Rat Leukemia Research Articles

Harnessing the heart's resistance to malignant tumors: cardiac-derived extracellular vesicles decrease fibrosarcoma growth and leukemia-related mortality in rodents.

The heart is known for its resistance to cancer. Although different conjectures have been proposed to explain this phenomenon, none has been tested. We propose that the heart microenvironment may exert anti-cancer properties. So, our objective was to test the anti-oncogenic potential of cardiac-derived extracellular vesicles (EVs).For that EVs secreted by cardiosphere-derived cells (CDCs, heart progenitor cells) were tested in vitro on fibrosarcoma HT1080. In vivo models comprised the xenograft HT1080 fibrosarcoma in athymic mice (n=35), and spontaneous acute lymphocyte leukemia in old rats (n=44). CDC-EVs were compared with two control groups: EVs secreted by bone-marrow derived mesenchymal stem cells (MSC-EVs) and phosphate-buffered saline (PBS).Injection of CDC-EVs led to a 2.5-fold decrease of fibrosarcoma growth in mice (p<0.01 and p<0.05 for human and rat EVs, respectively) vs PBS group. The effect was associated with 2-fold decrease of tumor cells proliferation (p<0.001) and 1.5-fold increase of apoptosis (p<0.05) in CDC-EV vs PBS mice. Salutary changes in tumor gene and protein expression were observed in CDC-EV animals. CDC-EVs reduced tumor vascularization compared with PBS (p<0.05) and MSC-EVs (p<0.01). Moreover, CDC-EVs increased leukemia-free survival (p<0.05) in old rats vs PBS. MiR-146, highly enriched in CDC-EVs, may be implicated in part of the observed effects. In conclusion, this study presents the first evidence that ties together the long-recognized enigma of the “heart immunity to cancer” with an antioncogenic effect of heart-derived EVs. These findings open up cancer as a new therapeutic target for CDC-EVs.

Abstract 132: African polyherbal formulation alleviates benzene-induced leukemia in Wistar rats

Abstract Background: Chemotherapy and radiotherapy are effective cancer treatment options but they are accompanied by serious side effects. Therefore, more effective therapies are sorely needed. This study investigated the chemotherapeutic effects of a polyherbal formulation on benzene induced leukemia in Wistar rats. The polyherbal formulation is composed of fruits such as Orange, Lime, Lemon, Pineapple, Grape and Vegetables like; Pumpkin leave, Garden egg in honey medium. Method: Leukemia was induced by injecting 0.2 ml of benzene solution intravenously through the tail at 48 h intervals for four weeks. Leukemia developed in 92% of rats within 2 to 4 weeks after the last benzene injection and further observed for leukemia development in appropriate rat groups. Leukemia burden was assessed using indicator parameters such as total leukocyte, red blood cell count, hematocrit, and hemoglobin concentration. Immunophenotyping enumeration of CD 19 expression also corroborated the leukemia assessment by providing specific cellular marker for differentiation of the developed leukemia type. Exactly 0.48 ml of the polyherbal formulation was administered orally by gavage using oral cannular once daily post leukemia induction for four weeks. Results: Leukemia induction reflected in significant reduction in hematocrit, hemoglobin concentration, red cell count and a marked and a significant increase in leukocyte count and CD 19 expression over the control (p &amp;lt; 0.05). There is significant difference between the leukemia group treated with polyherbal formulation and the leukemia positive control group untreated. Conclusion: This study reveals profound chemotherapeutic activity of the polyherbal formulation against experimentally developed leukemia by reversing the induced leukemia in the positive control rat group, hence the ability of the polyherbal formulation in alleviating the cancer symptoms. Table 1.Hematological parameters in polyherbal formulation treated leukemia bearing Wistar rats.Estimated parametersNegative control rats (Mean±SD)Leukemia Positive rats. (Mean±SD)Polyherbal formulation treated leukemic rats (Mean±SD)CD 19 (X 109/L)47.59±1.3989.41±1.7262.32 + 1.42*Total Leukocyte count (X 109/L)6.84 + 0.3011.08 + 0.317.45 + 0.65*Red blood cells(X 1012/L)6.30 + 0.383.5 + 0.385.10 + 0.03*Hematocrit (L/L)0.43 + 0.170.26. + 0.200.37 + 0.08*Hemoglobin (g/dl)13.62 + 0.696.63 + 0.9611.80 + 2.96*Platelets (X 109/L)554.80 + 92.23389.50 + 146.10425.10 + 60.8**Significance at p&amp;lt;0.05 Citation Format: Olufemi E. Akanni, Ayodeji Faremi, Aminat O. Agboola, Adekemi R. Akanni, Oluseyi E. Bamisaye. African polyherbal formulation alleviates benzene-induced leukemia in Wistar rats [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 132. doi:10.1158/1538-7445.AM2017-132

African Polyherbal Formulation Possesses Chemopreventive and Chemotherapeutic Effects on Benzene- Induced Leukemia in Wistar Rats

Chemotherapy and radiotherapy are effective cancer treatment options but they are accompanied by serious side effects and complications such as systemic cytotoxicity, chemo - or radio - resistance. Therefore, more effective therapies are sorely needed. This study investigated the chemopreventive and chemotherapeutic effects of a polyherbal formulation on benzene induced eukemia in Wistar rats. The polyherbal formulation is composed of fruits such as Orange, Lime, Lemon, Pineapple, Grape and Vege tables like; Pumpkin leave, Garden egg suspended in honey medium. Method s : Leukemia was induced by injecting 0.2 ml of benzene solution intravenously through the tail of young rats at 48 h intervals for four times. Leukemia developed in 92% of rats some w eeks after the last benzene injection and following up weekly observation for leukemia development in appropriate rat groups. Leukemia burden was assessed using indicator parameters such as total leukocyte, red blood cell count, hematocrit, and hemoglobin concentration. Exactly 0.48 ml of the polyherbal formulation was administered orally by gavage using oral cannular once daily post leukemia induction for four weeks. Results: Leukemia induction reflected in significant reduction in hematocrit, hemoglobin concentration, red cell count and a marked and a significant increase in leukocyte count over the control (p < 0.05). There is significant difference between the chemopreventive group and chemotherapeutic (leukemia positive group treated with poly - herbal f ormulation) and the leukemia positive control group untreated. Conclusion: This study reveals profound activity of the poly - herbal formulation at preventing and reversing experimentally developed leukemia in the rat groups, hence the ability of the polyhe rbal formulation in alleviating the cancer symptoms

Kigelia africana Stem Bark, Fruit and Leaf Extracts Alleviate Benzene-induced Leukaemia in Rats

Aims: This study was aimed at evaluating the potential leukaemia chemotherapeutic activities of various extracts of Kigelia africana stem bark; fruit and leaf by using a benzene-induced model of leukaemia to validate its folkloric use. Evidences from African herbal medicine and traditional applications have shown that Kigelia africana plant has several beneficial therapeutic properties against microbial infections and cancer cell lines. Study Design: Experimental study. Place and Duration of Study: Department of Biomedical Science, College of Health Sciences, Ladoke Akintola University of Technology, Osogbo, Nigeria and study was conducted between August, 2016 and November, 2016. Methodology: Rats were administered 0.2 mL of benzene solution intravenously through the tail 48-hourly for 4 weeks, tested periodically and observed till leukaemia developed prior to treatment with respective extract of the Kigelia africana stem bark, fruit and leaf in appropriate rat groups after leukaemia was confirmed with haematological protocols. Leukaemic rats were administered with 100 mg/mL contained in 0.5 mL stem bark, 0.5 mL fruit and 0.2 mL of the leaf extract orally by gavage using oral cannula once daily post leukemia induction for four weeks. Haematological parameters and white blood cells differential counts (lymphocytes) were assessed in both control and treatment groups to determine the leukaemia burden. Results: Kigelia africana treatment using the stem bark, fruit and leaf significantly (P < 0.05) alleviated the anaemia indices and reduced the marked leucocytosis usually associated with leukaemia toward the negative control level when compared with the leukaemia control group. Antileukaemic activity however appears to be highest in stem bark, and least in the leaf. Conclusion: This study revealed the potential of ethanol extracts of Kigelia africana stem bark, fruit and leaf to reverse leukaemic effects in benzene-induced leukaemia bearing wistar rats and this suggest that the extracts might be promising natural, non-toxic and anticancer agents.

Some adverse effects of cytarabine in leukemic rats

Some adverse effects of cytarabine in leukemic rats

Exploring simple, transparent, interpretable and predictive QSAR models for classification and quantitative prediction of rat toxicity of ionic liquids using OECD recommended guidelines

The present study explores the chemical attributes of diverse ionic liquids responsible for their cytotoxicity in a rat leukemia cell line (IPC-81) by developing predictive classification as well as regression-based mathematical models. Simple and interpretable descriptors derived from a two-dimensional representation of the chemical structures along with quantum topological molecular similarity indices have been used for model development, employing unambiguous modeling strategies that strictly obey the guidelines of the Organization for Economic Co-operation and Development (OECD) for quantitative structure–activity relationship (QSAR) analysis. The structure–toxicity relationships that emerged from both classification and regression-based models were in accordance with the findings of some previous studies. The models suggested that the cytotoxicity of ionic liquids is dependent on the cationic surfactant action, long alkyl side chains, cationic lipophilicity as well as aromaticity, the presence of a dialkylamino substituent at the 4-position of the pyridinium nucleus and a bulky anionic moiety. The models have been transparently presented in the form of equations, thus allowing their easy transferability in accordance with the OECD guidelines. The models have also been subjected to rigorous validation tests proving their predictive potential and can hence be used for designing novel and “greener” ionic liquids. The major strength of the present study lies in the use of a diverse and large dataset, use of simple reproducible descriptors and compliance with the OECD norms.

A case of zymbal gland carcinoma complicated with leukemia in Wistar rat

A one year old breeder male Wistar rat showed hard mass of 2x5x3 cm size on the right external ear canal and right side of the head. On cut surface, it comprised mainly of a yellowish friable material with occasional cystic spaces. Liver and spleen were enlarged. Microscopically, the neoplasm comprised polygonal cells arranged in islands and trabeculae supported by a fine to moderate fibrovascular stroma. Neoplastic islands and trabeculae form a peripheral layer of flattened to cuboidal epithelial cells with scanty amounts of eosinophilic, granular cytoplasm; round to oval nuclei with finely stippled chromatin and up to 4 distinct nucleoli (basaloid reserve cells); and a central area containing polygonal cells with abundant microvacuolated cytoplasm and round nuclei (sebaceous differentiation). Nuclei were round with finely stippled chromatin and one to two nucleoli. On microscopic examination, liver revealed centrilobular hepatocellular degeneration and infiltration of tumor cells in the sinusoidal and periportal areas while examination of spleen showed diffuse infiltration of round to oval neoplastic cells. It was diagnosed as a rare case of zymbal gland carcinoma complicated with leukemic changes in liver and spleen.

Abstract 4112: Inositol hexaphosphate (IP6) and inositol against large granular lymphocytic leukemia (LGLL)

Abstract Introduction: The anticancer activity of dietary IP6 and its parent compound inositol, either alone or in combination, has been shown in many different experimental models. Using rat leukemia RNK-16 cells, we investigated the in vitro efficacy of IP6 and inositol in large granular lymphocytic leukemia (LGLL) that develops spontaneously in male Fisher 344 rats and is very similar to human disease. RAS and ERK are constitutively active in LGLL in both humans and rats, and the PI3K/Akt pathway plays a major role in proliferation and survival of these leukemic cells. Therefore, we also investigated the effect of IP6 and inositol on these pathways. Methods: We have previously shown that after in vitro or in vivo administration, exogenous IP6 rapidly dephosphorylates to inositol. In this study, we first implemented the assay for the measurement of unlabeled intracellular myo-inositol by LC-MS/MS after in vitro exposure to IP6 and inositol. Cytotoxicity of IP6 and inositol against Fisher 344 RNK-16 cells alone and in combination with 4-OH-cyclophosphamide was measured by MTT cytotoxicity assay. Western blot analysis was used for protein expression. Results: Uptake of inositol increased more than 5-fold following exposure to 2 mM inositol, and no differences were observed between unsynchronized and synchronized cells. Inositol was relatively non-toxic (30% cytotoxicity at 100 mM), while IP6 had an IC50 of approximately 2 mM. 4-OH-cyclophosphamide had an IC50 of 180 µg/mL, and co-administration of either IP6 or inositol decreased its IC50 by 3-fold. While the expression and phosphorylation status of ERK1/2 were unaffected, p-Akt was completely inhibited between 5 and 60 min after 2 mM IP6 and between 15 and 60 min after inositol, by 2-fold. Furthermore, IP6 treatment caused a significant increase in the expression of the Cdk inhibitor p27Kip1 in RNK-16 cells (1.9-fold) 5 and 15 min. Conclusion: Our data data suggest the use of inositol and IP6 as chemopreventive agents in control of LGLL in humans or as adjuvant therapies. Further studies are justified and warranted. Support: P30-CA-47904 Citation Format: Ivana Vucenik, Jan H. Beumer, Julie L. Eiseman. Inositol hexaphosphate (IP6) and inositol against large granular lymphocytic leukemia (LGLL). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4112. doi:10.1158/1538-7445.AM2014-4112

Engineering therapeutic cancer vaccines that activate antitumor immunity.

Vaccination represents one the most effective methods of preventing disease. Because dendritic cells (DCs) are the most efficient antigen presenting cells, exploiting their plasticity is likely to yield improved therapeutic vaccines. Herein, we applied a novel DC-based vaccine (i.e., DC loaded with leukemia antigens that have been transfected with an IL-10 siRNA capable of coordinately activating DCs via TLR7/8) in a rat model of acute myeloid leukemia. Leukemic rats treated with this new vaccine had less leukemic cell mass in their bone marrows and less extramedullar dissemination of the leukemic disease examined postmortem compared with rats given the control vaccine. Collectively, the new strategy demonstrates the possible usefulness of dual siRNAs as an immunomodulatory drug with antileukemic properties.

Chemopreventive and anti-leukemic effects of ethanol extracts of Moringa oleifera leaves on wistar rats bearing benzene induced leukemia.

Pharmacological exploitation of natural compounds has continued to lead to development of non-synthetic and non-toxic anticancer agents that are promising at ameliorating the menace of neoplastic diseases such as leukemia. This study is an attempt to determine the chemopreventive and antileukemic activities of ethanol extracts of Moringa oleifera leaves on benzene induced leukemia bearing rats. Leukemia was induced by intravenous injection of 0.2 mL benzene solution 48 hourly for 4 weeks in appropriate rat groups. Ethanol extract of Moringa oleifera (EMO) leaves was administered at 0.2 mL of 100 mg/mL to respective treatment rat groups. A standard antileukemic drug (cyclophosphamide) was also used to treat appropriate rat groups. Clinical examination of liver and spleen with hematological parameters were employed to assess the leukemia burden following analysis of the rat blood samples on Sysmex KX-21N automated instrument. Leukemia induction reflected in severe anemia and a marked leukocytosis over the control/baseline group. Liver and spleen enlargements were also observed in group exposed to benzene carcinogen. The in vivo antioxidative potential of EMO was evaluated using Malondialdehyde (MDA) and reduced glutathione (GSH) levels. The liver MDA and GSH levels obtained in benzene induced leukemic rats treated with EMO compared favorably with those obtained in similar treatments with the standard drug (p< 0.05). The extract demonstrated chemopreventive and anti-leukemic activities as much as the standard anti-leukemic drug (p>0.05) by ameliorating the induced leukemic condition in the affected rat groups owing to its bioactive constituents. This study reveals that the extract might be an active, natural and non-toxic anticancer drug lead.

Derivation of Embryonic Stem Cell Lines from Parthenogenetically Developing Rat Blastocysts

This study was undertaken to establish rat embryonic stem (ES) cells from parthenogenetically developing blastocysts. Ten blastocysts were prepared by treatment of ovulated rat oocytes with ionomycin and cycloheximide, and three alkaline phosphatase-positive ES cell lines were established using the N2B27 medium supplemented with mitogen activated protein kinase kinase inhibitor PD0325901, glycogen synthase kinase 3 inhibitor CHIR99021, rat leukemia inhibitory factor, and forskolin. Expression of stem cell marker genes (Oct-4, rNanog, Fgf-4, and Rex-1) was confirmed in all three ES cell lines by reverse transcriptase-polymerase chain reaction (RT-PCR). Combined bisulfite restriction analysis showed that the differentially methylated region locus of five imprinted genes (H19, Meg3IG, Igf2r, Peg5, and Peg10) in these ES cells remained to be demethylated or was hypomethylated, which was similar to that in control ES cells established from normal blastocysts. Characteristics of the parthenogenetic blastocyst-derived ES cells were successfully transmitted to the next generation through a chimeric rat for one of the three ES cell lines. This is the first report on germline-competent (genuine) ES cells derived from parthenogenetically developing rat blastocysts.

Cancer Risk Among Tetrafluoroethylene Synthesis and Polymerization Workers

Tetrafluoroethylene (TFE), a compound used for the production of fluorinated polymers including polytetrafluoroethylene, increases the incidence of liver and kidney cancers and leukemia in rats and mice. This is the first time the cancer risk in humans has been explored comprehensively in a cohort mortality study (1950-2008) that included all polytetrafluoroethylene production sites in Europe and North America at the time it was initiated. A job-exposure matrix (1950-2002) was developed for TFE and ammonium perfluoro-octanoate, a chemical used in the polymerization process. National reference rates were used to calculate standardized mortality ratios (SMRs) and 95% confidence intervals. Among 4,773 workers ever exposed to TFE, we found a lower rate of death from most causes, as well as increased risks for cancer of the liver (SMR = 1.27; 95% confidence interval: 0.55, 2.51; 8 deaths) and kidney (SMR = 1.44; 95% confidence interval: 0.69, 2.65; 10 deaths) and for leukemia (SMR = 1.48; 95% confidence interval: 0.77, 2.59; 12 deaths). A nonsignificant upward trend (P = 0.24) by cumulative exposure to TFE was observed for liver cancer. TFE and ammonium perfluoro-octanoate exposures were highly correlated, and therefore their separate effects could not be disentangled. This pattern of findings narrows the range of uncertainty on potential TFE carcinogenicity but cannot conclusively confirm or refute the hypothesis that TFE is carcinogenic to humans.

At What Level of Collective Equipoise Does a Randomized Clinical Trial Become Ethical for the Members of Institutional Review Board/Ethical Committees?

Background:The conduct of a randomized controlled trial (RCT) is deemed ethical only if we are in state of “equipoise” as to which treatment would be most beneficial for the patients. Individual equipoise applies to an individual clinician or a member of ethical, institutional review board (IRB), whilst collective equipoise refers to the profession as a whole. It is argued that physicians are not bound by the equipoise but their actions are directed by the confines of the expert opinion. Experts can agree or disagree in various proportions on the merit of a given treatment. Hence, the collective equipoise will be often incomplete. In turn, the opinions of content expert in the field of the proposed trial influence the IRB members’ decision regarding trial approval.Methods:We conducted a survey of IRB members at University of South Florida and the IRB members attending the bioethics conference organized in Clearwater, Florida, USA. The survey was made available as hard copy (paper based) and included six hypothetical scenarios outlining clinical trials targeted at measuring the collective equipoise. We defined the collective equipoise as the situation when survey participants were equally split (50:50) in their decision regarding whether a proposed clinical trial would be ethical to conduct. The opinion of 100 experts in the field expressed as proportion of experts favoring treatment A vs. B in each of the five scenarios was made available to the participants.Results:The response rate of our survey was 33% (71/218). Fifty percent of the IRB members would approve an RCT addressing the efficacy of two drugs for the management of headache even if 80% of experts favor one treatment over another (median: 80%; third quartile: 80%). Similarly, half of participating IRB members would approve the study when the median distribution of equipoise among experts was 70% (70 in favor of treatment A vs. 30 in favor of treatment B) for treatment of leukemia, 60% for treatment of geriatric patients and 70% for treatment of newborns. Half of IRB members would approve the study when the median distribution of equipoise among experts was 70% for treatment for leukemia in dogs and 85% for leukemia in rats (and 25% of IRB members would approve such a study even if 100% of experts favors one treatment over another). None of the demographic features of respondents affected collective equipoise.Conclusions:This is the first study assessing collective equipoise among ethical committee/IRB members. Our study findings show that IRB members perceived that conduct of a trial enrolling humans is unethical when the equipoise level is beyond 80% (80:20 distribution of uncertainty). IRB members require a higher level of equipoise when it comes to testing a new drug in humans than in animals. A relatively high level of equipoise is needed for IRB members to be comfortable to approve trials involving life-threatening situations, children and elderly patients.

Anti-leukemic and immunomodulatory effects of fungal metabolites of Pleurotus pulmonarius and Pleurotus ostreatus on benzene-induced leukemia in Wister rats.

BackgroundThe use of natural bioactive compounds in conventional chemotherapy is a new direction in cancer treatment that is gaining more research attention recently. Bioactive polysaccharides and polysaccharide-protein complexes from some fungi (edible mushrooms) have been identified as sources of effective and non-toxic antineoplastic agents. Selected oyster mushrooms (Pleurotus pulmonarius and P. ostreatus being local [Nigeria] and exotic strains, respectively) were cultured on a novel medium of yeast extract supplemented with an ethanolic extract of Annona senegalensis, and the antileukemic potential of their metabolites was studied.MethodsLeukemia was successfully induced in Wister rats by intravenous injection (0.2 mL) of a benzene solution every 2 days for 3 consecutive weeks. The aqueous solution of fungal metabolites (20 mg/mL) produced by submerged fermentation was orally administered (0.2 mL) before, during, and after leukemia induction. Leukemia burden was assessed by comparing the hematological parameters at baseline and after leukemia induction. The immunomodulatory potential of the metabolites was assessed by using a phagocytic assay (carbon clearance method). The ability to enhance leukopoiesis was assessed by using the total leukocyte count.ResultsLeukemia induction resulted in significant anemia indices and leukocytosis (P<0.05) in the experimental rats. Both metabolites equally enhanced leukopoiesis and demonstrated phagocytic actions; P. ostreatus activity was significantly higher than that of P. pulmonarius (P<0.05).ConclusionThe metabolites exhibited profound antileukemic potential by suppressing leukemia and demonstrating immunotherapeutic activities on animals after oral administration in various experimental groups.

Abstract B81: Prostate 5LX® inhibits proliferation of prostate cancer cells through alteration of inflammatory pathways

Abstract Purpose: Inflammation has been recognized as a key factor in the development of age-related degenerative diseases including cancer. Among important pro-inflammatory factors, bioactive lipids derived from cyclooxygenase (COX) or lipoxygenase (LOX) enzymes have been implicated as key targets for treatment and prevention of cancers. Here we report the effect of a multi-herb product “Prostate 5LX” on metabolism of inflammatory mediators in human prostate cancer and rat macrophage cells and their association with anti-proliferative activity. Methods: The effect of Prostate 5LX and its individual components on eicosanoid metabolism in human prostate cancer PC3, LNCaP, rat leukemia RBL-1 and macrophage RAW267.4 cells were examined using a validated LC/MS/MS analytical method. The effect of this natural product on the expression of inflammation associated genes in RAW267.4 cells was measured using TaqMan inflammation array kits and RT-PCR. Protein expression of relevant enzymes was evaluated by Western blot using appropriate antibodies. Results: When PC3 cells were treated with as little as 50 μg (0.1μl) Prostate 5LX/ml, the relative formation of 5-LOX products, LTB4 and 5-HETE was reduced by 69.2% and 36.7%, respectively. Similarly, Prostate 5LX at this concentration almost totally blocked the formation of LTB4 and 5-HETE in RBL-1 cells which are known to have relatively higher levels of 5-LOX and are a good model for evaluation of 5-LOX activity. The effect of Prostate 5LX on inhibition of 5-LOX activity was concentration-dependent. Unexpectedly, the level of 12-HETE, a 12-LOX metabolite, in PC3 or RBL-1 cells treated with Prostate 5LX was also significantly reduced in a concentration-dependent manner compared to untreated controls. To further our understanding of the role of individual botanical extracts in contributing to these unique biological activities, the effect of individual extracts on 5-LOX and 12-LOX metabolism was evaluated against the parent product in RBL-1 cells. While Saw Palmetto (80 μg/ml), green tea (50 μg/ml) and rosemary (10 μg/ml) significantly inhibited formation of 5-HETE by 67.1±11.2%, 45.3±8.3% and 68.5± 12.1%, respectively, stinging nettle (50 μg/ml) extract inhibited production of 12-HETE by 46.1% in RBL-1 cells. Intriguingly, the inhibitory effect of Prostate 5LX in the concentration comparable to that used for those individual botanical extracts, was much more stronger than any of the individual components evidenced by a 97.1% reduced formation of 5-HETE. The expression of both 5-LOX and 12-LOX proteins was reduced by Prostate 5LX in PC3, LNCaP and RBL-1 cells. Furthermore, the expression of LTA4 hydrolyase and leukotriene receptors were also markedly reduced by Prostate 5LX in Raw 267.4 cells. Interestingly, the growth of prostate cancer PC3 and LNCaP cells were significantly inhibited by Prostate 5LX (250 μg/ml) and these inhibitory effects appeared to be mediated through inhibition of 5-LOX and 12-LOX pathways in LNCaP and PC3 cells, respectively. Conclusions: Taken together, these preliminary data suggest that Prostate 5LX is capable of inhibiting the proliferation of prostate cancer cells mediated by alteration of lipoxygenase pathways, especially 5- and 12-LOX pathways. Given that inflammation plays an important role in prostate cancer initiation and development, these data may provide new insight on how this natural product affects prostate health, particularly prevents the development of prostate cancer. Citation Information: Cancer Prev Res 2011;4(10 Suppl):B81.

Incomplete Chromatin Condensation in Enlarged Rat Myelocytic Leukemia Cells

The distinguishable morphologic features of nuclei of acute myelogenous leukemia cells with enlarged size and finely distributed nuclear chromatin indicate incomplete chromosome condensation that can be related to elevated gene expression. To confirm this, interphase chromosome structures were studied in exponentially growing rat myelomonocytic leukemia 1 cells isolated at the University of Debrecen (My1/De cells). This cell line was established from primary rat leukemia chemically induced by 7,12-dimethylbenz[a]anthracene treatment. The enlarged nuclei of My1/De cells allowed improved fluorescent visualization of chromosomal structures. Increased resolution revealed major interphase intermediates consisting of (1) veil-like chromatin, (2) chromatin ribbon, (3) chromatin funnel, (4) chromatin bodies, (5) elongated prechromosomes, (6) seal-ring, spiral shaped, and circular chromosomal subunits, (7) elongated, bent, u- and v-shaped prechromosomes, and (8) metaphase chromosomes. Results confirmed the existence of the chromatin funnel, the first visible interphase chromosome generated by the supercoiling of the chromatin ribbon. Other intermediates not seen previously included the spiral subunits that are involved in the chromonemic folding of metaphase chromosomes. The existence of spiral subunits favors the helical coil model of chromosome condensation. Incomplete chromatin condensation in leukemia cells throughout the cell cycle is an indication of euchromatization contributing to enhanced gene expression and is regarded as a leukemic factor.

Generation of Germline-Competent Rat Induced Pluripotent Stem Cells

BackgroundRecent progress in rat pluripotent stem cell technology has been remarkable. Particularly salient is the demonstration that embryonic stem cells (ESCs) in the rat (rESCs) can contribute to germline transmission, permitting generation of gene-modified rats as is now done using mouse ESCs (mESCs) or mouse induced pluripotent stem cells (iPSCs; miPSCs). However, determinations of whether rat iPSCs (riPSCs) can contribute to germ cells are not published. Here we report the germline competency of riPSCs.Methodology/Principal FindingsWe generated riPSCs by transducing three mouse reprogramming factors (Oct3/4, Klf4, and Sox2) into rat somatic cells, followed by culture in the presence of exogenous rat leukemia inhibitory factor (rLIF) and small molecules that specifically inhibit GSK3, MEK, and FGF receptor tyrosine kinases. We found that, like rESCs, our riPSCs can contribute to germline transmission. Furthermore we found, by immunostaining of testis from mouse-rat interspecific chimeras with antibody against mouse vasa homolog, that riPSCs can contribute to embryonic development with chimera formation in mice (rat-mouse interspecific chimeras) and to interspecific germlines.Conclusions/SignificanceOur data clearly demonstrate that using only three reprogramming factors (Oct3/4, Klf4, and Sox2) rat somatic cells can be reprogrammed into a ground state. Our generated riPSCs exhibited germline transmission in either rat-rat intraspecific or mouse-rat interspecific chimeras.

Therapeutic Efficacy of FTY720 In a Rat Model of LGL Leukemia

AbstractAbstract 776Natural kill cell large granular lymphocytic (NK-LGL) leukemia is a fatal disorder with death occurring in days to weeks following diagnosis. There is no known curative therapy for this disease. Therefore, there is an urgent unmet need for development of new therapeutics for this deadly leukemia. Fischer F344 rat LGL leukemia model has been established as an important experimental model for the study of NK-LGL leukemia progression and closely resembles human aggressive NK-LGL leukemia exhibiting clonal expansion of CD3-CD8a+ lymphocytes. FTY720, a new immunosuppressant, has been studied for its putative anti-cancer properties in the recent years. At four weeks after transplantation of leukemic LGL cell line, the rats displayed early signs of leukemia, including weight loss, rough hair coat and increased level of neutrophils. By week 5, circulating blasts, anemia, thrombocytopenia, and splenomegaly were observed. These leukemic rats were then injected intraperitoneally with 4.5mg/kg of FTY720 or PBS every day over a 4-week treatment period. Animals died within the next 1 to 2 weeks if treated with PBS. The median survival in PBS treated group was 41 days compared to 51 days in FTY720 treated group (Mantel-Cox test, p<0.0001). Importantly, 5 of 16 leukemic rats treated with FTY720 had maintenance of normal blood counts without circulating blasts suggesting achievement of complete clinical remission. The remaining eleven leukemic rats treated with FTY720 had a transient improvement as evidenced by reduction of white blood cell counts and elevated platelet counts after two weeks treatment. Subsequently, however, blast counts rose and animals died within the following one week. To further determine remission status, the five responsive animals were euthanized after cessation of the treatment. At necropsy, we found these rats had normal levels of CD3-CD8a+ LGL cells in the blood, marrow, and spleen. Of note, the eleven leukemic rats not achieving remission also displayed significant reduction of LGL cells in these tissues, to lesser extent. Examination of spleen sections from rats responsive to FTY720 showed normal splenic histology. In contrast, leukemic rats not achieving remission showed leukemic LGL infiltration of the red pulp and depletion of the white pulp. Interestingly, pro-apoptotic proteins Bax and Bak were dramatically increased while anti-apoptotic protein Mcl-1 was decreased in the spleens of the rats achieving remission; however, they remained unchanged in leukemic rats not achieving remission. These data indicate that in vivo therapeutic efficacy of FTY720 may be a consequence of modulation of anti-apoptosis signaling which led to resolution of leukemic cell infiltration. We then extended these studies to NK LGL from patients. Initial experiments demonstrated that FTY720 displayed dose- and time-dependent apoptotic cell death in PBMC from NK-LGL leukemia patients (CD3−CD56+>80%). In contrast, treatment with 10uM FTY720 did not induce significant cytotoxic effects in PBMC from normal donors, or normal NK cells. In addition, treatment of human and rat NK-LGL leukemia cells with FTY720 led to caspase-dependent apoptosis, generation of reactive oxygen species (ROS), and Mcl-1 degradation which did not occur at the transcriptional level. Of interest, inhibition of ROS rescued FTY720 induced apoptosis in leukemic NK cells. Moreover, efficient knockdown of Mcl-1 resulted in more than two fold increase in apoptotic cell death of NKL, a human NK-LGL leukemia cell line. Collectively, these results indicate efficacy of FTY720 in a rat model of NK LGL leukemia via production of ROS and decreased mcl-1 expression or signaling. Disclosures:No relevant conflicts of interest to declare.

Final Amended Safety Assessment of Hydroquinone as Used in Cosmetics

Hydroquinone is an aromatic compound that functions in cosmetics as an antioxidant, fragrance, reducing agent, or polymerization inhibitor. Hydroquinone is also used as a skin bleaching agent. Safety and toxicity information indicate that hydroquinone is dermally absorbed in humans from both aqueous and alcoholic formulations and is excreted mainly as the glucuronide or sulfate conjugates. Hydroquinone is associated with altered immune function in vitro and in vivo in animals and an increased incidence of renal tubule cell tumors and leukemia in F344 rats, but the relevance to humans is uncertain. Quantitatively, however, the use of hydroquinone in cosmetics is unlikely to result in renal neoplasia through this mode of action. Thus, hydroquinone is safe at concentrations of ≤1% in hair dyes and is safe for use in nail adhesives. Hydroquinone should not be used in other leave-on cosmetics.

Antileukemic action of (−)-epicatechin in the spleen of rats with acute myeloid leukemia

The aim of this study was to investigate whether (−)-epicatechin (EC) can induce DNA damage and apoptosis of cancer cells in the spleen of rat with acute myeloid leukemia. Healthy and leukemic rats were given EC by gavage at a dose of 40 mg/kg b.w. for 22 consecutive days. Spleen cells were subjected for analysis of DNA damage and apoptosis. The amount of DNA damage was estimated by the comet assay, while apoptosis was examined by flow cytometry using Annexin V staining. Leukemic cells were identified in the spleen cells by indirect immunofluorescence using RM-124 antibody followed by flow cytometry analysis. The results show that EC did not affect DNA damage in the splenocytes of healthy rats, but significantly increased the extent of DNA strand breaks in the spleen cells of leukemic animals. EC administration to leukemic rats induced a significant increase in the level of Annexin V-positive leukemic cells, but the level of non-leukemic Annexin V-positive cells remained unchanged in comparison to control. The percentage of leukemic cells decreased significantly under EC influence comparing to the untreated group. The results of the study reveal that EC could be used as an effective supplement of standard therapy against acute myeloid leukemia.