miRNA-6516-5p regulates the proliferation and migration of renal cancer cells by targeting ODC1 / 国际外科学杂志

Objective:To explore the expression of microRNA (miRNA)-6516-5p in renal cancer cell lines and the molecular mechanisms regulating the proliferation and migration of renal cancer cells.Methods:quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression of miR-6516-5p in renal cancer cell lines and normal proximal renal tubular epithelial cell lines. The liposome method was used to transiently transfect miR-6516-5p mimic and nonsense sequence (NC) into renal cancer cells with the lowest expression of miR-6516-5p, namely miR-6516-5p group and NC group. qRT-PCR was used to detect the expression of miR-6516-5p in transfected cells. CCK-8 and Transwell migration experiment were used to detect the proliferation and migration of transfected cells. Bioinformatics software and dual luciferase gene report experiment were used to predict and verify the regulation of miR-6516-5p on target gene, respectively. qRT-PCR and Western blotting were used to detect the expression of target gene in transfected cells. Measurement data were expressed as mean±standard deviation ( ± s), t-test was used for comparison between two groups, and one-way analysis of variance was used for comparison between multiple groups. Results:The expression of miR-6516-5p in renal cancer cell lines was significantly lower than that of normal proximal tubular epithelial cells ( P<0.01), and the expression of miR-6516-5p in 786-O cells was the lowest ( F=27.69, P<0.01). The expression of miR-6516-5p in 786-O cells in NC group and miR-6516-5p group was 1.01±0.08 and 9.91±1.16, respectively. Compared with the NC group, the expression of miR-6516-5p in 786-O cells in the miR-6516-5p group was significantly increased ( t=7.63, P<0.01). Up-regulation of miR-6516-5p can significantly inhibit the proliferation of 786-O cells ( P<0.05). The migration numbers of NC group and miR-6516-5p group were 85.65±8.77 and 28.05±6.20, respectively. Overexpression of miR-6516-5p could inhibit the migration of 786-O cells ( t=5.36, P< 0.01). The target gene of miR-6516-5p may be ornithine decarboxylase 1 ( ODC1), miR-6516-5p can significantly inhibit the luciferase activity of wild-type ODC1-3′UTR ( t=9.83, P<0.01). Up-regulation of miR-6516-5p can reduce the expression of ODC1 mRNA and protein in 786-O cells ( P<0.01). Conclusion:The expression of miR-6516-5p is reduced in renal cancer cell lines, miR-6516-5p inhibits the proliferation and migration of renal cancer 786-O cells by targeting ODC1, miR-6516-5p may become a potential molecular target of renal cancer.

Optimization of a cucurbit6uril-based real-time label-free method for analyzing the activity of ornithine decarboxylase / 生物工程学报

Ornithine decarboxylase (ODC) is a key enzyme in the biosynthetic pathway of polyamines and catalyzes the decarboxylation of ornithine to produce putrescine. Inhibition of ODC activity is a potential approach for the prevention and treatment of many diseases including cancer, as the expression levels and the activities of ODC in many abnormal cells and tumor cells are generally higher than those of normal cells. The discovery and evaluation of ODC inhibitors rely on the monitoring of the reaction processes catalyzed by ODC. There are several commonly used methods for analyzing the activity of ODC, such as measuring the yield of putrescine by high performance liquid chromatography, or quantifying the yield of isotope labelled carbon dioxide. However, the cumbersome operation and cost of these assays, as well as the difficulty to achieve high-throughput and real-time detection, hampered their applications. In this work, we optimized a real-time label-free method for analyzing the activity of ODC based on the macromolecule cucurbit[6]uril (CB6) and a fluorescent dye, DSMI (trans-4-[4-(dimethylamino) styryl]-1-methylpyridinium iodide). Finally, the optimized method was used to determine the activities of different ODC inhibitors with different inhibition mechanisms.

Research on anti-non-small cell lung cancer activity of ornithine decarboxylase anti-enzyme inhibitor-1 small molecule inhibitor / 中国药理学通报

Aim To investigate the inhibitory effect of small molecule inhibitors of ornithine decarboxylase inhibitor 1 (AZIN-1) on non-small cell lung cancer and its mechanism. Methods Cell proliferation was detected by Cell Counting Kit-8 (CCK-8). Apoptosis was analyzed by flow cytometry (PI/Annexin V-FITC double staining). The expression of ornithine decarboxylase (ODC), ornithine decarboxylase anti-enzyme-1 (AZ-1) and AZIN-1 was detected by Western blot. Cell cycle was analyzed by flow cytometry (PI single staining). The total polyamine content in cellswas measured by high performance liquid chromatography (HPLC). Results Small molecule inhibitor of AZIN-1 could significantly inhibit the proliferation of A549 cells, cause G0/G, cycle arrest, induce apoptosis of A549 cells, inhibit the expression of AZIN-1 and ODC, interfere with intracellular polyamine metabolism, and reduce total polyamine content in cells. Conclusions Small molecule inhibitor of AZIN-1 has significant growth inhibitory effect on A549 cells, and its mechanism may be related to the induction of apoptosis and interference with polyamine metabolism.

Exogenous spermidine ameliorates tubular necrosis during cisplatin nephrotoxicity / 대한해부학회지

The hallmark of cisplatin-induced acute kidney injury is the necrotic cell death in the kidney proximal tubules. However, an effective approach to limit cisplatin nephrotoxicity remains unknown. Spermidine is a polyamine that protects against oxidative stress and necrosis in aged yeasts, and the present study found that exogenous spermidine markedly attenuated tubular necrosis and kidney dysfunction, but not apoptosis, during cisplatin nephrotoxicity. In addition, exogenous spermidine potently inhibited oxidative/nitrative DNA damage, poly(ADP-ribose) polymerase 1 (PARP1) activation and ATP depletion after cisplatin injection. Conversely, inhibition of ornithine decarboxylase (ODC) via siRNA transfection in vivo significantly increased DNA damage, PARP1 activation and ATP depletion, resulting in acceleration of tubular necrosis and kidney dysfunction. Finally, exogenous spermidine removed severe cisplatin injury induced by ODC inhibition. In conclusion, these data suggest that spermidine protects kidneys against cisplatin injury through DNA damage and tubular necrosis, and this finding provides a novel target to prevent acute kidney injury including nephrotoxicity.

siRNA-mediated antizyme inhibitor down-regulates the expression of ornithine decarboxylase of prostate cancer cell PC3 / 基础医学与临床

Objective To evaluate whether down-regulating antizyme inhibitor(AZIN) can regulate the expression of ornithine decarboxylase(ODC) and the proliferation of prostate cancer cell PC3 or not.Methods siRNA-AZIN transfected prostate cancer cell PC3,the level of antizyme(AZ),AZIN and ODC were measured by RT-PCR and westernblot. MTT was used to measure the proliferation of cells. Results The mRNA level of AZIN declined(P<0.01);the protein level of AZIN and ODC declined(P<0.05). Knockdown of AZIN significantly inhibited the proliferation of PC3(P<0.05). Conclusions Transfecting siRNA-AZIN can decrease the level of AZIN, then the decline level of ODC inhibits the proliferation of PC3.

Biochemical Characterization and Antibiotic Susceptibility of Coagulase Negative Staphylococci Isolates from Exudates and Body Fluids

Coagulase negative Staphylococci (CoNS) are increasingly being recognized as significant nosocomial pathogens, partly due to the growing appreciation of this group of organisms as opportunistic pathogens or due to increase in the use of transient or permanent medical devices in seriously ill and immunocompromised patients. Aims and Objectives 1) Isolation of CoNS from exudates and body fluids. 2) Biochemical characterization of CoNS. 3) Antibiotic susceptibility pattern of CoNS. Method 180CoNS isolated from various exudates and body fluids such as pus, wound swabs, endotracheal secretions, sputum, branchialaspitate, and central lining tube were collected. All the CoNS isolates were processed in the Microbiology Laboratory and identified by colony morphology, gram staining, catalase, slide, tube coagulase test, anaerobic acid from mannitol, and deoxyribonuclease. Bacitracin (0.04 U) and furazolidone (100 μg) susceptibilities were done to exclude Micrococcus and Stomatococcus spp. The following biochemical tests were done for the speciation of the CoNS: urease test, phosphatase test, polymyxin B disc test, novobiocin disk test, ornithine decorboxylase test, mannitol to acid, Voges-Proskauertest, mannose fermentation, trehalose fermentation and antibiotic susceptibility testing. Result Out of 180 isolates, 78 are Staphylococcus epidermidis (43.3%), 63 are Staphylococcus hemolyticus (35%), 21 are Staphylococcus hominis (11.6%), and 18 are Staphylococcus lugdunensis (10.0%). Maximum number of CoNS were isolated from pus specimens (58.33%), followed by wound swabs (18.33%). A total of 164out of 180 strains were negative for both bound and free coagulase. A total of 60 strains were bound coagulase slow positive and free coagulase negative. S. epidermidis was the most frequent isolate and 68 S. epidermidis isolates were identified if ornithine decorboxylase was considered positive, while negative 10 S. epidermidis isolates required inclusion of trehalose and mannitol for speciation. Antibiotic susceptibility testing showed maximum resistance to penicillin (78.3) followed by chloramphenicol (41.6%). No resistance to vancomycin was seen. Conclusion: The study revealed S. epidermidis is the predominant CoNS from endotracheal secretions and also pus samples. S. hemolyticus was isolated from pus and central lining tubes, S. hominis and S. lugdunensis were isolated mainly from wound swabs. The present study suggests if coagulase-ve Staphylococci are repeatedly isolated from patients with infection they should be taken seriously and ABST done on these isolates for proper diagnosis and treatment especially in nosocomial infections.

Characterization of OAZ1 and its potential functions in goose follicular development

Background: Ornithine decarboxylase antizyme 1 (OAZ1) is an important regulator of polyamine synthesis and uptake. Our previous studies indicated that high OAZ1 expression in the ovaries of laying geese is responsible for poor egg production. In the present study, the molecular characterization of goose OAZ1 gene was analyzed, as well as the expression profile in various follicular tissues. Results: An 873-bp cDNA sequence of the OAZ1 gene (Accession No. KC845302) with a +1 frameshift site (+175T) was obtained. The sequence consisted of a 652-bp two overlapping open reading frames (a putative protein with 216 amino acids). The OAZ domain, OAZ signature and OAZ super family domain were prominent conserved regions among species. As the follicle size increased, OAZ1 abundance showed an increasing trend during follicular development, while it decreased during follicular regression. The level of OAZ1 mRNA expression was the lowest in the fifth largest preovulatory follicle, and was 0.65-fold compared to the small white follicle (P b 0.05). OAZ1 mRNA expression in the largest preovulatory and postovulatory follicle was 2.11- and 2.49-fold compared to the small white follicle, respectively (P b 0.05). Conclusions: The goose OAZ1 structure confirms that OAZ1 plays an important role in ornithine decarboxylase-mediated regulation of polyamine homeostasis. Our findings provide an evidence for a potential function of OAZ1 in follicular development, ovulation and regression.

Growth inhibition of human breast cancer cells and down-regulation of ODC1 and ADA genes by Nepeta binaloudensis

ABSTRACT Nepeta binaloudensis Jamzad, Lamiaceae, is a rare medicinal plant endemic to Iran. In spite of many studies about the chemical constituents and antibacterial effects of this species, no report has been provided about its cytotoxic and anticancer activities. In this study we have evaluated the effects of EtOH 70%, hexane and aqueous extracts of N. binaloudensis on the cell proliferation and n-hexane extract on the expression of adenosine deaminase and ornithine decarboxylase 1 genes in breast cancer cell lines (MCF-7, MDA-MB-231) compared to non-cancer line (MCF-10A). The cell lines were subjected to increasing doses of the extracts ranging from 10 to 320 µg/ml. Cell viability was quantified by MTS assay. Expression of adenosine deaminase and ornithine decarboxylase 1 genes was analyzed by real time PCR. N. binaloudensis inhibited the growth of malignant cells in a time and dose-dependent manner. Among extracts of N. binaloudensis, the hexane extract was found to be more toxic compared to other extracts. Results showed a marked decrease in the expression of ornithine decarboxylase 1 and adenosine deaminase genes in cancer cell lines. At 60 µg/ml concentration of N. binaloudensis hexane extract ornithine decarboxylase 1 and adenosine deaminase mRNA expression were reduced 4.9 fold and 3.5 fold in MCF-7 cell line and 3.6 fold and 2.6 fold in MDA-MB-231 cell line compared to control, respectively. The result of our study highlights the potential influences of N. binaloudensis hexane extract on ornithine decarboxylase 1 and adenosine deaminase genes expression in breast cancer cells and its relation to inhibition of cancer cell growth.

Studies on OAZI-1 protein complex in inducing specific antitumor effects in mice / 中国免疫学杂志

Objective:To analyze whether the OAZI-1 (ornithine decarboxylase antizyme inhibitor-1) protein complex isolated from tumor cells could induce specific antitumor effects in the experiment mice .Methods:OAZI-1 protein complexes were isolated from B16-F1 melanoma cells by immune magnetic beads coated with OAZI-1 antibody and used as the vaccine to immune the C 57BL/6 mice.After immunization,the mice were inoculated subcutaneously with live B 16-F1 cells and then tumor formation and growth were ob-served.ELISA was used to determine the level of cytokine IFN-γin the serum of immunized mice.Lactate dehydrogenase assay (LDH) was performed to evaluate killing effect of spleen lymphocytes on B 16-F1 cells.The mice immunized by purified OAZI-1 from prokaryotic expression and PBS were used as controls in the animal experiment .Results: Compared with the control mice ,the spleen lymphocytes ( effector cells ) from the mice inoculated with OAZI-1 protein complexes had stronger killing ability on B 16-F1 cells (target cells).At three different effector:target ratio (10:1,50:1,100:1),the killing ability of these spleen lymphocytes were 46.2%, 59.5%and 92.5% respectively,which was significantly higher than the spleen lymphocytes from the mice inoculated with purified AZIN-1 protein (36.1%,26.8% and 45.9%) or inoculated with PBS (24.6%,24.0% and 27.2%).In addition,the content of serum anti-tumor cytokine IFN-γwas also significantly higher in the mice inoculated with OAZI-1 protein complexes (538.3 pg/ml) than the mice inoculated with purified AZIN-1 ( 256.2 pg/ml ) or with PBS ( 131.0 pg/ml ) .When B16-F1 live cells were subcutaneously inoculated into the immunized mice described above ,the tumor formation rate was only 40%in the mice immunized with OAZI-1 protein complex ,but 100%in the mice immunized with PBS or purified OAZI-1.The growth of inoculated tumors in the mice immunized with OAZI-1 protein complex was also much slower than the control mice .Conclusion:The results in this study suggest that the OAZI-1 protein complex isolated from B 16-F1 tumor cells could contain some tumor antigens .When used as tumor vaccine to inoculate mice ,this complex can induce anti-tumor immune killing activity in experimental animals .

OAZI-1 enhances the immunogenicity of Melan-A vaccine in mice / 基础医学与临床

Objective To investigate whether ornithine decarboxylase antizyme inhibitor-1(OAZI-1) can enhance the immunogenicity of Melan-A and induce antitumor immune effect in the experimental animals.Methods The eukaryotic expression plasmid pcDNA3.1(-) /OAZI-1, pcDNA3.1 (-)/Melan-A and pcDNA3.1(-)/Melan-A-OAZI-1 were constructed and used to immunize BALB/c mice.The spleen lymphocytes were prepared from the immunized mice and then used to determine the lymphocyte subsets by flow cytometry assay and tumor-killing activity by LDH release assay.The blood samples were collected from the immunized mice and used to test the serum INF-γ by ELISA.Results The eukaryotic expression plasmid pcDNA3.1(-)/OAZI-1, pcDNA3.1(-)/Melan-A and pcDNA3.1(-)/Melan-A-OAZI-1 were successfully constructed.All three gene vaccines could increaseCD4+ T cell ratio (P<0.05), among of them, the ratio in the pcDNA3.1(-)/Melan-A-OAZI-1 and pcDNA3.1(-)/Melan-A immunized groups increased more significantly than other groups but no obvious differences was observed between these two groups.Similarly, all three gene vaccines could also increased CD8+T cells ratio significantly (P<0.05), but, comparing with all other groups, the highest increase was observed in the pcDNA3.1(-)/Melan-A-OAZI-1 immune group (P<0.05).The pcDNA3.1(-)/Melan-A-OAZI-1 gene vaccines significantly increased cytotoxic activity of the spleen lymphocyte in the immune mice(P<0.05).Among the three gene vaccines only pcDNA3.1(-)/Melan-A-OAZI-1 could significantly increased the INF-γ level in the mice serum (P<0.05).Conclusions OAZI-1 can improve antitumor immunity by promoting tumor antigen presentation.

Spermidine is protective against kidney ischemia and reperfusion injury through inhibiting DNA nitration and PARP1 activation / 대한해부학회지

Kidney ischemia and reperfusion injury (IRI) is associated with a high mortality rate, which is attributed to tubular oxidative and nitrative stresses; however, an effective approach to limit IRI remains elusive. Spermidine, a naturally occurring polyamine, protects yeast cells against aging through the inhibition of oxidative stress and necrosis. In the present study, spermidine supplementation markedly attenuated histological damage and kidney dysfunction during IRI. In addition, exogenous spermidine potently inhibited poly(ADP-ribose) polymerase 1 (PARP1) activation and DNA nitrative/oxidative stress following IRI. Conversely, inhibition of ornithine decarboxylase (ODC) via siRNA transfection in vivo significantly enhanced DNA nitration, PARP1 activation, and functional damage during IRI. Finally, in ODC knockdown kidneys, PARP1 inhibition attenuated histological and functional damage induced by IRI, but not DNA nitrative stress. In conclusion, these data suggest that spermidine protects kidneys against IRI through blocking DNA nitration and PARP1 activation and this finding provides a novel target for prevention of acute kidney injury including IRI.

Speciation of clinically significant coagulase negative staphylococci and their antibiotic resistant patterns in a tertiary care hospital.

Background: Coagulase negative Staphylococci (CONS) are normal human microbiota and sometimes cause infections, often associated with implanted devices, such as joint prosthesis, shunts and intravascular catheters, especially in very young, old and immunocompromised patients. These infections are difficult to treat because of the risk factors and the multiple drug resistant nature of the organisms. The study is undertaken to speculate CONS isolates from various clinical samples and to determine antibiotic susceptibility pattern of CONS by Kirby Bauer disc diffusion method. Methods: A total of 134 clinically significant CONS isolated from pus, urine, blood, fluid, sputum, ear swabs, endotracheal tube, ophthalmic, semen and nail samples. These isolates initially identified by colony morphology, Gram staining, catalase test, slide coagulase test, tube coagulase test and mannitol fermentation. Speciation of CONS was done by novobiocin resistance test, urease activity, ornithine decarboxylase and aerobic acid production from mannose. Results: S. epidermidis is the most frequent isolate 62 (46.3%) followed by S. saprophyticus 38(28.4%), S. haemolyticus 27(20.1%), S. lugdunensis 3(2.2%). S. warneri 3(2.2%), S. cohinii 1(0.7%). Antibiotic susceptibility testing of the isolates showed maximum resistance to penicillin 128 (95.5%) and ampicillin118 (88%) followed by erythromycin 96 (71.6%), cefoxitin 89 (66.4%), gentamicin 33(24.6%), piperacillin & tazobactam 31(23.8%), amoxicillin & clavulanic acid 25 (18.7%), linezolid 23 (17.2%), levofloxacin 9 (6.7%), vancomycin & teicoplanin 2 (1.5%), tigecycline 1 (0.7%). Conclusion: S. epidermidis is the more common isolate identified and CONS are often resistant to multiple antibiotics (Penicillin, ampicillin) & glycopeptides have been considered as the drugs of choice for the management of infections caused by these organisms.

Effect of difluoromethylorithine proliferation, apoptosis and cell cycle in different breast cell lines with different ODC G316A / 肿瘤研究与临床

Objective To investigate the proliferation,apoptosis and cell cycle and possible mechanisms of different breast cell lines by difluoromethylorithine (DMFO).Methods The growth of breast cancer MDA-435 (ODC GG) cell lines and SK-br3 (ODC AA) cell lines treated with DFMO were observed.The apoptosis and cell cycle were detected by flow cytometry.PCR was applied to detect the changes of A and G alleles of ODC G316A in MCF-7 cells treated with DFMO.Results The growth inhibition rates of MDA-435 and SK-br3 cells treated with 10 mmol/L and 20 mmol/L DFMO after 48 h were 24.1％ and 33.6 ％,46.3 ％ and 53.5 ％,respectively,and there was statistical significance (t =2.134,P =0.021,t =2.213,P =0.019).The growth inhibition rates of MDA-435 and SK-br3 treated with 10 mmol/L and 20 mmol/L DFMO after 72 h were 28.9 ％ and 35.7 ％,54.3 ％ and 65.4 ％,respectively,and there was statistical significance (t =2.434,P =0.015,t =2.489,P =0.013).The apoptosis rates of MDA-435 (ODC GG) and SK-br3 (ODC AA) cells both dealt with 20 mmol/L of DFMO after 24 h,48 h and 72 h were (7.58± 2.06) ％ and (13.88±3.45) ％ (t =2.047,P =0.041),(43.28±14.28) ％ and (59.96±16.42) ％ (t =3.680,P =0.000),(77.87±30.25) ％ and (93.08±32.15) ％ (t =3.293,P =0.000 1),respectively.The proportions of S stage cells MDA-435 (ODC GG) and SK-br3 (ODC AA) cells under the same condition after 24 h,48 h and 72 h were (13.25±2.38) ％ and (12.89±2.21) ％ (P ＞ 0.05),(21.43±3.12) ％ and (12.24±3.55) ％ (t =2.638,P =0.012),(16.32±3.23) ％ and (15.24±3.01) ％ (P ＞ 0.05),respectively.After the treatment by DFMO,the expression of ODC G316A allele A in breast cancer cell line MCF-7 (ODC AG) was reduced (t =3.708,P =0.000),and the expression of G had no significant changes.Conclusion The proliferation inhibition and apoptosis in breast cancer cells treated by DFMO is different in breast cancer cells with different genetic type of ODC G316A.DFMO can inhibit the activity of ODC,and the mechanism may be that DFMO could selectively bind to ODC G316A allele A.

Effects of Shengxue Pills on formaldehyde-induced cell damage in mice / 中草药

Objective: To explore the prophylactic and curative effects of Shengxue Pills on formaldehyde-induced cell damage of lung, liver, spleen, bone marrow, and peripheral blood lymphocytes in mice and the mechanisms. Methods: The mice were randomly divided into five groups: control, Shengxue Pill prophylaxis, Shengxue Pill therapy, and two model groups. ELISA was used to detect hydroxyl radicals, superoxide anion radical, and ornithine decarboxylase (ODC) activities in the lung, liver, and spleen cells. Bone marrow cells and peripheral blood lymphocyte micronucleus rate were observed with high power lens. Results: Hydroxyl radicals, superoxide anion radicals, and ODS activities in the cells of the organs in mice were investigated, and the number of bone marrow cells and peripheral blood lymphocyte micronucleus rate in mice in the prophylacitc and therapeutic groups were significantly greater than those in the model groups, respectively as well as in the normal group. Conclusion: Shengxue Pills could prevent and cure the formaldehyde-induced cell damages of the lung, liver, spleen, bone marrow, and peripheral blood lymphocytes of mice.

Prevention and rehabilitation of Shengxue Pills on mice injured by low-dose radiation and their mechanisms / 中草药

Objective: To study the prevention and rehabilitation of Shengxue Pills on liver, spleen, bone marrow cells, peripheral blood lymphocyte of mice injured by low-dose radiation and their mechanisms. Methods: Mice were randomly divided into five groups: control, Shengxue Pills prevention, Shengxue Pills rehabilitation, model 1 (corresponding to rehabilitation), and model 2 (corresponding to prevention) groups. Using the ELISA method to detect the activity of hydroxyl radical, superoxide anion radical, and ornithine decarboxylase (ODC) in liver and spleen cells. The bone marrow cells and peripheral lymphocyte micronucleus rate were observed under high power lens. Results: Compared with control group, the amounts of hydroxyl radical and superoxide anion radical in the two model groups were obviously increased. Compared with the corresponding model groups, the above indexes in prevention group and rehabilitation group were significantly decreased. Compared with the control group, the activity of ODC in mice of the two model groups was obviously enhanced, but bone marrow cells and peripheral lymphocyte micronucleus rate were increased. Compared with the corresponding model groups, the activity of ODC in mice of prevention and rehabilitation groups was weakened, and bone marrow cells and peripheral lymphocyte micronucleus rate were decreased. But each index of spleen cells showed no significant difference. Conclusion: Shengxue Pills could effectively prevent and rehabilitate the damage in liver cells, peripheral blood lymphocytes, and bone marrow cells of mice injured by low-dose radiation.

Effect of ornithine decarboxylase antizyme inhibitor-1 overexpression on the proliferation of B16-F1 mouse melanoma cells / 肿瘤

Objective: To study the effect of ornithine decarboxylase antizyme inhibitor-1 (OAZI-1) overexpression on the proliferation of B16-F1 mouse melanoma cells. Methods: OAZI-1 gene was cloned out from the cDNAs derived from H22 mouse hepatocellular carcinoma cells, and then subcloned into the eukaryotic expression vector pcDNA3.1(+). The recombinant plasmid pcDNA3.1(+)/OAZI-1 was transfected into B16-F1 cells with LipofectAMINE2000 reagents. The positive clone with OAZI-1 overexpression was identified by Western blotting and real-time fluorescent quantitative PCR. The effects of OAZI-1 overexpression on the cell proliferation and the cell cycle distribution were detected by MTT and flow cytometry. The level of polyamine was detected by reversed-phase high performance liquid chromatography. The chemiluminescence analysis was used to determine the activity of spermine oxidase (SMO). Results: The B16-F1 clone with OAZI-1 overexpression was obtained successfully and named as B16/over. The expression level of OAZI-1 mRNA was 3.6-fold higher in the B16/over cells than that in the control B16/3.1 cells transfected with pcDNA3.1(+). The level of ornithine decarboxylase antizyme (OAZ) mRNA was decreased, and the level of ornithine decarboxylase (ODC) mRNA was elevated in B16/ over cells. Overexpression of OAZI-1 in B16-F1 cells could promote the cell proliferation and influence the cell cycle distribution, including decreasing the cell number in G0/G1 phase and increasing the cell number in S and G2/M phases. In B16/over cells, the content of putrescine was increased, but the contents of spermidine and spermine were decreased, and the activity of SMO was elevated. Conclusion: Overexpression of OAZI-1 may promote the proliferation of B16-F1 cells by elevating the content of putrescine, which indicates that OAZI-1 may be a potential target for melanoma therapy. Copyright© 2011 by the Editorial Board of Tumor.

Hydroxydibenzoylmethane induces apoptosis through repressing ornithine decarboxylase in human promyelocytic leukemia HL-60 cells

Ornithine decarboxylase (ODC) is the rate-limiting enzyme in polyamine biosynthesis and a target for chemoprevention. Hydroxydibenzoylmethane (HDB), a derivative of dibenzoylmethane of licorice, is a promising chemopreventive agent. In this paper, we investigated whether HDB would inhibit the ODC pathway to enhance apoptosis in human promyelocytic leukemia HL-60 cells. We found ODC enzyme activity was reduced during HDB treatment. Overexpression of ODC in HL-60 parental cells could reduce HDB-induced apoptosis, which leads to loss of mitochondrial membrane potential (Deltapsim), through lessening intracellular ROS. Furthermore, ODC overexpression protected cytochrome c release and the activation of caspase-3 following HDB treatment. The results demonstrated HDB-induced apoptosis was through a mechanism of down-regulation of ODC and occurred along a ROS-dependent mitochondria-mediated pathway.

Protective role of ornithine decarboxylase (ODC)/polyamines system in the myocardium induced by ischemic preconditioning in rats / 中国病理生理杂志

AIM: To explore the protective role of ornithine decarboxylase (ODC)/polyamines system in the myocardium induced by ischemic preconditioning in rats.METHODS: The experiment model of simulating myocardial ischemia-reperfusion was replicated by Langendorff perfused rat heart. The hearts were randomly divided into six groups: control group, ischemic-reperfusion group (IR), weak ischemic preconditioning group (IPCw), strong ischemic preconditioning groups (IPCs) and inhibitor groups (DF-EG-IPCw and DF-EG-IPCs). The expression of ODC was quantified by Western blotting analysis. The contents of polyamines (putrescine, spermidine, spermine) in cardiac tissue were detected with high performance liquid chromatography. The hemodynamics was obtained using the PowerLab 8/SP TM data acquisition system. The infarct size was measured using triphenyltetrazolium chloride (TTC) staining and the apoptosis cardiomyocytes were observed under optic microscope after TUNEL method treatment. RESULTS: In contrast with control group, in IR group the putrescine contents increased, the expression of ODC was down-regulated, the contents of spermine and the total polyamine pool decreased (P<0.05). At the same time, the cardiac function declined, with an increase in myocardium infarct size and the apoptosis rate of cardiomyocytes (P<0.05). When compared with IR group in terms of LVDP, HR and CF, both IPCw and IPCs groups had significant improvements in cardiac functions (P<0.05). These two groups also had smaller myocardium infarct size (P<0.01) and apoptosis rate of cardiomyocytes (P<0.01). When compared with IR group, the expression of ODC, the contents of spermine and the total polyamine pool increased in both IPCw (P<0.05) and IPCs groups (P<0.01), but the putrescine contents declined. In the respective inhibitor groups of the weak and ischemic preconditioning, the expression of ODC and the levels of putrescine, spermidine, spermine and the total polyamine pool decreased remarkably (DF-EG-IPCw vs IPCw, P<0.05; DF-EG-IPCs vs IPCs, P<0.01), while the myocardium infarct size and apoptosis rate of cardiomyocyte were relatively bigger in both inhibitor groups (P<0.05). Also, the heart function decreased significantly in terms of LVDP, HR and CF compared to their matched ischemic preconditioning group (P<0.05).CONCLUSION: Ischemic preconditioning significantly up-regulates the ODC / polyamines system in Langendorff perfused rat hearts and provides protective effects on myocardium with ischemia/reperfusion injury. Inhibition of bio-synthesis of polyamine abolishes the cardiac function improvement and the decreases the infarct size and apoptosis rate induced by ischemic preconditioning. It reveals that the ornithine decarboxylase (ODC) /polyamines system may be involved in the protection of myocardium induced by IPC in rats.

INHIBITION OF GLUCOCORTICOID ON ORNITHINE DECARBOXYLASE DURING RAT LIVER REGENERATION / 解剖学报

Objective The regulation of ornithine decarboxylase (ODC) gene expression and enzyme activity by corticosterone, the main glucocorticoid in rat, during rat liver regeneration induced by partial hepatectomy (PH) was evaluated.Methods Bilateral adrenaleetomies (ADX) and sham-ADX were performed on ether-anesthetized rats 3 days before PH.Corticosterone in sesame oil was injected subcutaneously to adrenalectomied rats. ODC mRNA, ODC protein and enzyme activity were detected by RT-PCR, Western blotting and high performance liquid chromatography (HPLC), respectively. Results The ODC mRNA levels, protein accumulation and enzyme activity were lower in the intact liver compared to the regenerating liver.After PH, mRNA levels were remarkably enhanced in all groups (n=6 in each group) and peaked at 5 hours post-PH. Till 7 hours, the contents in all groups from high to low were ADX group,control group (Sham-ADX group), ADX treated with 10mg/kg and 40mg/kg body weight corticosterone group, respectively. ODC protein accumulation in ADX rats was higher than that in control rats (n=13, the same below), but it decreasod in corticosterone-treated (10mg/kg) rats until 24 hours post-PH, with a strong decline seen in 40mg/kg corticosterone-treated rats. ODC activity was rapidly promoted, and the highest levels were observed at 6 hours after PH in all groups (n=6 in each group). After corticosterone treatment, the activities declined significantly at 6 hours post-PH, with the lowest value found in the 40mg/kg group. Conclusion Corticosterone treatment results in dose-dependent decreases in ODC mRNA and enzyme protein both in the intact liver and the regenerating liver. The change in ODC activity is partially related to alterations of ODC mRNA and protein accumulation.

Adenovirus-mediated Expression of Both Antisense Ornithine Decarboxylase (ODC) and S-adenosylmethionine Decarboxylase (AdoMetDC)Inhibits Lung Cancer Cell Growth And Invasion In vitro and In vivo / 生物化学与生物物理进展

Polyamine biosynthesis is controlled primarily by ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase(AdoMetDC). Antisense ODC and AdoMetDC sequences were cloned into an adenoviral vector (Ad-ODC-AdoMetDCas). To evaluated the effect of recombinant adenovirus Ad-ODC-AdoMetDCas which can simultaneously express both antisense ornithine decarboxylase (ODC) and sadenosylmethionine decarboxylase (AdoMetDC), the human lung cancer cell line A-549, was infected with Ad-ODC-AdoMetDCas as well as with control vector. Viable cell counting, determination of polyamine concentrations, cell apoptosis,and Matrigel invasion assays were performed in order to assess properties of tumor growth and invasiveness. Furthermore,Ad-ODC-AdoMetDCas's anti-tumor effect was also evaluated in vivo in a nude mice xenograft model. It was demonstrated that adenovirus-mediated ODC and AdoMetDC antisense expression could inhibit tumor cell growth, lead to cell apoptosis and reduce tumor cell invasiveness. Polyamine levels were significantly decreased in Ad-ODC-AdoMetDCas-treated cells compared with controls.This adenovirus also induced tumor regression in established tumors in nude mice. It was suggested that as a new anticancer reagent,the recombinant adenovirus Ad-ODC-AdoMetDCas holds promising hope for the therapy of lung cancers.