Sergio Comincini

microRNA-17 regulates the expression of ATG7 and modulates the autophagy process, improving the sensitivity to temozolomide and low-dose ionizing radiation treatments in human glioblastoma cells

ATG7 is a key autophagy-promoting gene that plays a critical role in the regulation of cell death and survival of various cell types. We report here that microRNAs (miRNAs), a class of endogenous 22–24 nucleotide noncoding RNA molecules able to affect stability and translation of mRNA, may represent a novel mechanism for regulating ATG7 expression and therefore autophagy. We demonstrated that ATG7 is a potential target for miR-17, and this miRNA could negatively regulate ATG7 expression, resulting in a modulation of the autophagic status in T98G glioblastoma cells. Treatment of these tumor cells with the miR-17 mimic decreased, and with the antagomir increased, the expression of ATG7 protein. Dual luciferase reporter assay confirmed that a specific miR-17 binding sequence in the 3′-UTR of ATG7 contributed to the modulation of the expression of the gene by miR-17. Interestingly, our results showed that anti-miR-17 administration activated autophagy through autophagosome formation, as resulted by LC3B and ATG7 protein expression increase, and by the analysis of GFP-LC3 positive autophagosome vesicles in living cells. Furthermore, the autophagy activation by anti-miR-17 resulted in a decrease of the threshold resistance at temozolomide doses in T98G cells, while miR-17 modulation in U373-MG glioblastoma cells resulted in a sensitization to low ionizing radiation doses. Our study of the role of miR-17 in regulating ATG7 expression and autophagy reveals a novel function for this miRNA sequence in a critical cellular event with significant impacts in cancer development, progression and treatment.

Autophagy and ionizing radiation in tumors: The “survive or not survive” dilemma

Autophagy is a so‐called “self‐eating” system responsible for degrading long‐lived proteins and cytoplasmic organelles, whose products are recycled to maintain cellular homeostasis. This ability makes autophagy a good candidate for a survival mechanism in response to several stresses, including the tumor cell transformation. In particular, recent studies suggested that autophagy functions as a pro‐death mechanism within different tumor contexts. It is, however, widely reported that autophagy represents both a survival mechanism or contributes directly to cell death fate. This interplay of the autophagy functions has been observed in many types of cancers and, in some cases, autophagy has been demonstrated to both promote and inhibit antitumor drug resistance. From a therapeutical point of view, the effects of the modulation of the tumor cell autophagic status, in response to ionizing radiations, are presently of particular relevance in oncology. Accordingly, this review also provides a perspective view on future works for exploring the modulation of autophagic indices in tumor cells as a novel molecular‐based adjuvant strategy, in order to improve radiotherapy and chemotherapy effects in cancer patients. J. Cell. Physiol. 228: 1–8, 2013.

Different involvement of autophagy in human malignant glioma cell lines undergoing irradiation and temozolomide combined treatments.

Glioblastoma (GB) has a poor prognosis, despite current multimodality treatment. Beside surgical resection, adjuvant ionizing radiation (IR) combined with Temozolomide (TMZ) drug administration is the standard therapy for GB. This currently combined radio‐chemotherapy treatment resulted in glial tumor cell death induction, whose main molecular death pathways are still not completely deciphered. In this study, the autophagy process was investigated, and in vitro modulated, in two different GB cell lines, T98G and U373MG (known to differ in their radiosensitivity), after IR or combined IR/TMZ treatments. T98G cells showed a high radiosensitivity (especially at low and intermediate doses), associated with autophagy activation, assessed by Beclin‐1 and Atg‐5 expression increase, LC3‐I to LC3‐II conversion and LC3B‐GFP accumulation in autophagosomes of irradiated cells; differently, U373MG cells resulted less radiosensitive. Autophagy inhibition, using siRNA against BECN1 or ATG‐7 genes, totally prevented decrease in viability after both IR and IR/TMZ treatments in the radiosensitive T98G cells, confirming the autophagy involvement in the cytotoxicity of these cells after the current GB treatment, contrary to U373MG cells. However, rapamycin‐mediated autophagy, that further radiosensitized T98G, was able to promote radiosensitivty also in U373MG cells, suggesting a role of autophagy process in enhancing radiosensitivity. Taken together, these results might enforce the concept that autophagy‐associated cell death might constitute a possible adjuvant therapeutic strategy to enhance the conventional GB treatment. J. Cell. Biochem. 113: 2308–2318, 2012.

Genomic organization, comparative analysis, and genetic polymorphisms of the bovine and ovine prion Doppel genes (PRND).

Abstract. The doppel protein (Dpl) is a prion-like protein encoded by the gene PRND, which has been found downstream of the prion gene, PRNP, in human and mouse. This paper describes the isolation and structural organization of the bovine and ovine PRND genes, which are composed of two exons compared with the three of human and mouse. Intergenic distances between PRNP and PRND were covered by means of long-range PCR and found to be 16.8 and 20 kb, in cattle and sheep respectively. The 5′ and 3′ untranslated regions (UTR) were analyzed to identify transcription regulatory sequences and compared with those from the PRND and PRNP sequences published for other species. Three polymorphisms (R50H, N110H, and R132Q) were revealed in the cattle coding region; two synonymous substitutions (I12I, A26A) were found in sheep. None of the polymorphisms was significantly associated with either Bovine Spongiform Encephalopathy (BSE) in cattle or scrapie in sheep.

The PrP-like protein Doppel gene in sheep and cattle: cDNA sequence and expression

Abstract. cDNAs encoding the ovine and bovine prion protein-like protein Doppel (Dpl) have been cloned. Sequencing revealed cDNAs of 2.85 and 3.31 kb from ovine and bovine testicular tissue, in accordance with observations of single transcripts of 3.2 and 3.6 kb on Northern blots. Sequence alignments showed a very high degree of identity between the sheep and cattle Dpl cDNAs, except for a 0.4-kb stretch in the bovine 3′ untranslated region and the terminal 3′ end of the sequences. The expression pattern of the Dpl gene (Prnd) in adult tissues from both species was compared by Northern blot and RT-PCR analyses. The Prnd gene was expressed strongly in testicular tissue, while low levels of expression were seen in other tissues. The open reading frame of the ovine and bovine sequences encodes a 178-amino acid protein with 95% sequence identity between the two species. Predicted structural features are in close agreement with previous reports for mouse, human, and rat Dpl.

Emerging roles of microRNA in modulating cell-death processes in malignant glioma.

MicroRNAs (miRNAs) are small noncoding RNA molecules that regulate protein expression by cleaving or repressing the translation of target mRNAs. In mammals, their function mainly represses the mRNA transcripts via imperfect complementary sequences in the 3′UTR of target mRNAs. Several miRNAs have been recently reported to be involved in modulation of different genes in tumors, including glioblastoma, the most frequent brain tumor in adults. Despite the improvements in treatments, survival of patients remains poor, and glioblastoma is one of the most lethal form of human cancer. To define novel strategies against this tumor, emerging research investigated miRNAs involvement in glioblastoma. In particular, this review is focused on miRNAs involved on the two principal programmed cell‐death, apoptosis and autophagy, recently described from the literature. Moreover, the discovery of miRNAs role in glioma cell‐death pathways has also revealed a new category of therapeutic targets, fundamental for this kind of tumor. J. Cell. Physiol. 229: 277–286, 2014.

RAPD analysis of systematic relationships among the Cervidae

We investigated the possible application of RAPD (Random Amplified Polymorphic DNA) analysis to the study of the systematic relationships of five cervid taxa. Amplifications with eight different primers gave reproducible electrophoretic patterns which could be regarded as a data-set consisting of monomorphic and polymorphic characters. Some of these characters are species- and subspecies-specific. Band-sharing analysis and numerical taxonomy methods allowed us to generate a phenetic tree. Our results point out new possible systematic considerations within the examined taxa.

Autophagy is Modulated in Human Neuroblastoma Cells Through Direct Exposition to Low Frequency Electromagnetic Fields

In neurogenerative diseases, comprising Alzheimer’s (AD), functional alteration in autophagy is considered one of the pathological hallmarks and a promising therapeutic target. Epidemiological investigations on the possible causes undergoing these diseases have suggested that electromagnetic fields (EMF) exposition can contribute to their etiology. On the other hand, EMF have therapeutic implications in reactivating neuronal functionality. To partly clarify this dualism, the effect of low‐frequency EMF (LF‐EMF) on the modulation of autophagy was investigated in human neuroblastoma SH‐SY5Y cells, which were also subsequently exposed to Aβ peptides, key players in AD. The results primarily point that LF‐EMF induce a significant reduction of microRNA 30a (miR‐30a) expression with a concomitant increase of Beclin1 transcript (BECN1) and its corresponding protein. Furthermore, LF‐EMF counteract the induced miR‐30a up‐regulation in the same cells transfected with miR‐30a mimic precursor molecules and, on the other side, rescue Beclin1 expression after BECN1 siRNA treatment. The expression of autophagy‐related markers (ATG7 and LC3B‐II) as well as the dynamics of autophagosome formation were also visualized after LF‐EMF exposition. Finally, different protocols of repeated LF‐EMF treatments were assayed to contrast the effects of Aβ peptides in vitro administration. Overall, this research demonstrates, for the first time, that specific LF‐EMF treatments can modulate in vitro the expression of a microRNA sequence, which in turn affects autophagy via Beclin1 expression. Taking into account the pivotal role of autophagy in the clearance of protein aggregates within the cells, our results indicate a potential cytoprotective effect exerted by LF‐EMF in neurodegenerative diseases such as AD. J. Cell. Physiol. 229: 1776–1786, 2014.

Silencing of cellular prion protein (PrPC) expression by DNA-antisense oligonucleotides induces autophagy-dependent cell death in glioma cells.

Malignant gliomas are the most common and lethal primary central nervous system neoplasms. Several intriguing lines of evidence have recently emerged indicating that the cellular prion protein (PrPC) may exert neuro- and cyto-protective functions: PrPC overexpression protects cultured neurons and also tumor cell lines exposed to various pro-apoptotic stimuli while, on the contrary, PrPC silencing sensitizes Adriamycin-resistant human breast carcinoma cells to TRAIL-mediated cell death. In order to determine if PrPC is involved in the resistance of glial tumors to cell death, the effects of cellular prion protein downregulation by antisense approach were investigated in different human malignant glioma cell lines. PrPC downregulation induced profound morphological changes and significant cell death. In addition, a significant tumor volume reduction was noted after PrPC silencing in a EGFP-GL261 glioma murine model. Investigations of the molecular effects induced by PrPC silencing were carried out on T98G human glioma cells by analysing autophagic as well as typical apoptotic markers (nuclear morphology, caspase-3/7, p53 and PARP-1). The results indicated that apoptosis was not induced after PrPC downregulation while, on the contrary, electron microscopy analysis, and an accumulation of GFP-LC3-II in autophagosomal membranes of GFP-LC3 transfected cells, indicated a predominant activation of autophagy. PrPC silencing also led to induction of LC3-II, increase in Beclin-1 and a concomitant decrease in p62, Bcl-2 and in the phosphorylation of 4E-BP1, a target of mTOR autophagy signaling. In conclusion, our results show for the first time that interfering with the cellular prion protein expression could modulate autophagy-dependent cell death pathways in glial tumor cells.

Combined EGFR and autophagy modulation impairs cell migration and enhances radiosensitivity in human glioblastoma cells

Glioblastoma (GBM) remains the most aggressive and lethal brain tumor due to its molecular heterogeneity and high motility and invasion capabilities of its cells, resulting in high resistance to current standard treatments (surgery, followed by ionizing radiation combined with Temozolomide chemotherapy administration). Locus amplification, gene overexpression, and genetic mutations of epidermal growth factor receptor (EGFR) are hallmarks of GBM that can ectopically activate downstream signaling oncogenic cascades such as PI3K/Akt/mTOR pathway. Importantly, alteration of this pathway, involved also in the regulation of autophagy process, can improve radioresistance in GBM cells, thus promoting the aggressive phenotype of this tumor. In this work, the endogenous EGFR expression profile and autophagy were modulated to increase radiosensitivity behavior of human T98G and U373MG GBM cells. Our results primarily indicated that EGFR interfering induced radiosensitivity according to a decrease of the clonogenic capability of the investigated cells, and an effective reduction of the in vitro migratory features. Moreover, EGFR interfering resulted in an increase of Temozolomide (TMZ) cytotoxicity in T98G TMZ‐resistant cells. In order to elucidate the involvement of the autophagy process as pro‐death or pro‐survival role in cells subjected to EGFR interfering, the key autophagic gene ATG7 was silenced, thereby producing a transient block of the autophagy process. This autophagy inhibition rescued clonogenic capability of irradiated and EGFR‐silenced T98G cells, suggesting a pro‐death autophagy contribution. To further confirm the functional interplay between EGFR and autophagy pathways, Rapamycin‐mediated autophagy induction during EGFR modulation promoted further impairment of irradiated cells, in terms of clonogenic and migration capabilities. Taken together, these results might suggest a novel combined EGFR‐autophagy modulation strategy, to overcome intrinsic GBM radioresistance, thus improving the efficacy of standard treatments. J. Cell. Physiol. 229: 1863–1873, 2014.