Effect Of Difluoromethylornithine Research Articles

DFMO inhibition of neuroblastoma tumorigenesis.

Most high-risk neuroblastoma patients who relapse succumb to disease despite the existing therapy. We recently reported increased event-free and overall survival in neuroblastoma patients receiving difluoromethylornithine (DFMO) during maintenance therapy. The effect of DFMO on cellular processes associated with neuroblastoma tumorigenesis needs further elucidation. Previous studies have shown cytotoxicity with IC50 values >5-15 mM, these doses are physiologically unattainable in patients, prompting further mechanistic studies at therapeutic doses. We characterized the effect of DFMO on cell viability, cell cycle, apoptosis, neurosphere formation, and protein expression invitro using five established neuroblastoma cell lines (BE2C, CHLA-90, SHSY5Y, SMS-KCNR, and NGP) at clinically relevant doses of 0, 50, 100, 500, 1000, and 2500 μM. Limiting Dilution studies of tumor formation in murine models were performed. Statistical analysis was done using GraphPad and the level of significance set at p = 0.05. There was not a significant loss of cell viability or gain of apoptotic activity in the invitro assays (p > 0.05). DFMO treatment initiated G1 to S phase cell cycle arrest. There was a dose-dependent decrease in frequency and size of neurospheres and a dose-dependent increase in beta-galactosidase activity in all cell lines. Tumor formation was decreased in xenografts both with DFMO-pretreated cells and in mice treated with DFMO. DFMO treatment is cytostatic at physiologically relevant doses and inhibits tumor initiation and progression in mice. This study suggests that DFMO, inhibits neuroblastoma by targeting cellular processes integral to neuroblastoma tumorigenesis at clinically relevant doses.

Difluoromethylornithine Induces Apoptosis through Regulation of AP-1 Signaling via JNK Phosphorylation in Epithelial Ovarian Cancer.

Difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase (ODC), has promising activity against various cancers and a tolerable safety profile for long-term use as a chemopreventive agent. However, the anti-tumor effects of DFMO in ovarian cancer cells have not been entirely understood. Our study aimed to identify the effects and mechanism of DFMO in epithelial ovarian cancer cells using SKOV-3 cells. Treatment with DFMO resulted in a significantly reduced cell viability in a time- and dose-dependent manner. DFMO treatment inhibited the activity and downregulated the expression of ODC in ovarian cancer cells. The reduction in cell viability was reversed using polyamines, suggesting that polyamine depletion plays an important role in the anti-tumor activity of DFMO. Additionally, significant changes in Bcl-2, Bcl-xL, Bax protein levels, activation of caspase-3, and cleavage of poly (ADP-ribose) polymerase were observed, indicating the apoptotic effects of DFMO. We also found that the effect of DFMO was mediated by AP-1 through the activation of upstream JNK via phosphorylation. Moreover, DFMO enhanced the effect of cisplatin, thus showing a possibility of a synergistic effect in treatment. In conclusion, treatment with DFMO alone, or in combination with cisplatin, could be a promising treatment for ovarian cancer.

Abstract 4137: Inhibition of protein synthesis with polyamine depletion in high-risk neuroblastoma

Abstract The MYC family of oncogenes coordinately deregulate cellular programs that link cell cycle progression with the requisite biomass and energy creation. Neuroblastoma is a MYC-driven, commonly lethal pediatric tumor, with a high proportion of tumors containing MYCN amplification. Polyamine synthesis is a pathway downstream of MYC, with polyamines functioning, in part, to support protein synthesis. ODC1 encodes ornithine decarboxylase, the rate-limiting enzyme in polyamine synthesis, and we found that ODC1 is co-amplified with MYCN in a subset of high-risk neuroblastomas (~8%). Difluoromethylornithine (DFMO) is an irreversible inhibitor or ornithine decarboxylase and is an approved agent for the treatment of Trypanosomiasis. We have previously shown that DFMO inhibits tumor progression and synergizes with chemotherapy in murine mouse models of neuroblastoma, and we postulate that inhibition of protein synthesis is a predominant mechanism of anti-tumor activity. The effects of polyamine depletion on specific components of protein translational machinery have yet to be defined. Here, we show inhibition of global protein synthesis of up to 90% after treatment with various concentrations of DFMO in vitro, with a larger decrease in protein synthesis generally correlating with the presence of MYCN amplification. We found that colony formation is inhibited at DFMO exposures as low as 150 to 500 μM DFMO, which are achievable concentrations in adults based on previous clinical trials. We also show via isoelectric focusing that treatment of neuroblastoma cells in vitro with DFMO leads to a marked decrease in the proportion of hypusinated, or active, eIF5A (an elongation factor important in translation of polyproline stretches and possibly broader influence on translation initiation). To interrogate influences of polyamine depletion on the cap-dependent translation eIF4F complex, we evaluated for changes in the phosphorylation of 4E-BP1 in DFMO treatment conditions, though no changes in phosphorylation were seen compared to controls. Studies to further elucidate the protein synthetic components affected by polyamine depletion are ongoing, as are studies to assess for selective effects of DFMO on polyproline-containing proteins via eIF5A. Further elucidation of the mechanisms of DFMO activity will identify opportunities for drug synergy and provide a responder hypothesis to test in upcoming pivotal Phase 2 and 3 clinical trial of DFMO in the treatment of neuroblastoma. Citation Format: Andrea T. Flynn, Kangning Liu, Annette Vu, Michael D. Hogarty. Inhibition of protein synthesis with polyamine depletion in high-risk neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4137.

Abstract 3190: DFMO preferentially drives cancer stem cells in neuroblastoma towards senescence

Abstract Background: High-risk neuroblastoma (HRNB) is an aggressive form of the most common extracranial solid tumor cancer in children, often characterized by amplified levels of MYCN. HRNB patients in remission have a high relapse rate, resulting in poor survival. Recent studies have shown that cancer stem cells (CSCs) play a role in the progression, chemoresistance, and relapse of many cancers. Difluoromethylornithine (DFMO) is being evaluated to prevent relapse in clinical trials. Our current study examines the in vitro and in vivo effects of DFMO on tumor-initiating cells through evaluating CSC viability, CSC markers and senescence. Methods: BE(2)-C and SMS-KCNR established HRNB cell lines were injected subcutaneously at limiting cell dilutions into nude mice. DFMO was administered in the drinking water (2%, by volume) for 50 and 75 days for BE(2)-C and SMS-KCNR, respectively. Tumor formation was monitored, and LDA was performed. In a second xenograft model BE(2)-C cells were injected into nude mice, once tumors were palpable, the mice were given either normal drinking water (vehicle) or DFMO (2%)-containing water. Following 7 days of treatment, the tumors were resected and measured for volumes/weights as well as the harvested for Western blot analysis of CSC markers (OCT4, SOX2, NANOG, KLF4). BE(2)-C cells were cultured for 48 and 72h with or without 5mM DFMO and stained for SA-βGal activity as a marker of senescence. NB cell lines treated for 48 and 72h with a range of DFMO concentrations (0, 10μM, 50μM, 100μM, 1mM, 2.5mM, and 5mM) were harvested and labeled with cell surface antibodies for CSCs (CD133, CD114), senescence (CD148), differentiation (CD24), and NB (CD56) for 30min in the dark, and cell viability was determined by 7AAD staining immediately before flow cytometric analysis. Results: Limiting dilution analysis (LDA) on xenograft mice receiving DFMO showed both prevention of tumor formation (p value<0.001) and a decrease in frequency of tumor-initiating cells (p value p<0.04). Tumors harvested from mice treated with DFMO for 7 days showed decrease in volume and weight relative to control (p value <0.001) as well as a decrease in the presence of CSC markers shown by Western blot. Galactosidase (SA-βGal) staining of cultured HRNB cells showed the induction of senescence in HRNB cells when treated with DFMO by 72h (p value <0.001). Flow cytometric analysis determined that DFMO specifically targeted the CSC subpopulation at as low as 50uM concentrations (100uM p value<0.05); CSCs had decreased viability and higher levels of senescence-associated protein expression than the overall NB population (p value<0.05). Conclusions: Our results show the preferential induction of senescence and overall decrease in the CSC population at very low doses of DFMO. This may expose a novel treatment mechanism to inhibit CSC function and prevent relapse in HRNB patients. Citation Format: Tracey Avequin, Austin Goodyke, Joseph Zagorski, Tyler Maser, Giselle L. Saulnier Sholler. DFMO preferentially drives cancer stem cells in neuroblastoma towards senescence [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3190.

Abstract 1242: Ornithine decarboxylase as a therapeutic target in endometrial cancer

Abstract Endometrial cancer is the 4th most common cancer and the 6th deadliest cancer in US women. The American Cancer Society estimates there will be 60,050 new endometrial cancers in 2016, an increase of more than 10% from the previous year highlighting the need for more effective treatments and prevention. Ornithine Decarboxylase (ODC) a key enzyme in polyamine synthesis is often overexpressed in cancers and contributes to cell proliferation and tumor growth. Therefore, ODC and the polyamine pathway are considered rational targets for cancer treatment or prevention. We noted ubiquitous expression of ODC1 in our previously published endometrial cancer gene array data and confirmed this in the cancer genome atlas (TCGA), finding expression in all four molecular sub-types with highest expression in copy number high cancers which have the worst clinical outcomes. Therefore, we explored the association of ODC1 gene expression with clinical outcomes of overall survival (OS) and recurrence in the TCGA cohort and noted that elevated ODC1 was significantly related to OS (Wald test p=0.001) and recurrence (p=0.01). Importantly, we confirmed these observations using QRT-PCR in a validation cohort of 60 endometrial cancers and found that endometrial cancers with elevated ODC1 had significantly shorter recurrence-free intervals (p=5.59x10-5) and elevated hazard ratio=3.72. Similar to TCGA data we also noted a strong trend to worse OS ( p=0.00014) with elevated hazard ratio 3.81. Numerous studies including clinical trials have examined the chemopreventive and anti-tumor effects of difluoromethylornithine (DFMO), a specific inhibitor of ODC. We found that DFMO treatment significantly reduced cell proliferation, cell viability, and colony formation in human cell line models derived from undifferentiated, endometrioid, serous, MMT and clear cell endometrial cancers. In contrast, immortalized uterine endometrial epithelial cells (EM E6/E7 TERT1) were less sensitive to DFMO. To confirm the significant effects of DFMO in vitro we performed an in vivo study with human endometrial cancer (ACI-98) tumor-bearing athymic nude mice. Xenografted mice were either treated with 2% (w/v) DFMO supplied in drinking water or water only (n=10/group). DFMO significantly reduced the tumor burden in mice compared to controls (p=0.0023). ODC-regulated polyamines (putrescine [Put] and spermidine [Spd]) known activators of cell proliferation were strongly decreased in response to DFMO, in both tumor tissue ([Put] (p=0.0006), [Spd] (p<0.0001)) and blood plasma ([Put] (p<0.0001), [Spd] (p=0.0049)) of treated mice. Results of these studies indicate that some endometrial cancers appear particularly sensitive to DFMO. Our findings indicate that the polyamine pathway in endometrial cancers in general and specifically those most clinically relevant endometrial cancers could be targeted for effective treatment, chemoprevention or chemoprevention of recurrence. Citation Format: Hong Im Kim, Chad R. Schultz, Andrea L. Buras, Elizabeth Friedman, Alyssa M. Fedorko, Leigh G. Seamon, Gadisetti Chandramouli, André S. Bachmann, John I. Risinger. Ornithine decarboxylase as a therapeutic target in endometrial cancer [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 1242. doi:10.1158/1538-7445.AM2017-1242

Protein RS1 (RSC1A1) Downregulates the Exocytotic Pathway of Glucose Transporter SGLT1 at Low Intracellular Glucose via Inhibition of Ornithine Decarboxylase.

Na+-d-glucose cotransporter 1 (SGLT1) is rate-limiting for glucose absorption in the small intestine. Shortly after intake of glucose-rich food, SGLT1 abundance in the luminal membrane of the small intestine is increased. This upregulation occurs via glucose-induced acceleration of the release of SGLT1-containing vesicles from the trans-Golgi network (TGN), which is regulated by a domain of protein RS1 (RSC1A1) named RS1-Reg. Dependent on phosphorylation, RS1-Reg blocks release of vesicles containing SGLT1 or concentrative nucleoside transporter 1. The hypothesis has been raised that RS1-Reg binds to different receptor proteins at the TGN, which trigger release of vesicles with different transporters. To identify the presumed receptor proteins, two-hybrid screening was performed. Interaction with ornithine decarboxylase 1 (ODC1), the rate-limiting enzyme of polyamine synthesis, was observed and verified by immunoprecipitation. Binding of RS1-Reg mutants to ODC1 was characterized using surface plasmon resonance. Inhibition of ODC1 activity by RS1-Reg mutants and the ODC1 inhibitor difluoromethylornithine (DFMO) was measured in the absence and presence of glucose. In addition, short-term effects of DFMO, RS1-Reg mutants, the ODC1 product putrescine, and/or glucose on SGLT1 expressed in oocytes of Xenopus laevis were investigated. High-affinity binding of RS1-Reg to ODC1 was demonstrated, and evidence for a glucose binding site in ODC1 was provided. Binding of RS1-Reg to ODC1 inhibits the enzymatic activity at low intracellular glucose, which is blunted at high intracellular glucose. The data suggest that generation of putrescine by ODC1 at the TGN stimulates release of SGLT1-containing vesicles. This indicates a biomedically important role of ODC1 in regulation of glucose homeostasis.

Combination treatment of TRAIL, DFMO and radiation for malignant glioma cells.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has shown potent and cancer-selective killing activity and drawn considerable attention as a promising therapy for cancer. Another promising cancer therapy is difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase, which is oraly administered and well tolerated. Nevertheless, many types of cancer, including gliomas, have exhibited resistance to TRAIL-induced apoptosis and similarly the potency of DFMO should be enhanced to optimize therapeutic efficacy. In this study we sought to determine whether DFMO, in combination with TRAIL and radiation, could result in an enhanced anti-glioma effect in vitro. We investigated the effect of DFMO, TRAIL and radiation in various combinations on a panel of glioblastoma cell lines (A172, T98G, D54, U251MG). Viability and proliferation of the cells were examined with trypan blue exclusion assay, crystal violet and xCELLigence system. Apoptosis (Annexin-PI), cell cycle and activation of caspase-8 were tested with flow cytometry. BAD protein levels were determined by Western blot analysis. DFMO induced BAD overexpression. Combination treatment with DFMO, TRAIL and radiation significantly reduced cell viability in all cell lines tested. Increased induction of cell death and cell cycle arrest was confirmed with flow cytometry in A172 and D54 cell lines, while enhanced activation of annexin and caspase-8 was revealed in U251MG and T98G cells. The treatment of glioblastoma cell lines with combination of DFMO, TRAIL and radiation showed an enhanced effect. This combination treatment may represent a novel strategy for targeting glioblastoma.

Metabolomics of adherent mammalian cells by capillary electrophoresis-mass spectrometry: HT-29 cells as case study

In this work, the optimization of an effective protocol for cell metabolomics is described with special emphasis in the sample preparation and subsequent analysis of intracellular metabolites from adherent mammalian cells by capillary electrophoresis-mass spectrometry. As case study, colon cancer HT-29 cells, a human cell model to investigate colon cancer, are employed. The feasibility of the whole method for cell metabolomics is demonstrated via a fast and sensitive profiling of the intracellular metabolites HT-29 cells by capillary electrophoresis-time-of-flight mass spectrometry (CE-TOF MS). The suitability of this methodology is further corroborated through the examination of the metabolic changes in the polyamines pathway produced in colon cancer HT-29 cells by difluoromethylornithine (DFMO), a known potent ornithine decarboxylase inhibitor. The selection of the optimum extraction conditions allowed a higher sample volume injection that led to an increase in CE-TOF MS sensitivity. Following a non-targeted metabolomics approach, 10 metabolites (namely, putrescine, ornithine, gamma-aminobutyric acid (GABA), oxidized and reduced glutathione, 5′-deoxy-5′-(methylthio)adenosine, N-acetylputrescine, cysteinyl-glycine, spermidine and an unknown compound) were found to be significantly altered by DFMO (p<0.05) in HT-29 cells. In addition to the effect of DFMO on polyamine metabolism, minor modifications of other metabolic pathways (e.g., related to intracellular thiol redox state) were observed.

Effect of difluoromethylornithine and methotrexate on proliferation of human breast carcinoma cells

Introduction: In human colon cancer cells, the combination of difluoromethylornithine (DFMO) and methotrexate (MTX) has been shown to inhibit cellular proliferation. Objectives: To determine if the combination of DFMO and MTX inhibit proliferation of human breast cancer cells. Materials and methods: Sterile cell culture techniques, including harvesting and subculturing, were used in performing these experiments. Human breast carcinoma cells were treated alone and in combination with DFMO and MTX for 72hours. Vehicle control for MTX was dimethylsulfoxide at equivalent concentration. Results: Cell viability was inhibited with DFMO and MTX treatment alone. However, the combination treatment had no inhibitory effect. Conclusions: In human breast carcinoma cells, DFMO and MTX each inhibited cell growth by approximately 35% compared with untreated cells. However, in combination, DFMO and MTX resulted in no inhibition of cell growth. This drug combination is antagonistic and does not seem to be beneficial for therapeutic use. Other drug agents could be tested in combination to indicate inhibition of proliferation. Acknowledgements: Special thanks to our Dean, Dr Khaled Algumizi, our supervisor, Dr Sami Aldham, Dr Fahad Alateeq, Professor Patricia Thompson and her laboratory members: Julie Bickmeier, Ashley Vargas, Christina Preece and Manuel Snyder for all their help and effort.

The metabolism and pharmacokinetics of phospho-sulindac (OXT-328) and the effect of difluoromethylornithine.

Phospho-sulindac (PS; OXT-328) prevents colon cancer in mice, especially when combined with difluoromethylornithine (DFMO). Here, we explored its metabolism and pharmacokinetics. PS metabolism was studied in cultured cells, liver microsomes and cytosol, intestinal microsomes and in mice. Pharmacokinetics and biodistribution of PS were studied in mice. PS undergoes reduction and oxidation yielding PS sulphide and PS sulphone; is hydrolysed releasing sulindac, which generates sulindac sulphide (SSide) and sulindac sulphone (SSone), all of which are glucuronidated. Liver and intestinal microsomes metabolized PS extensively but cultured cells converted only 10% of it to PS sulphide and PS sulphone. In mice, oral PS is rapidly absorbed, metabolized and distributed to the blood and other tissues. PS survives only partially intact in blood; of its three major metabolites (sulindac, SSide and SSone), sulindac has the highest C(max) and SSone the highest t(1/2) ; their AUC(0-24h) are similar. Compared with conventional sulindac, PS generated more SSone but less SSide, which may contribute to the safety of PS. In the gastroduodenal wall of mice, 71% of PS was intact; sulindac, SSide and SSone together accounted for <30% of the total. This finding may explain the lack of gastrointestinal toxicity by PS. DFMO had no effect on PS metabolism but significantly reduced drug level in mouse plasma and other tissues. Our findings establish the metabolism of PS define its pharmacokinetics and biodistribution, describe its interactions with DFMO and largely explain its gastrointestinal safety.

Abstract A61: Pilot study of difluoromethylornithine (DFMO) in patients with Barrett's esophagus and dysplasia

Abstract A61 Background Patients with Barrett's esophagus (BE) and dysplasia are ideal candidates for chemopreventive strategies to reduce cancer risk. Difluoromethylornithine (DMFO) is a promising chemopreventive agent that inhibits polyamine synthesis involved in the regulation of cell growth and differentiation. We conducted a pilot study to examine the effects of DFMO on esophageal histopathology, mucosal polyamine content, and gene expression. Materials and Methods Patients (n=10) with histologically confirmed BE and low grade dysplasia were enrolled in a single-arm study of DFMO (0.5 g/m2/d) given continuously for 6 months. Esophagoscopy with biopsies (4 quadrants every 1 cm) was performed at baseline, 3 and 6 months, and at 12 months (where available). Grading of dysplasia was performed by a GI pathologist blinded to clinical/biomarker data. Audiology was also performed. Mucosal polyamine content was measured by high-performance liquid chromatography, and analyzed using paired t-tests. Gene expression profiling was performed using a cDNA two-color chip, and was compared between baseline and 6 months. Expression data was normalized using the two-channel fastlo routine and differential expression was computed using a Wilcoxon signed-rank test. Results After 6 months of DFMO treatment, histopathological response data demonstrate regression of dysplasia (n= 1), stable disease (n= 8), and progression of dysplasia (n= 1). Within the stable category, 2 patients with extensive low grade dysplasia showed only focal dysplasia based upon 4 or more biopsies at 6 months. Histolopathological improvements seen at 6 months were maintained at 12 months. Overall, DFMO was well tolerated. One patient had subclinical, unilateral ototoxicity. Modulation of polyamines (spermine, spermidine, putrescine, histamine) was shown only for putrescine which decreased with treatment (6 mo., p=0.07) and showed recovery to baseline at 12 months (p= 0.01). Gene expression profiling revealed modulated genes of potential biological importance (KLF5, KAL1, RFC5, TIMM8a, TC10) and of these, Krüppel-like factor 5 (KLF5) was downregulated whereas the others were upregulated. Conclusion &amp;gt;Further study of the chemopreventive effect of DFMO upon BE with dysplasia is suggested. DFMO treatment suppressed mucosal putrescine levels and downregulated KLF5, an important mediator of cell proliferation. Citation Information: Cancer Prev Res 2008;1(7 Suppl):A61.

Effect of difluoromethylornithine on chromosomes in living organisms and in tissue culture cells.

DL-α-difluoromethylornithine (DFMO), a specific inhibitor of polyamine biosynthesis, has been shown to have antitumor and antitrypanosome effects. Significantly, our earlier work indicates that DFMO can also induce chromosome aberrations in Chinese hamster ovary (CHO) cells in serumfree medium, where extensive polyamine depletion can be achieved. We show now that DFMO increases chromosome aberrations in baby hamster kidney (BHK) cells, provided that effective polyamine depletion is ensured by growing the cells in low serum (0.2 %) medium. However. African green monkey kidney (Vero) cells cultured similarly in the low serum medium did not show enhanced chromosomal damage after DFMO treatment. DFMO did not induce chromosome aberrations in human lymphocytes, human fibroblasts or BHK cells grown in ordinary medium containing 10 % serum. Administration of DFMO to Chinese hamsters and to patients with lymphomas was not found to damage the chromosomes in peripheral lymphocytes andlor in bone marrow cells.

The Effect of Difluoromethylornithine on Decreasing Prostate Size and Polyamines in Men: Results of a Year-Long Phase IIb Randomized Placebo-Controlled Chemoprevention Trial

Prostate cancer is a major health issue, and prevention of prostate cancer and/or its progression will yield benefits for men. Difluoromethylornithine (DFMO) is an antiproliferative agent, inhibiting ornithine decarboxylase, the first enzyme in the polyamine pathway, and has been studied as a therapeutic and chemopreventive agent. The prostate has high levels of tissue polyamines and has shown sensitivity to DFMO both in vitro and in vivo. Eighty-one men participated in a 1-year randomized trial of placebo or DFMO. Prostate volume determination and biopsy of the prostate for histology and polyamine content were done at baseline and after 12 months. Other biomarker variables were assessed, including total and free prostate-specific antigen and prostate-specific antigen doubling time. Compared with baseline, men receiving DFMO had a smaller increase in prostate volume (0.14 cm(3)) than those on placebo (2.95 cm(3); P = 0.0301) at 1 year. In addition, DFMO caused a 60.8% reduction of prostate putrescine levels compared with a 139.5% increase in the placebo arm (P = 0.0014). Stratification by ornithine decarboxylase genotype showed that DFMO reduced prostate volume (P = 0.029) and putrescine levels (P = 0.0053) in the AA + GA group but not in the GG group. There were no grade 3 or 4 toxicities. There was no clinical ototoxicity, with one subclinical grade 2 hearing decline on audiogram. In this randomized placebo-controlled trial, DFMO induced a decrease of prostate putrescine levels and rate of prostate growth. The potential of this compound for prostate cancer or hyperplasia should be further studied.

538: The Effect of Difluoromethylornithine on Prostate Size and Polyamines in Men at Familial Risk for Prostate Cancer: Results of a One Year, Randomized, Double Blind, Placebo Controlled Chemoprevention Trial

You have accessJournal of UrologyModerated Poster 16, Sunday, May 20, 2007, 3:30 - 5:30 pm1 Apr 2007538: The Effect of Difluoromethylornithine on Prostate Size and Polyamines in Men at Familial Risk for Prostate Cancer: Results of a One Year, Randomized, Double Blind, Placebo Controlled Chemoprevention Trial Anne R. Simoneau, Eugene W. Gerner, Argyrios Ziogas, Ray Nagle, Thomas E. Ahlering, and Frank L. Meyskens Anne R. SimoneauAnne R. Simoneau More articles by this author , Eugene W. GernerEugene W. Gerner More articles by this author , Argyrios ZiogasArgyrios Ziogas More articles by this author , Ray NagleRay Nagle More articles by this author , Thomas E. AhleringThomas E. Ahlering More articles by this author , and Frank L. MeyskensFrank L. Meyskens More articles by this author View All Author Informationhttps://doi.org/10.1016/S0022-5347(18)30778-XAboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail "538: The Effect of Difluoromethylornithine on Prostate Size and Polyamines in Men at Familial Risk for Prostate Cancer: Results of a One Year, Randomized, Double Blind, Placebo Controlled Chemoprevention Trial." The Journal of Urology, 177(4S), p. 179 © 2016 by American Urological AssociationFiguresReferencesRelatedDetails Volume 177Issue 4SApril 2007Page: 179 Advertisement Copyright & Permissions© 2016 by American Urological AssociationMetricsAuthor Information Anne R. Simoneau More articles by this author Eugene W. Gerner More articles by this author Argyrios Ziogas More articles by this author Ray Nagle More articles by this author Thomas E. Ahlering More articles by this author Frank L. Meyskens More articles by this author Expand All Advertisement PDF DownloadLoading ...

In vivo enhancement of herpes simplex virus thymidine kinase/ganciclovir cancer gene therapy with polyamine biosynthesis inhibition

We have earlier demonstrated that inhibition of polyamine biosynthesis with difluoromethylornithine (DFMO) can be used to enhance the cytotoxicity of herpes simplex virus thymidine kinase/ganciclovir (HSV-TK/GCV) gene therapy in different tumor cell lines. Here, the utility of this treatment combination was tested in vivo in a nude mouse tumor model. First, the effect of DFMO was verified by treating mice bearing subcutaneous 9L rat glioma tumors with 2% DFMO in drinking water. The drug treatment induced almost complete suppression of ornithine decarboxylase activity, and as a result, a strong decrease in intratumoral putrescine and spermidine concentrations, which were normalized 4 days after drug removal. Consequently, the tumors displayed a significant reduction in the proliferation activity that was increased to 20% higher than the normal level at day 4 and returned to normal level 7 days after DFMO removal. Next, 9L tumors with 30% of TK-GFP fusion gene positive cells were induced and the animals were given DFMO and GCV in 2 treatment schemes, with the drug administration periods overlapping either 5 or 2 days. The analysis of tumor size at the end of the treatment revealed that DFMO can enhance HSV-TK/GCV cytotoxicity when the overlap between DFMO and GCV was 5 days, but the result was not significant. However, the 2-day overlap scheme yielded a significantly (p < 0.05, ANOVA) enhanced antitumor effect. In conclusion, the data here confirms that a novel combination of 2 clinically relevant treatment modalities, polyamine deprivation and HSV-TK/GCV suicide gene therapy, can be used synergistically in vivo.

Effect of difluoromethylornithine on reperfusion injury after temporary middle cerebral artery occlusion

Polyamines have been shown to play an important role in the disturbance of the blood-brain barrier (BBB) in a number of pathological states including ischemia. BBB disturbances may be almost completely prevented by treating animals with the ornithine decarboxylase (ODC) inhibitor, alpha-difluoromethylornithine (DFMO). DFMO has been also shown to prevent N-Methyl-D-aspartate (NMDA) toxicity in tissue cultures. It has been suggested that the pathological disturbances in polyamine metabolism observed following cerebral ischemia, particularly the post-ischemic increase in putrescine, may contribute to the ischemic injury that is most evident in the CA1 subfield of the hippocampus. In this study, effects of DFMO in cerebral ischemia and reperfusion were examined. The results showed that inhibition of the polyamine system by DFMO decreased ischemic injury volume and brain tissue water content in a dose-dependent manner, without change in vital signs, including systemic arterial blood pressure, arterial partial oxygen pressure, regional cerebral blood flow and body temperature.

Metabolic Stability of α-Methylated Polyamine Derivatives and Their Use as Substitutes for the Natural Polyamines

Metabolically stable polyamine derivatives may serve as useful surrogates for the natural polyamines in studies aimed to elucidate the functions of individual polyamines. Here we studied the metabolic stability of alpha-methylspermidine, alpha-methylspermine, and bis-alpha-methylspermine, which all have been reported to fulfill many of the putative physiological functions of the natural polyamines. In vivo studies were performed with the transgenic rats overexpressing spermidine/spermine N(1)-acetyltransferase. alpha-Methylspermidine effectively accumulated in the liver and did not appear to undergo any further metabolism. On the other hand, alpha-methylspermine was readily converted to alpha-methylspermidine and spermidine; similarly, bis-alpha-methylspermine was converted to alpha-methylspermidine to some extent, both conversions being inhibited by the polyamine oxidase inhibitor N(1), N(2)-bis(2,3-butadienyl)-1,4-butanediamine. Furthermore, we used recombinant polyamine oxidase, spermidine/spermine N(1)-acetyltransferase, and the recently discovered spermine oxidase in the kinetic studies. In vitro studies confirmed that methylation did not protect spermine analogs from degradation, whereas the spermidine analog was stable. Both alpha-methylspermidine and bis-alpha-methylspermine overcame the proliferative block of early liver regeneration in transgenic rats and reversed the cytostasis induced by an inhibition of ornithine decarboxylase in cultured fetal fibroblasts.

Effect of difluoromethylornithine, a chemotherapeutic agent, on wound healing

Introduction: Difluoromethylornithine (DFMO) is a potential chemotherapeutic agent and currently in Phase II trials for colon and prostate malignancies. DFMO will potentially be used for chemotherapy in postoperative patients in low dosage. Therefore, we studied the effect of low dosage DFMO on wound healing. Methods: Two groups of twenty male Sprague Dawley rats underwent a midline dorsal incision with implantation of polyvinyl alcohol sponges into subcutaneous pockets. In Exp 1, ten rats were injected daily with DMFO (500 mg/kg IP) starting on day of wounding while controls (ten rats) received equivalent volume of normal saline. In Exp 2, DFMO and normal saline treatments were delayed until post-wounding day 3. All rats were sacrificed on day 10 post-wounding. Incisional wound breaking strength (WBS, g) and sponge hydroxyproline (OHP, g/100mg sponge) content (collagen deposition), were assessed. Efficacy of DFMO treatment was assessed by measuring wound fluid polyamines, putrescine (PUT) and spermidine (SPER)(M) Results: Treatment with DFMO starting on day 0 (Exp 1) resulted in a significant decrease in collagen deposition and wound breaking strength. Neither of the wound healing parameters was affected when treatment was begun 3 days post-wounding (Exp 2). Conclusion: Wound healing is decreased when DFMO treatment is started on the day of surgery. This suggests that DFMO treatment may play a critical role in the synthesis of collagen in early wound healing and chemotherapy with DFMO should be delayed for patients requiring surgery. TABLE—ABSTRACT P116GroupWBSOHPPUTSPEREXP 1Control409 ± 821435 ± 10211.6 ± 3.4NDDFMO340 ± 41∗1014 ± 67∗∗2.2 ± 0.4∗∗NDEXP 2Control376 ± 941568 ± 9015.3 ± 5.295.4 ± 18.6DFMO354 ± 411624 ± 1505.7 ± 5.4∗∗63.1 ± 12.8∗∗Mean ± standard deviation.∗P < 0.05 and∗∗P < 0.005 by t-test; Student’s t-test; ND—not determined.

Chemoprevention of mammary carcinogenesis in a transgenic mouse model by alpha-difluoromethylornithine (DFMO) in the diet is associated with decreased cyclin D1 activity.

Mechanisms underlying the chemopreventive effect of difluoromethylornithine (DFMO) on the development of mammary cancer were investigated utilizing the whey acidic protein promoter-T antigen transgenic mouse model of breast cancer progression. Mice were exposed to four different doses of DFMO in the diet (3.5, 4.9, 7.0 and 10 g/kg diet). Tumor latency was increased in a dose-dependent manner. DFMO at the highest dose significantly delayed tumor onset (131 days as compared to 109 days in control unexposed mice, P=0.018). Analyses of preneoplastic mammary tissue collected 1 month after DFMO treatment demonstrated that DFMO (10 g/kg diet) significantly increased the ratio of apoptotic to proliferative indices (P=0.013) and significantly reduced the percentage of cells demonstrating nuclear localized cyclin D1 (P=0.013). Nuclear localizations of p27, p21 and Stat5a were not affected. Inhibitory effects of DFMO on cell growth and survival were lost as the cells progressed to cancer. In conclusion, the chemopreventive effects of DFMO on mammary cancer progression were mediated by changes in both apoptosis and cell proliferation in preneoplastic cells. Alterations in cyclin D1 activity in preneoplastic cells could represent an early biomarker of chemopreventive action and are consistent with a mechanistic role for cyclin D1 in progression of mammary cancer.

Growth-inhibitory effects of the chemopreventive agent indole-3-carbinol are increased in combination with the polyamine putrescine in the SW480 colon tumour cell line.

BackgroundMany tumours undergo disregulation of polyamine homeostasis and upregulation of ornithine decarboxylase (ODC) activity, which can promote carcinogenesis. In animal models of colon carcinogenesis, inhibition of ODC activity by difluoromethylornithine (DFMO) has been shown to reduce the number and size of colon adenomas and carcinomas. Indole-3-carbinol (I3C) has shown promising chemopreventive activity against a range of human tumour cell types, but little is known about the effect of this agent on colon cell lines. Here, we investigated whether inhibition of ODC by I3C could contribute to a chemopreventive effect in colon cell lines.MethodsCell cycle progression and induction of apoptosis were assessed by flow cytometry. Ornithine decarboxylase activity was determined by liberation of CO2 from 14C-labelled substrate, and polyamine levels were measured by HPLC.ResultsI3C inhibited proliferation of the human colon tumour cell lines HT29 and SW480, and of the normal tissue-derived HCEC line, and at higher concentrations induced apoptosis in SW480 cells. The agent also caused a decrease in ODC activity in a dose-dependent manner. While administration of exogenous putrescine reversed the growth-inhibitory effect of DFMO, it did not reverse the growth-inhibition following an I3C treatment, and in the case of the SW480 cell line, the effect was actually enhanced. In this cell line, combination treatment caused a slight increase in the proportion of cells in the G2/M phase of the cell cycle, and increased the proportion of cells undergoing necrosis, but did not predispose cells to apoptosis. Indole-3-carbinol also caused an increase in intracellular spermine levels, which was not modulated by putrescine co-administration.ConclusionWhile indole-3-carbinol decreased ornithine decarboxylase activity in the colon cell lines, it appears unlikely that this constitutes a major mechanism by which the agent exerts its antiproliferative effect, although accumulation of spermine may cause cytotoxicity and contribute to cell death. The precise mechanism by which putrescine enhances the growth inhibitory effect of the agent remains to be elucidated, but does result in cells undergoing necrosis, possibly following accumulation in the G2/M phase of the cell cycle.