Targeting Cancer by Using Nanoparticles to Modulate RHO GTPase Signaling.

Functionalized nanomaterials have recently been introduced as efficient vehicles for targeted delivery of drugs and other tailored molecules to cancer cells. They emerge as new opportunities for addressing particular challenging targets such as RHO guanosine triphosphatases (GTPases), a group of signaling molecules involved in the progression of a variety of tumor types. RHO GTPases comprise a subfamily of the Ras superfamily of small GTPases. They are best known for their role in cell migration through the remodeling of the actin cytoskeleton. However, they are also key regulators of a broad number of cellular functions, ranging from proliferation to cell adhesion and differentiation. Not surprisingly, their dysregulation has been implicated in the development and progression of many types of cancer. The RHO GTPase subfamily includes 20 members that can be further separated into typical and atypical RHO GTPases. The typical RHO family members include the classical RHOA, RAC1 and CDC42 proteins, which cycle between an active GTP-bound and inactive GDP-bound conformation, under the coordinated action of three types of regulators: GEFs, GAPs and GDIs. Atypical RHO family members have small changes in key residues that alter their regulatory mechanisms. Nevertheless, both typical and atypical RHO GTPases contribute to cancer progression but, in contrast to Ras proteins, very few mutations have been found in tumors. In most cancers, it is the expression level and/or activity of RHO GTPases that is dysregulated. RHO GTPase signaling has thus long been seen as an attractive target for cancer treatment but their ubiquity and the lack of isoform-specific drugs have posed significant obstacles to the development of viable therapeutic strategies. Based on the success of recent nanomedicine approaches, this chapter reviews representative studies of how functionalized nanoparticles can be designed to target tumor-specific molecules and directly or indirectly modulate the expression and/or activity of particular RHO GTPases in cancer cells.

Vital Pulp Therapy in Permanent Mature Posterior Teeth with Symptomatic Irreversible Pulpitis: A Systematic Review of Treatment Outcomes.

Background and Objectives: Symptomatic irreversible pulpitis in permanent mature teeth is a common indication for nonsurgical root canal treatment (NSRCT), but contemporary studies have reported on vital pulp therapy (VPT) applied in such teeth as a less invasive treatment. This systematic review assessed the outcomes of VPT, including partial and full pulpotomy performed with hydraulic calcium silicate cements (HCSCs) in permanent mature posterior teeth diagnosed with symptomatic irreversible pulpitis. Materials and Methods: The PRISMA guidelines were followed. The search strategy included PubMed®, EMBASE, Cochrane library and grey literature electronic databases. The quality assessment of the identified studies followed the Cochrane Collaboration Risk of Bias, ROBINS-I and Newcastle-Ottawa Scale tools. Results: The search of primary databases identified 142 articles, of which 9 randomized controlled trials and 3 prospective cohort studies were selected for review. The risk-of-bias was assessed as 'high' or 'serious', 'fair', and 'low' for three, seven and two articles, respectively. One to five years after VPT using HCSCs, the success rates mostly ranged from 78 to 90%. Based on two articles, the outcomes of the VPT and NSRCT were comparable at one and five years. Despite the necessity for the intra-operative pulp assessment in VPT procedures, the majority of the studies did not fully report on this step or on the time needed to achieve hemostasis. Small sample sizes, of under 23 teeth, were reported in three studies. Conclusions: The reviewed 12 articles reported favorable outcomes of the VPT performed with HCSCs in permanent mature posterior teeth with symptomatic irreversible pulpitis, with radiographic success in the range of 81 to 90%. Two articles suggested comparable outcomes of the VPT and root canal treatment. Universal case selection and outcome criteria needs to be established for VPT when considered as an alternative to NSRCT. This evidence supports the need for further research comparing longer-term outcomes of both of the treatment modalities.

Long-Term Prognosis of Endodontic Microsurgery-A Systematic Review and Meta-Analysis.

Background and objectives: The long-term outcome of endodontic microsurgery (EMS) performed on root-filled teeth affected by post-treatment apical periodontitis (AP) has been a matter of debate, re-launched by the introduction of novel root-end filling materials which have been proven to improve the short-term outcome of EMS. The purpose of this systematic review and meta-analysis is to evaluate the clinical and radiographic long-term outcome of endodontic microsurgery in teeth diagnosed with secondary AP through radiographic evaluation. Materials and Methods: This systematic review and meta-analysis followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Inclusion and exclusion criteria were defined a priori to select the best longitudinal evidence. Only randomized clinical trials (RCT) and prospective clinical studies (PCS), with a follow-up ≥ 2-year, and exhibiting well-established clinical and radiographic outcome criteria, were selected. Results: A total of 573 articles were obtained, from which 10 fulfill inclusion criteria: 6 PCS and 4 RCT. Meta-analysis showed a pooled proportion of success rate of 91.3%, from an overall amount of 453 treated teeth included in RCT; from overall 839 included teeth in PCS, a pooled success rate of 78.4% was observed, with the follow-up time ranging from 2 to 13-years. Survival rate outcomes varied from 79 to 100% for the same follow-up period. Five prognostic factors with influence on the outcome were disclosed: smoking habits, tooth location and type, absence/presence of dentinal defects, interproximal bone level, and root-end filling material. Conclusions: High success rates and predictable results can be expected when EMS is performed by trained endodontists, allowing good prognosis and preservation of teeth affected by secondary AP.

The third dimension: new developments in cell culture models for colorectal research.

Cellular models are important tools in various research areas related to colorectal biology and associated diseases. Herein, we review the most widely used cell lines and the different techniques to grow them, either as cell monolayer, polarized two-dimensional epithelia on membrane filters, or as three-dimensional spheres in scaffold-free or matrix-supported culture conditions. Moreover, recent developments, such as gut-on-chip devices or the ex vivo growth of biopsy-derived organoids, are also discussed. We provide an overview on the potential applications but also on the limitations for each of these techniques, while evaluating their contribution to provide more reliable cellular models for research, diagnostic testing, or pharmacological validation related to colon physiology and pathophysiology.

Phosphorylation of SRSF1 by SRPK1 regulates alternative splicing of tumor-related Rac1b in colorectal cells.

The premessenger RNA of the majority of human genes can generate various transcripts through alternative splicing, and different tissues or disease states show specific patterns of splicing variants. These patterns depend on the relative concentrations of the splicing factors present in the cell nucleus, either as a consequence of their expression levels or of post-translational modifications, such as protein phosphorylation, which are determined by signal transduction pathways. Here, we analyzed the contribution of protein kinases to the regulation of alternative splicing variant Rac1b that is overexpressed in certain tumor types. In colorectal cells, we found that depletion of AKT2, AKT3, GSK3ß, and SRPK1 significantly decreased endogenous Rac1b levels. Although knockdown of AKT2 and AKT3 affected only Rac1b protein levels suggesting a post-splicing effect, the depletion of GSK3ß or SRPK1 decreased Rac1b alternative splicing, an effect mediated through changes in splicing factor SRSF1. In particular, the knockdown of SRPK1 or inhibition of its catalytic activity reduced phosphorylation and subsequent translocation of SRSF1 to the nucleus, limiting its availability to promote the inclusion of alternative exon 3b into the Rac1 pre-mRNA. Altogether, the data identify SRSF1 as a prime regulator of Rac1b expression in colorectal cells and provide further mechanistic insight into how the regulation of alternative splicing events by protein kinases can contribute to sustain tumor cell survival.

Pro-Inflammatory Cytokines Trigger the Overexpression of Tumour-Related Splice Variant RAC1B in Polarized Colorectal Cells.

An inflammatory microenvironment is a tumour-promoting condition that provides survival signals to which cancer cells respond with gene expression changes. One example is the alternative splicing variant Rat Sarcoma Viral Oncogene Homolog (Ras)-Related C3 Botulinum Toxin Substrate 1 (RAC1)B, which we previously identified in a subset of V-Raf Murine Sarcoma Viral Oncogene Homolog B (BRAF)-mutated colorectal tumours. RAC1B was also increased in samples from inflammatory bowel disease patients or in an acute colitis mouse model. Here, we used an epithelial-like layer of polarized Caco-2 or T84 colorectal cancer (CRC) cells in co-culture with fibroblasts, monocytes or macrophages and analysed the effect on RAC1B expression in the CRC cells by RT-PCR, Western blot and confocal fluorescence microscopy. We found that the presence of cancer-associated fibroblasts and M1 macrophages induced the most significant increase in RAC1B levels in the polarized CRC cells, accompanied by a progressive loss of epithelial organization. Under these conditions, we identified interleukin (IL)-6 as the main trigger for the increase in RAC1B levels, associated with Signal Transducer and Activator of Transcription (STAT)3 activation. IL-6 neutralization by a specific antibody abrogated both RAC1B overexpression and STAT3 phosphorylation in polarized CRC cells. Our data identify that pro-inflammatory extracellular signals from stromal cells can trigger the overexpression of tumour-related RAC1B in polarized CRC cells. The results will help to understand the tumour-promoting effect of inflammation and identify novel therapeutic strategies.

Subcutaneous Implantation Assessment of New Calcium-Silicate Based Sealer for Warm Obturation.

Calcium silicate-based sealers were recently introduced as a new class of endodontic sealers, with potential further benefits due to their bioactivity. The aim of this study was to evaluate the biocompatibility of two new hydraulic calcium silicate-based sealers, TotalFill BC Sealer (FKG, La Chaux-des-Fonds, Switzerland) and TotalFill BC Sealer HiFlow (FKG, La Chaux-des-Fonds, Switzerland) through subcutaneous implantation in connective tissue of rats. Subcutaneous implantation was performed in 16 young Wistar rats. Four polyethylene tubes were implanted in each animal, one empty to serve as a control, and three filled with tested sealers: AH Plus as reference (Dentsply DeTrey, Konstanz, Germany), TotalFill BC Sealer (BC) and TotalFill BC Sealer HiFlow (HiFlow). Eight rats were euthanized at 8 days and the remaining eight at 30 days. Hematoxylin-eosin staining was used to score the inflammatory reaction, macrophage infiltrate and to measure the thickness of the fibrous capsule. von Kossa staining was performed to evaluate the mineralization level. Kruskal-Wallis test followed by Dunn's post hoc test was used to analyze non-parametric data. To analyze the influence of the implantation time within each material, a Mann-Whitney U test was performed. At eight days post-implantation, AH Plus induced a more intense inflammatory reaction when compared both with the control (p ≤ 0.001) and BC (p ≤ 0.01). HiFlow presented a higher score of macrophage infiltrate than control (p ≤ 0.01) and BC (p ≤ 0.05). The fibrous capsule thickness in this period was significantly higher for the BC group when compared to control (p ≤ 0.01) and AH Plus (p ≤ 0.05). The mineralization potential was higher for the HiFlow group when compared with the control (p ≤ 0.001) and AH Plus (p ≤ 0.001). At 30 days post-implantation, the score for the inflammatory reaction remained higher for the AH Plus group when compared both to control (p ≤ 0.01) and BC (p ≤ 0.001). The macrophage infiltrate of the HiFlow was significantly higher than control (p ≤ 0.001) and AH Plus groups (p ≤ 0.01), additionally, the fibrous capsule of the BC (p ≤ 0.001) and HiFlow (p ≤ 0.01) groups were both thicker than control. Mineralization potential was observed only on BC (p ≤ 0.05) and HiFlow groups (p ≤ 0.001), when compared to control). BC exhibited the best biocompatibility performance of all tested sealers and HiFlow provided the greatest induction of mineralized tissues. Both TotalFill BC Sealer and TotalFill BC Sealer HiFlow are biocompatible and show potential bioactivity when implanted in the subcutaneous tissue. Bioactivity was not found in AH Plus.

Cytotoxicity and genotoxicity of MWCNT-7 and crocidolite: assessment in alveolar epithelial cells versus their coculture with monocyte-derived macrophages.

In the past years, several in vitro studies have addressed the pulmonary toxicity of multi-walled carbon nanotubes (MWCNT) and compared it with that caused by asbestos fibers, but their conclusions have been somewhat inconsistent and difficult to extrapolate to in vivo. Since cell coculture models were proposed to better represent the in vivo conditions than conventional monocultures, this work intended to compare the cytotoxicity and genotoxicity of MWCNT-7 (Mitsui-7) and crocidolite using A549 cells grown in a conventional monoculture or in coculture with THP-1 macrophages. Although a decrease in A549 viability was noted following exposure to a concentration range of MWCNT-7 and crocidolite, no viability change occurred in similarly exposed cocultures. Early events indicating epithelial to mesenchymal transition (EMT) were observed which could explain apoptosis resistance. The comet assay results were similar between the two models, being positive and negative for crocidolite and MWCNT-7, respectively. An increase in the micronucleus frequency was detected in the cocultured A549-treated cells with both materials, but not in the monoculture. On the other hand, exposure of A549 monocultures to MWCNT-7 induced a highly significant increase in nucleoplasmic bridges in which those were found embedded. Our overall results demonstrate that (i) both materials are cytotoxic and genotoxic, (ii) the presence of THP-1 macrophages upholds the viability of A549 cells and increases the aneugenic/clastogenic effects of both materials probably through EMT, and (iii) MWCNT-7 induces the formation of nucleoplasmic bridges in A549 cells.

Signaling Pathways Driving Aberrant Splicing in Cancer Cells.

Aberrant profiles of pre-mRNA splicing are frequently observed in cancer. At the molecular level, an altered profile results from a complex interplay between chromatin modifications, the transcriptional elongation rate of RNA polymerase, and effective binding of the spliceosome to the generated transcripts. Key players in this interplay are regulatory splicing factors (SFs) that bind to gene-specific splice-regulatory sequence elements. Although mutations in genes of some SFs were described, a major driver of aberrant splicing profiles is oncogenic signal transduction pathways. Signaling can affect either the transcriptional expression levels of SFs or the post-translational modification of SF proteins, and both modulate the ratio of nuclear versus cytoplasmic SFs in a given cell. Here, we will review currently known mechanisms by which cancer cell signaling, including the mitogen-activated protein kinases (MAPK), phosphatidylinositol 3 (PI3)-kinase pathway (PI3K) and wingless (Wnt) pathways but also signals from the tumor microenvironment, modulate the activity or subcellular localization of the Ser/Arg rich (SR) proteins and heterogeneous nuclear ribonucleoproteins (hnRNPs) families of SFs.

A molecular switch in the scaffold NHERF1 enables misfolded CFTR to evade the peripheral quality control checkpoint.

The peripheral protein quality control (PPQC) checkpoint removes improperly folded proteins from the plasma membrane through a mechanism involving the E3 ubiquitin ligase CHIP (carboxyl terminus of Hsc70 interacting protein). PPQC limits the efficacy of some cystic fibrosis (CF) drugs, such as VX-809, that improve trafficking to the plasma membrane of misfolded mutants of the CF transmembrane conductance regulator (CFTR), including F508del-CFTR, which retains partial functionality. We investigated the PPQC checkpoint in lung epithelial cells with F508del-CFTR that were exposed to VX-809. The conformation of the scaffold protein NHERF1 (Na(+)/H(+) exchange regulatory factor 1) determined whether the PPQC recognized "rescued" F508del-CFTR (the portion that reached the cell surface in VX-809-treated cells). Activation of the cytoskeletal regulator Rac1 promoted an interaction between the actin-binding adaptor protein ezrin and NHERF1, triggering exposure of the second PDZ domain of NHERF1, which interacted with rescued F508del-CFTR. Because binding of F508del-CFTR to the second PDZ of NHERF1 precluded the recruitment of CHIP, the coexposure of airway cells to Rac1 activator nearly tripled the efficacy of VX-809. Interference with the NHERF1-ezrin interaction prevented the increase of efficacy of VX-809 by Rac1 activation, but the actin-binding domain of ezrin was not required for the increase in efficacy. Thus, rather than mainly directing anchoring of F508del-CFTR to the actin cytoskeleton, induction of ezrin activation by Rac1 signaling triggered a conformational change in NHERF1, which was then able to bind and stabilize misfolded CFTR at the plasma membrane. These insights into the cell surface stabilization of CFTR provide new targets to improve treatment of CF.