Emiliano Fratini

Spermine Metabolism and Anticancer Therapy

The natural polyamines (PA), putrescine (PUT), spermidine (SPD) and spermine (SPM) are ubiquitous constituents of eukaryotic cells. The increase of PA in malignant and proliferating cells attracted the interest of scientists during last decades, addressing PA depletion as a new strategy to inhibit cell growth. Selective enzyme inhibitors were developed for decreasing PA metabolism and to act as chemotherapeutic anticancer agents. Indeed, the complexity of the PA homoeostasis overcomes the PA perturbation by a single enzyme to take effect therapeutically. Recently, an increasing interest has been posed on spermine-oxidase (SMO), the only catabolic enzyme able to specifically oxidise SPM. Interestingly, the absence of SPM is compatible with life, but its accumulation and degradation is lethal. Augmented SMO activity provokes an oxidative stress rendering cells prone to die, and appears to be important in the cell differentiation pathway. Extra-cellular SPM is cytotoxic, but its analogues are capable of inhibiting cell growth at low concentrations, most likely by intracellular SPM depletion. These pivotal roles seem to evoke the biological processes of stress response, wherein balance is mandatory to live or to die. Thus, altering SPM metabolism could allow a multi-tasking therapeutic strategy, addressed not only to inhibit PA metabolism. Several tetramines are presently in early phases (I and II) of clinical trials, and it will be a matter of a few more years to understand whether SPM-related therapeutic approaches would be of benefit for composite treatment protocols of cancer.

Spermine oxidase (SMO) activity in breast tumor tissues and biochemical analysis of the anticancer spermine analogues BENSpm and CPENSpm

BackgroundPolyamine metabolism has a critical role in cell death and proliferation representing a potential target for intervention in breast cancer (BC). This study investigates the expression of spermine oxidase (SMO) and its prognostic significance in BC. Biochemical analysis of Spm analogues BENSpm and CPENSpm, utilized in anticancer therapy, was also carried out to test their property in silico and in vitro on the recombinant SMO enzyme.MethodsBC tissue samples were analyzed for SMO transcript level and SMO activity. Students t test was applied to evaluate the significance of the differences in value observed in T and NT samples. The structure modeling analysis of BENSpm and CPENSpm complexes formed with the SMO enzyme and their inhibitory activity, assayed by in vitro experiments, were examined.ResultsBoth the expression level of SMO mRNA and SMO enzyme activity were significantly lower in BC samples compared to NT samples. The modeling of BENSpm and CPENSpm complexes formed with SMO and their inhibition properties showed that both were good inhibitors.ConclusionsThis study shows that underexpression of SMO is a negative marker in BC. The SMO induction is a remarkable chemotherapeutical target. The BENSpm and CPENSpm are efficient SMO inhibitors. The inhibition properties shown by these analogues could explain their poor positive outcomes in Phases I and II of clinical trials.

Increased spermine oxidase (SMO) activity as a novel differentiation marker of myogenic C2C12 cells.

Spermine oxidase (SMO) is a FAD-containing enzyme involved in animal cell polyamines (PA) homeostasis, selectively active on spermine and producing H(2)O(2), spermidine, and the 3-aminopropanal. In the present study, we have examined the SMO gene expression during the mouse myoblast C2C12 cell differentiation induced with two different stimuli by RT-PCR analysis, polysome-mRNP distribution and enzyme activity. SMO transcript accumulation and enzymatic activity increases during C2C12 cell differentiation and correlates with the decrease of spermine content. Many proteins are highly regulated during the phenotypic conversion of rapidly dividing C2C12 myoblasts into fully differentiated post-mitotic myotubes. The SMO gene induction represents a novel and additional marker of C2C12 cell differentiation. The sub-cellular localization of the SMOalpha and SMOmu splice variants is not involved in the differentiation processes. Nuclear localization of only the SMOmu protein was confirmed.

Reactive oxygen species spermine metabolites generated from amine oxidases and radiation represent a therapeutic gain in cancer treatments.

The most frequent interventions in cancer therapy are currently the destruction of cells by irradiation or administration of drugs both able to induce radical formation and toxic metabolites by enzyme-catalyzed reactions. The aim of this study was to determine the cell viability of cells undergoing a DNA damage threshold accomplished by ROS overproduction via both ectopic expression of murine spermine oxidase (mSMOX) and bovine serum amine oxidase (BSAO) enzymes. Low dose of X-irradiation delivers a challenging dose of damage as evaluated in proficient Chinese hamster AA8 cell line and both deficient transcription-coupled nucleotide excision repair (NER) UV61 cells and deficient base excision repair (BER) EM9 cells, at 6 and 24 h after exposure. The priming dose of ROS overexposure by mSMOX provokes an adaptive response in N18TG2, AA8 and EM9 cell lines at 24 h. Interestingly, in the UV61 cells, ROS overexposure by mSMOX delivers an earlier adaptive response to radiation. The enzymatic formation of toxic metabolites has mainly been investigated on wild-type (WT) and multidrug-resistant (MDR) cancer cell lines, using and spermine as substrate of the BSAO enzyme. MDR cells are more sensitive to the toxic polyamine metabolites than WT cells, thus indicating a new therapeutic strategy to overcome MDR tumors. Since SMOX in mammals is differentially activated in a tissue-specific manner and cancer cells can differ in terms of DNA repair and MDR capabilities, it could be of interest to simultaneously treat with very low dose of X-rays and/or to alter SMOX metabolism to generate a differential response in healthy and cancer tissues.

Dose-Dependent Onset of Regenerative Program in Neutron Irradiated Mouse Skin

Background Tissue response to irradiation is not easily recapitulated by cell culture studies. The objective of this investigation was to characterize, the transcriptional response and the onset of regenerative processes in mouse skin irradiated with different doses of fast neutrons. Methodology/Principal Findings To monitor general response to irradiation and individual animal to animal variation, we performed gene and protein expression analysis with both pooled and individual mouse samples. A high-throughput gene expression analysis, by DNA oligonucleotide microarray was done with three months old C57Bl/6 mice irradiated with 0.2 and 1 Gy of mono-energetic 14 MeV neutron compared to sham irradiated controls. The results on 440 irradiation modulated genes, partially validated by quantitative real time RT-PCR, showed a dose-dependent up-regulation of a sub-class of keratin and keratin associated proteins, and members of the S100 family of Ca2+-binding proteins. Immunohistochemistry confirmed mRNA expression data enabled mapping of protein expression. Interestingly, proteins up-regulated in thickening epidermis: keratin 6 and S100A8 showed the most significant up-regulation and the least mouse-to-mouse variation following 0.2 Gy irradiation, in a concerted effort toward skin tissue regeneration. Conversely, mice irradiated at 1 Gy showed most evidence of apoptosis (Caspase-3 and TUNEL staining) and most 8-oxo-G accumulation at 24 h post-irradiation. Moreover, no cell proliferation accompanied 1 Gy exposure as shown by Ki67 immunohistochemistry. Conclusions/Significance The dose-dependent differential gene expression at the tissue level following in vivo exposure to neutron radiation is reminiscent of the onset of re-epithelialization and wound healing and depends on the proportion of cells carrying multiple chromosomal lesions in the entire tissue. Thus, this study presents in vivo evidence of a skin regenerative program exerted independently from DNA repair-associated pathways.

Spermine metabolism and radiation-derived reactive oxygen species for future therapeutic implications in cancer: an additive or adaptive response

Abstract Destruction of cells by irradiation-induced radical formation is one of the most frequent interventions in cancer therapy. An alternative to irradiation-induced radical formation is in principle drug-induced formation of radicals, and the formation of toxic metabolites by enzyme catalyzed reactions. Thus, combination therapy targeting polyamine metabolism could represent a promising strategy to fight hyper-proliferative disease. The aim of this work is to discuss and evaluate whether the presence of a DNA damage provoked by enzymatic ROS overproduction may act as an additive or adaptive response upon radiation and combination of hyperthermia with lysosomotropic compounds may improve the cytocidal effect of polyamines oxidation metabolites. Low level of X-irradiations delivers challenging dose of damage and an additive or adaptive response with the chronic damage induced by spermine oxidase overexpression depending on the deficiency of the DNA repair mechanisms. Since reactive oxygen species lead to membrane destabilization and cell death, we discuss the effects of BSAO and spermine association in multidrug resistant cells that resulted more sensitive to spermine metabolites than their wild-type counterparts, due to an increased mitochondrial activity. Since mammal spermine oxidase is differentially activated in a tissue specific manner, and cancer cells can differ in term of DNA repair capability, it could be of interest to open a scientific debate to use combinatory treatments to alter spermine metabolism and deliver differential response.

A proposed integrated systems approach to the radiation biology of cosmic interest: biophysics and molecular characterization of tissues irradiated with 14 MeV neutrons

Low-dose exposure of ionizing radiation triggers cell-to-cell communications and tissue interplay alterations. These alterations may play a fundamental role in non-cancer effects, overwhelming the theory of the DNA centric approach. Neither the mechanisms of these effects are fully understood nor is it possible to dissect the real incidence of quality and quantity of incident radiation during in vivo exposure, overall for particulate high-linear energy transfer (LET) radiation. Moreover, the knowledge of particulate high-LET radiation is mandatory for the human deep space exploration and to gain efficiency in the dose/effect ratio for radiotherapy. The aim of this mini-review was to describe an integrated system approach to the radiation biology of cosmic interest which could be set up in the framework of a future Sino-Italy cooperation among participating laboratories. We propose, in particular, to deliver X-rays and neutron irradiation at ENEA-FNG (Frascati, Italy) and heavy ion irradiation at IMP-CAS (Lanzhou, China) to in vivo models. The integrated system approach will focus on the correlation between the quality and quantity of radiation exposure and its in vivo biological effects. Wide range molecular profiling will analyze mainly cell and DNA damages and cell-to-cell and tissues interplay, meanwhile biochemical and chemical specific composition will be detected by infrared spectroscopy. The recently characterized alteration of leptin metabolism is discussed in more detail to present a successful example of systemic approach to cosmic radiation biology.

Caves and other subsurface environments in the future exploration of Mars: the absence of natural background radiation as biology concern

The human deep space exploration focused on Mars, being the near eventual final destination. Mars surface habitation is considered a dangerous solution. Among others, radiation, micrometeorites and dust storms, are the major causes of risk for the human well-being during the mission. Caves on Mars can provide a natural shelter against these environmental hazards. Several practical applications address forward the use of caves as habitation opportunity. Within the caves, inflatable structures can be used, as opposed to the heavy structure surface buildings. For this reason, studies were taken to assess the possible areas for which caves could be used as human habitats as an alternative for an initial settlement of Mars. The results obtained not only suggest strong arguments for the use of caves, but also open questions for further quantification and understanding of current unknowns. In the present review, we focus on the hypothetical biological effects due to the cave shelter for natural background radiation that is of three orders of magnitude lower than Mars surface. Background radiation is the ubiquitous ionizing radiation that organisms experienced on the Earth, being present at the appearance of life on Earth. Nevertheless, both the effects of natural background radiation and its absence are not well known. We review the literature based on the effects of the absence of natural background to pursue a common scientific effort to gain scientific knowledge in this future deep space exploration scenario in a Sino-Italy cooperation, taking advantage of the JingPing cavern laboratory in the Sichuan region.