Teloxantron inhibits the processivity of telomerase with preferential DNA damage on telomeres.

Telomerase reactivation is one of the hallmarks of cancer, which plays an important role in cellular immortalization and the development and progression of the tumor. Chemical telomerase inhibitors have been shown to trigger replicative senescence and apoptotic cell death both in vitro and in vivo. Due to its upregulation in various cancers, telomerase is considered a potential target in cancer therapy. In this study, we identified potent, small-molecule telomerase inhibitors using a telomerase repeat amplification protocol assay. The results of the assay are the first evidence of telomerase inhibition by anthraquinone derivatives that do not exhibit G-quadruplex-stabilizing properties. The stability of telomerase in the presence of its inhibitor was evaluated under nearly physiological conditions using a cellular thermal shift assay. Our data showed that the compound induced aggregation of the catalytic subunit (hTERT) of human telomerase, and molecular studies confirmed the binding of the hit compound with the active site of the enzyme. The ability of new derivatives to activate DNA double-strand breaks (DSBs) was determined by high-resolution microscopy and flow cytometry in tumor cell lines differing in telomere elongation mechanism. The compounds triggered DSBs in TERT-positive A549 and H460 lung cancer cell lines, but not in TERT-negative NHBE normal human bronchial epithelial and ALT-positive U2OS osteosarcoma cell lines, which indicates that the induction of DSBs was dependent on telomerase inhibition. The observed DNA damage activated DNA damage response pathways involving ATM/Chk2 and ATR/Chk1 cascades. Additionally, the compounds induced apoptotic cell death through extrinsic and intrinsic pathways in lung cancer cells. Taken together, our study demonstrated that anthraquinone derivatives can be further developed into novel telomerase-related anticancer agents.

Telomere as a Therapeutic Target in Dedifferentiated Liposarcoma.

BACKGROUND: Well-differentiated (WD)/dedifferentiated (DD) liposarcoma (LPS) accounts for ~60% of retroperitoneal sarcomas. WDLPS and DDLPS divergently evolve from a common precursor and are both marked by the amplification of the 12q13-q15 region, leading to the abnormal expression of MDM2, CDK4, and HMGA2 genes. DDLPS is a non-lipogenic disease associated with aggressive clinical behavior. Patients have limited therapeutic options, especially for advanced disease, and their outcome remains largely unsatisfactory. This evidence underlines the need for identifying and validating DDLPS-specific actionable targets to design novel biology-driven therapies. METHODS: Following gene expression profiling of DDLPS clinical specimens, we observed the up-regulation of "telomere maintenance" (TMM) pathways in paired DD and WD components of DDLPS. Considering the relevance of TMM for LPS onset and progression, the activity of a telomeric G-quadruplex binder (RHPS4) was assessed in DDLPS patient-derived cell lines. RESULTS: Equitoxic concentrations of RHPS4 in DDLPS cells altered telomeric c-circle levels, induced DNA damage, and resulted in the accumulation of γ-H2AX-stained micronuclei. This evidence was paralleled by an RHPS4-mediated reduction of in vitro cell migration and induction of apoptosis/autophagy. CONCLUSIONS: Our findings support telomere as an intriguing therapeutic target in DDLPS and suggest G-quadruplex binders as innovative therapeutic agents.

On the Road to Fight Cancer: The Potential of G-quadruplex Ligands as Novel Therapeutic Agents.

Nucleic acid sequences able to adopt a G-quadruplex conformation are overrepresented within the human genome. This evidence strongly suggests that these genomic regions have been evolutionary selected to play a pivotal role in several aspects of cell biology. In the present review article, we provide an overview on the biological impact of targeting G-quadruplexes in cancer. A variety of small molecules showing good G-quadruplex stabilizing properties has been reported to exert an antitumor activity in several preclinical models of human cancers. Moreover, promiscuous binders and multiple targeting G-quadruplex ligands, cancer cell defense responses and synthetic lethal interactions of G-quadruplex targeting have been also highlighted. Overall, evidence gathered thus far indicates that targeting G-quadruplex may represent an innovative and fascinating therapeutic approach for cancer. The continued methodological improvements, the development of specific tools and a careful consideration of the experimental settings in living systems will be useful to deepen our knowledge of G-quadruplex biology in cancer, to better define their role as therapeutic targets and to help design and develop novel and reliable G-quadruplex-based anticancer strategies.

The Role of Alternative Lengthening of Telomeres Mechanism in Cancer: Translational and Therapeutic Implications.

Telomere maintenance mechanisms (i.e., telomerase activity (TA) and the alternative lengthening of telomere (ALT) mechanism) contribute to tumorigenesis by providing unlimited proliferative capacity to cancer cells. Although the role of either telomere maintenance mechanisms seems to be equivalent in providing a limitless proliferative ability to tumor cells, the contribution of TA and ALT to the clinical outcome of patients may differ prominently. In addition, several strategies have been developed to interfere with TA in cancer, including Imetelstat that has been the first telomerase inhibitor tested in clinical trials. Conversely, the limited information available on the molecular underpinnings of ALT has hindered thus far the development of genuine ALT-targeting agents. Moreover, whether anti-telomerase therapies may be hampered or not by possible adaptive responses is still debatable. Nonetheless, it is plausible hypothesizing that treatment with telomerase inhibitors may exert selective pressure for the emergence of cancer cells that become resistant to treatment by activating the ALT mechanism. This notion, together with the evidence that both telomere maintenance mechanisms may coexist within the same tumor and may distinctly impinge on patients' outcomes, suggests that ALT may exert an unexpected role in tumor biology that still needs to be fully elucidated.

The Oncogenic Signaling Pathways in BRAF-Mutant Melanoma Cells are Modulated by Naphthalene Diimide-Like G-Quadruplex Ligands.

Melanoma is the most aggressive and deadly type of skin cancer. Despite the advent of targeted therapies directed against specific oncogene mutations, melanoma remains a tumor that is very difficult to treat, and ultimately remains incurable. In the past two decades, stabilization of the non-canonical nucleic acid G-quadruplex structures within oncogene promoters has stood out as a promising approach to interfere with oncogenic signaling pathways in cancer cells, paving the way toward the development of G-quadruplex ligands as antitumor drugs. Here, we present the synthesis and screening of a library of differently functionalized core-extended naphthalene diimides for their activity against the BRAFV600E-mutant melanoma cell line. The most promising compound was able to stabilize G-quadruplexes that formed in the promoter regions of two target genes relevant to melanoma, KIT and BCL-2. This activity led to the suppression of protein expression and thus to interference with oncogenic signaling pathways involved in BRAF-mutant melanoma cell survival, apoptosis, and resistance to drugs. This G-quadruplex ligand thus represents a suitable candidate for the development of melanoma treatment options based on a new mechanism of action and could reveal particular significance in the context of resistance to targeted therapies of BRAF-mutant melanoma cells.

Comparative Assessment of Antitumor Effects and Autophagy Induction as a Resistance Mechanism by Cytotoxics and EZH2 Inhibition in INI1-Negative Epithelioid Sarcoma Patient-Derived Xenograft.

Epithelioid sarcoma (ES) is a rare mesenchymal malignancy marked by SMARCB1/INI1 deficiency. Retrospective clinical data report on the activity of anthracycline- and gemcitabine-based regimens. EZH2 inhibitors are currently being tested in clinical trials. Since comparisons of these agents are unlikely to be prospectively evaluated in the clinics, we took advantage of an INI1-deficient proximal-type ES patient-derived xenograft (PDX ES-1) to comparatively assess its preclinical antitumor activity. Mice were treated with doxorubicin and ifosfamide, singly or in combination, gemcitabine, and the EZH2 inhibitor EPZ-011989. Comparable antitumor activity (max tumor volume inhibition: ~90%) was caused by gemcitabine, EPZ-011989, and the doxorubicin-ifosfamide combination. The integration of RNAseq data, generated on tumors obtained from untreated and EPZ-011989-treated mice, and results from functional studies, carried out on the PDX-derived ES-1 cell line, revealed autophagy induction as a possible survival mechanism in residual tumor cells following EPZ-011989 treatment and identified HMGA2 as a main player in this process. Our data support the clinical use of gemcitabine and the doxorubicin-ifosfamide combination, confirm EZH2 as a therapeutic target in proximal-type ES, and suggest autophagy as a cytoprotective mechanism against EZH2 inhibition.

Distinct biological responses of metastatic castration resistant prostate cancer cells upon exposure to G-quadruplex interacting naphthalenediimide derivatives.

Small molecules able to bind non-canonical G-quadruplex DNA structures (G4) have been recently tested as novel potential agents for the treatment of prostate cancer thanks to their repression of aberrant androgen receptor gene. However, metastatic castration-resistant prostate cancer (mCRPC), a letal form of prostate cancer, is still incurable. Here we tested two naphthalenediimide derivatives, previously reported as multitarget agents, on a couple of relevant mCRPC cell models (DU145 and PC-3). We showed that these compounds interfere with the RAS/MEK/ERK and PI3K/AKT pathways. Interestingly, both these two biological processes depend upon Epidermal Growth Factor Receptor (EGFR) activation. By means of biological and analytical tools we showed that our compounds are efficient inducers of the structural transition of the EGFR promoter towards a G-quadruplex conformation, ultimately leading to a reduction of the receptor production. The overall result is an interesting cytotoxic profile for these two derivatives. Thanks to their activity at different steps, these compounds can open the way to novel therapeutic approaches for mCRPC that could contribute to escape resistance to selective treatments.

miR-205 enhances radiation sensitivity of prostate cancer cells by impairing DNA damage repair through PKCε and ZEB1 inhibition.

BACKGROUND: Radiotherapy is one of the main treatment options for non-metastatic prostate cancer (PCa). Although treatment technical optimization has greatly improved local tumor control, a considerable fraction of patients still experience relapse due to the development of resistance. Radioresistance is a complex and still poorly understood phenomenon involving the deregulation of a variety of signaling pathways as a consequence of several genetic and epigenetic abnormalities. In this context, cumulative evidence supports a functional role of microRNAs in affecting radioresistance, suggesting the modulation of their expression as a novel radiosensitizing approach. Here, we investigated for the first time the ability of miR-205 to enhance the radiation response of PCa models. METHODS: miR-205 reconstitution by a miRNA mimic in PCa cell lines (DU145 and PC-3) was used to elucidate miR-205 biological role. Radiation response in miRNA-reconstituted and control cells was assessed by clonogenic assay, immunofluorescence-based detection of nuclear γ-H2AX foci and comet assay. RNAi was used to silence the miRNA targets PKCε or ZEB1. In addition, target-protection experiments were carried out using a custom oligonucleotide designed to physically disrupt the pairing between the miR-205 and PKCε. For in vivo experiments, xenografts generated in SCID mice by implanting DU145 cells stably expressing miR-205 were exposed to 5-Gy single dose irradiation using an image-guided animal micro-irradiator. RESULTS: miR-205 reconstitution was able to significantly enhance the radiation response of prostate cancer cell lines and xenografts through the impairment of radiation-induced DNA damage repair, as a consequence of PKCε and ZEB1 inhibition. Indeed, phenocopy experiments based on knock-down of either PKCε or ZEB1 reproduced miR-205 radiosensitizing effect, hence confirming a functional role of both targets in the process. At the molecular level, miR-205-induced suppression of PKCε counteracted radioresistance through the impairment of EGFR nuclear translocation and the consequent DNA-PK activation. Consistently, disruption of miR-205-PKCε 3'UTR pairing almost completely abrogated the radiosensitizing effect. CONCLUSIONS: Our results uncovered the molecular and cellular mechanisms underlying the radiosensitizing effect of miR-205. These findings support the clinical interest in developing a novel therapeutic approach based on miR-205 reconstitution to increase PCa response to radiotherapy.

Down-Regulation of the Androgen Receptor by G-Quadruplex Ligands Sensitizes Castration-Resistant Prostate Cancer Cells to Enzalutamide.

Stabilization of the G-quadruplexes (G4s) within the androgen receptor (AR) gene promoter to block transcription may represent an innovative approach to interfere with aberrant AR signaling in castration resistant prostate cancer (CRPC). A library of differently functionalized naphthalene diimides (NDIs) was screened for their ability to stabilize AR G4s: the core-extended NDI (7) stood out as the most promising ligand. AR-positive cells were remarkably sensitive to 7 in comparison to AR-negative CRCP or normal prostate epithelial cells; 7 induced remarkable impairment of AR mRNA and protein amounts and significant perturbations in the expression levels of KLK3 and of genes involved in the activation of AR program via feedback mechanisms. Moreover, 7 synergistically interacted with Enzalutamide, an inhibitor of AR signaling used in second-line therapies. Overall, our data show that stabilization of AR G4s may represent an alternative treatment options for CRPC and other malignancies relying on aberrant androgen signaling.

Naphthalene diimide-derivatives G-quadruplex ligands induce cell proliferation inhibition, mild telomeric dysfunction and cell cycle perturbation in U251MG glioma cells.

In the present paper, the biological effects of three different naphthalene diimides (NDIs) G-quadruplex (G4) ligands (H-NDI-Tyr, H-NDI-NMe2, and tetra-NDI-NMe2) were comparatively evaluated to those exerted by RHPS4, a well-characterized telomeric G4-ligand, in an in vitro model of glioblastoma. Data indicated that NDIs were very effective in blocking cell proliferation at nanomolar concentrations, although displaying a lower specificity for telomere targeting compared to RHPS4. In addition, differently from RHPS4, NDIs failed to enhance the effect of ionizing radiation, thus suggesting that additional targets other than telomeres could be involved in the strong NDI-mediated anti-proliferative effects. In order to test telomeric off-target action of NDIs, a panel of genes involved in tumor progression, DNA repair, telomere maintenance, and cell-cycle regulation were evaluated at transcriptional and translational level. Specifically, the compounds were able to cause a marked reduction of TERT and BCL2 amounts as well as to favor the accumulation of proteins involved in cell cycle control. A detailed cytofluorimetric analysis of cell cycle progression by means of bromodeoxyuridine (BrdU) incorporation and staining of phospho-histone H3 indicated that NDIs greatly reduce the progression through S-phase and lead to G1 accumulation of BrdU-positive cells. Taken together, these data indicated that, besides effects on telomeres and oncogenes such as Tert and Bcl2, nanomolar concentrations of NDIs determined a sustained block of cell proliferation by slowing down cell cycle progression during S-phase. In conclusion, our data indicate that NDIs G4-ligands are powerful antiproliferative agents, which act through mechanisms that ultimately lead to altered cell-cycle control.

miR-380-5p-mediated repression of TEP1 and TSPYL5 interferes with telomerase activity and favours the emergence of an "ALT-like" phenotype in diffuse malignant peritoneal mesothelioma cells.

BACKGROUND: Understanding the molecular/cellular underpinnings of diffuse malignant peritoneal mesothelioma (DMPM), a fatal malignancy with limited therapeutic options, is of utmost importance for the fruitful management of the disease. In this context, we previously found that telomerase activity (TA), which accounts for the limitless proliferative potential of cancer cells, is prognostic for disease relapse and cancer-related death in DMPM patients. Consequently, the identification of factors involved in telomerase activation/regulation may pave the way towards the development of novel therapeutic interventions for the disease. Here, the capability of miR-380-5p, a microRNA negligibly expressed in telomerase-positive DMPM clinical specimens, to interfere with telomerase-mediated telomere maintenance and, hence, with cancer cell growth was assessed on preclinical models of DMPM. METHODS: DMPM cells were transfected with a miR-380-5p synthetic precursor, and the effects of miRNA replacement were evaluated in terms of growing capability, induction of apoptosis and interference with TA. Reiterated weekly transfections were also performed in order to analyse the phenotype arising upon prolonged miR-380-5p reconstitution in DMPM cells. RESULTS: The ectopic expression of miR-380-5p elicited a remarkable inhibition of TA and resulted in DMPM cell growth impairment and apoptosis induction. In particular, we demonstrated for the first time that these effects were the result of a molecular circuitry converging on telomerase associated protein 1 (TEP1), where the miRNA was able to target the gene both directly in unconventional targeting modality and indirectly via p53 accumulation consequent to miRNA-mediated downregulation of testis-specific protein, Y-encoded-like 5 gene. Moreover, miR-380-5p did not cause telomere attrition and cell growth arrest in long-term DMPM transfectants, which in turn showed slightly elongated telomeres and molecular features (e.g. c-circle DNA and reduced expression levels of chromatin remodeler ATRX) resembling an alternative lengthening of telomeres (ALT) phenotype. CONCLUSIONS: miR-380-5p interferes with TA in DMPM cells by targeting TEP1. Notably, in the long-term setting, miR-380-5p-mediated impairment of TA did not result in telomere attrition. Instead, a phenotype reminiscent of ALT emerged in DMPM cells as possible compensatory pathway that safeguards DMPM cell growth, an event that may be regarded as a potential resistance mechanism to anticancer therapies based on telomerase inhibitors.

CPAM type 2-derived mesenchymal stem cells: Malignancy risk study in a 14-month-old boy.

INTRODUCTION: The association between congenital pulmonary airway malformations (CPAM) and malignancy is reported in the literature. Interactions between the tumor, immune, and mesenchymal stromal/stem cells (MSCs) have been recognized as crucial for understanding tumorigenesis. We characterized MSCs isolated from CPAM lesions in order to define potential malignancy risks. METHODS: CPAM II pulmonary tissue was used for MSC expansion; a "healthy" lung section from the same child was used as a comparator. Morphology, immunophenotype, differentiation and immunological capacity, proliferative growth, gene signature telomerase activity, and in vivo tumorigenicity in nude mice were evaluated. RESULTS: MSCs were successfully isolated and propagated from CPAM tissue. CPAM-MSCs presented the typical MSC morphology and phenotype, while exhibiting high proliferative capacity, reaching confluence at a median time of 5 days as well as differentiation capabilities. CPAM-MSCs at early passages were not neoplastic and chromosomally normal, even though unbalanced chromosomal rearrangements were noted by molecular karyotype. CONCLUSIONS: CPAM-MSCs exhibited specific features similar to tumor derived MSCs. Whilst there was no evidence of malignant transformation in the cystic tissue, our results provide evidence that this abnormal tissue has malignant potential. MSCs are considered important players in the tumor microenvironment and they have been closely linked to regulation of tumor survival, growth, and progression. Thus, early lesion resection also in asymptomatic patients might be indicated to exclude that the microenvironment may be potentially permissive to cancer development.

Synthesis and Superpotent Anticancer Activity of Tubulysins Carrying Non-hydrolysable N-Substituents on Tubuvaline.

Synthetic tubulysins 24 a-m, containing non-hydrolysable N-substituents on tubuvaline (Tuv), were obtained in high purity and good overall yields using a multistep synthesis. A key step was the formation of differently N-substituted Ile-Tuv fragments 10 by using an aza-Michael reaction of azido-Ile derivatives 8 with the α,ß-unsaturated oxo-thiazole 5. A structure-activity relationship study using a panel of human tumour cell lines showed strong anti-proliferative activity for all compounds 24 a-m, with IC50 values in the sub-nanomolar range, which were distinctly lower than those of tubulysin A, vinorelbine and paclitaxel. Furthermore, 24 a-m were able to overcome cross-resistance to paclitaxel and vinorelbine in two tumour cell lines with acquired resistance to doxorubicin. Compounds 24 e and 24 g were selected as leads to evaluate their mechanism of action. In vitro assays showed that both 24 e and 24 g interfere with tubulin polymerization in a vinca alkaloid-like manner and prevent paclitaxel-induced assembly of tubulin polymers. Both compounds exerted antimitotic activity and induced apoptosis in cancer cells at very low concentrations. Compound 24 e also exhibited potent antitumor activity at well tolerated doses on in vivo models of diffuse malignant peritoneal mesothelioma, such as MESOII peritoneal mesothelioma xenografts, the growth of which was not significantly affected by vinorelbine. These results indicate that synthetic tubulysins 24 could be used as standalone chemotherapeutic agents in difficult-to-treat cancers.

Emerging Role of G-quadruplex DNA as Target in Anticancer Therapy.

DNA has represented the most exploited target for the development of anticancer agents. It is now established that DNA may assume a variety of non-B conformations. This evidence has generated a total novel wave of interest in DNA as a cancer-associated target, since its distinct non-B structures may be regarded as sites for selective therapeutic intervention. G-quadruplexes are peculiar non-B DNA conformations that may form within guaninerich nucleic acid sequences. They are generated by a core of two or more vertically stacked G-quartets (i.e., the square planar arrangement of four guanine residues) held together by intervening loops of variable length. The evidence that G-quadruplexes are highly polymorphic and overrepresented within human genome points out at such non-B DNA conformations as druggable sites amenable of targeting by small molecules. In the present paper we will provide a concise overview on the emerging role of G-quadruplex structures forming within telomeres, gene promoters and mitochondrial DNA as a promising therapeutic target in cancer. In this context, a variety of small molecules has been documented to have excellent G-quadruplex binding/stabilizing properties and to exert good antiproliferative and antitumor activity in several in vitro and in vivo models of human cancers. Pieces of evidence indicate that targeting G-quadruplexes may represent an innovative and fascinating approach for the therapeutic management of the neoplastic disease. However, several issues still need to be addressed both at chemical and biological level before G-quadruplex-interacting molecules will turn out into effective therapeutic agents. Nevertheless, this has been an exciting, though sometime subdued, field of research over the last century. The continued improvements in methodologies and the development of specific tools will contribute not only to achieve the design and development of potentially novel anticancer approaches but also to deepen our knowledge of G-quadruplex biology and, consequently, of cancer at molecular level.

Targeting of RET oncogene by naphthalene diimide-mediated gene promoter G-quadruplex stabilization exerts anti-tumor activity in oncogene-addicted human medullary thyroid cancer.

Medullary thyroid cancer (MTC) relies on the aberrant activation of RET proto-oncogene. Though targeted approaches (i.e., tyrosine kinase inhibitors) are available, the absence of complete responses and the onset of resistance mechanisms indicate the need for novel therapeutic interventions. Due to their role in regulation of gene expression, G-quadruplexes (G4) represent attractive targets amenable to be recognized or stabilized by small molecules. Here, we report that exposure of MTC cells to a tri-substituted naphthalene diimide (NDI) resulted in a significant antiproliferative activity paralleled by inhibition of RET expression. Biophysical analysis and gene reporter assays showed that impairment of RET expression was consequent to the NDI-mediated stabilization of the G4 forming within the gene promoter. We also showed for the first time that systemic administration of the NDI in mice xenotransplanted with MTC cells resulted in a remarkable inhibition of tumor growth in vivo. Overall, our findings indicate that NDI-dependent RET G4 stabilization represents a suitable approach to control RET transcription and delineate the rationale for the development of G4 stabilizing-based treatments for MTC as well as for other tumors in which RET may have functional and therapeutic implications.

miR-342 overexpression results in a synthetic lethal phenotype in BRCA1-mutant HCC1937 breast cancer cells.

Expression of miR-342 has been strongly correlated with estrogen receptor (ER) status in breast cancer, where it is highest in ER-positive and lowest in triple-negative tumors. We investigated the effects of miR-342 transfection in the triple-negative breast cancer cell lines MDA-MB-231 and HCC1937, the latter carrying a germ-line BRCA1 mutation. Reconstitution of miR-342 led to caspase-dependent induction of apoptosis only in HCC1937 cells, while overexpression of wild-type BRCA1 in HCC1937 cells counteracted miR-342-mediated induction of apoptosis, suggesting that miR-342 overexpression and the lack of functional BRCA1 result in a synthetic lethal phenotype. Moreover, siRNA-mediated depletion of BRCA1 in MDA-MB-231 cells expressing the wild-type protein led to apoptosis upon transfection with miR-342. Using an in silico approach and a luciferase reporter system, we identified and functionally validated the Baculoviral IAP repeat-containing 6 gene (BIRC6), which encodes the anti-apoptotic factor Apollon/BRUCE, as a target of miR-342. In our model, BIRC6 likely acts as a determinant of the miRNA-dependent induction of apoptosis in BRCA1-mutant HCC1937 cells. Together, our findings suggest a tumor-suppressive function of miR-342 that could be exploited in the treatment of a subset of BRCA1-mutant hereditary breast cancers.

MicroRNAs and the Response of Prostate Cancer to Anti-Cancer Drugs.

Despite considerable advances in early diagnosis, prostate cancer (PCa) remains the second leading cause of cancer-related deaths in men in western countries. In fact, although efficient therapies exist for early-stage disease, the treatment of advanced PCa remains unsuccessful mainly due to its poor responsiveness to anti-cancer agents. This evidence underlines the urgent need for the development of novel and more effective therapeutic approaches. In this context, the documented dysregulation of microRNAs (miRNAs)--which are short non-coding RNAs that regulate gene expression at post-transcriptional level- in PCa, together with their potential to simultaneously regulate multiple oncogenic/ tumor-suppressive pathways, has stimulated interest in defining a functional association between altered expression of specific miRNAs and the response of PCa to anti-cancer agents. The purpose of this review is to provide an overview on PCa-related miRNAs as potential novel therapeutic targets/tools, with a special focus on the role that they may play in conditioning the responsiveness of PCa to anti-cancer drugs.

Double stranded promoter region of BRAF undergoes to structural rearrangement in nearly physiological conditions.

The folding of oncogene promoters into non-canonical DNA secondary structures is considered a strategy to control gene expression. Herein, we focused on a 30 bases sequence located upstream of the transcription start site of BRAF (Braf-176) that contains 80% of guanines. We analyzed the structural behavior of the G- and C-rich strands. By the use of spectroscopic and electrophoretic techniques we confirmed that they actually fold into a predominant antiparallel G-quadruplex and into an i-motif, respectively, and that they can coexist at nearly physiological conditions. Finally, the influence of several factors (KCl, pH, PEG200) on the conversion of the double stranded form of the oncogene promoter into the two above mentioned non-canonical structures has been explored.

Redox-Sensitive PEG-Polypeptide Nanoporous Particles for Survivin Silencing in Prostate Cancer Cells.

We report the engineering of intracellular redox-responsive nanoporous poly(ethylene glycol)-poly(l-lysine) particles (NPEG-PLLs). The obtained particles exhibit no toxicity while maintaining the capability to deliver a small interfering RNA sequence (siRNA) targeting the anti-apoptotic factor, survivin, in prostate cancer cells. The redox-mediated cleavage of the disulfide bonds stabilizing the NPEG-PLL-siRNA complex results in the release of bioactive siRNA into the cytosol of prostate cancer PC-3 cells, which, in turn, leads to the effective silencing (∼59 ± 8%) of the target gene. These findings, obtained under optimal conditions, indicate that NPEG-PLLs may protect the therapeutic nucleic acid in the extracellular and intracellular environments, thus preventing the occurrence of competitive interactions with serum and cytosolic proteins as well as degradation by RNase. The intracellular trafficking and final fate of the NPEG-PLLs were investigated by a combination of deconvolution microscopy, fluorescence lifetime imaging microscopy, and super-resolution structured illumination microscopy. A significant impairment of cell survival was observed in cells concomitantly exposed to paclitaxel and siRNA-loaded NPEG-PLLs. Overall, our findings indicate that NPEG-PLLs represent a highly loaded depot for the delivery of therapeutic nucleic acids to cancer cells.

Water-soluble isoindolo[2,1-a]quinoxalin-6-imines: in vitro antiproliferative activity and molecular mechanism(s) of action.

Water-soluble isoindoloquinoxalin (IIQ) imines and the corresponding acetates were conveniently prepared from the key intermediates 2-(2'-aminophenyl)-2H-isoindole-1-carbonitriles obtained by a Strecker reaction between substituted 1,2-dicarbaldehydes and 1,2-phenylenediamines. Both series were screened by the National Cancer Institute (Bethesda, MD) and showed potent antiproliferative activity against a panel of 60 human tumor cell lines. Several of the novel compounds showed GI50 values at a nanomolar level on the majority of the tested cell lines. Among IIQ derivatives, methoxy substituents at positions 3 and 8 or/and 9 were especially effective in impairing cell cycle progression and inducing apoptosis in cancer cells. These effects were associated to IIQ-mediated impairment of tubulin polymerization at pharmacologically significant concentrations of tested compounds. In addition, impaired DNA topoisomerase I functions and perturbation in telomere architecture were observed in cells exposed to micromolar concentrations of IIQ derivatives. The above results suggest that IIQ derivatives exhibit multi-target cytotoxic activities.