Effect Of DFMO Research Articles

The role of Astragalus polysaccharides in promoting IEC-6 cell migration from polyamine-mediated Ca2+ regulation

Astragalus polysaccharide (APS) has a protective effect on injured intestinal mucosa by promoting intestinal cell migration, but the specific mechanism is unclear. The polyamine-mediated calcium signaling pathway is an important mechanism of cell migration, generally, and we tested the hypothesis that APS can protect damaged intestinal mucosa through the polyamine-mediated calcium signaling pathway. High-performance liquid chromatography (HPLC), infrared chromatography, cell scratch test, Western blot, co-immunoprecipitation, polyamine inhibitor (DFMO), si-Cav1, RhoA inhibitor (Rhosin) and Rac1 inhibitor (NSC23766) were used to detect the pharmacodynamic of APS. The results show that APS can promote cell migration. In addition, APS increased the formations of RhoA/TRPC1, Cav1/TRPC1, and Rac1/PLCγ-1 complexes as well as the expressions of TRPC1, PLCγ-1, RhoA, Cav1, and Rac1, and it reversed the inhibitory effect of DFMO on the above factors. APS also reversed the inhibitory effect of si-Cav1 on Cav1 expression, cytoplasmic Ca2+ concentrations ([Ca2+]cyt), and cell migration. Moreover, APS removed the inhibition of NSC23766 and Rhosin on [Ca2+]cyt and cell migration. In vivo study, the water extract of Astragalus membranaceus (WEA) (15 g/kg) reduced the indomethacin-induced injury of intestinal mucosa as well. These observations suggest that APS can treat gastrointestinal mucosal injury through the polyamine calcium signaling pathway.

ETMR-18. TARGETING Lin28 IN ETMR WITH ODC1 INHIBITOR DFMO

Embryonal tumor with multilayered rosettes (ETMR), is an aggressive brain tumor primarily occurring in young patients (<4 years of age) and characterized by C19MC amplification and Lin28 overexpression. These genetic hallmarks have been shown to participate in driving ETMR in a C19MC-Lin28-MYCN circuit. Reducing Lin28 disrupts this circuit and reduces cell viability in ETMR models. Investigation of therapeutic agents targeting this pathway is required to provide new treatment options for this deadly disease. We present data showing the effect of DFMO (α-difluoromethylornithine) in ETMR, an ODC1 inhibitor known to reduce Lin28 in neuroblastoma. DFMO treatment of the ETMR cell line BT-183 resulted in a significant reduction of intracellular Lin28 protein levels (P<0.05) as indicated by flow cytometry. In concert with this reduction in Lin28, there was a as significant reduction in viable cells (P<0.05), and the number of CD133+ cells were reduced 2-fold (P<0.05). High throughput drug testing of BT-183 identified a number of additional therapeutic agents with potential therapeutic efficacy for ETMR and combining these with cytostatic agent DFMO demonstrated the potential use of these drugs in combination. These in vitro data were complemented by testing of DFMO in an in vivo stereotaxic xenograft ETMR model, with inhibition of tumor burden monitored by bioluminescent imaging of the tumors. Together this work shows that Lin28 targeting agents such as DFMO merit further examination and integrating these types of agents into treatment strategies for ETMR may lead to better outcomes.

&lt;p&gt;Simultaneous Inhibition of Ornithine Decarboxylase 1 and Pyruvate Kinase M2 Exerts Synergistic Effects Against Hepatocellular Carcinoma Cells&lt;/p&gt;

PurposePreviously, we showed that lactate promoted the proliferation and mobility of hepatocellular carcinoma (HCC) cells by increasing the expression of ornithine decarboxylase 1 (ODC1). In this study, we determined the relationship between ODC1 and pyruvate kinase M2 (PKM2, a key lactate metabolism enzyme), and determined the combined effects of difluoromethylornithine (DFMO; an ODC1 inhibitor) and compound 3k (a PKM2 inhibitor) on HCC cells.MethodsFirst, the relationship between PKM2 and ODC1 was analyzed using Western blotting, Cell Counting Kit (CCK)-8 assays, transwell assays, bioinformatics, quantitative real-time fluorescent PCR (qRT-PCR), and immunohistochemical staining. Thereafter, the ODC1 inhibitor DFMO and the PKM2 inhibitor compound 3k were employed. Their combined effects on HCC cell proliferation and mobility were evaluated via CCK-8 assay, flow cytometry, a subcutaneous xenograft tumor model in mice, wound healing assays, and transwell assays. Additionally, the effects of DFMO and compound 3k on the epithelial–mesenchymal transition phenotype and the AKT/GSK-3β/β-catenin pathway were explored using Western blotting and immunofluorescence.ResultsPKM2 knockdown significantly decreased the ODC1 expression, and the proliferation and invasion of HCC cells, while ODC1 overexpression reversed the inhibitory effects of PKM2 knockdown. Similarly, inhibition of ODC1 also decreased the expression of PKM2 via reducing the c-myc-induced transcription. PKM2 was co-expressed with ODC1 in HCC samples, while simultaneously upregulated PKM2 and ODC1 led to the poorest survival outcome. DFMO and compound 3k synergistically inhibited HCC cell proliferation, induced apoptosis, and suppressed cell mobility, as well as the EMT phenotype and the AKT/GSK-3β/β-catenin pathway. The AKT activator SC79 reversed the inhibitory effects.ConclusionPKM2/ODC1 are involved in a positive feedback loop. The simultaneous inhibition of ODC1 and PKM2 using DFMO and compound 3k exerts synergistic effects against HCC cells via the AKT/GSK-3β/β-catenin pathway. Thus, DFMO combined with compound 3k may be a novel effective strategy for treating HCC.

P2.06-18 Targeting Polyamines for Treatment of Malignant Pleural Mesothelioma in Xenograft Models

Inhaling asbestos fibers is the commonest cause of malignant pleural mesothelioma (MPM). Although the import and use of asbestos have been restricted, the incidence of MPM is still rising due to a long lag time in malignant transformation. In 2004, the US Food and Drug Administration approved a combination of pemetrexed with cisplatin for treatment of unresectable MPM. However, overall prognosis is still extremely poor. As such, development of novel therapeutic options is urgently needed. Ornithine decarboxylase (ODC) is highly expressed in 211H and H226 MPM xenografts and clinical tumor samples. Upregulation of ODC increases polyamine production and enhances tumor growth. a-difluoromethylornithine (DFMO) is a specific ODC inhibitor which can suppress polyamines production. Recent preclinical studies have demonstrated the therapeutic effect of DFMO in colon cancers using xenograft model. However, therapeutic effect of DFMO in MPM has not yet been studied. This study aims to disclose the therapeutic effect of DFMO in MPM xenograft models. The findings from this study will provide scientific foundation for future design of clinical trials of DFMO for therapy for advanced MPM. Nude mice were subcutaneous inoculated with tumor cells [211H (biphasic) or H226 (epithelioid)]. Mice were treated with DFMO in drinking water when tumor size reached 50-100 mm3. Mice with tumor size >600mm3 were considered reaching humane endpoint. Spermidine levels, protein expression, cytokines concentrations and apoptosis were investigated by Dot plot, Western blot, ELISA and TUNEL assay respectively. DFMO suppressed tumor growth in both xenografts. DFMO increased median survival from 29 days in control arm to 41 days in treatment arm in mice with 211H xenografts (p = 0.0234), while from 30 days to 43.5 days in those with H226 xenografts (p = 0.0050). There was no synergism when combining DFMO (0.5% or 2%) with either cisplatin (1.25 or 2.5 mg/kg) or pemetrexed (75 or 150 mg/kg). The tumor suppressive effect of DFMO was more effective when compared with cisplatin or pemetrexed. Upon DFMO treatment, decrease in spermidine level, increase in nitrotyrosine content, and activation of apoptosis were observed in both xenografts. In addition, increase in nitrosocysteine level, increase in intratumoral IL-6, keratinocyte chemoattractant (KC) and TNF alpha as well as elevation of DNA lesion and downregulation of pAkt were induced by DFMO in H226 xenografts. DFMO may have a potential role in treating MPM. Acknowledgment: This research was supported by Hong Kong Pneumoconiosis Compensation Fund Board, HKSAR and YC Chan Scientist Award.

Therapeutic targeting of polyamines in malignant pleural mesothelioma xenograft models.

e20060 Background: Although the use of asbestos has been restricted, the incidence of malignant pleural mesothelioma (MPM) is still rising. The US FDA approved a combination of pemetrexed with cisplatin for treatment of unresectable MPM. And development of novel adjuvant therapeutic options for resected early-stage disease is also urgently needed. From our preliminary data, ornithine decarboxylase (ODC) is highly expressed in MPM xenografts and clinical tumor samples. Upregulation of ODC increases polyamines production and enhances tumor growth. a-difluoromethylornithine (DFMO) is a specific ODC inhibitor. This study aims to disclose the adjuvant (minimal residual disease setting) and therapeutic (metastatic setting) effects of DFMO in MPM xenografts. Methods: In adjuvant therapy setting, nude mice were fed with DFMO in drinking water 7 days before subcutaneous inoculation of 200,000 tumor cells. In therapeutic setting, 107 corresponding cells were injected subcutaneously into nude mice which were randomized for treatment after established tumor growth. Mice with tumor size &gt; 600mm3 were considered reaching humane endpoint. Spermidine levels, protein expression, cytokines concentration, and apoptosis were investigated by Dot plot, Western blot, ELISA, and TUNEL assay respectively. Results: In adjuvant therapy setting, DFMO suppressed tumor growth and increased median survival in both 211H and H226 xenografts. In H226 xenografts, 43% of treated mice have not yet reached humane endpoint, mimicking long-term survival. Upon DFMO treatment, decrease in spermidine level, increase in nitrotyrosine content, and activation of apoptosis were observed in both xenografts. In addition, increase in nitrosocysteine level, intratumoral IL-6, keratinocyte chemoattractant and TNFα, DNA lesions and inhibition of Akt/mTOR pathway were induced by DFMO in H226 xenografts, which may explain higher potency of DFMO in these xenografts. In therapeutic setting, DFMO also suppressed tumor growth in both xenografts with similar mechanisms. Conclusions: DFMO may have a potential role as adjuvant therapy in MPM especially epithelioid mesothelioma.

P2.06-19 Targeting Polyamines as Potential Adjuvant Therapy in Malignant Pleural Mesothelioma Xenograft Models

Inhaling asbestos fibers is one of the commonest of malignant pleural mesothelioma (MPM). Although the import and use of asbestos have been restricted, the incidence of MPM is still rising due to a long lag time in malignant transformation. In 2004, the US Food and Drug Administration approved a combination of pemetrexed with cisplatin for treatment of unresectable MPM. At the same time, development of novel adjuvant therapeutic options for resected early-stage disease is also urgently needed. Ornithine decarboxylase (ODC) is highly expressed in 211H and H226 MPM xenografts and clinical tumor samples. Upregulation of ODC increases polyamine production and enhances tumor growth. a-difluoromethylornithine (DFMO) is a specific ODC inhibitor. Recent preclinical studies have demonstrated the adjuvant effect of DFMO in colon cancers using xenograft model. However, adjuvant effect of DFMO in MPM has not yet been studied. This study aims to disclose the adjuvant effect of DFMO in MPM xenograft models. The findings from this study will provide scientific foundation for future design of clinical trials of DFMO for adjuvant therapy in early disease for advanced MPM. Nude mice were fed with DFMO in drinking water 7 days before subcutaneous inoculation of 200,000 tumor cells (211H (biphasic) or H226 (epithelioid)). Mice with tumor size >600mm3 were considered reaching humane endpoint. Spermidine levels, protein expression, cytokines concentrations, NFkB translocation and apoptosis were investigated by Dot plot, Western blot, ELISA, immunofluorescence staining and TUNEL assay respectively. DFMO suppressed tumor growth in both xenografts. DFMO increased median survival from 49.5 days in control arm to 65 days in treatment arm in mice with 211H xenografts (p = 0.08), while from 44 days to 120 days in those with H226 xenografts (p = 0.0002). In H226 xenograft model, 43% of treated mice have not yet reached humane endpoint, mimicking long-term survival. Upon DFMO treatment, decrease in spermidine level, increase in nitrotyrosine content, nuclear translocation of NFkB, elevation of serum IL-6 and activation of apoptosis were observed in both xenografts. In addition, increase in nitrocysteine level, decrease in serum keratinocyte chemoattractant (KC), increase in serum TNF alpha, elevation of DNA lesion and inhibition of Akt/mTOR pathway were induced by DFMO in H226 xenografts, which may explain higher potency of DFMO in this xenograft. DFMO may have a potential role as adjuvant therapy in MPM especially epithelioid mesothelioma. Acknowledgment: This research was supported by Hong Kong Pneumoconiosis Compensation Fund Board, HKSAR.

Polysaccharide extracts of Astragalus membranaceus and Atractylodes macrocephala promote intestinal epithelial cell migration by activating the polyamine-mediated K+ channel

Astragalus membranaceus (Radix Astragali, RA) and Atractylodes macrocephala (Rhizoma Atractylodis Macrocephalae, RAM) are often used to treat gastrointestinal diseases. In the present study, we determined the effects of polysaccharides extracts from these two herbs on IEC-6 cell migration and explored the potential underlying mechanisms. A migration model with IEC-6 cells was induced using a single-edged razor blade along the diameter of cell layers in six-well polystyrene plates. The cells were grown in control media or media containing spermidine (5 μmol·L−1, SPD), alpha-difluoromethylornithine (2.5 mmol·L−1, DFMO), 4-Aminopyridine (40 μmol·L−1, 4-AP), the polysaccharide extracts of RA or RAM (50, 100, or 200 mg·L−1), DFMO plus SPD, or DFMO plus polysaccharide extracts of RA or RAM for 12 or 24 h. Next, cytosolic free Ca2+ ([Ca2+]cyt) was measured using laser confocal microscopy, and cellular polyamine content was quantified with HPLC. Kv1.1 mRNA expression was assessed using RT-qPCR and Kv1.1 and RhoA protein expressions were measured with Western blotting analysis. A cell migration assay was carried out using Image-Pro Plus software. In addition, GC-MS was introduced to analyze the monosaccharide composition of both polysaccharide extracts. The resutls showed that treatment with polysaccharide extracts of RA or RAM significantly increased cellular polyamine content, elevated [Ca2+]cyt and accelerated migration of IEC-6 cells, compared with the controls (P < 0.01). Polysaccharide extracts not only reversed the inhibitory effects of DFMO on cellular polyamine content and [Ca2+]cyt, but also restored IEC-6 cell migration to control level (P < 0.01 or < 0.05). Kv1.1 mRNA and protein expressions were increased (P < 0.05) after polysaccharide extract treatment in polyamine-deficient IEC-6 cells and RhoA protein expression was increased. Molar ratios of D-ribose, D-arabinose, L-rhamnose, D-mannose, D-glucose, and D-galactose was 1.0 : 14.1 : 0.3 : 19.9 : 181.3 : 6.3 in RA and 1.0 : 4.3 : 0.1 : 5.7 : 2.8 : 2.2 in RAM. In conclusion, treatment with RA and RAM polysaccharide extracts stimulated migration of intestinal epithelial cells via a polyamine-Kv1.1 channel activated signaling pathway, which facilitated intestinal injury healing.

PH gradient-liquid chromatography tandem mass spectrometric assay for determination of underivatized polyamines in cancer cells

Altered levels of polyamines in biological specimens have been suggested as potential biomarkers for cancer. Difluoromethylornithine (DFMO, an irreversible inhibitor of ornithine decarboxylase) is reported to modulate polyamines to potentially attenuate proliferation of neuroblastoma cells. A clinical trial is being conducted to evaluate DFMO in various cancers. To determine the pharmacodynamics effect of DFMO, an analytical assay is needed to accurately measure the changes in polyamines in cancer cells. In this study, a novel pH gradient LC-ESI-MS/MS method was developed and validated for the quantitation of polyamines (putrescine, spermidine and spermine) in cancer cells. To separate polar and basic polyamines, a multi-mode column composed of ODS and weak ionic ligands was used. The pH gradient was generated from pH 5.3 to pH 2.7 with 2 mM ammonium acetate and 0.4% acetic acid in 10% acetonitrile as mobile phase. The detection of polyamines was performed utilizing multiple reaction monitoring on electrospray ionization mass spectrometry operated in positive ion mode. A pH gradient method increased resolution and decreased peak width of conventional analytical assays, resulting in 10–250-fold higher detection limits. Mobile phases without ion-pairing reagents were LC-MS compatible and eliminated possible signal suppression and MS contamination. The developed method was successfully applied to the analysis of polyamines in neuroblastoma and leukemia cells treated with DFMO. Putrescine levels were significantly (p < 0.001) decreased in CCRF-CEM (3.68 vs 1.23 ng/mg protein), SK-N-BE(2) (1.98 vs 1.31 ng/mg protein) and CHLA-20 (2.06 vs 0.90 ng/mg protein) cells treated with DFMO relative to vehicle control. The assay will provide a useful tool in determining the pharmacodynamic effect of DFMO in cancer clinical trials.

Atractylenolide I stimulates intestinal epithelial repair through polyamine-mediated Ca2+ signaling pathway

BackgroundAn impairment of the integrity of the mucosal epithelial barrier can be observed in the course of various gastrointestinal diseases. The migration and proliferation of the intestinal epithelial (IEC-6) cells are essential repair modalities to the healing of mucosal ulcers and wounds. Atractylenolide I (AT-I), one of the major bioactive components in the rhizome of Atractylodes macrocephala Koidz. (AMR), possesses multiple pharmacological activities. This study was designed to investigate the therapeutic effects and the underlying molecular mechanisms of AT-I on gastrointestinal mucosal injury. MethodsScratch method with a gel-loading microtip was used to detect IEC-6 cell migration. The real-time cell analyzer (RTCA) system was adopted to evaluate IEC-6 cell proliferation. Intracellular polyamines content was determined using high performance liquid chromatography (HPLC). Flow cytometry was used to measure cytosolic free Ca2+ concentration ([Ca2+]c). mRNA and protein expression of TRPC1 and PLC-γ1 were determined by real-time PCR and Western blotting assay respectively. ResultsTreatment of IEC-6 cells with AT-I promoted cell migration and proliferation, increased polyamines content, raised cytosolic free Ca2+ concentration ([Ca2+]c), and enhanced TRPC1 and PLC-γ1 mRNA and protein expression. Depletion of cellular polyamines by DL-a-difluoromethylornithine (DFMO, an inhibitor of polyamine synthesis) suppressed cell migration and proliferation, decreased polyamines content, and reduced [Ca2+]c, which was paralleled by a decrease in TRPC1 and PLC-γ1 mRNA and protein expression in IEC-6 cells. AT-I reversed the effects of DFMO on polyamines content, [Ca2+]c, TRPC1 and PLC-γ1 mRNA and protein expression, and restored IEC-6 cell migration and proliferation to near normal levels. ConclusionOur data demonstrate that AT-I stimulates intestinal epithelial cell migration and proliferation via the polyamine-mediated Ca2+ signaling pathway. Therefore, AT-I may have the potential to be further developed as a promising therapeutic agent to treat diseases associated with gastrointestinal mucosal injury, such as inflammatory bowel disease and peptic ulcer.

Abstract 2625: Simultaneous targeting of ODC and 5-LOX/COX block the tobacco carcinogen-induced lung adenoma progression to adenocarcinoma in A/J mice

Abstract Increased polyamine synthesis and inflammation have long been associated with intraepithelial neoplasia and their progression to malignant tumor growth, including lung cancer. Targeting multiple pathways simultaneously with low-dose combinations may be an effective approach to modulate different pathways and their downstream signaling, which may result in an increased efficacy and reduced side effects than a single-agent high dose strategy. The aim of the present study was to investigate the effects of DFMO (ODC inhibitor) and licofelone, a dual 5-LOX-COX inhibitor, individually and in combination, on 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone NNK-induced lung adenoma and progression to adenocarcinoma in female A/J mice. At 6 weeks of age, mice (25 /group) were fed AIN-76A-modified diet, and one week later, lung tumors were induced with a single intraperitoneal (i.p.) injection of 10 μmol NNK/mouse. Three weeks after the NNK treatment, groups of mice were fed with either control or experimental diets containing DFMO (1500 or 3000 ppm) or Licofelone (200 or 400 ppm) or combination of low doses of DFMO and Licofelone. Mice were killed after 17 or 34 weeks of drug exposure and tumors were evaluated via histopathology and lung tumors were assayed for modification of various biomarkers of proliferation and apoptosis. Results suggest that both DFMO and licofelone showed dose-dependent inhibition of NNK-induced lung adenoma progression to adenocarcinoma. At high dose DFMO and Licofelone showed 46% and 55% of adenocarcinoma inhibition. Importantly, low dose combination of DFMO and licofelone showed more pronounced effects at both 17 or 34 weeks in inhibiting the total adenocarcinomas (adenoma and adenocarcinoma progression) by &amp;gt;65% and somewhat in a synergistic manner as compared to individual low doses of DFMO and licofelone. Combination-treated lung tumors exhibited modulation of ODC pathway key components (Arg1, Oat, Oaz, SRM, SMS, and SAT) along with decreased proliferation (PCNA, cyclin D1 and Cyclin A) and increased expression of p53, p21 and p27 compared to tumors from mice fed with control diet. These data suggest that targeting ODC plus 5-LOX/COX decreases the progression of adenoma to adenocarcinoma. Furthermore, adenoma progression delay by combination of DFMO and Licofelone is associated with decreased tumor invasive markers such as MMTPs and EMT markers. In conclusion, targeting two or more pathways is an effective chemopreventive approach for high-risk lung cancer individual's particularly former tobacco smokers with lung hyperplasia and adenomas. (Supported by Kerley-Cade Chair Endowment and NCI-N01-CN-53300) Citation Format: Gaurav Kumar, Jagan Mohan R. Patolla, Venkateshwar Madka, Altaf Mohammed, Li Qian, Yuting Zhang, Laura Biddick, Anil Singh, Allison Gillaspy, Stanley Lightfoot, Levy Kopelovich, Vernon E. Steele, Chinthalapally V. Rao. Simultaneous targeting of ODC and 5-LOX/COX block the tobacco carcinogen-induced lung adenoma progression to adenocarcinoma in A/J mice. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2625.

Targeting Ornithine Decarboxylase by α-Difluoromethylornithine Inhibits Tumor Growth by Impairing Myeloid-Derived Suppressor Cells.

α-Difluoromethylornithine (DFMO) is currently used in chemopreventive regimens primarily for its conventional direct anticarcinogenesic activity. However, little is known about the effect of ornithine decarboxylase (ODC) inhibition by DFMO on antitumor immune responses. We showed in this study that pharmacologic blockade of ODC by DFMO inhibited tumor growth in intact immunocompetent mice, but abrogated in the immunodeficient Rag1(-/-) mice, suggesting that antitumor effect of DFMO is dependent on the induction of adaptive antitumor T cell immune responses. Depletion of CD8(+) T cells impeded the tumor-inhibiting advantage of DFMO. Moreover, DFMO treatment enhanced antitumor CD8(+) T cell infiltration and IFN-γ production and augmented the efficacy of adoptive T cell therapy. Importantly, DFMO impaired Gr1(+)CD11b(+) myeloid-derived suppressor cells (MDSCs) suppressive activity through at least two mechanisms, including reducing arginase expression and activity and inhibiting the CD39/CD73-mediated pathway. MDSCs were one primary cellular target of DFMO as indicated by both adoptive transfer and MDSC-depletion analyses. Our findings establish a new role of ODC inhibition by DFMO as a viable and effective immunological adjunct in effective cancer treatment, thereby adding to the growing list of chemoimmunotherapeutic applications of these agents.

95 ROLE OF POLYAMINES IN BOVINE PRE-IMPLANTATION DEVELOPMENT

Polyamines are involved in trophectoderm attachment and outgrowth, but little is known about their role in earlier stages of development. The objective of this study was to evaluate the effects of an inhibitor of polyamine synthesis (difluoromethylornithine, DFMO) on development (blastocyst formation and hatching) and cell allocation to the trophectoderm (TE, CDX2-positive) and inner cell mass (ICM, SOX2-positive) in the bovine embryo. Cumulus-oocyte complexes (COCs) were recovered from slaughterhouse ovaries and matured for 24 h in a defined maturation medium (5.0 mM glucose, 0.6 mM cysteine, 0.5 mM cysteamine, 0.1 IU mL–1 FSH, 50 ng mL–1 EGF, and 2.5 mg mL–1 recombinant human albumin). Frozen-thawed spermatozoa were processed by gradient centrifugation and co-incubated (2 × 106 mL–1) with COC [10 COC/50 µL; 7.5 µg mL–1 heparin, 2 mM caffeine, and 8.0 mg mL–1 fatty-acid free (FAF) BSA] for 20 to 22 h. After removing cumulus cells, zygotes were cultured (10 embryos/20 µL) in a medium for cleavage stage bovine embryos (0.5 mM glucose, 0.3 mM pyruvate, 6.0 mM lactate, 0.25 mM citrate, 1.0 mM alanyl-glutamine, 0.25 × MEM nonessential and essential amino acids, 5 µM EDTA, and 8.0 mg mL–1 FAF BSA). After 72 h, embryos with &gt;4 cells were randomly allocated (5 embryos/20 µL) to a culture medium for compaction and blastocyst formation (3.0 mM fructose, 0.1 mM pyruvate, 6.0 mM lactate, 0.5 mM citrate, 1.0 mM alanyl-glutamine, 1× MEM nonessential amino acids, 0.5× MEM essential amino acids, 0.075 mM myo-inositol, and 8.0 mg mL–1 FAF BSA) containing 0 (control), 5, or 10 mM DFMO. Embryonic development was evaluated at 192 h post-insemination (96 h in the second medium containing DFMO treatments), and hatching or hatched blastocysts were fixed for analysis of cell allocation. All data were analysed by ANOVA and P &lt; 0.05 was considered significant. Blastocyst formation and hatching (% of embryos cultured in the presence of treatments) were both inhibited (P &lt; 0.05) when embryos (n = 157/treatment) were cultured with 5 (39.5 ± 3.9%, 14.6 ± 2.8%) or 10 (39.5 ± 3.9%, 14.0 ± 2.8%) mM DFMO compared with embryos cultured without DFMO (53.5 ± 4.0%, 26.1 ± 3.5%). The number of TE cells was also reduced (P &lt; 0.05) in the presence of 5 (121.4 ± 7.2) and 10 (123.6 ± 6.7) mM DFMO compared with embryos cultured without DFMO (152.4 ± 9.7), but the number of ICM cells (45.2 to 54.0) and the total number of cells (TE+ICM, 168.8 to 201.1) were not affected (P &gt; 0.05). In a second experiment (n = 163 to 165/treatment), the negative effects of DFMO on hatching (17.0 ± 2.9%; P &lt; 0.05, v. control, 30.7 ± 3.6%) could be partially reversed when embryos were cultured with both 10 mM DFMO and an exogenous polyamine (100 µM putrescine, 23.0 ± 3.3% DFMO+Put; P &gt; 0.05 v. control). The number of TE cells for embryos cultured with DFMO+Put (153.9 ± 8.7) was intermediate between embryos cultured with (138.0 ± 6.9) or without DFMO (control, 161.6 ± 8.7), but these differences were not significant (P &gt; 0.05). These results provide the first evidence of a role for polyamines during blastocyst formation and hatching of bovine embryos, with specific effects on trophectoderm proliferation and hatching.

Eflornithine (DFMO) prevents progression of pancreatic cancer by modulating ornithine decarboxylase signaling.

Ornithine decarboxylase (ODC) is the key rate-limiting enzyme in the polyamine synthesis pathway and it is overexpressed in a variety of cancers. We found that polyamine synthesis and modulation of ODC signaling occurs at early stages of pancreatic precursor lesions and increases as the tumor progresses in Kras-activated p48(Cre/+)-LSL-Kras(G12D/+) mice. Interest in use of the ODC inhibitor eflornithine (DFMO) as a cancer chemopreventive agent has increased in recent years since ODC was shown to be transactivated by the c-myc oncogene and to cooperate with the ras oncogene in malignant transformation of epithelial tissues. We tested the effects of DFMO on pancreatic intraepithelial neoplasias (PanIN) and their progression to pancreatic ductal adenocarcinoma (PDAC) in genetically engineered Kras mice. The Kras(G12D/+) mice fed DFMO at 0.1% and 0.2% in the diet showed a significant inhibition (P < 0.0001) of PDAC incidence compared with mice fed control diet. Pancreatic tumor weights were decreased by 31% to 43% (P < 0.03-0.001) with both doses of DFMO. DFMO at 0.1% and 0.2% caused a significant suppression (27% and 31%; P < 0.02-0.004) of PanIN 3 lesions (carcinoma in situ). DFMO-treated pancreas exhibited modulated ODC pathway components along with decreased proliferation and increased expression of p21/p27 as compared with pancreatic tissues derived from mice fed control diet. In summary, our preclinical data indicate that DFMO has potential for chemoprevention of pancreatic cancer and should be evaluated in other PDAC models and in combination with other drugs in anticipation of future clinical trials.

Atractylodes macrocephala Koidz stimulates intestinal epithelial cell migration through a polyamine dependent mechanism

Ethnopharmacological relevanceAtractylodes macrocephala Koidz (AMK), a valuable traditional Chinese herbal medicine, has been widely used in clinical practice for treating patients with disorders of the digestive system. AMK has shown noteworthy promoting effect on improving gastrointestinal function and immunity, which might represent a promising candidate for the treatment of intestinal mucosa injury. The aim of this study was to investigate the efficacy of AMK on intestinal mucosal restitution and the underlying mechanisms via intestinal epithelial (IEC-6) cell migration model. Materials and methodsA cell migration model of IEC-6 cells was induced by a single-edge razor blade along the diameter of the cell layers in six-well polystyrene plates. After wounding, the cells were grown in control cultures and in cultures containing spermidine (5μM, SPD, reference drug), alpha-difluoromethylornithine (2.5mM, DFMO, polyamine inhibitor), AMK (50, 100, and 200mg/L), DFMO plus SPD and DFMO plus AMK for 12h. The polyamines content was detected by high-performance liquid chromatography (HPLC) with pre-column derivatization. The Rho mRNAs expression levels were assessed by Q-RT-PCR. The Rho and non-muscle myosin II proteins expression levels were analyzed by Western blot. The formation and distribution of non-muscle myosin II stress fibers were monitored with immunostaining techniques using specific antibodies and observed by confocal microscopy. Cell migration assay was carried out using inverted microscope and the Image-Pro Plus software. All of these indexes were used to evaluate the effectiveness of AMK. Results(1) Treatment with AMK caused significant increases in cellular polyamines content and Rho mRNAs and proteins expression levels, as compared to control group. Furthermore, AMK exposure increased non-muscle myosin II protein expression levels and formation of non-muscle myosin II stress fibers, and resulted in an acceleration of cell migration in IEC-6 cells. (2) Depletion of cellular polyamines by DFMO resulted in a decrease of cellular polyamines levels, Rho mRNAs and proteins expression, non-muscle myosin II protein formation and distribution, thereby inhibiting IEC-6 cell migration. AMK not only reversed the inhibitory effects of DFMO on the polyamines content, Rho mRNAs and proteins expression, non-muscle myosin II protein formation and distribution, but also restored cell migration to control levels. ConclusionsThe results obtained from this study revealed that AMK significantly stimulates the migration of IEC-6 cells through a polyamine dependent mechanism, which could accelerate the healing of intestinal injury. These findings suggest the potential value of AMK in curing intestinal diseases characterized by injury and ineffective repair of the intestinal mucosa in clinical practice.

Inhibition of Polyamine Formation Antagonizes Vascular Smooth Muscle Cell Proliferation and Preserves the Contractile Phenotype

The polyamines putrescine, spermidine and spermine play essential roles in cell proliferation and migration, two processes involved in the development of vascular disease. Thus, intervention with polyamine formation may represent a way to inhibit unwanted vascular smooth muscle cell (VSMC) proliferation. The aim of the present study was to assess the importance of polyamines for VSMC proliferation and vascular contractility. The rate-limiting step in polyamine biosynthesis is catalysed by ornithine decarboxylase (ODC). Treatment with α-difluoromethylornithine (DFMO), an irreversible inhibitor of ODC, reduced DNA synthesis in primary rat VSMCs in a concentration-dependent manner with an IC50 value of 100μM. Moreover, DFMO reduced VSMC migration assessed in a scratch assay. The DFMO-induced attenuation of VSMC proliferation was associated with lowered cellular amount of polyamines. The antiproliferative effect of DFMO was specific because supplementation with polyamines reversed the effect of DFMO on proliferation and normalized cellular polyamine levels. Isometric force recordings in cultured rat tail artery rings showed that DFMO counteracts the decrease in contractility caused by culture with foetal bovine serum as growth stimulant. We conclude that inhibition of polyamine synthesis by DFMO may limit the first wave of cell proliferation and migration, which occurs in the acute phase after vascular injury. Besides its antiproliferative effect, DFMO may prevent loss of the smooth muscle contractile phenotype in vascular injury.

Atractylodes macrocephala Koidz promotes intestinal epithelial restitution via the polyamine—Voltage-gated K+ channel pathway

Ethnopharmacological relevanceAtractylodes macrocephala Koidz (AMK) has been used widely as a digestive and tonic in traditional Chinese medicine. AMK has shown noteworthy promoting effect on intestinal epithelial cell migration, which might represent a promising candidate for the treatment of intestinal mucosa injury. The aim of this study was to investigate the efficacy of AMK on intestinal mucosal restitution and the underlying mechanisms via IEC-6 cell migration model. Materials and methodsA wounding model of IEC-6 cells was induced by a single-edge razor blade along the diameter of six-well polystyrene plates. The cells were grown in control cultures and in cultures containing spermidine (5μmol/L, SPD, reference drug), alpha-difluoromethylornithine (2.5mmol/L, DFMO, polyamine inhibitor), AMK (50, 100, and 200μg/mL), DFMO plus SPD and DFMO plus AMK for 24h. The membrane potential (MP) and cytosolic free Ca2+ concentration ([Ca2+]cyt) were detected by flow cytometry, and polyamines content was determined via high-performance liquid chromatography (HPLC). The expression of Kv1.1 mRNA and protein levels were assessed by RT-qPCR and Western blot analysis, respectively. Cell migration assay was carried out using the Image-Pro Plus software. All of these indexes were used to evaluate the effectiveness of AMK. Results(1) Treatment with AMK caused significant increases in cellular polyamines content, membrane hyperpolarization, an elevation of [Ca2+]cyt and an acceleration of cell migration in IEC-6 cells, as compared to control group. (2) AMK not only reversed the inhibitory effects of DFMO on the polyamines content, MP, and [Ca2+]cyt but also restored IEC-6 cell migration to control levels. (3) The Kv1.1 mRNA and protein expression were significantly increased by AMK treatment in control and polyamine-deficient IEC-6 cells. ConclusionsThe results of our current studies revealed that treatment with AMK significantly stimulates the migration of intestinal epithelial cells through polyamine-Kv1.1 channel signaling pathway, which could promote the healing of intestinal injury. These results suggest the potential usefulness of AMK to cure intestinal disorders characterized by injury and ineffective repair of the intestinal mucosa.

AMXT-1501, a novel polyamine transport inhibitor, synergizes with DFMO in inhibiting neuroblastoma cell proliferation by targeting both ornithine decarboxylase and polyamine transport

Neuroblastoma (NB) is associated with MYCN oncogene amplification occurring in approximately 30% of NBs and is associated with poor prognosis. MYCN is linked to a number of genes including ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis. ODC expression is elevated in many forms of cancer including NB. Alpha-difluoromethylornithine (DFMO), an ODC inhibitor, is currently being used in a Phase I clinical trial for treatment of NB. However, cancer cells treated with DFMO may overcome their polyamine depletion by the uptake of polyamines from extracellular sources. A novel polyamine transport inhibitor, AMXT-1501, has not yet been tested in NB. We propose that inhibiting ODC with DFMO, coupled with polyamine transport inhibition by AMXT-1501 will result in enhanced NB growth inhibition. Single and combination drug treatments were conducted on three NB cell lines. DFMO IC50 values ranged from 20.76 to 33.3 mM, and AMXT-1501 IC50 values ranged from 14.13 to 17.72 µM in NB. The combination treatment resulted in hypophosphorylation of retinoblastoma protein (Rb), suggesting growth inhibition via G1 cell cycle arrest. Increased expression of cleaved PARP and cleaved caspase 3 in combination-treated cells starting at 48 hr suggested apoptosis. The combination treatment depleted intracellular polyamine pools and decreased intracellular ATP, further verifying growth inhibition. Given the current lack of effective therapies for patients with relapsed/refractory NB and the preclinical effectiveness of DFMO with AMXT-1501, this combination treatment provides promising preclinical results. DFMO and AMXT-1501 may be a potential new therapy for children with NB.

Local inhibition of ornithine decarboxylase reduces vascular stenosis in a murine model of carotid injury

Polyamines are organic polycations playing an essential role in cell proliferation and differentiation, as well as in cell contractility, migration and apoptosis. These processes are known to contribute to restenosis, a pathophysiological process often occurring in patients submitted to revascularization procedures. We aimed to test the effect of α-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase, on vascular cell pathophysiology in vitro and in a rat model of carotid arteriotomy-induced (re)stenosis. The effect of DFMO on primary rat smooth muscle cells (SMCs) and mouse microvascular bEnd.3 endothelial cells (ECs) was evaluated through the analysis of DNA synthesis, polyamine concentration, cell viability, cell cycle phase distribution and by RT-PCR targeting cyclins and genes belonging to the polyamine pathway. The effect of DFMO was then evaluated in arteriotomy-injured rat carotids through the analysis of cell proliferation and apoptosis, RT-PCR and immunohistochemical analysis of differential gene expression. DFMO showed a differential effect on SMCs and on ECs, with a marked, sustained anti-proliferative effect of DFMO at 3 and 8 days of treatment on SMCs and a less pronounced, late effect on bEnd.3 ECs at 8 days of DFMO treatment. DFMO applied perivascularly in pluronic gel at arteriotomy site reduced subsequent cell proliferation and preserved smooth muscle differentiation without affecting the endothelial coverage. Lumen area in DFMO-treated carotids was 49% greater than in control arteries 4 weeks after injury. Our data support the key role of polyamines in restenosis and suggest a novel therapeutic approach for this pathophysiological process.

Abstract 2765: Targeting both ornithine decarboxylase and polyamine transport inhibits tumor growth in neuroblastoma.

Abstract Background: Neuroblastoma (NB) is associated with MYCN oncogene amplification occurring in approximately 30% of NBs and is associated with poor prognosis. MYCN is linked to a number of genes including ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis. ODC expression is elevated in NB. Alpha-difluoromethylornithine (DFMO), an ODC inhibitor, is currently being used in a Phase I clinical trial for treatment of NB. However, cancer cells treated with DFMO may overcome their polyamine depletion by the uptake of polyamines from extracellular sources. A novel polyamine transport inhibitor, AMXT-1501, has not yet been tested on NB. We proposed that inhibiting ODC with DFMO, coupled with polyamine transport inhibition by AMXT-1501, would result in increased NB growth inhibition. Methods: Single and combination drug treatments were conducted on three NB cell lines in vitro and in a xenograft mouse model in vivo. Cell viability was measured using Calcein AM fluorescent assay. Real-time cell proliferation was measured using the xCELLigence system. Western blot analysis was used to measure cleaved and full PARP, cleaved and full caspase 3, phosphorylated Rb and MYCN levels. ATP level per cell was measured using CyQuant fluorescent DNA assay combined with the Cell Titer GLO luminescent cell viability assay. Reverse-phase HPLC was used to measure polyamine levels in cells. Results: DFMO IC50 values ranged from 20.76 to 33.3 mM, and AMXT-1501 IC50 values ranged from 14.13 to 17.72 μM in NB cell lines. Low dose combination treatment (2.5 μM AMXT-1501, 2.5 mM DFMO) synergistically inhibited cell viability and proliferation in vitro. Combination treatment decreased MYCN expression and resulted in hypophosphorylation of pRb, suggesting cell growth inhibition. Increased expression of cleaved PARP and cleaved caspase 3 in combination-treated cells starting at 48 hours suggested apoptosis. The combination treatment resulted in intracellular polyamine pool depletion and decreased intracellular ATP. The combination treatment also significantly reduced tumor size in NB xenograft mice in vivo. Conclusion: Given the current lack of effective therapies for relapsed/refractory NB patients and the preclinical effectiveness of DFMO with AMXT-1501, this combination treatment provides promising preclinical results. DFMO and AMXT-1501 may be a potential new therapy for children with NB. Citation Format: Ann M. Kendzicky, Ping Zhao, Katherine Samal, Lisette P. Yco, Heather McClung, Eugene W. Gerner, André S. Bachmann, Giselle L. Sholler. Targeting both ornithine decarboxylase and polyamine transport inhibits tumor growth in neuroblastoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2765. doi:10.1158/1538-7445.AM2013-2765

Comparative effects of difluoromethylornithine and siberian ginseng root tincture on radiation-induced carcinogenesis in rats and their lifespan

The effects of α-difluoromethylornithine (DFMO) and Siberian ginseng root tincture (SGRT) on radiation-induced carcinogenesis in rats and their lifespan have been studied. It has been shown that both DFMO and SGRT increased the survival rate as well as decreased the rate and multiplicity of malignant and benign tumors in irradiated rats. The beneficial effect of DFMO on the survival rate and its anticarcinogenic action are more pronounced than SGRT.