Intraperitoneal Injection Of 5-fluorouracil Research Articles

Anise (Pimpinella anisum L.) attenuates azoxymethane-induced colorectal cancer by antioxidant, anti-inflammatory, and anti-apoptotic pathways in rats.

Herbal medicine is one of the most common fields explored for combating colon cancers, and Pimpinella anisum L. seeds (PAS) have been utilized widely as medicinal agents because of their increased essential oil (trans-anethole) contents. In this essence, our study investigates the toxic effect and chemoprotective potentials of PAS against azoxymethane (AOM)-induced colon cancer in rats. The toxicity trial for PAS conducted by clustering fifteen rats into three groups (five rats each): A, normal control had 10% Tween 20; B, ingested with 2g/kg PAS; and C, supplemented with 4g/kg PAS. The in vivo cancer trial was performed by using 30 rats (Sprague-Dawley) that were randomly adapted in five steel cages (six rats each): group A, normal controls received two subcutaneous injections of normal saline 0.09% and ingested orally 10% Tween 20; groups B-E, rats received two injections of 15mg/kg of azoxymethane (AOM) subcutaneously in 2weeks and treated orally with 10% Tween 20 (group B) or intraperitoneal injection of 5-fluorouracil (35mg/kg) (group C), or orally given 200mg/kg PAS (group D) and 400mg/kg PAS (group E) for 8weeks. After the scarification of rats, the colon tissues were dissected for gross and histopathological evaluations. The acute toxicity trial showed the absence of any toxic signs in rats even after 14days of ingesting 4g/kg of PAS. The chemoprotective experiment revealed significant inhibitory potentials (65.93%) of PAS (400mg/kg) against aberrant crypto foci incidence that could be correlated with its positive modulation of the immunohistochemically proteins represented by a significant up-regulation of the Bax protein and a decrease of the Bcl-2 protein expressions in colon tissues. Furthermore, PAS-treated rats had notably lower oxidative stress in colon tissues evidenced by decreased MDA levels and increased antiradical defense enzymes (SOD, CAT, and GPx). The outcomes suggest 400mg/kg PAS as a viable additive for the development of potential pharmaceuticals against colorectal cancer.

Thrombopoietin receptor agonist antibody for treating chemotherapy-induced thrombocytopenia

BackgroundThrombocytopenia is a common complication in cancer patients undergoing chemotherapy. Chemotherapy-induced thrombocytopenia (CIT) leads to dose reduction and treatment delays, lowering chemotherapy efficacy and survival rate. Thus, rapid recovery and continuous maintenance of platelet count during chemotherapy cycles are crucial in patients with CIT. Thrombopoietin (TPO) and its receptor, myeloid proliferative leukemia (MPL) protein, play a major role in platelet production. Although several MPL agonists have been developed to regulate thrombopoiesis, none have been approved for the management of CIT due to concerns regarding efficacy or safety. Therefore, the development of effective MPL agonists for treating CIT needs to be further expanded.MethodsAnti-MPL antibodies were selected from the human combinatorial antibody phage libraries using phage display. We identified 2R13 as the most active clone among the binding antibodies via cell proliferation assay using BaF3/MPL cells. The effect of 2R13 on megakaryocyte differentiation was evaluated in peripheral blood CD34+ cells by analyzing megakaryocyte-specific differentiation markers (CD41a+ and CD42b+) and DNA ploidy using flow cytometry. The 2R13-induced platelet production was examined in 8- to 10-week-old wild-type BALB/c female mice and a thrombocytopenia mouse model established by intraperitoneal injection of 5-fluorouracil (150 mg/kg). The platelet counts were monitored twice a week over 14 days post-initiation of treatment with a single injection of 2R13, or recombinant human TPO (rhTPO) for seven consecutive days.ResultsWe found that 2R13 specifically interacted with MPL and activated its signaling pathways. 2R13 stimulated megakaryocyte differentiation, evidenced by increasing the proportion of high-ploidy (≥ 8N) megakaryocytes in peripheral blood-CD34+ cells. The platelet count was increased by a single injection of 2R13 for up to 14 days. Injection of 5-fluorouracil considerably reduced the platelet count by day 4, which was recovered by 2R13. The platelets produced by 2R13 sustained a higher count than that achieved using seven consecutive injections of rhTPO.ConclusionsOur findings suggest that 2R13 is a promising therapeutic agent for CIT treatment.

Wumei pills attenuates 5-fluorouracil-induced intestinal mucositis through Toll-like receptor 4/myeloid differentiation factor 88/nuclear factor-κB pathway and microbiota regulation

BACKGROUNDRadiotherapy and chemotherapy can kill tumor cells and improve the survival rate of cancer patients. However, they can also damage normal cells and cause serious intestinal toxicity, leading to gastrointestinal mucositis[1]. Traditional Chinese medicine is effective in improving the side effects of chemotherapy. Wumei pills (WMP) was originally documented in the Treatise on Exogenous Febrile Diseases. It has a significant effect on chronic diarrhea and other gastrointestinal diseases, but it is not clear whether it affects chemotherapy-induced intestinal mucositis (CIM).AIMTo explore the potential mechanism of WMP in the treatment of CIM through experimental research.METHODSWe used an intraperitoneal injection of 5-fluorouracil (5-Fu) to establish a CIM mouse model and an oral gavage of WMP decoction (11325 and 22650 mg/kg) to evaluate the efficacy of WMP in CIM. We evaluated the effect of WMP on CIM by observing the general conditions of the mice (body weight, food intake, spleen weight, diarrhea score, and hematoxylin and eosin stained tissues). The expression of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), IL-1β, and myeloperoxidase (MPO), as well as the Toll-like receptor 4/myeloid differentiation factor 88/nuclear factor-κB (TLR4/MyD88/NF-κB) signaling pathway proteins and tight junction proteins (zonula occludens-1, claudin-1, E-cadherin, and mucin-2) was determined. Furthermore, intestinal permeability, intestinal flora, and the levels of short-chain fatty acids (SCFA) were also assessed.RESULTSWMP effectively improved the body weight, spleen weight, food intake, diarrhea score, and inflammatory status of the mice with intestinal mucositis, which preliminarily confirmed the efficacy of WMP in CIM. Further experiments showed that in addition to reducing the levels of TNF-α, IL-1β, IL-6, and MPO and inhibiting the expression of the TLR4/MyD88/NF-κB pathway proteins, WMP also repaired the integrity of the mucosal barrier of mice, regulated the intestinal flora, and increased the levels of SCFA (such as butyric acid).CONCLUSIONWMP can play a therapeutic role in CIM by alleviating inflammation, restoring the mucosal barrier, and regulating gut microbiota.

Glutaryl Melatonin Niosome Gel for Topical Oral Mucositis: Anti- Inflammatory and Anticandidiasis.

Glutaryl melatonin, which is synthesized from melatonin and is a pineal glandderived neurohormone with anti-inflammatory and anti-oxidant properties, was comparatively investigated for its potential use as a topical anti-inflammatory agent. Glutaryl melatonin, synthesized and screened for in vitro anti-candidiasis and in vitro and in vivo anti-inflammatory activities, was formulated as a niosome gel for topical oral evaluation in 5- fluorouracil-induced oral mucositis in mice. In vitro anti-fungal activity in Candida albicans, in vitro anti-inflammatory activity in Escherichia coli liposaccharide-induced RAW cells and in vivo anti-inflammatory activity using a croton oilinduced ear edema model in ICR mice were investigated. Mucositis in mice (n= 6/group, 10-week-old mice) was induced by intraperitoneal injections of 5-fluorouracil, and the mice were subjected to a topical oral application of niosome gel containing melatonin (2% w/w) or glutaryl melatonin (2% w/w) and were compared with mice subjected to blank, fluocinolone acetonide (0.5% w/w) and control conditions. Glutaryl melatonin, at a 14.2 mM concentration, showed the highest fungicidal effect on C. albicans using the broth dilution method, indicating a nonsignificant difference from 1 μM of nystatin (p = 0.05). Nitric oxide, interleukin-6 and tumor necrosis factors were analyzed by ELISA. Liposaccharide-induced RAW cells were significantly reduced by glutaryl melatonin (p < 0.01). Ear edema inhibition of glutaryl melatonin was significant 1 h after application compared with that of melatonin (p = 0.03). Food consumption and body weight of the 5-fluorouracil-treated mice were significantly lower than those of the normal mice before all treatments (p < 0.05). Differences in the amount of licking behavior, which were observed in the control group for 5 min, were noticeable in the 5- fluorouracil-treated mice but not in the mice treated with the glutaryl melatonin niosome gel. Glutaryl melatonin exhibited mild anti-candidiasis and anti-inflammatory properties. The incorporation of glutaryl melatonin in a niosome gel formulation, demonstrated the potential for topical oral applications to reduce oral discomfort caused by 5-fluorouracil treatment in mice.

Patchouli alcohol attenuates 5-fluorouracil-induced intestinal mucositis via TLR2/MyD88/NF-kB pathway and regulation of microbiota

Intestinal mucositis causes great suffering to cancer patients who undergo chemotherapy and radiotherapy. Owing to the uncertain side effects of anticancer drugs to attenuate patients’ intestinal mucositis, many studies focused on traditional Chinese medicine (TCM). Patchouli alcohol (PA) is an active compound extracted from Pogostemon cablin, and has potent gastrointestinal protective effect. However, whether PA has an effect on intestinal mucositis is still unknown. Therefore, we established a rat model of intestinal mucositis via intraperitoneal injection of 5-fluorouracil, and intragastrically administrated PA (10, 20, and 40 mg/kg) to evaluate the effect of PA on intestinal mucositis. The routine observation (body weight, food intake, and diarrhea) in rats was used to detect whether PA had an effect on intestinal mucositis. Levels of inflammatory cytokines (TNF-α, IL-1β, IL-6, IL-10, and MPO), mucosal barrier proteins (zonula occludens -1 (ZO-1), claudin-1, occludin, myosin light chain (MLC), and mucin-2) and intestinal microbiota were determined to elucidate the underlying mechanism of PA action on intestinal mucositis in rats. The results showed that PA could effectively improve body weight, food intake, and diarrhea in intestinal mucositis rats, preliminary confirming PA efficacy. Further experiments revealed that PA not only decreased the levels of TNF-α, IL-1β, IL-6, and MPO but also increased the level of IL-10 significantly. In addition, the expression of mucosal barrier proteins and microbiota community were also improved after PA treatment in diseased rats. Hence, PA may prevent the development and progression of intestinal mucositis by improving inflammation, protecting mucosal barrier, and regulating intestinal microbiota.

CURCUMA LONGA L. HAS THERAPEUTIC EFFECT IN CHEMOTHERAPY-INDUCED ORAL MUCOSITIS IN HAMSTERS DECREASING ANGIOGENESIS AND TRANSFORMING GROWTH FACTOR-β1

Objectives We explored the effects of mucoadhesive formulation with Curcuma longa L. extract (MCL) on oral mucositis induced by 5-fluorouracil in hamsters. Study Design Seventy-two golden Syrian hamsters separated into 4 groups: control group, placebo group (no active substance), positive control group (AdMuc topical chamomile), and MCL group (topical use of mucoadhesive Curcuma longa). The animals received an intraperitoneal injection of 5-fluorouracil on days 0 and 2. On days 3 and 4, the buccal mucosa was scratched, and therapy was initiated on day 5. The animals received 2 daily applications of the product according to the experimental group. Wound area was calculated and histologic sections of 3 μm were stained by hematoxylin and eosin for semiquantitative analysis of re-epithelization and degree of tissue inflammation. Immunohistochemistry was used for transforming growth factor β1 (TGF-β1) and CD31 analysis. Results The MCL group showed a greater clinical reduction of the mucositis lesions at day 8, a higher degree of re-epithelization, and a lower inflammatory process, with a lower angiogenesis and TGF-β1 epithelial labeling compared with the placebo and control groups (P Conclusions MCL Curcuma longa L. has a therapeutic effect accelerating the healing of oral mucositis reducing the inflammatory response and increasing re-epithelization.

Experimental study on the efficacy and toxicity of intraperitoneal injection of 5-fluorouracil to treat PDX model of pseudomyxoma peritonei

Objective To evaluate the efficacy and toxicity of intraperitoneal injection of 5-fluorouracil (5-Fu) in patient derived xenograft (PDX) model of pseudomyxoma peritonei (PMP). Methods One PMP tumor from surgery was used to establish PMP PDX models, which were equally randomized into control (no treatment), vehicle (normal saline, 0.2 ml) and treatment (5-Fu, 50 mg/kg, 0.2 ml) groups. Experimental peritoneal cancer index (ePCI) score, body weight, and pathological examination were performed to evaluate the efficacy and toxicity. Results PDX models replicating human PMP were established and diagnosed as high grade mucinous carcinoma peritonei with signet ring cells. Average ePCI score of treatment group was significantly lower than control and vehicle groups [(0.9±1.2) points vs. (3.6±1.7) points vs. (2.7±1.1) points, F=9.253, P<0.01]. Histopathological study revealed obvious tumor necrosis and reduced signet ring cells in the treatment group. The Ki-67 label index of treatment group was significantly lower than control group and vehicle group [(57.3±15.1)% vs. (72.2±15.1)% vs. (70.0±14.0)%, F=4.923, P<0.05]. The body weight of treatment group was significantly lower than control group and vehicle group [(19.7±2.8) g vs. (22.7±1.8) g vs. (21.9±1.7) g, F=4.654, P<0.05]. Major toxicities in the treatment group were animal death (10.0%), congestive splenomegaly (88.9%), lymphocyte accumulation (77.8%), bile canaliculus hyperplasia and obstruction (22.2%), cholestasis (100.0%), and acidophilic body (55.6%) in the liver. Conclusion In the PDX model of human PMP, intraperitoneal injection of 5-Fu could inhibit tumor proliferation and progression, with lowered ePCI score and reduced Ki-67, and toxicity was mainly on liver. Key words: Pseudomyxoma peritonei; PDX model; 5-Fluorouracil; Intraperitoneal injection; Organ toxicity

Study of the chemical structure of exopolysaccharide produced from streptomycete and its effect as an attenuate for antineoplastic drug 5-fluorouracil that induced gastrointestinal toxicity in rats

Chemotherapeutic medications, including 5 – fluorouracil (5FU), are the same old technique to most cancers and are associated with numerous peripheral toxicities. We investigated exopolysaccharide (EPSST) produced from the isolated streptomycete of the Mediterranean Sea for the capability to lower the severity of mucositis in vivo. The streptomycete was isolated from Mediterranean Sea sediment from the beaches of Port Said Governorates, Egypt and identified morphologically, physiologically, and biochemically and confirmed by molecularly 16S rDNA analysis. The EPSST was extracted from the supernatant of streptomycete by using 4 volumes chilled ethanol and then the functional groups, MW, and chemical evaluation have been detected via Fourier-transform infrared (FTIR), and high-performance liquid chromatography (HPLC). In addition, antioxidant activity was measured through the usage of 2, 2-diphenyl-1-picrylhydrazyl (DPPH). Thirty-two male rats (180–200 g) were randomly divided into a control group (normal saline), intraperitoneal injection of 5-fluorouracil (5-FU, 150 mg/kg), normal rats were treated with EPSST and 5-FU + EPSST group. These groups were continued up to the day of sacrifice (28 days post treatments). The isolated strain became recognized based totally on 16S rDNA sequence as Streptomyce sp. with accession number SAMN08349905. The chemical evaluations of EPSST were galacturonic, glucose, galactose, mannose, and arabinose with a relative ratio of 2.1: 1: 5.37: 1.62: 1.29 individually, with an average molecular weight (Mw) 9.687 × 103 g/mol. Also, the EPSST contained uronic acid (16%) and sulfate (12.149%) and no protein was detected. EPSST inhibited the DPPH radical activity. The findings of this study propose that EPSST inhibits 5-FU-induced mucositis through adjustment of oxidative stress, apoptosis, inflammatory factors, activation of antioxidant enzymes. The clinical administration of EPSST may recover the chemotherapy-induced intestinal dysfunction, consequently increasing the clinical efficiency of chemotherapy. In addition, the administration of EPSST reduced 5-FU-induced histopathological incongruities such as neutrophil infiltration, loss of cellular integrity, and villus and crypt distortion. The clinical administration of EPSST may recover the chemotherapy-induced intestinal dysfunction, consequently increasing the clinical efficiency of chemotherapy.

Effects of Matricaria Recutita (L.) in the Treatment of Oral Mucositis.

Objective The objective of this study was to review the effects of the Matricaria recutita (L.) in the treatment of oral mucositis. Methodology The online search was performed in the period from June 2016 to April 2018 by means of databases LILACS (Latin American and Caribbean Center on Health Sciences Information), SciELO (Scientific Electronic Library Online), and MEDLINE (Medical Literature Analysis and Retrieval System Online). The consultation was restricted to the years 1991 to 2018 with the aim of elucidating the effects of Matricaria recutita in the treatment of oral mucositis. Results The final sample consisted of 21 studies, of which 10 were developed in animals and 11 in humans, published from 1991 to 2017, with a total sample of 644 patients. The total number of patients treated with Matricaria included in 11 studies was 364, while in the control groups the total number was 280. In experimental studies, animal models used were rats and the sample size ranged between 36 and 105 animals submitted to the induction of oral mucositis, where 4 studies used an intraperitoneal injection of 5-fluorouracil, while 7 induced lesion in the mucosa. From the data collected, it should be noted that both studies with humans and with animals showed significant effects. In this way, there is strong evidence for the discussion on the therapy; however, it should be noted that more studies are developed in order to clarify the most appropriate protocol for the prevention and treatment of injuries. Conclusion According to the results found in this study, Matricaria recutita appeared to be a promising alternative for the treatment of oral mucositis. However, due to the great variability in the various types of intervention, more controlled double-blind randomized clinical studies are necessary to ensure the best protocol for treating oral mucositis.

Metaplastic Cells in the Stomach Arise, Independently of Stem Cells, via Dedifferentiation or Transdifferentiation of Chief Cells

Spasmolytic polypeptide-expressing metaplasia (SPEM) develops in patients with chronic atrophic gastritis due to infection with Helicobacter pylori; it might be a precursor to intestinal metaplasia and gastric adenocarcinoma. Lineage tracing experiments of the gastric corpus in mice have not established whether SPEM derives from proliferating stem cells or differentiated, post-mitotic zymogenic chief cells in the gland base. We investigated whether differentiated cells can give rise to SPEM using a nongenetic approach in mice. Mice were given intraperitoneal injections of 5-fluorouracil, which blocked gastric cell proliferation, plus tamoxifen to induce SPEM. Based on analyses of molecular and histologic markers, we found SPEM developed even in the absence of cell proliferation. SPEM therefore did not arise from stem cells. In histologic analyses of gastric resection specimens from 10 patients with adenocarcinoma, we found normal zymogenic chief cells that were transitioning into SPEM cells only in gland bases, rather than the proliferative stem cell zone. Our findings indicate that SPEM can arise by direct reprogramming of existing cells-mainly of chief cells.

Noninvasive assessment of hepatic sinusoidal obstructive syndrome using acoustic radiation force impulse elastography imaging: A proof-of-concept study in rat models.

To determine the feasibility of acoustic radiation force impulse (ARFI) elastography in the evaluation of hepatic sinusoidal obstruction syndrome (SOS) in rat models. Rat SOS models of various severities were created by monocrotaline gavage (n = 40) or by intraperitoneal injection of 5-fluorouracil, leucovorin and oxaliplatin (FOLFOX) (n = 16). Liver shear-wave velocity (SWV) was measured using ARFI elastography. Liver samples were analysed for the SOS score, steatosis, lobular inflammation and fibrosis. The liver SWV was significantly elevated in the SOS models (1.29-2.24 m/s) compared with that of the matched control rats (1.01-1.09; p≤.09; veFor seven FOLFOX-treated rats which were longitudinally followed-up, the liver SWV significantly increased at 7 weeks (1.32±0.13 m/s) compared with the baseline (1.08±0.1 m/s, p=.015) and then significantly declined after a 2-week, treatment-free period (1.15±0.13 m/s; p=.048). Multivariate analysis revealed that the SOS score (p<.001) and lobular inflammation (p=.044) were independently correlated with the liver SWV. Liver SWV is elevated in SOS in proportion to the degree of sinusoidal injury and lobular inflammation in rat SOS models. ARFI elastography has potential as an examination for diagnosis, severity assessment and follow-up of SOS. • Liver SWV using ARFI elastography was significantly elevated in SOS rat. • Sinusoidal injury and lobular inflammation grades had correlation with liver SWV. • ARFI elastography has potential for diagnosis, severity assessment, and follow-up of SOS.

Морфологические особенности ткани селезенки после введения фторурацила и криоконсервированных ядросодержащих клеток кордовой крови

We studied the effect of administered cryopreserved nucleated cells of human cord blood (cCBNCs) on murine spleen tissue morphology under hemo- and immunosuppression induced by a single intraperitoneal injection of 5-fluorouracil (5-FU) antimetabolite of nucleic metabolism in the maximum tolerated dose. Progressing changes, typical for a damage caused by toxic substances, such as: congestion, exudation and leukocytes infiltration of spleen pulp; accumulation of macrophages either with phagocytized lymphocytes or their fragments; degenerative and necrotic changes in tissue elements of the pulp and follicles were revealed in spleen following 5‑FU introduction within entire observation period (16 days). A high degree of murine spleen reactivity in response to the administration of cryopreserved cord blood cells was found. To day 2 after cCBNCs administration we observed structural rearrangements in spleen, and already 10 days later 5-FU antimetabolite toxic injury the organ structure was back to the norm. Probl Cryobiol Cryomed 2016; 26(3): 249–258

Effects of acute chemotherapy-induced mucositis on spontaneous behaviour and the grimace scale in laboratory rats.

Intestinal mucositis is a frequent side-effect of chemotherapy treatment. Many oncological research programs aim to identify novel treatments for this distressing condition, and these programs frequently use rat models. Little is known about the presence and progression of pain in these models and how this can best be treated by analgesic therapy. We used a number of behaviour-based methods of pain assessment to determine which tools were best suited for pain identification. Baseline measures for behavioural assessment, rat grimace score and sociability were determined through analysis of continuously recorded video data and an applied social interaction test (n = 16). Mucositis was then induced by intraperitoneal injection of 5-fluorouracil (150 mg/kg) and further behavioural analyses undertaken. An assessment of enrichment interaction was also made by determining the mass of a plastic chew toy gnawed both pre- and post-chemotherapy injection. Behavioural scoring was performed 1, 6, 12, 24 and 48 h after injection, with facial expression being scored at the 12, 24 and 48 h time-points. Sociability testing was performed once during the post-injection period. No significant differences were found in grimace scores between baseline and later daily measures. Behaviours similar to those previously reported post-laparotomy were observed. Writhing, twitching and back-arching behaviours were most evident in rats affected by mucositis and were increased in frequency (respective P values: 0.002, 0.004 and 0.008) 48 h after chemotherapy injection compared with baseline, implying that pain onset occurred around this time-point. Social investigatory behaviour was also increased (P = 0.002) following disease onset. Each day, rats post-5FU injection gnawed a greater percentage of their Nylabone enrichment by weight than the saline-injected control rats (P = 0.046). These data suggest that, of the tools tested, behavioural assessment scoring may find greatest utility in rodent models of intestinal mucositis and should be investigated further.

Inhibitory effect and its mechanism of CS055 on human colon carcinoma transplanted tumor

Objective To explore the inhibitory effect and its mechanism of CS055[a selective histone deacetylase inhibitor(HDACi)]on human colon carcinoma in nude mice. Methods The xenograft model was established successfully.Fifty-five mice were divided into five groups and eleven blocks by randomized complete-block design.The mice were respectively gavaged by CS055 10. 0 mg/(kg·d)in the first group,5 mg/ (kg·d)in the second group and 2. 5 mg/(kg·d)in the third group, while the mice in fourth group were treated by intraperitoneal injection of 5-fluorouracil in a dose of 25. 0 mg/(kg·d)and those in the fifth group were gavaged by negative-control-drug 0. 1 ml/(10 g·d)as negative control group.After treatment, all nude mice were killed,and all the subcutaneous transplanted tumors were stripped and measured by calipers and scales.Inhibitory rate of tumor weight and tumor volume was calculated.Tissue hematoxylin and eosin (HE)staining and transmission electron microscopy detection were used to detect the tumor cell apoptosis. Western blotting was used to detect the expression of histone deacetylase 1(HDAC1), HDAC2, acetylated histone-H3(ac-H3), p21, cyclin dependent kinase 4(CDK4), and cysteinyl aspartate-specific protease (Caspase)-3. Results Compared to negative control group, the respective inhibition rate of tumor weight in high-dose group, mediate-dose group, low-dose group, and 5-fluorouracil group was 15. 36%(P> 0. 05),40. 27%(P 0. 05)and 55. 63%(P 0. 05), 40. 47%(P 0. 05) and 55. 74%(P<0. 01)respectively.CS055 could significantly inhibit the expression of HDAC1 and HDAC2,upregulate the level of acetylation of histone H3 and the expression of p21,Caspase-3, while downregulate the expression of CDK4 protein. Conclusion CS055 can inhibit the growth of human colon carcinoma xenograft tumor probably by inhibiting the expression of HDAC1 and HDAC2, upregulating the level of histone H3 acetylation and the expression of p21 protein, downregulating the expression of CDK4 protein, which promotes tumor cell cycle arrest, and upregulating the expression of Caspase-3 protein, which induces LoVo cells apoptosis. Key words: Colorectal cancer; Histone deacetylase inhibitor; Apoptosis; Nude mice

C/EBPβ Promotes Initial Expansion and Exhaustion of Hematopoietic Stem and Progenitor Cells in Response to Hematopoietic Stresses

Our previous findings have revealed the requirement of CCAAT Enhancer Binding Protein (C/EBPb), a leucine zipper transcription factor, in granulopoiesis (Hirai et al. Nat Immunol, 2006). During emergency situations such as infection, C/EBPb is involved in the sufficient supply of granulocytes through amplification of hematopoietic stem and progenitor cells (HSPCs) (Satake et al. J Immunol, 2012). In addition, we have shown that C/EBPb is upregulated by downstream signaling of BCR-ABL and promotes myeloid expansion and exhaustion of leukemic stem cells in chronic phase chronic myeloid leukemia (Hayashi et al. Leukemia, 2013). These observations suggested that C/EBPb plays important roles in regulation of normal and leukemic HSPCs. In this study, we focus on the functions of C/EBPb in normal HSPCs under stressed conditions.At steady state, the frequencies of HSPCs in the bone marrow (BM) of C/EBPb knockout (KO) mice were identical to those in the BM of wild type (WT) mice. It suggests that C/EBPb has little impact on the emergence or maintenance of HSPCs during steady state.To investigate function of C/EBPb in HSPCs, competitive repopulation assay was performed. Total BM cells from either WT or KO mice (CD45.2+) and the equal number of competitor cells from the BM of CD45.1+ WT mice were transplanted into lethally irradiated recipient WT mice (CD45.1+), and the chimerism of CD45.2+ cells in the peripheral blood (PB) of the recipient mice was monitored once a month. Chimerism of KO cells in the recipient mice was significantly lower than that of WT cells at 1 month after transplantation (52.2 ± 10.3% vs 37.8 ± 8.8%, p < 0.0000001, n = 37 vs 36) and the differences were maintained thereafter (Figure 1), suggesting that C/EBPb is required at early time points after transplantation.In order to elucidate the early events which make difference in the chimerism, homing ability was assessed first. Sixteen hours after transplantation of lineage depleted WT or KO BM cells (CD45.2+) together with lineage negative CD45.1+ WT BM cells, the frequencies of CD45.2+ WT and KO donor cells in the c-kit+ Sca1+ lineage- (KSL) fraction were identical. Then we compared the initial expansion of HSPCs. Purified 1000 KSL cells from either WT or KO mice (CD45.2+) were transplanted to lethally irradiated recipient WT mice (CD45.1+ / CD45.2+) together with the equal number of competitor KSL cells from WT mice (CD45.1+). The ratio of CD45.2+ KO cells to CD45.1+ competitors in the KSL fraction of the recipient mice was significantly lower than that of CD45.2+ WT cells at 4 weeks after transplantation (6.76 ± 2.35 vs 2.84 ± 1.16, p = 0.040, n = 4 vs 4). These results suggest that C/EBPb is required for initial expansion of HSPCs rather than for homing after transplantation.Next, we investigated the roles of C/EBPb in maintenance of HSPCs under stressed conditions. By staining of intracellular C/EBPb in combination with multi-color flow cytometric analysis, we found that C/EBPb is upregulated at protein level in KSL cells of WT mice 5 days after intraperitoneal injection of 5-fluorouracil (5-FU). Then the recipient mice were repetitively administered with 5-FU (150mg/kg i.p.) after BM transplantation in a competitive way. As mentioned above, the chimerism of KO cells in PB of recipient mice was significantly lower than those of WT mice at 1 month after transplantation. Interestingly, the chimerism of KO cells gradually increased by repetitive administration of 5-FU and even overtook those of WT cells 5 months after transplantation (Figure 2). In accordance with the changes observed in the PB, the chimerism of KO cells in the KSL fraction in the BM of recipient mice was significantly higher than those of WT cells (70.7 ± 25.3% vs 12.1 ± 9.78%, p = 0.016, n = 5 vs 4) 5 months after transplantation, suggesting that WT HSPCs exhausted earlier than KO HSPCs in response to hematopoietic stress.From these findings, we conclude that C/EBPb is required for initial expansion and exhaustion of HSPCs after hematopoietic stresses. We are currently investigating the molecular targets of C/EBPb and its clinical significance in the pathogenesis of leukemia. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The assessment of general well-being using spontaneous burrowing behaviour in a short-term model of chemotherapy-induced mucositis in the rat.

Mucositis is a common and serious side-effect experienced by cancer patients during treatment with chemotherapeutic agents. Consequently, programmes of research focus on the elucidation of novel therapeutics for alleviation of mucositis symptoms, and these frequently use animal models. However, although these models are assumed to be painful and distressing to the animal, endpoints are difficult to determine. The aim of this study was to evaluate whether a change in burrowing behaviour could provide an indication of disease onset and potentially be applied as a humane endpoint. Baseline burrowing behaviour was measured in healthy animals on three occasions by determining the weight of gravel displaced from a hollow tube. Mucositis was then induced in the same animals by intraperitoneal injection of 5-fluorouracil (150 mg/kg) and burrowing behaviour recorded over three consecutive days. Standard measures of disease progression, including body weight loss and clinical score, were also made. The presence of mucositis was confirmed at necropsy by findings of decreased duodenal and colon lengths, and reduced liver, spleen and thymus weights in comparison with non-treated control animals. Histological score of the jejunum and ileum was also significantly increased. Mucositis onset coincided with a decrease in mean burrowing behaviour which was progressive, however this result did not achieve statistical significance (P = 0.66).We conclude that burrowing may be a useful indicator of inflammation in the mucositis model, although this requires further characterization. Pre-selection of animals into treatment groups based on their prior burrowing performance should be pursued in further studies.

Platelet-rich fibrin has a healing effect on chemotherapy-induced mucositis in hamsters

The aim of this study was to evaluate the healing effect of topically applied platelet-rich fibrin (PRF) on experimental oral mucositis induced by chemotherapy in hamsters. Oral mucositis was induced in 93 Syrian golden hamsters by an intraperitoneal injection of 5-fluorouracil, which was followed by light scratching of the cheek pouch. The hamsters were randomly divided into a PRF group, a fibrin group, and an untreated control group. The recovery stage of oral mucositis was evaluated through daily weighing, measurements of the ulcer area, histopathologic analysis, and a myeloperoxidase activity assay. The PRF group exhibited significant improvements in the size and histologic features of the ulcer and in the myeloperoxidase activity compared with the control group (P < .05). The current findings suggest the consideration for future clinical trials in humans.

Protective effect of bilberry extract as a pretreatment on induced oral mucositis in hamsters

This study evaluated the effect of standardized bilberry extract containing anthocyanosides on chemotherapy-induced oral mucositis in hamsters. Twenty-four hamsters were randomly chosen and assigned to groups. Groups A and B were pretreated with deionized water, whereas group C was pretreated with bilberry extract daily for 7 days. Groups B and C underwent chemotherapy by intraperitoneal injections of 5-fluorouracil (days 4, 9, and 14); group A received normal saline. Potentiation of oral mucositis was achieved by scratching both cheek pouches of all animals with needles (days 5, 6, and 14). The pouches were histopathologically examined on day 17 after visual examination and blood sampling by cardiac puncture. The bilberry extract group showed significantly lower oral mucositis clinical and histopathologic scores (P < .05) and less percentile of mean daily weight reductions compared with animals receiving vehicle. Frequent administration of bilberry extract had a protective effect on oral mucosal damage induced by 5-fluorouracil in an animal model.

Androgen Treatment Mitigates Hematopoietic Cell Telomere Attrition In Vivo

AbstractAbstract 516We have previously shown that sex hormones up-regulate telomerase gene expression and telomerase activity in cultured human peripheral blood leukocytes and bone marrow (BM) CD34 cells (Calado et al. Blood 2009). In the current study, we tested the effects of androgens in the preservation of telomeres in vivo using mouse models. We first confirmed that mouse BM cells in vitro responded to 2–5mM testosterone, a primary androgen, with a 1.3–3.8 fold increase (P<0.05) in telomerase RNA expression in BM cells from mice heterozygous for the telomerase RNA gene deletion (Terc+/−) and in wild type controls but had no effect on BM cells from Terc−/− knockout mice. We then treated normal B6, Terc−/−, and Terc+/− mice with testosterone (in corn oil emulsion, 350 micrograms/mouse once weekly by subcutaneous injection) and found 1.3 – 2.1 fold up-regulation in telomerase mRNA expression in blood leukocytes from Terc+/+ and Terc+/− but not Terc−/− mice, suggesting that androgens can potentially up-regulate telomerase activity in vivo.To directly test androgen effect on telomere repair, we injected testosterone (350 micrograms/mouse/week) or corn oil into B6, Terc+/− and Terc−/− mice; there was no testosterone effect on telomere length in either B6 or Terc−/− mice at steady state after six months of treatment. In Terc+/− mice, however, testosterone treatment produced gain of telomere length relative to corn oil controls at four (1.94 ± 1.18 vs 0.49 ± 1.32 Kbs, P<0.4247) and six (2.08 ± 0.52 vs 0.18 ± 0.58 Kbs, P<0.0256) months, indicating that androgen treatment can extend telomeres in vivo under specific conditions. As telomere erosion is minimal under regular conditions in wild-type mice, especially during a short period of time, we hypothesized that androgen effects might be more evident under circumstances of hematopoietic stress and the requirement for accelerated cell replication. Thus, we tested the effect of testosterone on telomere maintenance in three different circumstances. First, we used a BM transplantation model in which we transplanted limiting numbers (5 × 105) of BM cells from Terc+/+, Terc+/−, or Terc−/− donors into lethally-irradiated B6-CD45.1 recipients, and then exposed recipients to weekly injections of testosterone or corn oil for four months. There was no effect of testosterone treatment in recipients of Terc+/+ and Terc−/− BM cells. However, testosterone injection led to telomere gain in recipients of Terc+/− BM cells at three (2.90 ± 0.84 vs 0.79 ± 0.89 Kbs, P<0.0947) and four (2.00 ± 0.61 vs 0.15 ± 0.65 Kbs, P<0.0446) months, respectively (Fig 1A). Second, we used a chemical stress model in which Terc+/− mice received three monthly intraperitoneal injections of 5-fluorouracil at 100 micrograms/gram of body weight; animals were divided into testosterone or corn oil treatment groups. At four months, testosterone treatment resulted in gain of telomere length (1.39 ± 0.60, 0.73 ± 0.50, 1.51 ± 0.62 and 0.97 ± 0.54 Kbs) while telomere attrition occurred in corn oil control animals (−0.03 ± 0.68, −0.93 ± 0.58, −0.37 ± 0.60 and 0.11 ± 0.62 Kbs; Fig 1B), showing a significant overall testosterone effect (P<0.0007) on telomere maintenance. Third, we used repeated irradiation in which Terc+/− mice received three monthly sub-lethal (6 Gys) doses of radiation accompanied by testosterone or corn oil injections. Again, corn oil control mice showed marked telomere attrition (−0.99 ± 0.28, −1.55 ± 0.43, −1.16 ± 0.38 and −0.23 ± 0.36 Kbs) whereas mice that received testosterone had gain or reduced attrition of telomere length (0.46 ± 0.32, −0.51 ± 0.50, −0.03 ± 0.43 and 0.83 ± 0.41 Kbs; P<0.0019; Fig 1C). When the same repeated irradiation protocol was applied to a small group of old Terc+/− mice at 21–26 months of age, telomere attrition was apparent in corn oil control animals at two to four months (−1.50 ± 1.17, −2.68 ± 0.62 and −1.75 ± 1.27 Kbs) but testosterone prevented telomere loss (1.27 ± 0.83, 0.20 ± 0.44 and1.21 ± 1.04 Kbs; P<0.0082; Fig 1D). We conclude that androgens are relatively inactive under normal, steady state conditions, but testosterone at pharmacologic doses can protect telomeres under conditions associated with hematopoietic stress. One implication of these results is the potential utility of sex hormone replacement or pharmacologic administration in patients at risk of secondary hematologic malignancies after chemo- and radiation therapies. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Subcutaneous injection containing IL-3 and GM-CSF ameliorates stab wound-induced brain injury in rats

Macrophage-like cells densely accumulate in stab wound-induced brain lesions in rats. Many of these cells express the macrophage marker Iba1 and the oligodendrocyte progenitor cell marker NG2 chondroitin sulfate proteoglycan (NG2), and have been termed BINCs (brain Iba1 +/NG2 + cells). Results from our previous study showed that BINCs elicit neuroprotective action, and agents inducing BINC activation or proliferation are expected to ameliorate traumatic brain injuries (TBIs). In the present study, TBI was established by inserting a needle into the cerebrum and moving the needle in a longitudinal, fan-like movement. Isolated BINCs from these stab lesions expressed mRNAs encoding receptors for interleukin-3 (IL-3) and granulocyte/macrophage colony-stimulating factor (GM-CSF). When this mixture of cytokines was added to the cultured BINCs, expression of mRNAs encoding insulin-like growth factor-1, hepatocyte growth factor, and proliferating cell nuclear antigen increased. The cytokine mixture induced enhanced wound healing in BINCs-brain cell co-cultures in vitro. Stab wounds in the rats resulted in significant brain tissue loss at 2 months post-lesion. However, tissue loss was reduced by 40% when the combination of IL-3 and GM-CSF was subcutaneously injected 7 times (once per day) beginning at 2 or 3 days post-lesion (dpl). BINCs are highly proliferative and an intraperitoneal injection of 5-fluorouracil (5FU) at 2 dpl eliminated the BINCs, resulting in death of the rats. The cytokine mixture injection significantly reduced mortality of the 5FU-treated rats. These results suggest that the combination of IL-3 and GM-CSF serves as a promising agent to ameliorate TBI via action on BINCs.