BrdU Labeling Index Research Articles

Evaluation of kinetic effects of Gliotoxin in different breast cancer cell lines.

In the present research, the antiproliferative properties of Gliotoxin, which is obtained from marine fungus and thought to be a promising metabolite, on MCF-7 and MDA-MB-231 breast cancer cells, which have different molecular subtypes, were evaluated. Different cell kinetic parameters were employed for this aim. In experiments, cell viability, cell index, mitotic index, BrdU labeling index, and apoptotic index were assessed. Gliotoxin concentrations of 1.5625 µM, 3.125 µM, and 6.25 µM were used in studies for both cell lines. As a result of the values obtained from cell viability and xCELLigence Real-Time Cell Analysis (RTCA) System, 1.5625 µM concentration was determined as IC50 dose. This concentration was applied to all other parameters and anticancer activities were observed.

Apoptotic effects of Phlomis armeniaca mediated biosynthesized silver nanoparticles in monolayer (2D) and spheroid (3D) cultures of human breast cancer cell lines.

The purpose of current research was to assess the apoptotic effects of biofabrication silver nanoparticles (AgNPs) mediated by the aqueous extract of Phlomis armeniaca on human breast cancer cells (MCF-7 and MDA-MB-231) in monolayer (2D) and spheroid (3D) cultures. The biosynthesized AgNPs were characterized by UV-Vis spectrophotometer (the peaks of resonances at 432nm), scanning electron microscopy (SEM) and energy-dispersive X-ray spectrometry (EDS). 1-20µM/mL AgNPs were applied to MCF-7 and MDA-MB-231 cell lines to determine IC50 values at 24, 48 and 72nd h and were found to be 10µM/mL for both cell lines. Immunohistochemical staining results of BrdU, TUNEL, caspase-3 and Endo G in both 2D and 3D cultures and gene expression levels of caspases (caspase-3, -8 and -9) and Endo G were evaluated. Moreover, the total oxidant/antioxidant status (TOS-TAS) due to AgNPs application in both cell culture mediums was evaluated. AgNPs treatment results in both cell lines in both 2D and 3D cultures showed a significant decrease in the BrdU labeling index, while large amounts of cells were labelled with TUNEL and Endo G. In 2D culture, Endo G expression increased in MCF-7 cells at 48 and 72nd hours, while it increased significantly in MDA-MB-231 cells at all hours. OSI results show that ROS production is increased in cell medium treated with AgNPs. In conclusion, AgNPs mediated by Phlomis armeniaca, synthesized by a green method, successfully induced damage to mitochondria, resulting in cell cycle arrest and consequent cell proliferation blockade and death in both MCF-7 and MDA-MB-231 cells.

Antiproliferative Effects of EGFR inhibitor Cetuximab and PARP Inhibitor Combination on Non-Small Cell Lung Cancer Cell Line A549 and Cervical Cancer Cell Line HeLa.

In this study, the efficacy of Cetuximab and Parp inhibitor (Parp 1 inhibitor) used in targeted therapies, alone or in combination, on non-small cell lung cancer cell line A549 and cervical cancer cell line HeLa cells were evaluated. For this purpose different cell kinetic parameters were used. Cell viability, mitotic index, BrdU labelling index and apoptotic index were evaluated in experiments. In single applications Cetuximab at concentrations ranging from 1 mg/ml to 10 mg/ml and Parp inhibitor at concentrations 5 µM -7 µM - 10 µM were applied. IC50 concentration of Cetuximab for A549 was 1 mg/ml, the IC50 concentration of Cetuximab for HeLa was 2 mg/ml, the IC50 concentration of Parp inhibitor for A549 was 5 µM, and the IC50 concentration of Parp inhibitor for HeLa was 7 µM. In both single and combinations, there was a significant decrease in cell viability, mitotic index, BrdU labelling index and there was a significant increase in apoptotic index. A comparison of cetuximab, PARPi and combination applications showed the superiority of combined applications over single applications in all cell kinetic parameters used.

Antitumor Activity of Tubulin-Binding Agent MPC-6827 on Different Types of Cancer Cell Lines.

In this study, the antitumor effects of tubulin-binding agent MPC-6827 on HeLa, MCF-7 and A549 cell lines originated from cervix carcinoma, metastatic breast adenocarcinoma and adenocarcinomic human alveolar basal epithelial cells respectively were determined. Cell index, BrdU labelling index, mitotic index and apoptotic index were evaluated in experiments. In cell index experiment 2 nM, 4 nM, 6 nM, 8 nM, 10 nM MPC-6827 applied to three cell lines. These parameters showed that 4 nM was the optimum concentration for HeLa and A549 cells, while 2 nM was the optimum concentration for MCF-7 cells. The use of optimum concentrations for each cell line has shown that while there was a significant decrease in mitotic index, BrdU labelling index, there was a significant increase in apoptotic index.

Quantification of Hepatocellular Mitoses in a Toxicological Study in Rats Using a Convolutional Neural Network.

Convolutional neural networks (CNNs) have been recognized as valuable tools for rapid quantitative analysis of morphological changes in toxicologic histopathology. We have assessed the performance of CNN-based (Halo-AI) mitotic figure detection in hepatocytes in comparison with detection by pathologists. In addition, we compared with Ki-67 and 5-bromodesoxyuridin (BrdU) immunohistochemistry labeling indices (LIs) obtained by image analysis. Tissues were from an exploratory toxicity study with a glycogen synthase kinase-3 (GSK-3) inhibitor. Our investigations revealed that (1) the CNN achieved similarly accurate but faster results than pathologists, (2) results of mitotic figure detection were comparable to Ki-67 and BrdU LIs, and (3) data from different methods were only moderately correlated. The latter is likely related to differences in the cell cycle component captured by each method. This highlights the importance of considering the differences of the available methods upon selection. Also, the pharmacology of our test item acting as a GSK-3 inhibitor potentially reduced the correlation. We conclude that hepatocyte cell proliferation assessment by CNNs can have several advantages when compared with the current gold standard: it relieves the pathologist of tedious routine tasks and contributes to standardization of results; the CNN algorithm can be shared and iteratively improved; it can be performed on routine histological slides; it does not require an additional animal experiment and in this way can contribute to animal welfare according to the 3R principles.

Cell proliferation analysis is a reliable predictor of lack of carcinogenicity: Case study using the pyrethroid imiprothrin on lung tumorigenesis in mice

In the mouse carcinogenicity study, an apparent increase in lung adenocarcinoma was observed in male mice at 7000 ppm. Based on the overall evaluation of toxicology, oncology, pathology and statistics, we concluded that the apparent increase in lung tumors is not relevant for evaluation of carcinogenicity of imiprothrin (Regul Toxicol Pharmacol, 105, 1–14, 2019). To investigate whether imiprothrin has any mitogenic effect on mouse Club cells, the present study examined its effects on replicative DNA synthesis of Club cells and lung histopathology in male mice treated with imiprothrin for 7 days at 3500 and 7000 ppm in the diet. Isoniazid, a known mouse lung mitogen and tumor inducer, was also examined at 1000 ppm in the diet as a positive control of Club cell mitogenesis and morphological changes. Neither imiprothrin nor isoniazid caused any necrotic changes in lung by light or electron microscopy. There were no increases observed in the bromodeoxyuridine (BrdU) labeling index in the imiprothrin groups, while there was a statistically significant increase in the BrdU labeling index in the isoniazid group. These findings demonstrate that imiprothrin does not induce mouse Club cell proliferation or morphologic changes, supporting our previous conclusion described above. Thus, imiprothrin should not be classified as a carcinogen. Furthermore, this study indicates that short-term studies focusing on cell proliferation can be reliable for predicting a lack of carcinogenic potential of test chemicals.

Combining BrdU-Labeling to Detection of Neuronal Markers to Monitor Adult Neurogenesis in Hydra.

The nervous system is produced and maintained in adult Hydra through the continuous production of nerve cells and mechanosensory cells (nematocytes or cnidocytes). De novo neurogenesis occurs slowly in intact animals that replace their dying nerve cells, at a faster rate in animals regenerating their head as a complete apical nervous system is built in few days. To dissect the molecular mechanisms that underlie these properties, a precise monitoring of the markers of neurogenesis and nematogenesis is required. Here we describe the conditions for an efficient BrdU-labeling coupled to an immunodetection of neuronal markers, either regulators of neurogenesis, here the homeoprotein prdl-a, or neuropeptides such as RFamide or Hym-355. This method can be performed on whole-mount animals as well as on macerated tissues when cells retain their morphology. Moreover, when antibodies are not available, BrdU-labeling can be combined with the analysis of gene expression by whole-mount in situ hybridization. This co-immunodetection procedure is well adapted to visualize and quantify the dynamics of de novo neurogenesis. Upon continuous BrdU labeling, the repeated measurements of BrdU-labeling indexes in specific cellular populations provide a precise monitoring of nematogenesis as well as neurogenesis, in homeostatic or developmental conditions.

Crucial role of Reg I from acinar-like cell cluster touching with islets (ATLANTIS) on mitogenesis of beta cells in EMC virus-induced diabetic mice

Recently, we reported the presence of distinct cell clusters named acinar-like cell clusters touching Langerhans islets with thin interstitial surrounding (ATLANTIS) in human pancreas. A morphological study in humans demonstrated that ATLANTIS and islet cell clusters are found together in the microenvironment enclosed by a common basement membrane, and ATLANTIS releases vesicles containing Regenerating gene protein (REG Iα) to islet cell clusters.We examined 1) the presence or absence of ATLANTIS in homozygous Reg I (mouse homologue of human REG Iα) deficient (Reg I−/-) and wild-type mice, and 2) the possible role of ATLANTIS in the regeneration of beta cell clusters after encephalomyocarditis (EMC) virus (D-variant) infection in Reg I−/- and wild-type mice.ATLANTIS was found in both wild-type and Reg I−/- mice. In both groups, mean blood glucose increased transiently to greater than 14.0 mmol/L at 5 days after EMC virus infection and recovered to baseline at 12 days. At 12 days after EMC virus infection, lower BrdU labeling indices were observed in islet beta cells of Reg I−/- mice compared to wild-type mice. Beta cell volume 12 days after EMC virus infection in Reg I−/- mice did not differ from that of wild-type mice.These results suggest that Reg I, which is released from ATLANTIS to islet beta cell clusters, has a crucial role in beta cell regeneration in EMC virus-induced diabetes. The presence of mechanism(s) other than that mediated by Reg I in beta cell restoration after destruction by EMC virus was also suggested.

852 Short-term ozone exposure stimulates both IGF1R and EGFR to promote diabetic wound healing

Ozonated oil, containing the potent disinfectant and oxygen donor ozone (O3), improves wound oxygenation and closure after topical application. However, the mechanism of O3 action on skin cells during wound healing is largely unknown. We hypothesized that IGF1R/EGFR crosstalk and activation of downstream signaling pathways are required for the observed acceleration in wound healing. In a diet-induced obese diabetic mouse model, topical application of O3 oil visibly promoted healing with a 2-3-fold reduction in the epidermal gap and 1.7-fold increase in granulation tissue compared with vehicle-treated mice at 8 days after initiation (when wounds in nondiabetic mice had closed). Immunohistochemical staining showed a 2-2.5 fold increase in BrdU labeling index (p<.01) and a 1.9-2.3 fold increase in keratinocyte (KC) migration (p<.05) at the wound edge at 4-8 days post wounding. Brief exposure of monolayer cultured normal KCs to O3 (20 uM, 2 h) daily increased cell proliferation in high glucose (HG; p<.001) within 24h and normal glucose (NG: p<.01 NG vs. HG+O3) by 72h in WST assays. Exposure to O3 also enhanced cell migration (p<.001, HG vs. HG+O3 by 24h and p<.05 NG vs. NG+O3 by 60h in mouse KCs and p<.01 HG vs. HG+O3 by 6h and p<.05 NG vs. NG+O3 by 18h in human KCs) in scratch assays. Under NG condition, brief O3 exposure increased EGF-stimulated p-ERK by 1.7-fold, p-AKT at Thr 308 and p-PI3K by 2.1-fold and p-AKT at Ser 473 by 2.9-fold, and increased IGF-1 stimulated p-ERK by 1.5-fold. Interestingly, stimulation with EGF or IGF-1 reversed HG-induced inhibition on PI3K/Akt and ERK activation in the presence of O3, suggesting that brief O3 exposure ameliorate skin cell function under HG condition. The increase in KC proliferation and migration caused by O3 exposure was completely inhibited by disrupting EGFR and IGF1R simultaneously. These findings suggest that topical exposure to O3 requires activation of both EGFR and IGF1R to accelerate wound healing.

Determination of Apoptotic Effect of Juglone on Human Bladder Cancer TCC-SUP and RT-4 Cells: An In Vitro Study.

This study aimed to investigate the effects of juglone on the human bladder carcinoma cell lines TCC-SUB and RT-4 in monolayer and spheroid cultures. Cells were treated with juglone at 24, 48, and 72 h of incubation. The activity of caspase-3 was detected in vitro using a caspase-3 colorimetric assay kit according to the manufacturer's instructions. The bromodeoxyuridine (BrdU) labeling index was used to determine the cells of the synthesis phase. The terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay was used to determine the death of cells in both the monolayer and spheroid cultures. The control group had a large S-phase fraction and many of the TCC-SUB and RT-4 cells nuclei were observed to be positive for BrdU. The dead cell count was higher in the TCC-SUB and RT-4 cell lines with juglone applied than in the controls. We conclude that juglone significantly inhibits the proliferation and induces the apoptosis of TCC-SUB and RT-4 cells in vitro.

Skin sensitization of 3, 4-bis (4'-aminofurazano-3') furoxan in mice evaluated by BrdU-ELISA local lymph node assay

Objective: To investigate skin sensitization of 3, 4-bis (4'-aminofurazano-3') furoxan (DATF) in mice using 5-bromo-2'-deoxyuridine enzyme-linked immunosorbent assay (BrdU-ELISA) . Methods: A total of 30 specific pathogen-free BALB/C mice were randomly divided into high-, medium-, and low-dose DATF groups, positive control group, and solvent control group, with six mice in each group. The mice in the high-, medium-, and low-dose DATF groups were treated with 50%, 25%, and 10% (0.5, 0.25, and 0.10 g/ml) DATF solution, those in the positive control group were treated with 1% 2, 4-dinitrochlorobenzol (DNCB) , and those in the solvent control group were treated with acetone/olive oil (4∶1) . After treatment, retroauricular lymph nodes were collected and cell suspension was prepared. ELISA was used to measure the level of cell proliferation after the addition of 5-bromo-2'-deoxyuridine (BrdU) , and the BrdU labeling index (LI) and test substance concentration at a stimulation index (SI) of 1.6 (EC1.6) were calculated. Results: There were no significant differences in auricular thickness between groups (P>0.05) , and DAFT did not have skin irritation. Compared with the solvent control group, the high-dose DATF group and the positive control group showed significant increases in the weight of lymph nodes (P<0.05) . Compared with the solvent control group, all the other groups showed significant increases in BrdU LI (P<0.01) . The low-, medium-, and high-dose DATF groups had SIs of 6.1, 8.8, and 12.1, respectively, and the EC1.6 of DATF was 2.2%, which suggested that DATF had strong sensitization. Conclusion: DATF has strong skin sensitization in mice, with reference to the guideline of Organization for Economic Co-operation and Development Item No. 442B (OECD TG 442B) .

Evaluation of gamma ray-induced gastrointestinal tract morphological and proliferative activity changes in rhesus monkeys.

To provide support for future pharmacology and preclinical studies, we have established a stable nonhuman primate animal model to demonstrate the histopathological changes in the gastrointestinal tract following gamma ray irradiation. In this study, 12 healthy rhesus monkeys were divided into 2 groups (control and radiation groups). Animals in the radiation group were exposed to gamma rays (cobalt 60 source) at a dose level of 6.5 Gy total body irradiation bilaterally (i.e. 3.25 Gy on each side). Control animals were sham exposed using identical procedures. After a 5-day in-life observation period, gastrointestinal tract tissues (esophagus, stomach, duodenum, jejunum, ileum, colon, and rectum) were collected and fixed in 10% neutral-buffered formalin for subsequent hematoxylin and eosin and 5-bromo-2-deoxyuridine (BrdU) immunohistochemistry processing. The results showed that the esophagus was undergoing degeneration without obvious inflammatory changes, while the stomach and duodenum exhibited both degeneration and inflammation. From the jejunum to the rectum, late-stage inflammation with glandular regeneration, as well as a high-level BrdU labeling index, was present.

Abstract 4071: Targeted disruption of T-cell protein tyrosine phosphatase in mouse epidermis reveals its critical role in chemically-induced skin carcinogenesis

Abstract Environmental factors such as chemical toxicants contribute to the development of skin cancer by creating mutations in housekeeping genes and proto-oncogenes which disrupt intracellular signaling mechanisms. One vital signaling mechanism is tyrosine phosphorylation signaling. Phosphotyrosine signaling is regulated by the counter-activities of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). Recently we reported TC-PTP (PTPN2) is critical regulator of STAT3 signaling in mouse keratinocytes. To assess the role of TC-PTP in skin carcinogenesis, we generated epidermal-specific TC-PTP-deficient (K14Cre.PTPN2fl/fl) transgenic mice. Loss of TC-PTP led to a desensitization to tumor initiator 7,12-dimethylbenz[a]anthracene (DMBA)-induced apoptosis both in vivo epidermis and in vitro keratinocytes. TC-PTP deficiency in epidermis resulted in a significant increase in epidermal thickness and hyperproliferation (assessed by bromodeoxyuridine (BrdU) labeling index) compared to control mice following treatment with the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA). Consistently, primary keratinocytes derived from TC-PTP-deficient mice showed a faster growth rate. To further investigate the role of TC-PTP in skin carcinogenesis, TC-PTP-deficient mice were subjected to two-stage skin carcinogenesis analysis. TC-PTP-deficient mice showed a shortened latency of tumor development and significantly increased numbers of tumors compared to control mice, demonstrating TC-PTP deficiency predisposes mice to skin carcinogenesis. The data reveals TC-PTP may be a novel potential target for the prevention of skin cancer due to its role in the regulation of proliferation and apoptosis in epidermis. Citation Format: Hyunseung Lee, Mihwa Kim, Liza D. Morales, Thomas J. Slaga, John DiGiovanni, Dae J. Kim. Targeted disruption of T-cell protein tyrosine phosphatase in mouse epidermis reveals its critical role in chemically-induced skin carcinogenesis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4071.

Portal vein arterialization promotes liver regeneration after extended partial hepatectomy in a rat model.

In the current study, we sought to establish a novel rat model of portal vein arterialization (PVA) and evaluate its impact on liver regeneration after extended partial hepatectomy (PH). A total of 105 Sprague-Dawley rats were randomly assigned to three groups: 68% hepatectomy (the PH group), portal arterialization after 68% hepatectomy (the PVA group), and right nephrectomy only (the control group). Liver regeneration rate (LRR), 5-bromo-2-deoxyuridine (BrdU) labeling index, and liver functions were assessed on postoperative day 2, 7, 14 and 28. The 28-day survival rates were compared among the three groups. The 28-day survival rates were similar in all groups (P = 0.331), and the anastomotic patency was 100%. The LRR in the PVA group was significantly higher than that of the PH group within postoperative 14 days (P < 0.05). The PVA and PH group had increased serum alanine aminotransferase levels (232 ± 61 U/L and 212 ± 53 U/L, respectively) compared with the control group (101 ± 13 U/L) on postoperative day 2, whereas from postoperative day 7 to day 28 there were no differences among the three groups. Serum albumin values were higher after the PVA procedure within postoperative day 14, which gradually became comparable on postoperative day 28 among the three groups. The peaks of BrdU labeling index appeared on postoperative day 2 in all rats, and the PVA procedure was associated with increased BrdU labeling index from postoperative day 7 to 28. The 28-day survival of the PVA rats was comparable. Our findings demonstrate that the PVA procedure utilizing portal vein trunk-renal artery microvascular reconstruction promotes remnant liver regeneration and confers beneficial effects on maintaining and even optimizing liver function after extended partial hepatectomy in rats.

Topical Naltrexone as Treatment for Type 2 Diabetic Cutaneous Wounds.

Objective: Type 2 diabetes (T2D) is associated with impaired cutaneous wound healing and can result in ulceration, infection, and/or amputation. More than 25 million people in the United States have T2D and are vulnerable to epithelial-related complications. Current therapies are limited in their efficacy. New treatments for full-thickness cutaneous wounds that focus on underlying diabetic pathways are needed. Approach: Topical application of the opioid receptor antagonist naltrexone (NTX) dissolved in cream reverses delayed wound closure in type 1 diabetic rat by the acceleration of reepithelialization and enhancement of angiogenesis and remodeling. NTX blocks the opioid growth factor (OGF)-OGF receptor (OGFr) axis and upregulates DNA synthesis and cell proliferation. To investigate whether NTX is an effective therapy for T2D wound closure, genetically obese mice (db/db) and normal C57Bl/6J mice received full-thickness cutaneous wounds. Wounds (5 mm in diameter) were treated topically three times daily with 10-5 M NTX or sterile saline dissolved in cream and photographed every 2 days. Results: Wounds in db/db mice treated with saline were 11-92% larger than those in normal mice throughout the 2-week observation. Topical NTX therapy in T2D mice reduced the residual wound size by 13-30% between days 8 and 14 relative to diabetic mice receiving saline. Reepithelialization and DNA synthesis, as analyzed by epithelial thickness and BrdU labeling indexes, respectively, were accelerated in NTX-treated wounds. Innovation and Conclusion: These data suggest that the OGF-OGFr axis plays a role in epithelial-related complications of T2D and that blockade of this pathway by NTX may be an effective treatment for wound repair.

Comparative uterotrophic effects of endoxifen and tamoxifen in ovariectomized Sprague-Dawley rats.

Endoxifen (4-hydroxy-N-desmethyl-tamoxifen), one of the major active metabolites of tamoxifen, has substantially greater estrogen antagonist properties and antiproliferative effects in breast tumor cells than tamoxifen, a mixed estrogen agonist/antagonist. An associated risk of endometrial cancer and hyperplasia has been linked to the estrogen agonist properties of tamoxifen. We evaluated endoxifen using a classic uterotrophic effects method. Rats were given endoxifen or tamoxifen orally for 3 days. Estradiol was the positive control. Endoxifen and tamoxifen plasma levels exceeded those previously observed clinically. Uterine weight was 3-fold higher in the estradiol group than in the tamoxifen or endoxifen groups, which did not differ from vehicle controls. Tamoxifen and endoxifen caused a greater increase in luminal epithelial cell height than estradiol. Both tamoxifen and endoxifen produced an increase in the stromal BrdU labeling index (LI) that was ≤ estradiol and inversely related to dose, but did not affect luminal epithelial cell BrdU LI. As expected, estradiol increased luminal epithelial cell proliferation. These results indicate that endoxifen induces uterotrophic effects, but is less potent than estradiol in eliciting these effects. Given prior preclinical observations that endoxifen has superior antitumor activity than tamoxifen, the observations of similar uterine effects suggest that the endoxifen risk/benefit ratio may be superior to tamoxifen.

Xanthine crystals induced by topiroxostat, a xanthine oxidoreductase inhibitor, in rats, cause transitional cell tumors

The present study was performed to elucidate the underlying mechanism of transitional cell tumors found in the carcinogenicity testing of topiroxostat, a xanthine oxidoreductase inhibitor, in which topiroxostat was orally given to F344 rats at 0.3, 1, and 3mg/kg for 2years. In the urinary bladder, transitional cell papillomas and/or carcinomas were seen in males receiving 0.3, 1, and 3mg/kg (1/49, 3/49, and 10/50, respectively). In the kidney, transitional cell papillomas and/or carcinomas in the pelvis were seen in 2/50 males and 1/50 females receiving 3mg/kg. In the mechanistic study by 52-week oral treatment with topiroxostat at 3mg/kg to F344 male rats, with and without citrate, simple and papillary transitional cell hyperplasias of the urinary bladder epithelium were observed in 5/17 in the topiroxostat-alone treatment group, along with xanthine-induced nephropathy, in contrast to neither xanthine crystals nor lesions in urinary organs by co-treatment group with citrate. As for sex differences of urinary bladder tumors, the BrdU labeling index for epithelial cells of the urinary bladder by 5-week oral treatment with topiroxostat at 10mg/kg to F344 rats was increased in males only, showing consistency with histopathological findings. Therefore, the present study indicates that transitional cell tumors induced by topiroxostat in rats were due to physical stimulation to transitional cells of xanthine crystals/calculi and provides that other factors were not implicated in this tumorigenesis. Furthermore, the present study suggests that such tumors do not predict for humans since topiroxostat-induced xanthine deposition is a rodent-specific event.

Orally administered nicotine induces urothelial hyperplasia in rats and mice

Tobacco smoking is a major risk factor for multiple human cancers including urinary bladder carcinoma. Tobacco smoke is a complex mixture containing chemicals that are known carcinogens in humans and/or animals. Aromatic amines a major class of DNA-reactive carcinogens in cigarette smoke, are not present at sufficiently high levels to fully explain the incidence of bladder cancer in cigarette smokers. Other agents in tobacco smoke could be excreted in urine and enhance the carcinogenic process by increasing urothelial cell proliferation. Nicotine is one such major component, as it has been shown to induce cell proliferation in multiple cell types in vitro. However, in vivo evidence specifically for the urothelium is lacking. We previously showed that cigarette smoke induces increased urothelial cell proliferation in mice. In the present study, urothelial proliferative and cytotoxic effects were examined after nicotine treatment in mice and rats. Nicotine hydrogen tartrate was administered in drinking water to rats (52ppm nicotine) and mice (514ppm nicotine) for 4 weeks and urothelial changes were evaluated. Histopathologically, 7/10 rats and 4/10 mice showed simple hyperplasia following nicotine treatment compared to none in the controls. Rats had an increased mean BrdU labeling index compared to controls, although it was not statistically significantly elevated in either species. Scanning electron microscopic visualization of the urothelium did not reveal significant cytotoxicity. These findings suggest that oral nicotine administration induced urothelial hyperplasia (increased cell proliferation), possibly due to a mitogenic effect of nicotine and/or its metabolites.

Inhibition Of BTK Activity In Myeloma Cells Within a Supportive Microenvironment Promotes Their Growth But Suppresses Metastasis

Bruton’s Tyrosine Kinase (BTK), a TEC family kinase, is expressed by hematopoietic cells and is indispensable for B cell maturation and migration. We and others recently demonstrated that BTK is expressed in MM cells and mediates their homing to bone through chemotaxis to SDF-1, a chemokine highly produced in the bone marrow (BM) (Tai et al,Blood 2012; Bam et al.,AJH 2013). The aim of this study was to unravel the role of BTK in MM cell adhesion, growth and metastasis in our in vitro and in vivo experimental systems. BTK activity was suppressed using lentiviral particles containing BTK or scramble shRNA, or using the clinical BTK inhibitor, ibrutinib. Primary MM cells passaged in our animal model or IL6-dependent INA6 MM line were also infected with EGFP/luciferase construct for tracking purposes and monitoring tumor growth. Knockdown of BTK in INA6 cells diminished their homotypic cell aggregation growth pattern and reduced adhesion to fibronectin compared to control cells by 38±6% (p&lt;0.01). In vivo we exploited our SCID-rab model whereby SCID mice were implanted with a piece of rabbit femur on each flank and luciferase-expressing INA6 MM cells containing scramble or BTK shRNA were injected directly into the right-side implanted bone. We previously demonstrated that MM cells grow restrictively in the implanted bone area and are capable of metastasis to the secondary implanted bone but not murine organs (Yata et al, Leukemia 2004). MM growth in the primary bone site of BTK shRNA group (n=13) was higher than in the scramble shRNA group (n=12) by 4±1 folds (p&lt;0.04) assessed by live-animal imaging. Overall MM burden was also higher in the BTK shRNA group by 4.3 folds (p&lt;0.03) and 2.3 folds (p&lt;0.002) detected by measurement of circulating human immunoglobulins and soluble syndecan-1 levels, respectively. In contrast, metastasis to the secondary implanted bones assessed by ex-vivo imaging 10 weeks after engraftment was lower by 37±11 folds (p&lt;0.002) in the BTK shRNA group compared to control scramble shRNA group. In both groups, metastatic tumor cells were identified only in the secondary implanted bones. MM cells extracted from the primary myelomatous bones retained their differences in BTK expression between the two groups. Cell cycle analysis on extracted MM cells revealed decreased sub-G phase (apoptotic cells) and increased in vivo BrdU labeling index in the BTK knockdown MM cells compared to control MM cells. Gene array analysis revealed BTK target genes including upregulation of factors associated with lymphoid malignancies (e.g. BCL11A, TIAM1, RAB31) and underexpression of genes mediating cell adhesion and migration (e.g. LPXN, DBN1, F11R, CT45) in the BTK knockdown cells compared to control cells. In vitro, we established a novel coculture system of MM cells with healthy adult human BM that first has been cultivated with MM patients’ serum for 7 days. Primary MM cells (n=20) were consistently capable of long-term (7 days) survival and growth in this coculture system. Growth of BTK-expressing MM cells (n=7) in the coculture was significantly increased by 61%-90% in the presence of 5 μM (p&lt;0.02) or 10 μM (p&lt;0.01) ibrutinib. Cocultured MM cells treated with 5 μM Ibrutinib had increased BrdU labeling index compared to control MM cells as assessed by flow cytometry. We also observed reduced expression of BTK in MM cells from focal lesion compared to interstitial marrow in paired clinical samples (n=106, p&lt;3.24E-05). These data suggest that BTK plays a role in MM cell homotypic and heterotypic adhesion, and that inhibition of BTK activity in MM cells within a supportive microenvironment promotes their proliferation and growth but suppresses metastasis. Disclosures: No relevant conflicts of interest to declare.

Grape seed extract and resveratrol prevent 4‐nitroquinoline 1‐oxide induced oral tumorigenesis in mice by modulating AMPK activation and associated biological responses

Preventive measures against oral carcinogenesis are urgently warranted to lower the high morbidity and mortality associated with this malignancy worldwide. Here, we investigated the chemopreventive efficacy of grape seed extract (GSE) and resveratrol (Res) in 4-nitroquinoline-1-oxide (4NQO)-induced tongue tumorigenesis in C57BL/6 mice. Following 8 weeks of 4NQO exposure (100 µg/ml in drinking water), mice were fed with either control AIN-76A diet or diet containing 0.2% GSE (w/w) or 0.25% Res (w/w) for 8 subsequent weeks, while continued on 4NQO. Upon termination of the study at 16 weeks, tongue tissues were histologically evaluated for hyperplasia, dysplasia, and papillary lesions, and then analyzed for molecular targets by immunohistochemistry. GSE and Res feeding for 8 weeks, moderately decreased the incidence, but significantly prevented the multiplicity and severity of 4NQO-induced preneoplastic and neoplastic lesions, without any apparent toxicity. In tongue tissues, both 4NQO + GSE and 4NQO + Res treatment correlated with a decreased proliferation (BrdU labeling index) but increased apoptotic death (TUNEL-positive cells) as compared to the 4NQO group. Furthermore, tongue tissues from both the 4NQO + GSE and 4NQO + Res groups showed an increase in activated metabolic regulator phospho-AMPK (Thr172) and decreased autophagy flux marker p62. Together, these findings suggest that GSE and Res could effectively prevent 4NQO-induced oral tumorigenesis through modulating AMPK activation, and thereby, inhibiting proliferation and inducing apoptosis and autophagy, as mechanisms of their efficacy.