NOX-1 proteins in high-grade, superficially and deeply invasive carcinomas (pT(1) and >pT(2)) as well as in carcinoma in situ, but not in low-grade and noninvasive phenotypes (pT(a)). These findings indicate an essential role for ALKBH8 in urothelial carcinoma cell survival mediated by NOX-1-dependent ROS signals, further suggesting new therapeutic strategies in human bladder cancer by inducing JNK/p38/gammaH2AX-mediated cell death by silencing of ALKBH8.

Abstract: Heparan sulfate proteoglycan syndecan-1 (CD138) is well known to be associated with cell proliferation, adhesion and migration in various types of malignancies. In the present study, we focused on the role of syndecan-1 in human prostate cancer. Immunohistochemical analysis revealed either no or rare expression of syndecan-1 in normal secretory glands and prostate cancer cells at hormone naïve status, whereas the expression was significantly increased in viable cancer cells following neo-adjuvant hormonal therapy. Syndecan-1 expression was much higher in the androgen independent prostate cancer cell lines DU145 and PC3, rather than the androgen-dependent LNCaP, but the level in LNCaP was up-regulated in response to long-term culture under androgen deprivation. Silencing of syndecan-1 by siRNA transfection reduced endogenous production of reactive oxygen species through down-regulating NADPH oxidase 2 and induced apoptosis in DU145 and PC3 cells. Consistently, NADPH oxidase 2 knockdown induced apoptosis to a similar extent. Subcutaneous inoculation of PC3 cells in nude mice demonstrated the reduction of tumor size by localized injection of syndecan-1 siRNA in the presence of atelocollagen. Moreover, the mouse model and chorioallantoic membrane assay demonstrated significant inhibition of vascular endothelial growth factor and tumor angiogenesis by silencing of syndecan-1. In conclusion, syndecan-1 might participate in the process of androgen-dependent to -independent conversion, and be a new target molecule for hormone resistant prostate cancer therapy.

Abstract: Peroxynitrite is a powerful oxidant capable of nitrating phenolic moieties, such as tyrosine or tyrosine residues in proteins and increases after traumatic brain injury (TBI). First, we tested the hypothesis that TBI increases nitrotyrosine (NT) immunoreactivity in the brain by measuring the number of NT-immunoreactive neurons in the cerebral cortex and hippocampus of rats subjected to parasagittal fluid-percussion TBI. Second, we tested the hypothesis that treatment with L-arginine, a substrate for nitric oxide synthase, further increases NT immunoreactivity over TBI alone. Rats were anesthetized with isoflurane and subjected to TBI, sham TBI, or TBI followed by treatment with L-arginine (100 mg/kg). Twelve, 24, or 72 h after TBI, brains were harvested. Coronal sections (10 microm) were incubated overnight with rabbit polyclonal anti-NT antibody, rinsed, and incubated with a biotinylated secondary antibody. The antigen-antibody complex was visualized using a peroxidase-conjugated system with diaminobenzidine as the chromagen. The number of NT-positive cortical and hippocampal neurons increased significantly in both ipsilateral and contralateral hemispheres up to 72 h after TBI compared with the sham-injured group. Remarkably, treatment with L-arginine reduced the number of NT-positive neurons after TBI in both cortex and hippocampus. Our results indicate that L-arginine actually prevents TBI-induced increases in NT immunoreactivity.

Abstract: Although most vaccines target foreign infectious agents, therapeutic cancer vaccines target both well-established and metastatic tumor cells expressing tumor antigens. Active immunotherapy is intended to enhance or activate the immunosurveillance of an individual through a therapeutic vaccine. Renal cell carcinoma (RCC) is one of the most immunoresponsive cancers in humans, which in turn makes it an ideal candidate for immune based therapies.

Abstract: Precise and objective measurements of tumor response have yet to be standardized in the mouse orthotopic bladder cancer model. In this study, we used image analysis and green fluorescent protein (GFP) to objectively measure tumor size in response to chemotherapy. KU-7 human bladder cancer cells transfected with GFP were intravesically inoculated into 8-week-old female nude mice. Fourteen days after tumor cell inoculation, the mice were assigned into a control (PBS) group or a doxorubicin (conc. 1.0 mg/ml) treatment group and received a single instillation of treatment. Fourteen days after treatment, the bladders were surgically exposed and fluorescent images were captured and later analyzed using image analysis. Bladders were processed for histological examination. Tumor incidence determined by GFP expression and histology was 100 and 80%, respectively, in the doxorubicin treatment group. A 9-fold (histology) vs. 12-fold (GFP expression) difference in tumor regression measured by tumor area (P<0.05) and a 5-fold (histology) vs. 9-fold (GFP expression) difference in tumor regression measured by the percent of tumor area in the bladder (P<0.001) were observed in the doxorubicin treatment group. Our findings suggest that using image analysis provides a precise, sensitive and objective means to measure tumor growth and treatment response in the mouse orthotopic bladder cancer model in lieu of histological methods. Consequently, the number of mice required in an experiment can be reduced since tissue samples are not needed for histology, thus making tissue samples readily available for additional assays in both a labor-effective and cost-effective manner.

Abstract: The tumor suppressor gene MMAC/PTEN located on chromosome10q23.3 has dual phosphatase activity in the phosphoinositide-3-kinase signaling pathway and inhibits Akt activation, a serine-threonine kinase, which is involved in proliferative and antiapoptotic pathways. Furthermore, MMAC/PTEN is frequently inactivated in a variety of tumors including prostate cancer. In this study, we generated a new type of gene transfer drug, GelaTen, which is a microsphere of cationized gelatin hydrogels incorporating PTEN plasmid DNA. Using our previously reported radiation-resistant PC3-Bcl-2 human prostate cancer cells (PTEN deleted), we examined the efficacy of GelaTen to force the expression of PTEN in vivo to inhibit tumor growth after intratumoral injection alone or with irradiation. Combinational therapy with GelaTen and irradiation improved both the in vitro and in vivo efficacy of growth inhibition compared with GelaTen or irradiation alone. These data show that GelaTen gene therapy, enabling radiosensitization, can potentially treat prostate cancers that have MMAC/PTEN gene alterations associated with radioresistance.

Abstract: The Akt/mammalian target of the rapamycin (mTOR) signaling pathway represents a promising target for cancer therapy. The phosphorylation status of Akt and of mTOR's phosphorylation target eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) is often used to assess the activity of Akt and mTOR signaling. The purpose of this study was to determine whether primary tumors differ from their metastasis in their expression of pAkt and p4E-BP1.

Abstract: Chelation of excessive neuronal zinc ameliorates zinc neurotoxicity and reduces subsequent neuronal injury. To clarify the molecular mechanisms of this neuroprotective effect, we used a focused cDNA array of stress-response genes with zinc chelation (calcium EDTA) in our rat model of fluid percussion brain injury at 2 h, 24 h, and 7 days after injury. In parallel experiments, we compared neuronal cell death in TUNEL-stained brain sections in traumatized rats with and without calcium EDTA treatment. Zinc chelation induced the expression of several neuroprotective genes; neuroprotective gene expression correlated with substantially decreased numbers of TUNEL-positive cells.

Abstract: The authors recently reported that transgenic mice (hGAS) expressing pharmacologic levels of progastrin (PG) (> 10 nM to 100 nM) exhibited increased susceptibility to colon carcinogenesis in response to azoxymethane (AOM). It is not known whether PG functions as a cocarcinogen at the concentrations observed in patients with hypergastrinemia (approximately 1.0 nM).

Abstract: Aberrant crypt foci (ACFs) may be the earliest recognizable histologic precursor lesion for colon cancer. ACF may develop from a complex of events, including the development of cryptal hyperproliferation, defects in the rate of apoptosis, and abnormalities in cellular adhesion. In this study, we hypothesized that human ACF would exhibit discrete differences in cell adhesion proteins compared with normal mucosa of biologic markers associated with colon cancer. ACFs were isolated from resected colon mucosa from 45 patients undergoing surgery for colon cancer. We evaluated the protein expression of 3 biologic markers that may be related to the progression of aberrant crypt foci to tumors: carcinoembryonic antigen, E-cadherin, and sialyl Tn antigen. In general, ACFs located near cancers in the right colon were more often hyperplastic than dysplastic; this was more noticeable in the left colon. Carcinoembryonic antigen expression was found to be more intense in apical portions of ACF crypts, with sialyl Tn antigen moderately increased, whereas E-cadherin diffusely stained throughout crypts within ACFs. There are significant biologic changes in potential tumor markers that accompany the early transformation of the normal glandular epithelium, some of which are expressed very early in the colon at the stage of appearance of ACF.

Abstract: KIT gain of function mutations play an important role in the pathogenesis of gastrointestinal stromal tumors (GISTs). Imatinib is a selective tyrosine kinase inhibitor of ABL, platelet-derived growth factor receptor (PDGFR), and KIT and represents a new paradigm of targeted therapy against GISTs. Here we report for the first time that, after imatinib treatment, an additional specific and novel KIT mutation occurs in GISTs as they develop resistance to the drug. We studied 12 GIST patients with initial near-complete response to imatinib. Seven harbored mutations in KIT exon 11, and 5 harbored mutations in exon 9. Within 31 months, six imatinib-resistant rapidly progressive peritoneal implants (metastatic foci) developed in five patients. Quiescent residual GISTs persisted in seven patients. All six rapidly progressive imatinib-resistant implants from five patients show an identical novel KIT missense mutation, 1982T-->C, that resulted in Val654Ala in KIT tyrosine kinase domain 1. This novel mutation has never been reported before, is not present in pre-imatinib or post-imatinib residual quiescent GISTs, and is strongly correlated with imatinib resistance. Allelic-specific sequencing data show that this new mutation occurs in the allele that harbors original activation mutation of KIT.

Abstract: We recently reported that transgenic mice overexpressing progastrin were at a higher risk for developing colon cancers in response to azoxymethane (AOM), whereas mice overexpressing gastrin-17 were at a reduced risk. To examine further the role of gastrins in colon carcinogenesis, we generated gastrin gene knockout mice (GAS-KO).

Abstract: Protein kinase C (PKC) has been implicated in colon carcinogenesis in humans and in rodent models. However, little is known about the specific role of individual PKC isozymes in this process. We recently demonstrated that elevated expression of PKC betaII in the colonic epithelium induces hyperproliferation in vivo (N. R. Murray et al., J. Cell Biol., 145: 699-711, 1999). Because hyperproliferation is a major risk factor for colon cancer, we assessed whether specific alterations in PKC betaII expression occur during azoxymethane-induced colon carcinogenesis in mice. An increase in PKC betaII expression was observed in preneoplastic lesions (aberrant crypt foci, 3.7-fold) compared with saline-treated animals, and in colon tumors (7.8-fold; P = 0.011) compared with uninvolved colonic epithelium. In contrast, PKC alpha and PKC betaI (a splicing variant of PKC betaII) expression was slightly decreased in aberrant crypt foci and dramatically reduced in colon tumors. Quantitative reverse transcription-PCR analysis revealed that PKC mRNA levels do not directly correlate with PKC protein levels, indicating that PKC isozyme expression is likely regulated at the posttranscriptional/translational level. Finally, transgenic mice expressing elevated PKC betaII in the colonic epithelium exhibit a trend toward increased colon tumor formation after exposure to azoxymethane. Taken together, our results demonstrate that elevated expression of PKC betaII is an important early, promotive event that plays a role in colon cancer development.

Abstract: Recent studies show that nonamidated gastrins (Gly-gastrin and progastrin) stimulate colonic proliferation. However, the role of nonamidated vs. amidated gastrins in colon carcinogenesis has not been defined. We measured intermediate markers of carcinogenesis in transgenic mice overexpressing either progastrin (hGAS) or amidated gastrin (INS-GAS) in response to azoxymethane (AOM). The hGAS mice showed significantly higher numbers of aberrant crypt foci (140-200% increase) compared with that in wild-type (WT) and INS-GAS mice (P < 0.05) after AOM treatment. The bromodeoxyuridine-labeling index of colonic crypts also was significantly elevated in hGAS mice vs. that in WT and INS-GAS mice. The results therefore provide evidence for a mitogenic and cocarcinogenic role of nonamidated gastrins (progastrin), which is apparently not shared by the amidated gastrins. Although nonamidated gastrins are now believed to mediate mitogenic effects via novel receptors, amidated gastrins mediate biological effects via different receptor subtypes, which may explain the difference in the cocarcinogenic potential of nonamidated vs. amidated gastrins. In conclusion, our results provide strong support for a cocarcinogenic role for nonamidated gastrins in colon carcinogenesis.

Abstract: 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is the most abundant heterocyclic amine carcinogen in the human diet and is a colon carcinogen in the rat. N-Acetyltransferase-2 (NAT2) catalyzes the conversion of PhIP and other heterocyclic amines to a DNA-reactive form. NAT2 has a polymorphic distribution in humans and other mammals, including rats. The rapid NAT2 genotype has been shown to be associated with increased colorectal cancer risk in some, but not all, human epidemiological studies. This investigation was designed to study the role of acetylator genotype in PhIP-induced colon carcinogenesis using aberrant crypt foci (ACF) as an intermediate biomarker. Five-week-old male, rapid-acetylator Fischer 344 (F344) rats and slow-acetylator Wistar-Kyoto (WKY) rats were fed the semipurified AIN76A diet with 0.01% PhIP, 0.04% PhIP, or no PhIP (control) for 8 weeks. PhIP induced ACF in both rapid- and slow-acetylator rats; 0.04% PhIP induced more ACF than 0.01% PhIP. There was no difference in the number of ACF between rapid- and slow-acetylator rats that were fed 0.01% PhIP. However, 0.04% PhIP induced 2-fold higher ACF and a greater dose-dependent increase in PhIP-induced ACF in the rapid-acetylator F344 rats compared with the slow-acetylator WKY rats. The results support human epidemiological studies showing higher risk for colorectal cancer in rapid acetylators who frequently consume meat that is very well done.

Abstract: We assessed the effects of 78 potential chemopreventive agents in the F344 rat using two assays in which the inhibition of carcinogen-induced aberrant crypt foci (ACF) in the colon was the measure of efficacy. In both assays ACF were induced by the carcinogen azoxymethane (AOM) in F344 rats by two sequential weekly injections at a dose of 15 mg/kg. Two weeks after the last AOM injection, animals were evaluated for the number of aberrant crypts detected in methylene blue stained whole mounts of rat colon. In the initiation phase protocol agents were given during the period of AOM administration, whereas in the post-initiation assay the chemopreventive agent was introduced during the last 4 weeks of an 8 week assay, a time when ACF had progressed to multiple crypt clusters. The agents were derived from a priority listing based on reports of chemopreventive activity in the literature and/or efficacy data from in vitro models of carcinogenesis. During the initiation phase carboxyl amidoimidazole, p-chlorphenylacetate, chlorpheniramine maleate, D609, diclofenac, etoperidone, eicosatetraynoic acid, farnesol, ferulic acid, lycopene, meclizine, methionine, phenylhexylisothiocyanate, phenylbutyrate, piroxicam, 9-cis-retinoic acid, S-allylcysteine, taurine, tetracycline and verapamil were strong inhibitors of ACF. During the post-initiation phase aspirin, calcium glucarate, ketoprofen, piroxicam, 9-cis-retinoic acid, retinol and rutin inhibited the outgrowth of ACF into multiple crypt clusters. Based on these data, certain phytochemicals, antihistamines, non-steroidal anti-inflammatory drugs and retinoids show unique preclinical promise for chemoprevention of colon cancer, with the latter two drug classes particularly effective in the post-initiation phase of carcinogenesis.

Abstract: Processing intermediates of preprogastrin (gly-gastrin and progastrin), termed nonamidated gastrins, are mitogenic for several cell types including colonic epithelial cells. However, presently it is not known if nonamidated gastrins play a role in colon carcinogenesis and if the effects are similar to those of amidated gastrins.

Abstract:

Abstract: We assessed the effects of 41 potential chemopreventive agents in the F344 rat using the inhibition of carcinogen-induced aberrant crypt foci (ACF) in the colon as the measure of efficacy. ACF were induced by the carcinogen azoxymethane in F344 rats by two sequential weekly injections at a dose of 15 mg/kg. Two weeks after the last azoxymethane injection, animals were evaluated for the number of aberrant crypts detected in methylene blue-stained whole mounts of rat colon. The 41 agents were derived from a priority listing that was based on reports of chemopreventive activity in the literature and/or efficacy data from in vitro models of carcinogenesis. The list of agents included representative examples of phytochemicals, vitamins, minerals, inhibitors of proliferation, inducers of Phase 1 and Phase 2 metabolism systems, nonsteroidal anti-inflammatory agents, and differentiation agents. Eighteen agents were positive in the assay, significantly reducing the incidence of ACF at least in one of two doses tested. As a chemical class, the nonsteroidal anti-inflammatory drugs, which included ibuprofen, ketoprofen, piroxicam, and indomethacin, were most active; other less potent agents were arginine, butylated hydroxyanisole, curcumin, diallyl sulfide, difluoromethylornithine, 18 beta-glycyrrhetinic acid, indole-3-carbinol, oltipraz, purpurin, rutin, and the sodium salts of butyrate, selenite, and thiosulfate. Twenty-three agents did not inhibit ACF; included among these were several agents that promoted the development of ACF at one or both doses tested: benzyl isothiocyanate,calcium glucarate, catechin, dihydroepiandosterone, fluocinolone acetonide,folic acid, levamisole, 2-mercaptoethanesulfonic acid, nordihydroguiaretic acid, potassium glucarate, propyl gallate, beta-sitosterol, sodium cromolyn, sodium molybdate, and sulfasalazine. The aberrant crypt assay demonstrates reasonable specificity and sensitivity in predicting which agents are likely to prevent colon cancer.

Abstract: Aberrant crypts are aggregates of single to multiple colonic crypts evidencing hallmarks of dysplasia and may be the earliest detectable pathological lesions for colon cancer. The aberrant crypt assay has been developed in 2 protocols. In one, putative chemoprevention agents are tested for inhibitory effects when administered concomitantly with a carcinogen. In the other, the objective of this study, aberrant crypts were induced in F344 rats by parenteral injection of the colon carcinogen azoxymethane (AOM) and allowed to develop for 4 weeks, when an average of 90-100 aberrant crypt foci per colon were found in the methylene blue-stained colon. Then, during the second 4 weeks of the experiment, aberrant crypts were allowed to further develop to a frequency of > 150 foci per colon, a time when multi-crypt foci were observed. During this time we tested the inhibitory effects of 4 analgesic drugs and 2 differentiation agents for effects of aberrant crypt growth and development. We found the non-steroidal anti-inflammatory drugs piroxicam, aspirin and ibuprofen, but not acetaminophen, to be effective in suppressing aberrant crypt formation or the progression to foci of multiple aberrant crypts. Treatment with chemosuppressing agents 13-cis-retinoic acid (13-cRA) and 4-hydroxyphenretinamide (4-HPR), known differentiating agents, however, did suppress expansion of aberrant crypt foci, with 13-cRA being the much more potent agent.

Abstract: Micronuclei frequency, a marker of genotoxicity, was studied within a trial of alpha-tocopherol for chemoprevention of oral leukoplakia. Oral swabs were obtained from two sites, the leukoplakia lesion and normal-appearing mucosa, at baseline and following 24 weeks of therapy with 400 international units of alpha-tocopherol twice daily. These specimens were analyzed for micronuclei frequency. The major risk factors for oral carcinogenesis in the group studied were cigarette smoking and alcohol consumption. alpha-tocopherol therapy produced a significant reduction in micronuclei frequencies in specimens from both the visible lesions (P < 0.01) and the normal-appearing mucosa (P < 0.01). The micronuclei frequencies, both at baseline and following therapy, were greater in specimens taken from the lesion than in those from the normal-appearing mucosa. Although these results indicate that alpha-tocopherol has a beneficial effect in oral carcinogenesis, there was no significant clinical or histological response associated with the change in micronuclei frequency. Micronuclei frequency has not yet been validated as a biomarker for cancer incidence, and consequently, its utility as an intermediate end point for chemoprevention trials is not known. Determining clinical significance of micronuclei frequency patterns in oral carcinogenesis and chemoprevention will require further study.

Abstract: Biomarkers are being sought that could serve as surrogate end points for chemoprevention trials. Micronuclei, cytoplasmic fragments of DNA, have been proposed as a biomarker and studied in oral pre-malignancy. This study evaluated micronuclei frequency in a randomized chemoprevention trial of oral pre-malignancy. A recent clinical trial evaluated the responses of pre-malignant oral lesions to 3 months of therapy with isotretinoin followed by 9 months of either low-dose isotretinoin or beta-carotene. For 57 study participants, micronuclei were counted in mucosal scrapings of the lesion and in normal-appearing mucosa at baseline and following 3 months and 12 months of therapy. Micronuclei counts were higher in scrapings from the lesion than in the normal-appearing mucosa. Following 3 months of isotretinoin, the micronuclei counts in scrapings of the lesion were significantly reduced. With treatment, the mean micronuclei count declined at 3 months. In a randomized comparison, both isotretinoin and beta-carotene maintained the suppression of micronuclei. The change in micronuclei count was not associated with the clinical or histological response to treatment. Chemoprevention treatment with isotretinoin led to a reduction in frequency of micronuclei, a marker of recent DNA injury, which was then maintained by both isotretinoin and beta-carotene.

Abstract: Bronchial micronuclei, small fragments of extra-nuclear DNA formed during cell division, provide a non-specific but quantifiable marker of DNA damage. Micronuclei have been used to assess carcinogen exposure and as an intermediate endpoint in chemoprevention trials. As part of an ongoing chemoprevention trial, heavy smokers underwent screening bronchoscopy, with biopsies taken at 6 standardized sites. Micronuclei counts were obtained for each site in each of the 40 volunteers found to have squamous metaplasia. Unlike squamous metaplasia, the average micronuclei counts among these heavy smokers were not associated with smoking history. Micronuclei counts were also not associated with the presence or extent of metaplasia. A striking degree of intra-individual variability was observed by comparing the micronuclei counts from different biopsy sites within individuals. The findings suggest that use of micronuclei from single sites may be misleading as a marker of carcinogen exposure or as an estimate of cancer risk. Serial measurements in individuals may provide the most useful information concerning carcinogenic exposure and the impact of chemopreventive agents.