Fluorescence contrast-enhanced proliferative lesion imaging by enema administration of indocyanine green in a rat model of colon carcinogenesis.

The fluorescent contrast agent indocyanine green (ICG) is approved by the Food and Drug Administration for clinical applications. We previously reported that cultured human colon tumor cells preferentially take up ICG by endocytic activity in association with disruption of their tight junctions. The present study explored ICG availability in fluorescence imaging of the colon to identify proliferative lesions during colonoscopy. The cellular uptake of ICG in cultured rat colon tumor cells was examined using live-cell imaging. Colon lesions in rats administered an ICG-containing enema were further assessed in rats with azoxymethane-induced colon carcinogenesis, using in vivo endoscopy, ex vivo microscopy, and immunofluorescence microscopy. The uptake of ICG by the cultured cells was temperature-dependent. The intracellular retention of the dye in the membrane trafficking system suggested endocytosis as the uptake mechanism. ICG administered via enema accumulated in colon proliferative lesions ranging from tiny aberrant crypt foci to adenomas and localized in proliferating cells. Fluorescence endoscopy detected these ICG-positive colonic proliferative lesions in vivo. The immunoreactivity of the tight-junction molecule occludin was altered in the proliferative lesions, suggesting the disruption of the integrity of tight junctions. These results suggest that fluorescence contrast-enhanced imaging following the administration of an ICG-containing enema can enhance the detection of mucosal proliferative lesions of the colon during colonoscopy. The tissue preference of ICG in the rat model evaluated in this study can be attributed to the disruption of tight junctions, which in turn promotes endocytosis by proliferative cells and the cellular uptake of ICG.

Promoter-region hypermethylation and expression downregulation of Yy1 (Yin yang 1) in preneoplastic liver lesions in a thioacetamide rat hepatocarcinogenesis model.

Thioacetamide (TAA) has been used to develop a rodent model for hepatocarcinogenesis. To determine the genes with epigenetic modifications in early hepatocarcinogenesis, we did a genome-wide scan for hypermethylated promoter regions using CpG island microarrays in TAA-promoted rat liver tissue. Eight genes were selected based on the microarray profile; of these, Yy1 and Wdr45b were confirmed to be hypermethylated by methylation-specific polymerase chain reaction (PCR) and pyrosequencing and downregulated by real-time reverse transcription PCR. Non-neoplastic liver cells had nuclear Yy1 immunoreactivity, while preneoplastic foci with glutathione S-transferase placental form (GST-P) immunoreactivity had decreased Yy1 immunoreactivity. The incidence of these foci was proportional to the dose of TAA administered. Co-expression analysis of gene products downstream of Yy1 revealed increased nuclear phospho-c-Myc(+) foci as well as nuclear and cytoplasmic p21(Cip1+) foci in Yy1(-) or GST-P(+) foci in response to TAA-promotion dose. Although the absolute number of cells was low, the incidence of death receptor 5(-) foci was increased in Yy1(-) foci in proportion to the TAA dose. Yy1(-)/GST-P(+) foci revealed a higher number of proliferating cell nuclear antigen (PCNA)-immunoreactive cells than Yy1(+)/GST-P(+) foci, while cleaved caspase-3(+) cells were unchanged between Yy1(-)/GST-P(+) and Yy1(+)/GST-P(+) foci. In the case of Wdr45b, most GST-P(+) foci were Wdr45b(-) and were not increased by TAA promotion. These results suggest involvement of Yy1 in the epigenetic gene regulation at the early stages of TAA promoted cell proliferation and concomitant cell cycle arrest in preneoplastic lesions.

Tumor suppression effects of bilberry extracts and enzymatically modified isoquercitrin in early preneoplastic liver cell lesions induced by piperonyl butoxide promotion in a two-stage rat hepatocarcinogenesis model.

To investigate the protective effect of bilberry extracts (BBE) and enzymatically modified isoquercitrin (EMIQ) on the hepatocarcinogenic process involving oxidative stress responses, we used a two-stage hepatocarcinogenesis model in N-diethylnitrosamine-initiated and piperonyl butoxide (PBO)-promoted rats. We examined the modifying effect of co-administration with BBE or EMIQ on the liver tissue environment including oxidative stress responses, cell proliferation and apoptosis, and phosphatase and tensin homolog (PTEN)/Akt and transforming growth factor (TGF)-ß/Smad signalings on the induction mechanism of preneoplastic lesions during early stages of hepatocellular tumor promotion. PBO increased the numbers and area of glutathione S-transferase placental form (GST-P)(+) liver cell foci and the numbers of Ki-67(+) proliferating cells within GST-P(+) foci. Co-administration of BBE or EMIQ suppressed these effects with the reductions of GST-P(+) foci (area) to 48.9-49.4% and Ki-67(+) cells to 55.5-61.4% of the PBO-promoted cases. Neither BBE nor EMIQ decreased microsomal reactive oxygen species induced by PBO. However, only EMIQ suppressed the level of thiobarbituric acid-reactive substances to 78.4% of the PBO-promoted cases. PBO increased the incidences of phospho-PTEN(-) foci, phospho-Akt substrate(+) foci, phospho-Smad3(-) foci and Smad4(-) foci in GST-P(+) foci. Both BBE and EMIQ decreased the incidences of phospho-PTEN(-) foci in GST-P(+) foci to 59.8-72.2% and Smad4(-) foci to 62.4-71.5% of the PBO-promoted cases, and BBE also suppressed the incidence of phospho-Akt substrate(+) foci in GST-P(+) foci to 75.2-75.7% of the PBO-promoted cases. These results suggest that PBO-induced tumor promotion involves facilitation of PTEN/Akt and disruptive TGF-ß/Smad signalings without relation to oxidative stress responses, but this promotion was suppressed by co-treatment with BBE or EMIQ through suppression of cell proliferation activity of preneoplastic liver cells.

Modifying effects of liver tumor promotion in rats subjected to co-administration of indole-3-carbinol and phenobarbital.

Indole-3-carbinol (I3C) and phenobarbital (PB) are cytochrome P450 (CYP) 1A and CYP2B inducers, respectively, and have liver tumor-promoting effects in rats. In this study, we investigated the modifying effects on tumor promotion by I3C and PB co-administration. Six-week-old male F344 rats received a single intraperitoneal injection of N-diethylnitrosamine for initiation treatment. Two weeks after the initiation, rats were given no tumor-promoting agents (DEN alone), I3C (2,500 or 5,000 ppm in diet), PB (60 or 120 ppm in drinking water), or 2,500 ppm I3C + 60 ppm PB for 6 weeks. One week after the I3C/PB treatments, all animals underwent a two-thirds partial hepatectomy. The number and area of liver cell foci positive for glutathione S-transferase placental form (GST-P(+) foci) were not significantly fluctuated in the PB+I3C group in the isoadditive statistical model. On the contrary, the mRNA levels of Cyp2b1/2 and Nqo1 were suppressed and enhanced, respectively, in the PB+I3C group in the isoadditive model, but there was no enhancement in the microsomal reactive oxygen species (ROS) production, thiobarbituric acid-reactive substance levels, and Ki-67(+) cell ratio in this group. The results suggest that the co-administration of I3C and PB causes no modifying effects in liver tumor promotion in rats.

Immunohistochemical cellular distribution of proteins related to M phase regulation in early proliferative lesions induced by tumor promotion in rat two-stage carcinogenesis models.

We have previously reported that 28-day treatment with hepatocarcinogens increases liver cells expressing p21(Cip1), a G1/S checkpoint protein, and M phase proteins, i.e., nuclear Cdc2, Aurora B, phosphorylated-Histone H3 (p-Histone H3) and heterochromatin protein 1α (HP1α), in rats. To examine the roles of these markers in the early stages of carcinogenesis, we investigated their cellular distribution in several carcinogenic target organs using rat two-stage carcinogenesis models. Promoting agents targeting the liver (piperonyl butoxide and methapyrilene hydrochloride), thyroid (sulfadimethoxine), urinary bladder (phenylethyl isothiocyanate), and forestomach and glandular stomach (catechol) were administered to rats after initiation treatment for the liver with N-diethylnitrosamine, thyroid with N-bis(2-hydroxypropyl)nitrosamine, urinary bladder with N-butyl-N-(4-hydroxybutyl)nitrosamine, and forestomach and glandular stomach with N-methyl-N'-nitro-N-nitrosoguanidine. Numbers of cells positive for nuclear Cdc2, Aurora B, p-Histone H3 and HP1α increased within preneoplastic lesions as determined by glutathione S-transferase placental form in the liver or phosphorylated p44/42 mitogen-activated protein kinase in the thyroid, and hyperplastic lesions having no known preneoplastic markers in the urinary bladder, forestomach and glandular stomach. Immunoreactive cells for p21(Cip1) were decreased within thyroid preneoplastic lesions; however, they were increased within liver preneoplastic lesions and hyperplastic lesions in other organs. These results suggest that M phase disruption commonly occur during the formation of preneoplastic lesions and hyperplastic lesions. Differences in the expression patterns of p21(Cip1) between thyroid preneoplastic and proliferative lesions in other organs may reflect differences in cell cycle regulation involving G1/S checkpoint function between proliferative lesions in each organ.

Suppressive effect of liver tumor-promoting activities in rats subjected to combined administration of phenobarbital and piperonyl butoxide.

Phenobarbital (PB) is a cytochrome P450 (CYP) 2B inducer, and piperonyl butoxide (PBO) is a CYP1A/2B inducer. These inducers have liver tumor-promoting effects in rats. In this study, we performed a rat two-stage liver carcinogenesis bioassay to examine the tumor-promoting effect of PB and PBO co-administration. Male rats received an intraperitoneal injection of N-diethylnitrosamine (DEN) for initiation. Two weeks after DEN administration, rats were given PB (60 or 120 ppm in drinking water), PBO (1,250 or 2,500 ppm in diet) or 60 ppm PB+1,250 ppm PBO for 6 weeks. One week after the PB/PBO treatment, all rats were subjected to a two-thirds partial hepatectomy. To evaluate the effect of the combined administration, we used two statistical additive models. In the isoadditive model, the average values of the area of GST-P positive foci in the PB+PBO group were significantly lower than those in the High PB or High PBO groups. In the heteroadditive model, the net values of Cyp1a1 mRNA level and microsomal reactive oxygen species (ROS) production in the PB+PBO group were significantly lower than the sum of those in the Low PB or Low PBO groups. On the contrary, there was no interactive effect in the PCNA-positive hepatocyte ratio, mRNA levels of Cyp2b1/2, Gstm3, Gpx2 and Nqo1, and the level of thiobarbituric acid-reactive substances in the PB+PBO group. These results suggest that PB and PBO co-administration causes suppressive effects in liver tumor-promoting activity in rats resulting from inhibited microsomal ROS production because of suppression of CYP1A induction.

Increased cellular distribution of vimentin and ret in the cingulum of rat offspring after developmental exposure to decabromodiphenyl ether or 1,2,5,6,9,10-hexabromocyclododecane.

To determine effects of developmental exposure to brominated flame retardants (BFRs), weak thyroid hormone disruptors, on white matter development, white matter-specific global gene expression analysis was performed using microdissection techniques and microarrays in male rats exposed maternally to decabromodiphenyl ether (DBDE), one of the representative BFRs, at 10, 100 or 1000 ppm. Based on previous gene expression profiles of developmental hypothyroidism and DBDE-exposed cases, vimentin(+) immature astrocytes and ret proto-oncogene (Ret)(+) oligodendrocytes were immunohistochemically examined after developmental exposure to representative BFRs, i.e., DBDE, 1,2,5,6,9,10-hexabromocyclododecane (HBCD; 100, 1000 or 10,000 ppm) and tetrabromobisphenol A (TBBPA; 100, 1000 or 10,000 ppm). Vimentin(+) and Ret(+) cell populations increased at ≥ 100 ppm and ≥ 10 ppm DBDE, respectively. Vimentin(+) and Ret(+) cells increased at ≥ 1000 ppm HBCD, with no effect of TBBPA. The highest dose of DBDE and HBCD revealed subtle fluctuations in serum thyroid-related hormone concentrations. Thus, DBDE and HBCD may exert direct effects on glial cell development at ≥ middle doses. At high doses, hypothyroidism may additionally be an inducing mechanism, although its contribution is rather minor.

Inhibitory effect of α-lipoic acid on thioacetamide-induced tumor promotion through suppression of inflammatory cell responses in a two-stage hepatocarcinogenesis model in rats.

To investigate the protective effect of α-lipoic acid (a-LA) on the hepatocarcinogenic process promoted by thioacetamide (TAA), we used a two-stage liver carcinogenesis model in N-diethylnitrosamine (DEN)-initiated and TAA-promoted rats. We examined the modifying effect of co-administered a-LA on the liver tissue environment surrounding preneoplastic hepatocellular lesions, with particular focus on hepatic macrophages and the mechanism behind the decrease in apoptosis of cells surrounding preneoplastic hepatocellular lesions during the early stages of hepatocellular tumor promotion. TAA increased the number and area of glutathione S-transferase placental form (GST-P)(+) liver cell foci and the numbers of proliferating and apoptotic cells in the liver. Co-administration with a-LA suppressed these effects. TAA also increased the numbers of ED2(+), cyclooxygenase-2(+), and heme oxygenase-1(+) hepatic macrophages as well as the number of CD3(+) lymphocytes. These effects were also suppressed by a-LA. Transcript levels of some inflammation-related genes were upregulated by TAA and downregulated by a-LA in real-time RT-PCR analysis. Outside the GST-P(+) foci, a-LA reduced the numbers of apoptotic cells, active caspase-8(+) cells and death receptor (DR)-5(+) cells. These results suggest that hepatic macrophages producing proinflammatory factors may be activated in TAA-induced tumor promotion. a-LA may suppress tumor-promoting activity by suppressing the activation of these macrophages and the subsequent inflammatory responses. Furthermore, a-LA may suppress tumor-promoting activity by suppressing the DR5-mediated extrinsic pathway of apoptosis and the subsequent regeneration of liver cells outside GST-P(+) foci.

Liver tumor promoting effect of orphenadrine in rats and its possible mechanism of action including CAR activation and oxidative stress.

Orphenadrine (ORPH), an anticholinergic agent, is a cytochrome P450 (CYP) 2B inducer. CYP2B inducers are known to have liver tumor-promoting effects in rats. In this study, we performed a rat two-stage liver carcinogenesis bioassay to examine the tumor-promoting effect of ORPH and to clarify its possible mechanism of action. Male rats were given a single intraperitoneal injection of N-diethylnitrosamine (DEN) as an initiation treatment. Two weeks after DEN administration, rats were fed a diet containing ORPH (0, 750, or 1,500 ppm) for 6 weeks. One week after the ORPH-administration rats were subjected to two-thirds partial hepatectomy for the acceleration of hepatocellular proliferation. The number and area of glutathione S-transferase placental form-positive foci significantly increased in the DEN-ORPH groups. Real-time RT-PCR revealed increased mRNA expression levels of Cyp2b1/2, Mrp2 and Cyclin D1 in the DEN-ORPH groups and of Gpx2 and Gstm3 in the DEN-High ORPH group. Microsomal reactive oxygen species (ROS) production and oxidative stress markers such as thiobarbituric acid-reactive substances and 8-hydroxydeoxyguanosine were increased in the DEN-High ORPH group. Immunohistochemically, constitutively active/androstane receptor (CAR) were clearly localized in the nuclei of hepatocytes in the DEN-ORPH groups. These results suggest that ORPH causes nuclear translocation of CAR resulting in the induction of the liver tumor-promoting activity. Furthermore, oxidative stress resulting from ROS production is also involved in the liver tumor-promoting activity of ORPH.

Enhanced liver tumor promotion activity in rats subjected to combined administration of phenobarbital and orphenadrine.

Phenobarbital (PB) and orphenadrine (ORPH) are cytochrome P450 (CYP) 2B inducers and have liver tumor-promoting effects in rats. In this study, we performed a rat two-stage liver carcinogenesis bioassay to examine the tumor-promoting effect of PB and ORPH co-administration. Twelve male rats per group were given an intraperitoneal injection of N-diethylnitrosamine (DEN) for initiation. Two-week after DEN administration, rats were given PB (60 or 120 ppm in drinking water), ORPH (750 or 1,500 ppm in diet) or 60 ppm PB+750 ppm ORPH for 6-week. One-week after the PB/ORPH treatment, all rats were subjected to two-thirds partial hepatectomy. To evaluate the effect of the combined administration, we used two statistical models: a heteroadditive model and an isoadditive model. In the heteroadditive model, the net values of the number and area of glutathione S-transferase placental form (GST-P) positive foci, Cyp2b1/2, Gstm3 and Gpx2 mRNA levels, microsomal reactive oxygen species (ROS) production and thiobarbituric acid-reactive substances level in the PB+ORPH group were significantly higher than the sum of the net values of those in the Low PB and Low ORPH groups. In the isoadditive model, the average values of the area of GST-P positive foci and PCNA positive hepatocyte ratio and Gstm3 mRNA level in the PB+ORPH group were significantly higher than the average values of those in the High PB and High ORPH groups. These results suggest that PB and ORPH co-administration causes synergistic effects in liver tumor-promoting activity in rats resulting from oxidative stress due to enhanced microsomal ROS production.

Methacarn as a whole brain fixative for gene and protein expression analyses of specific brain regions in rats.

For molecular analysis in anatomically-specific brain regions for rodent studies, it is necessary to establish a fast and accurate procedure for tissue sampling to achieve high integrity and expression fidelity of extracted molecules. The present study was performed to examine suitability of whole brain fixation with methacarn and subsequent tissue sampling using punch-biopsy devices for gene expression analysis in rats. After fixation, each specific region, i.e., hippocampal dentate gyrus, corpus callosum, cingulate cortex or cerebellar vermis was collected, and the integrity and variability of expression data of extracted total RNAs and polypeptides were examined. Methacarn fixation, acetone fixation, and unfixed tissues were compared. Methacarn fixation resulted in high integrity of total RNAs sufficient for conducting global expression analysis and superior in terms of uniformity in the integrity among brain regions to that of acetone fixation. Extracted polypeptide after methacarn fixation revealed similar integrity to that without fixation or with acetone fixation. Methacarn fixation resulted in lower mRNA expression variability between samples than acetone fixation in microarray analysis. The fidelity of polypeptide expression was mostly equivalent between methacarn and acetone fixation in 2-dimensional differential in-gel electrophoresis, although the expression levels of a small number of polypeptides from acetone-fixed tissues were affected. These results suggest that whole brain fixation with methacarn retains advantages for global analyses of mRNAs and polypeptides in rodent studies.

Aberrant activation of M phase proteins by cell proliferation-evoking carcinogens after 28-day administration in rats.

We have previously reported that hepatocarcinogens increase liver cells expressing p21(Cip1), a G1 checkpoint protein and M phase proteins after 28-day treatment in rats. This study aimed to identify early prediction markers of carcinogens available in many target organs after 28-day treatment in rats. Immunohistochemical analysis was performed on Ki-67, p21(Cip1) and M phase proteins [nuclear Cdc2, phospho-Histone H3 (p-Histone H3), Aurora B and heterochromatin protein 1α (HP1α)] with carcinogens targeting different organs. Carcinogens targeting thyroid (sulfadimethoxine; SDM), urinary bladder (phenylethyl isothiocyanate), forestomach (butylated hydroxyanisole; BHA), glandular stomach (catechol; CC), and colon (2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and chenodeoxycholic acid) were examined using a non-carcinogenic toxicant (caprolactam) and carcinogens targeting other organs as negative controls. All carcinogens increased Ki-67(+), nuclear Cdc2(+), p-Histone H3(+) or Aurora B(+) carcinogenic target cells, except for both colon carcinogens, which did not increase cell proliferation. On the other hand, p21(Cip1+) cells increased with SDM and CC. HP1α responded only to BHA. Results revealed carcinogens evoking cell proliferation concurrently induced cell cycle arrest at M phase or showing chromosomal instability reflecting aberration in cell cycle regulation, irrespective of target organs, after 28-day treatment. Therefore, M phase proteins may be early prediction markers of carcinogens evoking cell proliferation in many target organs.

Expression patterns of cell cycle proteins in the livers of rats treated with hepatocarcinogens for 28 days.

Some hepatocarcinogens induce cytomegaly, which reflects aberrant cell cycling and increased ploidy, from the early stages of administration to animals. To clarify the regulatory molecular mechanisms behind cell cycle aberrations related to the early stages of hepatocarcinogenesis, we performed gene expression analysis using microarrays and real-time reverse transcription polymerase chain reaction followed by immunohistochemical analysis in the livers of rats treated with the cytomegaly inducing hepatocarcinogens thioacetamide (TAA), fenbendazole, and methyleugenol, the cytomegaly non-inducing hepatocarcinogen piperonyl butoxide (PBO), or the non-carcinogenic hepatotoxicants acetaminophen and α-naphthyl isothiocyanate, for 28 days. Gene expression profiling showed that cell cycle-related genes, especially those of G(2)/M phase, were mostly upregulated after TAA treatment. Immunohistochemical analysis was performed on cell cycle proteins that were upregulated by TAA treatment and on related proteins. All hepatocarcinogens, irrespective of their cytomegaly inducing potential, increased liver cells immunoreactive for p21(Cip1), which acts on cells arrested in G(1) phase, and for Aurora B or Incenp, which is suggestive of an increase in a cell population with chromosomal instability caused by overexpression. PBO did not induce cell proliferation after 28-day treatment. Hepatocarcinogens that induced cell proliferation after 28-day treatment also caused an increase in p53(+) cells in parallel with increased apoptotic cells, as well as increased population of cells expressing M phase-related proteins nuclear Cdc2, phospho-Histone H3, and HP1α. These results suggest that hepatocarcinogens may increase cellular populations arrested in G1 phase or showing chromosomal instability after 28-day treatment. Hepatocarcinogens that induce cell cycle facilitation may cause M phase arrest accompanied by apoptosis.

Involvement of PTEN/Akt signaling and oxidative stress on indole-3-carbinol (I3C)-induced hepatocarcinogenesis in rats.

We previously reported that indole-3-carbinol (I3C) had hepatocellular tumor-promoting activity in a short-term (8 weeks) two-stage liver carcinogenesis model in rats. It was suggested that this effect was related to the production of reactive oxygen species (ROS) caused by cytochrome P450 1A (CYP1A) induction. In the present study, 0.5% I3C was administered to DEN-initiated rats for 26 weeks to examine the effect of prolonged administration of I3C and to clarify the possible mechanisms of I3C-induced hepatocarcinogenesis. The number and area of GST-P positive foci, ROS production, TBARS level, 8-OHdG content and mRNA levels of Ahr and Nrf2 gene batteries significantly increased in the DEN-I3C group compared with the DEN-alone group. Furthermore, some GST-P positive preneoplastic foci progressed to hepatocellular adenomas with the prolongation of I3C administration. Lack of PTEN and phospho-Smad2/3 expression and translocations of PDPK1 and phospho-Akt substrates to underneath the cell membrane were observed in the majority of hepatocellular adenomas. In addition, the number of Ki-67 positive cells increased in adenomas compared with the preneoplastic foci. These results suggest that the administration of I3C for 26 weeks in DEN-initiated rats induces tumor progression from hepatocellular altered foci to hepatocellular adenomas by ROS-mediated Akt activation that inhibits the TGF-ß/Smad signaling and results in the increased cell proliferation.

Effect of enzymatically modified isoquercitrin on preneoplastic liver cell lesions induced by thioacetamide promotion in a two-stage hepatocarcinogenesis model using rats.

To investigate the protective effect of enzymatically modified isoquercitrin (EMIQ) on the hepatocarcinogenic process, we used a two-stage hepatocarcinogenesis model in N-diethylnitrosamine-initiated and thioacetamide (TAA)-promoted rats. We examined the modifying effect of co-administration with EMIQ on the liver tissue environment including hepatic macrophages and lymphocytes and on the induction mechanism of preneoplastic cell apoptosis during early stages of hepatocellular tumor promotion. TAA increased the number and area of glutathione S-transferase placental form (GST-P)(+) liver cell foci and the numbers of proliferating and apoptotic cells in randomly selected areas in liver sections. Co-administration with EMIQ suppressed these effects. TAA also increased the numbers of ED2(+), cyclooxygenase-2(+), and heme oxygenase-1(+) liver cells, as well as the number of CD3(+) lymphocytes. These effects were also suppressed by EMIQ. EMIQ increased liver levels of thiobarbituric acid-reactive substance and 8-hydroxydeoxyguanosine, and TUNEL(+) apoptotic cells, death receptor 5 (DR5)(+) cells and 4-hydroxy-2-nonenal(+) cells within GST-P(+) foci. Outside the GST-P(+) foci, EMIQ decreased the numbers of apoptotic cells and DR5(+) cells. These results suggest that TAA-induced tumor promotion involves activation of hepatic macrophages producing proinflammatory factors. EMIQ may suppress the TAA-induced tumor-promoting activity by an anti-inflammatory mechanism mediated by suppressing the activation of these macrophages. Furthermore, EMIQ may suppress tumor-promoting activity differentially between the inside and outside of GST-P(+) foci. Within GST-P(+) foci, EMIQ facilitates the apoptosis of preneoplastic cells through the upregulation of DR5. Outside the GST-P(+) foci, EMIQ suppresses apoptosis and the subsequent regeneration of non-transformed liver cells.

Direct progression of capsular invasive carcinomas from subcapsular focal hyperplasias induced by hypothyroidism-mediated tumor promotion in a rat two-stage thyroid carcinogenesis model.

PURPOSE: Some goitrogens promote thyroid carcinogenesis in rats in an initiation-promotion model; this model frequently produces carcinomas that invade fibrously thickened capsules, termed capsular invasive carcinomas (CICs). The present study tested a hypothesis that CICs originate from parenchymal proliferative lesions located beneath the capsule. METHODS: Cell proliferation activity, cell-cycle kinetics and cellular invasion were immunohistochemically examined in subcapsular proliferative lesions in male F344 rats treated with an anti-thyroid agent, propylthiouracil or sulfadimethoxine, during the tumor-promotion phase after initiation with N-bis(2-hydroxypropyl)nitrosamine. RESULTS: Focal follicular cell hyperplasias (FFCHs) were the most commonly observed parenchymal proliferative lesions. Subcapsular FFCHs located near CICs showed more Ki-67(+) cells in the capsular side than the contrary parenchymal center side. Most of these FFCHs located near CICs showed accumulated immunoreactivity for cyclin A, cyclin D, cyclin E and cyclin-dependent kinase-2, whereas most subcapsular FFCHs located elsewhere did not show such accumulated expression of cell-cycle molecules. Subcapsular FFCHs immunoreactive at the capsular front for tenascin-C, a tumor invasion marker of extracellular matrix protein, showed high proliferation activity. CONCLUSIONS: Subcapsular FFCH-forming cells can potentially spread directly into the fibrously thickened capsule to form CICs by accelerating cell-cycle activity.

In vivo imaging of tissue-remodeling activity involving infiltration of macrophages by a systemically administered protease-activatable probe in colon cancer tissues.

This study evaluated the detection of tumors using in vivo imaging with a commercially available and systemically administered protease-activatable fluorescent probe, ProSense. To this end, we analyzed the delivery and uptake of ProSense as well as the target protease and its cellular source in a mouse xenograft tumor model. In vivo and ex vivo multi wavelength imaging revealed that ProSense signals accumulated within tumors, with preferential distribution in the vascular leakage area that correlates with vasculature development at the tumor periphery. Immunohistochemically, cathepsin B, which is targeted by ProSense, was specifically localized in macrophages. The codistribution of tenascin C immunoreactivity and gelatinase activity provided evidence of tissue-remodeling at the tumor periphery. Furthermore, in situ zymography revealed extracellular ProSense cleavage in such areas. Colocalization of cathepsin B expression and ProSense signals showing reduction by addition of cathepsin B inhibitor was confirmed in cultured macrophage-derived RAW264.7 cells. These results suggest that increased tissue-remodeling activity involving infiltration of macrophages is a mechanism that may be responsible for the tumor accumulation of ProSense signals in our xenograft model. We further confirmed ProSense signals at the tumor margin showing cathepsin B(+) macrophage infiltration in a rat colon carcinogenesis model. Together, these data demonstrate that systemically administered protease-activatable probes can effectively detect cancer invasive fronts, where tissue-remodeling activity is high to facilitate neoplastic cell invasion.

Fluctuations in cell proliferation, apoptosis, and cell cycle regulation at the early stage of tumor promotion in rat two-stage carcinogenesis models.

We previously demonstrated that 28-day administration of carcinogens that evoked cell proliferation as determined by immunoreactivity for Ki-67 or minichromosome maintenance 3 (Mcm3), in many target organs, increased the numbers of topoisomerase (Topo) IIα(+), ubiquitin D (Ubd)(+), and TUNEL(+) apoptotic cells. We also found increased co-expression of Topo IIα and Ubd, suggestive of increased spindle checkpoint disruption at the M phase. To investigate the roles of these markers in the early stages of carcinogenesis, we examined distribution changes in several carcinogenic target organs using rat initiation-promotion models. Promoting agents targeting the liver (piperonyl butoxide, methapyrilene hydrochloride), thyroid (sulfadimethoxine), urinary bladder (phenylethyl isothiocyanate), and forestomach and glandular stomach (catechol) were administered to rats after initiation treatment for each target organ. Numbers of Ki-67(+), Mcm3(+), Topo IIα(+) and TUNEL(+) cells increased within preneoplastic lesions as determined by glutathione S-transferase placental form in the liver or phospho-p44/42 mitogen-activated protein kinase in the thyroid, and hyperplastic lesions having no known preneoplastic markers in the urinary bladder, forestomach and glandular stomach. On the other hand, Ubd(+) cells did not increase within these preneoplastic lesions, but increased within hyperplastic lesions. These results suggest that both cell proliferation and apoptosis may be involved in the formation of preneoplastic lesions in the liver and thyroid examined here; however, spindle checkpoint disruption may not be involved in this process. Changes in hyperplastic lesions of the urinary bladder, forestomach and glandular stomach are similar to the 28-day carcinogen-treated cases, suggestive of the hyperplastic cellular character before the preneoplastic state.

Enhanced liver tumor promotion but not liver initiation activity in rats subjected to combined administration of omeprazole and ß-naphthoflavone.

Omeprazole (OPZ) and ß-naphthoflavone (BNF) are cytochrome P450 (CYP)1A inducers and have liver tumor promoting effects. In this study, we investigated the co-promoting and co-initiating effects of OPZ and BNF in rats. In Experiment 1, male rats were subjected to partial hepatectomy (PH), and given oral doses of 138 or 276 mg/kg OPZ, 0.125% or 0.25% BNF or 138 mg/kg OPZ+0.125% BNF (n = 9~12) for 6 weeks after N-diethylnitrosamine (DEN) initiation. In Experiment 2, male rats were treated with oral doses of 138 or 276 mg/kg OPZ, 0.03% or 0.06% BNF or 138 mg/kg OPZ+0.03% BNF (n = 11~12) for 9 days starting 1 week before initiating treatment. As an initiating treatment, 2-Amino-3,4-dimethylimidazo[4,5-f]quinolone (MeIQx) was orally administered 12 hr after PH. The rats were fed a basal diet for 15 days, followed by a diet containing 0.015% 2-acetylaminofluorene for the next 10 days with a single oral dose of carbon tetrachloride. In Experiment 1, the number and area of glutathione S-transferase placental form-positive foci in the OPZ+BNF group were significantly higher than the average values of the High OPZ or the High BNF group. The expression of cyclooxygenase-2 (Cox-2) and COX-2 protein in the liver significantly increased in the OPZ+BNF group. In Experiment 2, liver initiation activity was not enhanced by the co-administration of OPZ+BNF. The results of our studies suggest that the co-administration of OPZ and BNF results in synergistic effects in the liver tumor promotion probably owing to increased COX-2 expression, but no modifying effect in the liver initiation activity of MeIQx in rats.

Liver tumor promoting effect of omeprazole in rats and its possible mechanism of action.

Omeprazole (OPZ), a proton pump inhibitor, is a cytochrome P450 (CYP) 1A1/2 inducer. Some CYP1A inducers are known to have liver tumor promoting effects in rats and the ability to enhance oxidative stress. In this study, we performed a two-stage liver carcinogenesis bioassay in rats to examine the tumor promoting effect of OPZ (Experiment 1) and to clarify a possible mechanism of action (Experiment 2). In Experiment 1, male F344 rats were subjected to a two-third partial hepatectomy, and treated with 0, 138 or 276 mg/kg OPZ by oral gavage once a day for six weeks after an intraperitoneal injection of N-diethylnitrosamine (DEN). Liver weights significantly increased in the DEN+OPZ groups, and the number and area of glutathione S-transferase placental form (GST-P) positive foci significantly increased in the DEN+276 mg/kg OPZ group. In Experiment 2, the same experiment as Experiment 1 was performed, but the dosage of OPZ was 0 or 276 mg/kg. The number and area of GST-P positive foci as well as liver weights significantly increased in the DEN+276 mg/kg OPZ group. The number of proliferative cell nuclear antigen (PCNA)-positive cells also significantly increased in the same group. Real-time RT-PCR showed that the expression of AhR battery genes including Cyp1a1, Cyp1a2, Ugt1a6 and Nqo1, and Nrf2 battery genes including Gpx2, Yc2, Akr7a3, Aldh1a1 Me1 and Ggt1 were significantly upregulated in this group. However, the production of microsomal reactive oxygen species (ROS) and formation of thiobarbituric acid-reactive substances (TBARS) decreased, and 8-hydroxydeoxyguanosine (8-OHdG) content remained unchanged in this group. These results indicate that OPZ, CYP1A inducer, is a liver tumor promoter in rats, but oxidative stress is not involved in the liver tumor promoting effect of OPZ.