DFMO Research Articles

Targeting ornithine decarboxylase reverses the LIN28/Let-7 axis and inhibits glycolytic metabolism in neuroblastoma.

LIN28 has emerged as an oncogenic driver in a number of cancers, including neuroblastoma (NB). Overexpression of LIN28 correlates with poor outcome in NB, therefore drugs that impact the LIN28/Let-7 pathway could be beneficial in treating NB patients. The LIN28/Let-7 pathway affects many cellular processes including the regulation of cancer stem cells and glycolytic metabolism. Polyamines, regulated by ornithine decarboxylase (ODC) modulate eIF-5A which is a direct regulator of the LIN28/Let-7 axis. We propose that therapy inhibiting ODC will restore balance to the LIN28/Let-7 axis, suppress glycolytic metabolism, and decrease MYCN protein expression in NB. Difluoromethylornithine (DFMO) is an inhibitor of ODC in clinical trials for children with NB. In vitro experiments using NB cell lines, BE(2)-C, SMS-KCNR, and CHLA90 show that DFMO treatment reduced LIN28B and MYCN protein levels and increased Let-7 miRNA and decreased neurosphere formation. Glycolytic metabolic activity decreased with DFMO treatment in vivo. Additionally, sensitivity to DFMO treatment correlated with LIN28B overexpression (BE(2)-C>SMS-KCNR>CHLA90). This is the first study to demonstrate that DFMO treatment restores balance to the LIN28/Let-7 axis and inhibits glycolytic metabolism and neurosphere formation in NB and that PET scans may be a meaningful imaging tool to evaluate the therapeutic effects of DFMO treatment.

Delayed administration of alpha-difluoromethylornithine prevents hippocampus-dependent cognitive impairment after single and combined injury in mice.

Radiation exposure due to radiological terrorism and military circumstances are a continuing threat for the civilian population. In an uncontrolled radiation event, it is likely that there will be other types of injury involved, including trauma. While radiation combined injury is recognized as an area of great significance, overall there is a paucity of information regarding the mechanisms underlying the interactions between irradiation and other forms of injury, or what countermeasures might be effective in ameliorating such changes. The objective of this study was to determine if difluoromethylornithine (DFMO) could reduce the adverse effects of single or combined injury if administered beginning 24 h after exposure. Eight-week-old C57BL/J6 young-adult male mice received whole-body cesium-137 ((137)Cs) irradiation with 4 Gy. Immediately after irradiation, unilateral traumatic brain injury was induced using a controlled cortical impact system. Forty-four days postirradiation, animals were tested for hippocampus-dependent cognitive performance in the Morris water maze. After cognitive testing, animals were euthanized and their brains snap frozen for immunohistochemical assessment of neuroinflammation (activated microglia) and neurogenesis in the hippocampal dentate gyrus. Our data show that single and combined injuries induced variable degrees of hippocampus-dependent cognitive dysfunction, and when given 24 h post trauma, DFMO treatment ameliorated those effects. Cellular changes including neurogenesis and numbers of activated microglia were generally not associated with the cognitive changes. Further analyses also revealed that DFMO increased hippocampal protein levels of the antioxidants thioredoxin 1 and peroxiredoxin 3 compared to vehicle treated animals. While the mechanisms responsible for the improvement in cognition after DFMO treatment are not yet clear, these results constitute a basis for further development of DFMO as a countermeasure for ameliorating the of risks for cognitive dysfunction in individuals subjected to trauma and radiation combined injury.

Abstract B54: Difluoromethylornithine treatment affects the LIN28/Let-7 axis resulting in reduced glycolytic metabolism in neuroblastoma

Abstract Background: Overexpression of LIN28 correlates with poor outcome in neuroblastoma (NB). The LIN28/Let-7 axis affects many cellular processes including cell differentiation and glycolytic metabolism. MYCN overexpression correlates with decreased Let-7 miRNA which results in an increase in LIN28 protein. Recent studies have shown that ODC inhibition decreases LIN28 levels. We propose that therapy targeting ODC will affect the LIN28/Let-7 axis, thus suppressing the glycolytic metabolic activity of NB tumor cells. We also propose that cells overexpressing LIN28 will have greater sensitivity to Difluoromethylornithine (DFMO) treatment which inhibits ODC and decreases cellular polyamines. Methods: Two MYCN high-expression cell lines, BE(2)-C and SMS-KCNR, and one MYCN low-expression cell line, CHLA90, were grown in RPMI-1640 medium with 10% fetal bovine serum for 24 hours prior to treatment with DFMO. Cells were treated with 5 mM or 10 mM DFMO from 48-96 hours followed by cell viability assay, ATP per cell, and western blot analysis and 6 hours for qPCR analysis. Cell viability was measured using Calcein AM fluorescence assay. IC50 values were calculated using GraphPad software. ATP per cell was measured by combining Cell Titer GLO luminescence assay with CyQuant fluorescence assay. Western blot analysis was used to measure LIN28B and MYCN protein levels. TaqMan PCR reagents were used to measure Let-7 miRNA levels using qPCR analysis. SMS-KCNR cells were injected subcutaneously into nude mice for in vivo xenograft studies. Mice were drugged with 2% DFMO in drinking water when tumors reached 200mm3. Tumor volumes were measured using both caliper and micro-CT, and tumor glycolytic metabolism was determined by Maximum Standard Uptake Value (SUVMax) in the tumors through longitudinal 18F-FDG micro PET/CT scans on days 19 and 32. Results: treatment with high and low dose DFMO resulted in decreased LIN28B protein levels in all three cell lines at 48, 72, and 96 hours timepoints. MYCN protein levels decreased in MYCN high-expression cell lines, BE(2)-C and SMS-KCNR, with high and low dose DFMO treatments, but did not change in MYCN low-expression cell line CHLA90. Let-7 miRNA levels were increased in both MYCN high-expression cell lines after 6 hours of high dose DFMO treatment and no change was seen in CHLA90 cells. Sensitivity to DFMO correlated with LIN28B expression levels in all three cell lines (BE(2)-C>SMS-KCNR>CHLA90). BE(2)-C cells were most sensitive to DFMO treatment with an IC50 of 3.01 mM followed by SMS-KCNR cells (10.61 mM), and CHLA90 cells, which showed resistance (25.76 mM). In addition, ATP per cell levels were most significantly reduced in BE(2)-C cells after treatment with DFMO followed by SMS-KCNR and CHLA90 cells. In vivo 18F-FDG PET/CT studies showed decreased SUVMax in the DFMO treatment group indicating reduced glycolytic metabolism. Conclusions: Treatment with DFMO reverses the LIN28/Let-7 axis with a decrease in LIN28B protein expression and an increase in Let-7 miRNA expression. This axis has been shown to play a role in metabolic activity of cells. Decreased metabolic activity and decreased tumor growth is seen in NB cells both in vitro and in vivo following DFMO treatment. NB cell lines with higher levels of LIN28B and MYCN expression are more sensitive to DFMO treatment. These studies suggest that targeting of the LIN28/Let-7 pathway may offer a new method of treating neuroblastoma. Citation Format: Ann Kendzicky, Maria Rich, Anderson Peck, Zhao Ping, Elizabeth VanSickle, Heather McClung, Anthony Chang, Giselle Sholler. Difluoromethylornithine treatment affects the LIN28/Let-7 axis resulting in reduced glycolytic metabolism in neuroblastoma. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr B54.

Abstract 681: Heat shock-mediated suppression of ODC protein expression in colorectal cancer cells

Abstract Polyamines are small, positively charged metabolites that have important roles in several cancer hallmarks, including cell proliferation, invasion, and angiogenesis. Difluoromethylornithine (DFMO) is an FDA-approved drug that is in clinical trials for treatment of colorectal cancer. DFMO functions by inhibiting ornithine decarboxylase (ODC), which catalyzes the rate-limiting step in polyamine biosynthesis. By reducing polyamine levels, DFMO has shown much promise as an anti-cancer chemotherapeutic. One potential limitation to DFMO is that it causes a profound increase in ODC expression, which we suspect may counteract the intended use of the drug. However, strategies to suppress ODC expression would theoretically increase DFMO efficacy. We have discovered that transient hyperthermia (heat shock, 42°C) causes a profound and rapid suppression in ODC protein levels in RKO colorectal cancer cells. To investigate the responsible mechanism, we began by examining ODC mRNA expression and stability. Hyperthermia caused only a moderate reduction in ODC transcript levels. Also, mRNA stability, which was assessed using actinomycin D, was not reduced by heat shock. We next examined the effect of hyperthermia on the control of ODC protein turnover, which is known to be mediated by ODC antizyme (OAZ1). We performed real-time PCR to assess the effect of heat shock on OAZ1 expression. Data reveal a significant increase in the level of OAZ1 transcript. Since heat shock is known to activate the transcription factor HSF1, we next investigated the role of HSF1 in OAZ1 induction. Using siRNA to silence HSF1, we found that the induction of OAZ1 by heat shock is fully dependent on HSF1 expression. In addition to heat shock, HSF1 is also activated by various chemotherapeutics, but most notably by Hsp90 inhibitors. We therefore tested if Hsp90 inhibitors, geldanamycin and 17-AAG could be used to induce OAZ1 and promote ODC turnover. These results show that both Hsp90 inhibitors caused a significant activation of HSF1, an induction in OAZ1 expression, and decrease in ODC protein levels. We therefore propose that Hsp90 inhibitors may be effective in attenuating ODC expression and polyamine metabolism. We suggest that they may be used in combination with DFMO to prevent the compensatory increase in ODC levels and increase anti-cancer effects. Citation Format: Christina T.K. Wales, Aaron T. Jacobs. Heat shock-mediated suppression of ODC protein expression in colorectal cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 681. doi:10.1158/1538-7445.AM2014-681

Abstract 2135: Chemoprevention of tobacco carcinogen-induced lung cancer by DFMO and Licofelone administered individually or in combination in female A/J mice

Abstract Lung cancer is the leading cause of cancer death among both men and women in the US. Inflammatory eicosanoids and leukotrienes derived from cyclooxygenase (COX)-2 and 5-lipoxygenase (LOX) pathway positively influences lung tumor growth. Overexpression of ornithine decarboxylase (ODC) enzyme activity and polyamines has been associated with proliferation and progression of lung cancer. Thus, targeting both tumor inflammatory molecules and polyamines synthesis may provide additive/synergistic antitumor effects. To overcome clinical toxicity of high dose difluoromethyl ornithine (DFMO), an inhibitor of ODC; and to target both COX-2/5LOX, we tested low dose DFMO combined with Licofelone, a novel dual inhibitor COX-2/5-LOX, in lung cancer model. At 7 weeks of age, mice (25 mice/group) were treated with 10 μmol 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) /mouse to induce the lung tumors. Three weeks after the NNK treatment, mice were fed either control or experimental diets containing 200, 400 ppm licofelone or 1500 and 3000 ppm DFMO or low-dose combinations of both agents. Mice were killed at 20 weeks (10 mice/group) and/or 36 weeks (15 mice/group) after NNK-treatment for lung tumor evaluation. Administration of 200 and 400 ppm licofelone significantly suppressed lung tumor (adenoma + adenocarcinoma) formation by >32 to 35% (p43 to 47% (p25 to 30% (p44% (p53% (p60% (p<0.0001) respectively at 17 and 34 weeks exposure. These results suggest that low-dose combinations of licofelone and DFMO inhibit the NNK-induced lung adenocarcinoma formation synergistic/additive manner. Immunohistochemistry analysis revealed that lung tumors from mice exposed to licofelone plus DFMO showed significantly reduced proliferating cell nuclear antigen (PCNA)-positive index, increased accumulation of TUNEL positive cells as compared to lung tumors from NNK-treated mice fed with control diet. These results demonstrate the targeting lung tumor COX-LOX with ODC may provide better efficacy when compared to individual targets on tobacco carcinogen-induced lung adenocarcinomas formation. {Supported by NIH, NCI grants NO1-CN-53300 and Kerley-Cade Chair}. Citation Format: Jagan Mohan Reddy Patlolla, Levy Kopelovich, Li Qian, Laura Biddick, Yuting Zhang, Michael K. Sadeghi, Dhimant Desai, Shantu Amin, Stan Lightfoot, Vernon E. Steele, Chinthalapally V. Rao. Chemoprevention of tobacco carcinogen-induced lung cancer by DFMO and Licofelone administered individually or in combination in female A/J mice. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2135. doi:10.1158/1538-7445.AM2014-2135

A method to improve the efficacy of topical eflornithine hydrochloride cream

Context: Facial hirsutism is a cosmetic concern for women and can lead to significant anxiety and lack of self-esteem. Eflornithine cream is indicated for the treatment of facial hirsutism. However, limited success rate and overall patient's satisfaction, even with a long-term and high-frequency application, leave room for improvement.Objective: The objective of this study is to test the effect of microneedle treatment on the in vitro skin permeation and the in vivo efficacy of eflornithine cream in a mouse model.Materials and method: In vitro permeation study of eflornithine was performed using Franz diffusion cell. In vivo efficacy study was performed in a mouse model by monitoring the re-growth of hair in the lower dorsal skin of mice after the eflornithine cream was applied onto an area pretreated with microneedles. The skin and the hair follicles in the treated area were also examined histologically.Results and discussion: The hair growth inhibitory activity of eflornithine was significantly enhanced when the eflornithine cream was applied onto a mouse skin area pretreated with microneedles, most likely because the micropores created by microneedles allowed the permeation of eflornithine into the skin, as confirmed in an in vitro permeation study. Immunohistochemistry data revealed that cell proliferation in the skin and hair follicles was also significantly inhibited when the eflornithine cream was applied onto a skin area pretreated with microneedles.Conclusion: The integration of microneedle treatment into topical eflornithine therapy represents a potentially viable approach to increase eflornithine's ability to inhibit hair growth.

Synthesis, Characterization, Crystal Structure and Antimicrobial Evaluation of a Novel –M–X–M–X– Type Infinite Chain 1D Cu(II) Complex with Eflornithine Hydrochloride Hydrate as Ligand

A novel –M–X–M–X– type infinite chain 1D copper(II) complex of Eflornithine, Dichloro-[2-amino-5-ammonio-2-(difluoromethyl)pentanoate]copper(II) hydrate, [Cu(C6H12F2N2O2)Cl2]·H2O 1 has been synthesized and characterized by elemental analysis, spectroscopic techniques (UV/Vis and FT-IR), TGA and X-ray diffraction. Single-crystal X-ray diffraction analysis of the complex 1 showed the structure to be monoclinic with space group Cc, a = 13.1295(15) A, b = 12.1859(14) A, c = 8.1927(9) A, β = 118.359(3) A, V = 1153.5(2) A3, Z = 4. The complex exhibits a quadratic planar coordination of the Cu-atom. The Cu(II) centre is coordinated by two chloride atoms, an oxygen atom of the carboxyl- and a nitrogen atom of the amino-group, respectively, forming a quadratic planar geometry. The terminal amino group of the ligand is protonated to form NH3+ while the carboxylic moiety is deprotonated to form Zwitterionic eflornithine ligand, with the coordination of the metal at the nitrogen atom of the second amino group. The compound has –M–X–M–X– infinite 1D chain polymeric structure. Two neigbouring Cu(EFL)Cl chain units are bridged by an Cl− ion, forming a –Cu–Cl–Cu–Cl– linear chain structure along C-axis. The antibacterial activities of the complex on Escherichia coli (E. coli), Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa) were investigated and found to be active at higher concentration than the parent ligand.

Antizyme (AZ) regulates intestinal cell growth independent of polyamines.

Since antizyme (AZ) is known to inhibit cell proliferation and to increase apoptosis, the question arises as to whether these effects occur independently of polyamines. Intestinal epithelial cells (IEC-6) were grown in control medium and medium containing 5 mM difluoromethylornithine (DFMO) to inhibit ODC, DFMO + 5 µM spermidine (SPD), DFMO + 5 µM spermine (SPM), or DFMO + 10 µM putrescine (PUT) for 4 days and various parameters of growth were measured along with AZ levels. Cell counts were significantly decreased and mean doubling times were significantly increased by DFMO. Putrescine restored growth in the presence of DFMO. However, both SPD and SPM when added with DFMO caused a much greater inhibition of growth than did DFMO alone, and both of these polyamines caused a dramatic increase in AZ. The addition of SPD or SPM to media containing DFMO + PUT significantly inhibited growth and caused a significant increase in AZ. IEC-6 cells transfected with AZ-siRNA grew more than twice as rapidly as either control cells or those incubated with DFMO, indicating that removal of AZ increases growth in cells in which polyamine synthesis is inhibited as well as in control cells. In a separate experiment, the addition of SPD increased AZ levels and inhibited growth of cells incubated with DFMO by 50%. The addition of 10 mM asparagine (ASN) prevented the increase in AZ and restored growth to control levels. These results show that cell growth in the presence or absence of ODC activity and in the presence or absence of polyamines depends only on the levels of AZ. Therefore, the effects of AZ on cell growth are independent of polyamines.

Polyamine-Blocking Therapy Reverses Immunosuppression in the Tumor Microenvironment

Correcting T-cell immunosuppression may unleash powerful antitumor responses; however, knowledge about the mechanisms and modifiers that may be targeted to improve therapy remains incomplete. Here, we report that polyamine elevation in cancer, a common metabolic aberration in aggressive lesions, contributes significantly to tumor immunosuppression and that a polyamine depletion strategy can exert antitumor effects that may also promote immunity. A polyamine-blocking therapy (PBT) that combines the well-characterized ornithine decarboxylase (ODC) inhibitor difluoromethylornithine (DFMO) with AMXT 1501, a novel inhibitor of the polyamine transport system, blocked tumor growth in immunocompetent mice but not in athymic nude mice lacking T cells. PBT had little effect on the proliferation of epithelial tumor cells, but it increased the number of apoptotic cells. Analysis of CD45(+) tumor immune infiltrates revealed that PBT decreased levels of Gr-1(+)CD11b(+) myeloid suppressor cells and increased CD3(+) T cells. Strikingly, in a model of neoadjuvant therapy, mice administered with PBT one week before surgical resection of engrafted mammary tumors exhibited resistance to subsequent tumor rechallenge. Collectively, our results indicate that therapies targeting polyamine metabolism do not act exclusively as antiproliferative agents, but also act strongly to prevent immune escape by the tumor. PBT may offer a general approach to heighten immune responses in cancer.

Preventive Effects of NSAIDs, NO-NSAIDs, and NSAIDs Plus Difluoromethylornithine in a Chemically Induced Urinary Bladder Cancer Model

Urinary bladder cancer prevention studies were performed with the nonsteroidal anti-inflammatory drugs (NSAID) naproxen (a standard NSAID with a good cardiovascular profile), sulindac, and their nitric oxide (NO) derivatives. In addition, the effects of the ornithine decarboxylase inhibitor, difluoromethylornithine (DFMO), alone or combined with a suboptimal dose of naproxen or sulindac was examined. Agents were evaluated at their human equivalent doses (HED), as well as at lower doses. In the hydroxybutyl(butyl)nitrosamine (OH-BBN) model of urinary bladder cancer, naproxen (400 or 75 ppm) and sulindac (400 ppm) reduced the incidence of large bladder cancers by 82%, 68%, and 44%, respectively, when the agents were initially given 3 months after the final dose of the carcinogen; microscopic cancers already existed. NO-naproxen was highly effective, whereas NO-sulindac was inactive. To further compare naproxen and NO-naproxen, we examined their effects on gene expression in rat livers following a 7-day exposure. Limited, but similar, gene expression changes in the liver were induced by both agents, implying that the primary effects of both are mediated by the parent NSAID. When agents were initiated 2 weeks after the last administration of OH-BBN, DFMO at 1,000 ppm had limited activity, a low dose of naproxen (75 ppm) and sulindac (150 ppm) were highly and marginally effective. Combining DFMO with suboptimal doses of naproxen had minimal effects, whereas the combination of DMFO and sulindac was more active than either agent alone. Thus, naproxen and NO-naproxen were highly effective, whereas sulindac was moderately effective in the OH-BBN model at their HEDs.

Polyamine Transport Inhibitors: Design, Synthesis, and Combination Therapies with Difluoromethylornithine

The development of polyamine transport inhibitors (PTIs), in combination with the polyamine biosynthesis inhibitor difluoromethylornithine (DFMO), provides a method to target cancers with high polyamine requirements. The DFMO+PTI combination therapy results in sustained intracellular polyamine depletion and cell death. A series of substituted benzene derivatives were evaluated for their ability to inhibit the import of spermidine in DFMO-treated Chinese hamster ovary (CHO) and L3.6pl human pancreatic cancer cells. Several design features were discovered which strongly influenced PTI potency, sensitivity to amine oxidases, and cytotoxicity. These included changes in (a) the number of polyamine chains appended to the ring system, (b) the polyamine sequence, (c) the attachment linkage of the polyamine to the aryl core, and (d) the presence of a terminal N-methyl group. Of the series tested, the optimal design was N(1),N(1'),N(1″)-(benzene-1,3,5-triyltris(methylene))tris(N(4)-(4-(methylamino)butyl)butane-1,4-diamine, 6b, which contained three N-methylhomospermidine motifs. This PTI exhibited decreased sensitivity to amine oxidases and low toxicity as well as high potency (EC50 = 1.4 μM) in inhibiting the uptake of spermidine (1 μM) in DFMO-treated L3.6pl human pancreatic cancer cells.

Polyamine Depletion Attenuates Isoproterenol-Induced Hypertrophy and Endoplasmic Reticulum Stress in Cardiomyocytes

Background/Aim: Polyamines (putrescine, spermidine and spermine) play an essential role in cell growth, differentiation and apoptosis. Hypertrophy is accompanied by an increase in polyamine synthesis and endoplasmic reticulum stress (ERS) in cardiomyocytes. The present study was undertaken to elucidate the molecular interactions between polyamines, ERS and cardiac hypertrophy. Methods: Myocardial hypertrophy was simulated by incubating cultured neonatal rat cardiomyocytes in 100 nM isoproterenol (ISO). Polyamine deletion was achieved using 0.5 mM difluoromethylornithine (DFMO). Hypertrophy was estimated using cell surface area measurements, total protein concentrations and atrial natriuretic peptide (ANP) gene expression. Apoptosis was measured using flow cytometry and transmission electron microscopy. Expression of ornithine decarboxylase (ODC) and spermidine/spermine N1-acetyltransferase (SSAT) were analyzed via real-time PCR and Western blotting. Protein expression of ERS and apoptosis factors were analyzed using Western blotting. Results: DFMO (0.5 mM and 2 mM) treatments significantly attenuated hypertrophy and apoptosis induced by ISO in cardiomyocytes. DFMO also decreased lactate dehydrogenase (LDH) and malondialdehyde (MDA) level in the culture medium. In addition, DFMO (0.5 mM) down regulated the expression of ODC, glucose-regulated protein 78 (GRP78), C/EBP homologous protein (CHOP), cleaved caspase-12, and Bax and up regulated the expression of SSAT and Bcl-2. Finally, these changes were partly reversed by the addition of exogenous putrescine (0.5 mM). Conclusion: The data presented here suggest that polyamine depletion could inhibit cardiac hypertrophy and apoptosis, which is closely related to the ERS pathway.

Determination and Validation of Eflornithine Hydrochloride by (Rp-Hplc) Reversed Phase High Performance Liquid Chromatography

A simple highly sensitive, rapid, precise, accurate and specific RP-HPLC method was developed for quantification of eflornithine hydrochloride (2-difluoromethyl-DL-ornithine; DFMO) in its commercial formulation. In this RP-HPLC method, the separation was performed using BDS Hypersil 5µ C18 (150 X 4.6 mm) column at room temperature by using methanol: 2% of Glacial acetic acid in water (80:20 v/v) as mobile phase. The flow rate was 0.8 mL min<SUP>-1</SUP> with UV detection at 290 nm. The retention time of DFMO was 4.3. Linearity was observed over concentration range of 50-100 µg mL<SUP>-1</SUP> for DFMO. The LOD and LOQ were found to be 0.008438 µg mL<SUP>-1</SUP> and 0.028126 µg mL<SUP>-1</SUP> respectively. The accuracy of the proposed method was determined by recovery studies and found to be 100.5 % for DFMO. The proposed method was validated for ICH guidelines like linearity, limit of detection, accuracy, precision, ruggedness, robustness, and system suitability.

Topical combination of diclofenac, calcipotriol, and difluoromethylornithine has beneficial effects comparable to 5-fluorouracil for the treatment of non-melanoma skin cancer in mice

Non-melanoma skin cancer (NMSC) is the most common form of skin cancer. Owing to the significant adverse effects of existing treatments, alternatives are needed. The aim of this study was to evaluate the use of topically administered combination therapy and 5-flurouracil (5-FU) for the treatment of UVB induced NMSC in a mouse model. Ninety-six SKH-1 mice were randomized to one placebo group and two treatment groups (diclofenac+calcitriol+difluoromethylornithine (DFMO) and 5-FU). After UVB radiation for 20 weeks, the mice with tumours were treated topically for the following 17 weeks. Both treatments significantly reduced the number of tumours, number of mice with tumours as well as tumour area size compared with placebo. As the clinical use of 5-FU may induce more adverse effects, a combination of diclofenac+calcitriol+DFMO could be a promising alternative. Human studies are warranted to determine the beneficial effects and possible adverse effects of this new treatment.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Polyamine depletion inhibits the autophagic response modulating Trypanosoma cruzi infectivity

Autophagy is a cell process that in normal conditions serves to recycle cytoplasmic components and aged or damaged organelles. The autophagic pathway has been implicated in many physiological and pathological situations, even during the course of infection by intracellular pathogens. Many compounds are currently used to positively or negatively modulate the autophagic response. Recently it was demonstrated that the polyamine spermidine is a physiological inducer of autophagy in eukaryotic cells. We have previously shown that the etiological agent of Chagas disease, the protozoan parasite Trypanosoma cruzi, interacts with autophagic compartments during host cell invasion and that preactivation of autophagy significantly increases host cell colonization by this parasite. In the present report we have analyzed the effect of polyamine depletion on the autophagic response of the host cell and on T. cruzi infectivity. Our data showed that depleting intracellular polyamines by inhibiting the biosynthetic enzyme ornithine decarboxylase with difluoromethylornithine (DFMO) suppressed the induction of autophagy in response to starvation or rapamycin treatment in two cell lines. This effect was associated with a decrease in the levels of LC3 and ATG5, two proteins required for autophagosome formation. As a consequence of inhibiting host cell autophagy, DFMO impaired T. cruzi colonization, indicating that polyamines and autophagy facilitate parasite infection. Thus, our results point to DFMO as a novel autophagy inhibitor. While other autophagy inhibitors such as wortmannin and 3-methyladenine are nonspecific and potentially toxic, DFMO is an FDA-approved drug that may have value in limiting autophagy and the spread of the infection in Chagas disease and possibly other pathological settings.

Polyamines are oncometabolites that regulate the LIN28/let‐7 pathway in colorectal cancer cells

Polyamine metabolism is a highly coordinated process that is essential for normal development and neoplastic growth in mammals. Although polyamine metabolism is a validated pathway for prevention of carcinogenesis, the mechanisms by which polyamines elicit their tumorigenic effects are poorly understood. In this study, we investigated the role of polyamine metabolism in colon cancer by screening a non-coding RNA (ncRNA) platform to identify polyamine responsive signaling nodes. We report that multiple non-coding RNAs are altered by polyamine depletion including induction of microRNA (miRNA) let-7i, a member of the tumor suppressive let-7 family. The let-7 family targets several RNAs for translational repression, including the growth-associated transcription factor HMGA2 and is negatively regulated by the pluripotency factor LIN28. Depletion of polyamines using difluoromethylornithine (DFMO) or genetic knockdown of the polyamine-modified eukaryotic translation initiation factor 5A isoforms 1 and 2 (eIF5A1/2) resulted in robust reduction of both HMGA2 and LIN28. Locked nucleic acid (LNA) oligonucleotides targeting the seed region of the let-7 family rescued the expression of HMGA2, but not LIN28, in both DFMO-treated and eIF5A1/2 knockdown cultures. Our findings suggest that polyamines are oncometabolites that influence specific aspects of tumorigenesis by regulating pluripotency associated factors, such as LIN28, via an eIF5A-dependent but let-7-independent mechanism while the expression of proliferation-related genes regulated by let-7, such as HMGA2, is mediated through microRNA mediated repression. Therefore, manipulating polyamine metabolism may be a novel method of targeting the LIN28/let-7 pathway in specific disease states.

Elevated polyamines in urothelial cells from OAB subjects mediate oxotremorine-evoked rapid intracellular calcium rise and delayed acetylcholine release

Increased polyamine signaling in bladder urothelial cells (BUC) may play a role in the pathophysiology of overactive bladder (OAB). We quantitated intracellular polyamine levels in cultured BUC from OAB and asymptomatic (NB) subjects. We assessed whether polyamines modulated rapid intracellular calcium ([Ca(2+)]i) changes and delayed acetylcholine (ACh) release evoked by oxotremorine (OXO, a muscarinic agonist). BUC were cultured from cystoscopic biopsies. High-performance liquid chromatography (HPLC) quantitated intracellular putrescine, spermidine, and spermine levels. Five-millimeter difluoromethylornithine (DFMO), and one-millimeter methylglyoxalbisguanylhydrazone (MGBG) treatments were used to deplete intracellular polyamines. Ten micrometers of OXO were used to increase [Ca(2+)]i levels (measured by fura 2 microfluorimetry) and trigger extracellular ACh release (measured by ELISA). Polyamine levels were elevated in OAB compared with NB BUC (0.5 ± 0.15 vs. 0.16 ± 0.03 nmol/mg for putrescine, 2.4 ± 0.21 vs. 1.01 ± 0.13 nmol/mg for spermidine, and 1.90 ± 0.27 vs. 0.86 ± 0.26 nmol/mg for spermine; P < 0.05 for all comparisons). OXO evoked greater [Ca(2+)]i rise in OAB (205.10 ± 18.82% increase over baseline) compared with in NB BUC (119.54 ± 13.01%; P < 0.05). After polyamine depletion, OXO evoked [Ca(2+)]i rise decreased in OAB and NB BUC to 43.40 ± 6.45 and 38.82 ± 3.5%, respectively. OXO tended to increase ACh release by OAB vs. NB BUC (9.02 ± 0.1 vs. 7.04 ± 0.09 μM, respectively; P < 0.05). Polyamine depletion reduced ACh release by both OAB and NB BUC. In conclusion, polyamine levels were elevated twofold in OAB BUC. OXO evoked greater increase in [Ca(2+)]i and ACh release in OAB BUC, although these two events may be unrelated. Depletion of polyamines caused OAB BUC to behave similarly to NB BUC.

Development and Validation of Newer High Performance Thin Layer Chromatographic Method for Quantification of Eflornithine Hydrochloride in Pharmaceutical Formulations

A new, simple, sensitive, precise and robust high performance thin layer chromatography (HPTLC) method was developed for the estimation of eflornithine hydrochloride (DFMO) in pharmaceutical dosage forms. Estimation was performed on TLC aluminum plates precoated with silica gel 60F254 as stationary phase. Linear ascending development was carried out in twin trough glass chamber saturated with mobile phase consisting of methanol: chloroform: acetic acid: 1% triethylamine (4:6:0.1:0.5 v/v/v) at room temperature (25±2°C). Analysis of the plate in absorbance mode at 220nm was carried out. The system was found to give compact spots for DFMO with Rf (Retardation factor) value of 0.55 respectively. The data for calibration plots showed good linear relationship with correlation coefficient of 0.999 in the concentration range of 300–800ng mL-1 for DFMO respectively. The values of limit of detection (LOD) were 0.6238ng mL-1 and limit of quantification (LOQ) were 1.8903ng mL-1 for DFMO respectively. The accuracy of the method was 100.44%. The precision demonstrated a relative standard deviation of less than 2.5%. The results were satisfactory when compared with the literature. This new method was validated according to the International Conference on Harmonization (ICH) guidelines which include linearity, precision, accuracy, specificity, robustness, detection and quantitation limits. The developed methods found to be sufficiently precise and reproducible for established conditions and after validation may be used for routine analysis of eflornithine hydrochloride in pharmaceutical formulations.

A New Spectrophotometric Method for the Determination of Eflornithine Hydrochloride in Parenteral Formulation

This work describes the development, validation and stable studies of a new, simple and reliable UV spectroscopy procedure for the analysis of eflornithine hydrochloride. A new rapid, precise, accurate, specific and simple UV Spectrophotometric analytical method was validated to assay eflornithine hydrochloride (DFMO) in parenteral. Measurements were taken at λmax 283 using ethanol as the solvent. Beer’s law is obeyed in the concentration ranges 4 – 32µg mL<SUP>-1</SUP> with a correlation coefficient (r) equal to 1. The values of limit of detection (LOD) were 0.0675µg mL<SUP>-1</SUP> and limit of quantification (LOQ) were 0.2045µg mL<SUP>-1</SUP> for DFMO respectively. The value of molar absorptivity and sandell’s sensitivity was 0.3918 × 104L mol<SUP>-1</SUP> cm<SUP>-1</SUP> and 0.1633µg cm<SUP>-2</SUP> respectively. The developed method was validated as per International Conference of Harmonization guidelines with respect to specificity, linearity, limit of detection, limit of quantification, accuracy, precision and robustness. The accuracy of the method was 100.23%. The precision demonstrated a relative standard deviation of less than 1%. The results were satisfactory when compared with the literature. The proposed method might be applied in routine quality control in the pharmaceutical industries since it is precise, accurate, simple and economic. Commercial formulation and laboratory prepared mixtures were successfully analyzed using the developed methods.