microRNAs (miRNAs) are small noncoding RNAs that regulate gene expression by targeting messenger RNA (mRNA) transcripts. Recently, a miRNA expression profile of human tumors has been characterized by ...an overall miRNA downregulation. Explanations for this observation include a failure of miRNA post-transcriptional regulation, transcriptional silencing associated with hypermethylation of CpG island promoters and miRNA transcriptional repression by oncogenic factors. Another possibility is that the enzymes and cofactors involved in miRNA processing pathways may themselves be targets of genetic disruption, further enhancing cellular transformation. However, no loss-of-function genetic alterations in the genes encoding these proteins have been reported. Here we have identified truncating mutations in TARBP2 (TAR RNA-binding protein 2), encoding an integral component of a DICER1-containing complex, in sporadic and hereditary carcinomas with microsatellite instability. The presence of TARBP2 frameshift mutations causes diminished TRBP protein expression and a defect in the processing of miRNAs. The reintroduction of TRBP in the deficient cells restores the efficient production of miRNAs and inhibits tumor growth. Most important, the TRBP impairment is associated with a destabilization of the DICER1 protein. These results provide, for a subset of human tumors, an explanation for the observed defects in the expression of mature miRNAs.
Within tumors, Cancer Stem Cell (CSC) subpopulation has an important role in maintaining growth and dissemination while preserving high resistance against current treatments. It has been shown that, ...when CSCs are eliminated, the surrounding Differentiated Cancer Cells (DCCs) may reverse their phenotype and gain CSC-like features to preserve tumor progression and ensure tumor survival. This strongly suggests the existence of paracrine communication within tumor cells. It is evidenced that the molecular crosstalk is at least partly mediated by Extracellular Vesicles (EVs), which are cell-derived membranous nanoparticles that contain and transport complex molecules that can affect and modify the biological behavior of distal cells and their molecular background. This ability of directional transport of small molecules prospects EVs as natural Drug Delivery Systems (DDS). EVs present inherent homing abilities and are less immunogenic than synthetic nanoparticles, in general. Currently, strong efforts are focused into the development and improvement of EV-based DDS. Even though EV-DDS have already reached early phases in clinical trials, their clinical application is still far from commercialization since protocols for EVs loading, modification and isolation need to be standardized for large-scale production. Here, we summarized recent knowledge regarding the use of EVs as natural DDS against CSCs and cancer resistance.
Colorectal cancer (CRC) is a heterogeneous disease with high incidence and mortality worldwide. The efficacy of conventional CRC chemotherapy is hampered by poor drug solubility and bioavailability ...and suboptimal pharmacokinetic profiles. In this work, camptothecin (CPT), a potent anticancer drug, was loaded into an amphiphilic chitosan modified with PEG and oleic acid, to reduce CRC progression after oral administration. While CPT-loaded micelles presented anticancer activity against HCT116, Caco-2 and HT29 CRC cell lines in vitro, empty micelles demonstrated a safe profile when incubated with human blood cells and colorectal cancer cell lines. In a more complex 3D CRC multicellular spheroid model, CPT-loaded micelles also exhibited a significant effect on the spheroid's metabolic activity and size reduction. Remarkably, in vivo studies performed in a HCT116 xenograft model, showed a significant reduction on the tumor growth during and after treatment with CPT-loaded micelles. Moreover, in a more biological relevant in vivo model of chemically-induced CRC, orally administered CPT-loaded micelles demonstrated a significant reduction on tumor incidence and inflammation signs. The findings here reported indicate that CPT-loaded into chitosan-based micelles, by improving drug solubility, alongside its safety profile for normal tissues, may have a promising role CRC chemotherapy.
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•CPT-loaded micelles demonstrated in vitro anticancer activity against CRC cancer cell lines and 3D intestinal spheroids.•In an in vivo xenograft model, chitosan micelles were able to delay tumor growth.•In a CRC animal model, drug-loaded micelles decreased tumor development and progression after oral administration.
The global impairment of mature microRNAs (miRNAs) is emerging as a common feature of human tumors. One interesting scenario is that defects in the nuclear export of precursor miRNAs (pre-miRNAs) ...might occur in transformed cells. Exportin 5 (XPO5) mediates pre-miRNA nuclear export and herein we demonstrate the presence of XPO5-inactivating mutations in a subset of human tumors with microsatellite instability. The XPO5 genetic defect traps pre-miRNAs in the nucleus, reduces miRNA processing, and diminishes miRNA-target inhibition. The XPO5 mutant form lacks a C-terminal region that contributes to the formation of the pre-miRNA/XPO5/Ran-GTP ternary complex and pre-miRNAs accumulate in the nucleus. Most importantly, the restoration of XPO5 functions reverses the impaired export of pre-miRNAs and has tumor-suppressor features.
► Exportin-5 inactivating mutations occur in tumors with microsatellite instability ► Exportin-5 mutant form traps precursor microRNAs in the nucleus ► Exportin-5 mutations decrease miRNA processing efficiency and target inhibition ► Exportin-5 shows tumor-suppressor features in vitro and in vivo
Advanced cancer is still considered an incurable disease because of its metastatic spread to distal organs and progressive gain of chemoresistance. Even though considerable treatment progress and ...more effective therapies have been achieved over the past years, recurrence in the long-term and undesired side effects are still the main drawbacks of current clinical protocols. Moreover, a majority of chemotherapeutic drugs are highly hydrophobic and need to be diluted in organic solvents, which cause high toxicity, in order to reach effective therapeutic dose. These limitations of conventional cancer therapies prompted the use of nanomedicine, the medical application of nanotechnology, to provide more effective and safer cancer treatment. Potential of nanomedicines to overcome resistance, ameliorate solubility, improve pharmacological profile, and reduce adverse effects of chemotherapeutical drugs is thus highly regarded. Their use in the clinical setting has increased over the last decade. Among the various existing nanosystems, nanoparticles have the ability to transform conventional medicine by reducing the adverse effects and providing a controlled release of therapeutic agents. Also, their small size facilitates the intracellular uptake. Here, we provide a closer review of clinical prospects and mechanisms of action of nanomedicines to overcome drug resistance. The significance of specific targeting towards cancer cells is debated as well.
•F127 is efficiently carboxylated through the maleic anhydride method (PM F127:COOH).•Carboxylated F127 is efficiently conjugated with Cetuximab (PM F127:COOH:Cet).•Gelatin allows the ...complexation/transfection of siRNA (F127:COOH:Gelatin:siRNA:Cet).•F127:COOH:Gelatin:siRNA:Cet shows high silencing efficacy without cell toxicity.•Cetuximab functionalized PM internalized faster in EGFR overexpressing cancer cells.
New polymeric biomaterials and nanomedicines targeting cancer cells are highly required because of their potential clinical applications. Gene therapy enjoys high popularity as advanced therapy due to its high specificity; however, clinical translation is scarce because the lack of efficient and safe delivery methods. Here we address the design and development of a new nanosized targeting system consisting on Cetuximab-conjugated micelles (PM) of Pluronic® F127 (F127) and Gelatin for efficient delivery of small interfering RNA (siRNA) into epidermal growth factor receptor (EGFR) overexpressing breast cancer cells. Chemical modification by carboxylation of F127 is required prior to conjugation with Cetuximab and PM development. PM presenting appropriate physicochemical features (<40 nm) and an efficiently modified surface with Cetuximab were generated. Gelatin garnished the system with the cationic groups (ζ = +30 mV) essential for the complexation of siRNA, allowing high entrapment efficiency, and an efficient gene silencing of 70% with low cytotoxicity and proper hemocompatibility. Cetuximab-functionalized PM showed higher rate of cell internalization in EGFR expressing cells than non-functionalized PM, making them a new promising tool for targeted breast cancer treatment.
More than 50% of patients with Dukes C colorectal cancer have disease recurrence and die within 5 years after surgical removal of their primary tumor. It is currently not possible to distinguish ...patients with good and bad prognosis. SMAD4 is an important tumor suppressor gene that mediates transforming growth factor-beta superfamily signaling and is located in chromosome 18q21, a region with frequent genetic losses in these tumors. Allelic imbalance in 18q has been linked to poor prognosis in a subset of colorectal cancer patients. Therefore, we generated a tissue microarray containing triplicate tumor samples from 86 Dukes C patients and used immunohistochemistry to assess the relative expression level of SMAD4 and its value as a prognostic marker. In addition, SMAD4 was screened for mutations and two polymorphic microsatellite markers were used to assess the presence of allelic imbalance in these tumors. Patients with tumors expressing high SMAD4 levels had significantly better overall (P < 0.025) and disease-free (P < 0.013) survival than patients with low levels. This identifies SMAD4 as a prognostic marker for Dukes C colorectal cancer. Although all tumors with absent SMAD4 staining showed allelic imbalance in 18q21, tumors with 18q21 allelic imbalance as a group showed no difference in SMAD4 levels compared with tumors without allelic imbalance, suggesting that additional mechanisms of SMAD4 down-regulation exist. In addition, although SMAD4 mutations were found in five tumors, they were not associated with shorter survival. In conclusion, the level of expression of SMAD4 was found to be a more sensitive marker than 18q21 allelic imbalance and SMAD4 mutations, which were of no prognostic significance for these patients.
Obtaining high levels of pure proteins remains the main bottleneck of many scientific and biotechnological studies. Among all the available recombinant expression systems, Escherichia coli ...facilitates gene expression by its relative simplicity, inexpensive and fast cultivation, well-known genetics and the large number of tools available for its biotechnological application. However, recombinant expression in E. coli is not always a straightforward procedure and major obstacles are encountered when producing many eukaryotic proteins and especially membrane proteins, linked to missing posttranslational modifications, proteolysis and aggregation. In this context, many conventional and unconventional eukaryotic hosts are under exploration and development, but in some cases linked to complex culture media or processes. In this context, alternative bacterial systems able to overcome some of the limitations posed by E. coli keeping the simplicity of prokaryotic manipulation are currently emerging as convenient hosts for protein production. We have comparatively produced a “difficult-to-express” human protein, the lysosomal enzyme alpha-galactosidase A (hGLA) in E. coli and in the psychrophilic bacterium Pseudoalteromonas haloplanktis TAC125 cells (P. haloplanktis TAC125). While in E. coli the production of active hGLA was unreachable due to proteolytic instability and/or protein misfolding, the expression of hGLA gene in P. haloplanktis TAC125 allows obtaining active enzyme. These results are discussed in the context of emerging bacterial systems for protein production that represent appealing alternatives to the regular use of E. coli and also of more complex eukaryotic systems.
Aggresomes are protein aggregates found in mammalian cells when the intracellular protein degradation machinery is over-titered. Despite that they abound in cells producing recombinant proteins of ...biomedical and biotechnological interest, the physiological roles of these protein clusters and the functional status of the embedded proteins remain basically unexplored. In this work, we have determined for the first time that, like in bacterial inclusion bodies, deposition of recombinant proteins into aggresomes does not imply functional inactivation. As a model, human α-galactosidase A (GLA) has been expressed in mammalian cells as enzymatically active, mechanically stable aggresomes showing higher thermal stability than the soluble GLA version. Since aggresomes are easily produced and purified, we propose these particles as novel functional biomaterials with potential as carrier-free, self-immobilized catalyzers in biotechnology and biomedicine.
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