Antonella Borrelli

A recombinant MnSOD is radioprotective for normal cells and radiosensitizing for tumor cells

Organisms exposed to ionizing radiation are mainly damaged by free radicals, which are generated by the radiolysis of water contained in the cells. Recently a significant reduction of tissue injury from irradiation damage was demonstrated by using MnSOD-plasmid/liposome treatments in the protection of murine lung. In this study we show that a new active recombinant human MnSOD (rMnSOD), easily administered in vivo, not only exerts the same radioprotective effect on normal cells and organisms as any MnSOD, but it is also radiosensitizing for tumor cells. In addition, we show how healthy animals, exposed to lethal doses of ionizing radiation and daily injections with rMnSOD, were protected from radiodamage and were still alive 30 days after the irradiation, while animals treated with only PBS solution, in the absence of rMnSOD, died after 7-8 days from the radiotreatments. The molecular analysis of all irradiated tissues revealed that the antiapoptotic AVEN gene appeared activated only in the animals treated in the presence of rMnSOD. The data suggest that rMnSOD deserves to be considered as a pharmaceutical tool for making radiotherapy more selective on cancer cells and to prevent and/or cure the accidental damage derived from exposure to ionizing radiation.

Tumor suppressive activity of a variant isoform of manganese superoxide dismutase released by a human liposarcoma cell line

A cell line derived from a pleiomorphic liposarcoma, named LSA, was previously reported to secrete (a) factor(s) exhibiting oncotoxic properties. The present article describes the isolation, purification and sequence analysis of a protein released by LSA cells into conditioned culture medium. This protein proved to be a variant isoform of manganese superoxide dismutase (MnSOD), hence its designation as LSA‐type‐MnSOD. This LSA‐type‐SOD differed from conventional SODs in its secretion by producer cells, contrasting with the normal localization of SODs in the mitochondrial matrix. Interestingly, during the protein purification process, LSA‐type‐SOD cosegregated with a cytotoxic activity directed against a number of tumor cell lines, as determined under in vitro conditions. This cytopathic effect was most likely due to LSA‐type‐SOD, since it could be fully reproduced using recombinant SOD that was expressed from cDNA clones isolated from LSA cells mRNA preparations and henceforth designated L‐rSOD. In addition to its manifestation in cell lines kept in tissue culture, the oncotoxicity of LSA‐type‐SOD was further reflected in a remarkable capacity of this protein for suppression of mammary tumors in Balb‐C‐FRIII mice. Animals subcutaneously injected with L‐rSOD in the tumor area showed a complete disruption of established mammary carcinomas, as monitored by nuclear magnetic resonance (NMR) scanning. Moreover, metastatic spreading, which was readily detected in the control group, was suppressed in the treated animals. Altogether these data suggest that LSA‐type‐SOD interferes with survival and spreading of neoplastically transformed cells and deserves to be future validated as a therapeutic agent against cancer, either alone or in combination with conventional treatments.

Biophysical and biochemical characterization of a liposarcoma-derived recombinant MnSOD protein acting as an anticancer agent.

A recombinant MnSOD (rMnSOD) synthesized by specific cDNA clones derived from a liposarcoma cell line was shown to have the same sequence as the wild‐type MnSOD expressed in the human myeloid leukaemia cell line U937, except for the presence of the leader peptide at the N‐terminus. These results were fully confirmed by the molecular mass of rMnSOD as evaluated by ES/MS analysis (26662.7 Da) and the nucleotide sequence of the MnSOD cDNA. The role of the leader peptide in rMnSOD was investigated using a fluorescent and/or 68Gallium‐labelled synthetic peptide. The labelled peptide permeated MCF‐7 cells and uptake could be inhibited in the presence of an excess of oestrogen. In vivo it was taken up by the tumour, suggesting that the molecule can be used for both therapy and diagnosis. The in vitro and in vivo pharmacology tests confirmed that rMnSOD is only oncotoxic for tumour cells expressing oestrogen receptors. Pharmacokinetic studies in animals performed with 125I‐ and 131I‐labelled proteins confirmed that, when administered systemically, rMnSOD selectively reached the tumour, where its presence was unambiguously demonstrated by scintigraphic and PET scans. PCR analysis revealed that Bax gene expression was increased and the Bcl2 gene was down regulated in MCF7 cells treated with rMnSOD, which suggests that the protein induces a pro‐apoptotic mechanism.

CDK/CCN and CDKI Alterations for Cancer Prognosis and Therapeutic Predictivity

The regulation of cell growth and division occurs in an accurate sequential manner. It is dictated by the accumulation of cyclins (CCNs) and cyclin-dependent kinases (CDKs) complexes and degradation of CCNs. In human tumors, instead, the cell cycle is deregulated, causing absence of differentiation and aberrant cell growth. Oncogenic alterations of CCNs, CDKs, and CDKIs have been reported in more than 90% of human cancers, and the most frequent are those related to the G1 phase. Several molecular mechanisms, including gene overexpression, chromosomal translocations, point mutations, insertions and deletions, missense and frame shift mutation, splicing, or methylation, may be responsible for these alterations. The cell cycle regulators are involved in tumor progression given their association with cancers characterized by higher incidence of relapses and chemotherapy resistance. In the last decade anticancer drug researches focused on new compounds, able to target molecules related to changes in genes associated with tumor status. Recently, the studies have focused on the restoration of cell cycle control modulating molecular targets involved in cancer-cell alterations. This paper aims to correlate alterations of cell cycle regulators with human cancers and therapeutic responsivity.

The Functional Role of MnSOD as a Biomarker of Human Diseases and Therapeutic Potential of a New Isoform of a Human Recombinant MnSOD

Reactive oxygen species (ROS) are generated as a consequence of metabolic reactions in the mitochondria of eukaryotic cells. This work describes the role of the manganese superoxide dismutase (MnSOD) as a biomarker of different human diseases and proposes a new therapeutic application for the prevention of cancer and its treatment. The paper also describes how a new form of human MnSOD was discovered, its initial application, and its clinical potentials. The MnSOD isolated from a human liposarcoma cell line (LSA) was able to kill cancer cells expressing estrogen receptors, but it did not have cytotoxic effects on normal cells. Together with its oncotoxic activity, the recombinant MnSOD (rMnSOD) exerts a radioprotective effect on normal cells irradiated with X-rays. The rMnSOD is characterized by the presence of a leader peptide, which allows the protein to enter cells: this unique property can be used in the radiodiagnosis of cancer or chemotherapy, conjugating radioactive substances or chemotherapic drugs to the leader peptide of the MnSOD. Compared to traditional chemotherapic agents, the drugs conjugated with the leader peptide of MnSOD can selectively reach and enter cancer cells, thus reducing the side effects of traditional treatments.

Role of gut microbiota and oxidative stress in the progression of non-alcoholic fatty liver disease to hepatocarcinoma: Current and innovative therapeutic approaches

Non-alcoholic fatty liver disease (NAFLD) represents the most common chronic liver disease in industrialized countries. NAFLD progresses through the inflammatory phase of non-alcoholic steatohepatitis (NASH) to fibrosis and cirrhosis, with some cases developing liver failure or hepatocellular carcinoma (HCC). Liver biopsy remains the gold standard approach to a definitive diagnosis of NAFLD and the distinction between simple steatosis and NASH. The pathogenesis of NASH is still not clear. Several theories have been proposed ranging from the “Two Hit Theory” to the “Multiple Hit Theory”. However, the general consensus is that the gut microbiota, oxidative stress, and mitochondrial damage play key roles in the pathogenesis of NASH. The interaction between the gut epithelia and some commensal bacteria induces the rapid generation of reactive oxygen species (ROS). The main goal of any therapy addressing NASH is to reverse or prevent progression to liver fibrosis/cirrhosis. This problem represents the first “Achilles’ heel” of the new molecules being evaluated in most ongoing clinical trials. The second is the inability of these molecules to reach the mitochondria, the primary sites of energy production and ROS generation. Recently, a variety of non-pharmacological and pharmacological treatment approaches for NASH have been evaluated including vitamin E, the thiazolidinediones, and novel molecules related to NASH pathogenesis (including obeticholic acid and elafibranor). Recently, a new isoform of human manganese superoxide dismutase (MnSOD) was isolated and obtained in a synthetic recombinant form designated rMnSOD. This protein has been shown to be a powerful antioxidant capable of mediating ROS dismutation, penetrating biological barriers via its uncleaved leader peptide, and reducing portal hypertension and fibrosis in rats affected by liver cirrhosis. Based on these distinctive characteristics, it can be hypothesized that this novel recombinant protein (rMnSOD) potentially represents a new and highly efficient adjuvant therapy to counteract the progression from NASH to HCC.

The leader peptide of a human rec. MnSOD as molecular carrier which delivers high amounts of Cisplatin into tumor cells inducing a fast apoptosis in vitro

The leader peptide of a recombinant MnSOD (rMnSOD‐Lp) constitutes the carrier that allows rMnSOD to penetrate tumor cells. A synthetic preparation of rMnSOD‐Lp was 68Ga labeled (rMnSOD‐Lp‐68Ga) and injected into animals bearing spontaneous mammary cancers, followed by PET examinations, which demonstrated unambiguously the tumor sites in all the animals, suggesting that if rMnSOD‐Lp was able to transport the radioisotope into tumor cells, it would also be able to deliver cytotoxic molecules. The rMnSOD‐Lp was, therefore, conjugated to cisplatin (rMnSOD‐Lp‐CC) and added to cultured tumor cells. Equal concentrations of cisplatin were used for the tests. After treating the ovarian cancer cells with 11.1 μg of cisplatin alone, analysis by atomic absorbance spectrophotometry was able to detect only 6 ng of platinum, whereas when the same cells were treated with the same amount of cisplatin conjugated to leader peptide rMnSOD, 387 ng of platinum were detected, i.e., an amount 80 times greater. Only the tumor cells died following treatment with rMnSOD‐Lp‐CC; molecular analysis revealed that its addition generated an increasing expression of Erk‐2 and Bax products, which could be inhibited only by a selective MAP/ERK kinase inhibitor (PD98059), revealing that rMnSOD‐Lp‐CC has an apoptotic function, exactly as occurs when using the cisplatin alone. Data are statistically significant and indicate that by using rMnSOD‐Lp‐CC, the cisplatin can be transformed from an agent with antireplicative activity into a specific and selective antitumor molecule, increasing its therapeutic index. We think that rMnSOD‐Lp‐CC deserves to be considered as a new antitumor agent.

Recombinant Mitochondrial Manganese Containing Superoxide Dismutase Protects Against Ochratoxin A-Induced Nephrotoxicity

Ochratoxin A (OTA) is a natural mycotoxin, involved in the development of important human and animal diseases. In this work we have studied the role of oxidative stress in the development of OTA nephrotoxicity and the effect of a new recombinant mitochondrial manganese containing superoxide dismutase (rMnSOD) to prevent kidney damage induced by OTA. Blood pressure, glomerular filtration rate and renal histology were analyzed in control rats and in OTA treated rats. In addition, lipid peroxidation, catalase and superoxide dismutase productions were measured. Our data showed that animals treated with OTA presented hypertension and reduction of glomerular filtration rate (GFR). These effects are most probably related to an increase in the reactive oxygen species (ROS) productions. In fact, we have shown that treatment with rMnSOD restored the levels of blood pressure and GFR simultaneously. Moreover, we have noted that OTA induced alteration on glomerular and tubular degeneration and interstitial infiltrates and that use of rMnSOD combined with OTA prevent this renal histological damage confirming the potential therapeutic role in the treatment of rMnSOD OTA nephrotoxicity. J. Cell. Biochem. 117: 1352–1358, 2016.

Cell Penetrating Peptides as Molecular Carriers for Anti-Cancer Agents

Cell membranes with their selective permeability play important functions in the tight control of molecular exchanges between the cytosol and the extracellular environment as the intracellular membranes do within the internal compartments. For this reason the plasma membranes often represent a challenging obstacle to the intracellular delivery of many anti-cancer molecules. The active transport of drugs through such barrier often requires specific carriers able to cross the lipid bilayer. Cell penetrating peptides (CPPs) are generally 5–30 amino acids long which, for their ability to cross cell membranes, are widely used to deliver proteins, plasmid DNA, RNA, oligonucleotides, liposomes and anti-cancer drugs inside the cells. In this review, we describe the several types of CPPs, the chemical modifications to improve their cellular uptake, the different mechanisms to cross cell membranes and their biological properties upon conjugation with specific molecules. Special emphasis has been given to those with promising application in cancer therapy.

Effect of rMnSOD on survival signaling in pediatric high risk T-cell acute lymphoblastic leukaemia

Manganese superoxide dismutase (MnSOD) is a mitochondrial enzyme that defends against oxidative damage due to reactive oxygen species (ROS). A new isoform of MnSOD with cytotoxic activity was recently discovered in liposarcoma cells. Here, we tested the effectiveness of a recombinant form of this isoform (rMnSOD) on leukemic T cells, Jurkat cells, and lymphocytes. Our results confirm that leukemic T cells can internalize rMnSOD and that rMnSOD causes apoptosis of 99% of leukemic cells without showing toxic effects on healthy cells. Using light and electron microscopy, we determined that an rMnSOD concentration of 0.067 μM most effective on apoptosis induction. Western blot analysis showed that treatment with 0.067 μM rMnSOD resulted in high expression of the pro‐apoptotic protein Bax and low expression of the anti‐apoptotic protein Bcl‐2 in leukemia cells. Concerning signal transduction pathway no influence was observed after treatment except for Jurkat cells showing a slightly decreased expression of ERK phosphorylation. These results suggest that rMnSOD may be an effective and non‐toxic treatment option for T‐cell leukemia. J. Cell. Physiol. 230: 1086–1093, 2015.