Stable Dual miR-143 and miR-506 Upregulation Inhibits Proliferation and Cell Cycle Progression.

The mainstays of lung cancer pathogenesis are cell cycle progression dysregulation, impaired apoptosis, and unregulated cell proliferation. While individual microRNA (miR) targeting or delivering is a promising approach that has been extensively studied, combination of miR targeting can enhance therapeutic efficacy and overcome limitations present in individual miR regulations. We previously reported on the use of a miR-143 and miR-506 combination via transient transfections against lung cancer. In this study, we evaluated the effect of miR-143 and miR-506 under stable deregulations in A549 lung cancer cells. We used lentiviral transductions to either up- or downregulate the two miRs individually or in combination. The cells were sorted and analyzed for miR deregulation via qPCR. We determined the miR deregulations' effects on the cell cycle, cell proliferation, cancer cell morphology, and cell motility. Compared to the individual miR deregulations, the combined miR upregulation demonstrated a miR-expression-dependent G2 cell cycle arrest and a significant increase in the cell doubling time, whereas the miR-143/506 dual downregulation demonstrated increased cellular motility. Furthermore, the individual miR-143 and miR-506 up- and downregulations exhibited cellular responses lacking an apparent miR-expression-dependent response in the respective analyses. Our work here indicates that, unlike the individual miR upregulations, the combinatorial miR treatment remained advantageous, even under prolonged miR upregulation. Finally, our findings demonstrate potential advantages of miR combinations vs. individual miR treatments.

Murine Dermal Lymphatic Endothelial Cell Isolation.

The lymphatic system participates in the regulation of immune surveillance, lipid absorption, and tissue fluid balance. The isolation of murine lymphatic endothelial cells is an important process for lymphatic research, as it allows the performance of in vitro and biochemical experiments on the isolated cells. Moreover, the development of Cre-lox technology has enabled the tissue-specific deficiency of genes that cannot be globally targeted, leading to the precise determination of their role in the studied tissues. The dissection of the role of certain genes in lymphatic physiology and pathophysiology requires the use of lymphatic-specific promoters, and thus, the experimental verification of the expression levels of the targeted genes. Methods for efficient isolation of lymphatic endothelial cells from wild-type or transgenic mice enable the use of ex vivo and in vitro assays to study the mechanisms regulating the lymphatic functions and the identification of the expression levels of the studied proteins. We have developed, standardized and present a protocol for the efficient isolation of murine dermal lymphatic endothelial cells (DLECs) via magnetic bead purification based on LYVE-1 expression. The protocol outlined aims to equip researchers with a tool to further understand and elucidate important players of lymphatic endothelial cell functions, especially in facilities where fluorescence-activated cell sorting equipment is not available.

Targeting endothelial permeability in the EPR effect.

The characteristics of the primary tumor blood vessels and the tumor microenvironment drive the enhanced permeability and retention (EPR) effect, which confers an advantage towards enhanced delivery of anti-cancer nanomedicine and has shown beneficial effects in preclinical models. Increased vascular permeability is a landmark feature of the tumor vessels and an important driver of the EPR. The main focus of this review is the endothelial regulation of vascular permeability. We discuss current challenges of targeting vascular permeability towards clinical translation and summarize the structural components and mechanisms of endothelial permeability, the principal mediators and signaling players, the targeted approaches that have been used and their outcomes to date. We also critically discuss the effects of the tumor-infiltrating immune cells, their interplay with the tumor vessels and the impact of immune responses on nanomedicine delivery, the impact of anti-angiogenic and tumor-stroma targeting approaches, and desirable nanoparticle design approaches for greater translational benefit.

Lyophilized liposomal formulation of a peptidomimetic-Dox conjugate for HER2 positive breast and lung cancer.

Nanocarrier-mediated administration of chemotherapeutic drugs can increase the therapeutic index of drugs by reducing off-target site toxicity. Ligand-targeted drug delivery can be utilized to deliver chemotherapeutic drugs to cancer cells selectively and specifically. Here we report the evaluation of a lyophilized formulation of a liposome containing a peptidomimetic-doxorubicin conjugate for targeted delivery of doxorubicin to HER2-positive cancer cells. The lyophilized liposomal formulation exhibited improved release of peptidomimetic-doxorubicin conjugate at pH 6.5 compared to 7.4 and improved cellular uptake in cancer cells at pH 6.5. In vivo studies indicated that pH-sensitive formulation exhibited site-specific formulation delivery and improved anticancer efficacy than free doxorubicin. The findings suggested that combining a lyophilized pH-sensitive liposomal formulation containing trehalose as lyoprotectant with a targeting ligand coupled cytotoxic agent is a potential method for cancer chemotherapy while maintaining long-term stability at 4 °C of the liposome formulation.

Endothelial-Specific Targeting of RhoA Signaling via CD31 Antibody-Conjugated Nanoparticles.

Existing vascular endothelial growth factor-oriented antiangiogenic approaches are known for their high potency. However, significant side effects associated with their use drive the need for novel antiangiogenic strategies. The small GTPase RhoA is an established regulator of actin cytoskeletal dynamics. Previous studies have highlighted the impact of endothelial RhoA pathway on angiogenesis. Rho-associate kinase (ROCK), a direct RhoA effector, is potently inhibited by Fasudil, a clinically relevant ROCK inhibitor. Here, we aimed to target the RhoA signaling in endothelial cells by generating Fasudil-encapsulated CD31-targeting liposomes as a potential antiangiogenic therapy. The liposomes presented desirable characteristics, preferential binding to CD31-expressing HEK293T cells and to endothelial cells, inhibited stress fiber formation and cytoskeletal-related morphometric parameters, and inhibited in vitro angiogenic functions. Overall, this work shows that the nanodelivery-mediated endothelial targeting of RhoA signaling can offer a promising strategy for angiogenesis inhibition in vascular-related diseases. SIGNIFICANCE STATEMENT: Systemic administration of antiangiogenic therapeutics induces side effects to non-targeted tissues. This study, among others, has shown the impact of the RhoA signaling in the endothelial cells and their angiogenic functions. Here, to minimize potential toxicity, this study generated CD31-targeting liposomes with encapsulated Fasudil, a clinically relevant Rho kinase inhibitor, and successfully targeted endothelial cells. In this proof-of-principle study, the efficient Fasudil delivery, its impact on the endothelial signaling, morphometric alterations, and angiogenic functions verify the benefits of site-targeted antiangiogenic therapy.

Chlorotoxin and Lung Cancer: A Targeting Perspective for Drug Delivery.

In the generational evolution of nano-based drug delivery carriers, active targeting has been a major milestone for improved and selective drug accumulation in tissues and cell types beyond the existing passive targeting capabilities. Among the various active targeting moieties, chlorotoxin, a peptide extracted from scorpions, demonstrated promising tumor cell accumulation and selection. With lung cancer being among the leading diagnoses of cancer-related deaths in both men and women, novel therapeutic methodologies utilizing nanotechnology for drug delivery emerged. Given chlorotoxin's promising biological activity, we explore its potential against lung cancer and its utilization for active targeting against this cancer's tumor cells. Our analysis indicates that despite the extensive chlorotoxin's research against glioblastoma, lung cancer research with the molecule has been limited, despite some promising early results.

MiRNAs as Anti-Angiogenic Adjuvant Therapy in Cancer: Synopsis and Potential.

Angiogenesis is a key mechanism for tumor growth and metastasis and has been a therapeutic target for anti-cancer treatments. Intensive vascular growth is concomitant with the rapidly proliferating tumor cell population and tumor outgrowth. Current angiogenesis inhibitors targeting either one or a few pro-angiogenic factors or a range of downstream signaling molecules provide clinical benefit, but not without significant side effects. miRNAs are important post-transcriptional regulators of gene expression, and their dysregulation has been associated with tumor progression, metastasis, resistance, and the promotion of tumor-induced angiogenesis. In this mini-review, we provide a brief overview of the current anti-angiogenic approaches, their molecular targets, and side effects, as well as discuss existing literature on the role of miRNAs in angiogenesis. As we highlight specific miRNAs, based on their activity on endothelial or cancer cells, we discuss their potential for anti-angiogenic targeting in cancer as adjuvant therapy and the importance of angiogenesis being evaluated in such combinatorial approaches.

Oral Delivery of Nucleic Acids with Passive and Active Targeting to the Intestinal Tissue Using Polymer-Based Nanocarriers.

Despite the apparent advantages for long-term treatment and local therapies against intestinal diseases, the oral delivery of nucleic acids has been challenging due to unfavorable physiological conditions for their stability. In this study, a novel nanodelivery system of PEG-PCL nanoparticles with encapsulated nucleic acids-mannosylated PEI (Man-PEI) complexes was developed for intestinal delivery. We complexed model nucleic acids with Man-PEI at the optimal N/P ratio of 20:1 for in vitro and in vivo analyses. Cells were transfected in vitro and analyzed for gene expression, receptor-mediated uptake, and PEG-PCL nanoparticles' toxicity. We also evaluated the nucleic acid's stability in the nanocarrier during formulation, and under simulated gastrointestinal environments or the presence of nucleases. Finally, we assessed the biodistribution for the PEG-PCL nanoparticles with encapsulated complexes and their ability to transfect intestinal cells in vivo. Nucleic acids complexed with Man-PEI were protected from degradation against nucleases. In comparison to the parent compound PEI, Man-PEI transfected the cells with an overall higher potency. Competition assay indicated receptor-mediated endocytosis promoted by mannose receptors. The PEG-PCL nanoparticles with Man-PEI/plasmid complexes indicated minimal cytotoxicity. The nanocarrier successfully protected the complexes in a simulated gastric fluid environment and released them in a simulated intestinal fluid environment, promoted by the presence of lipases. The oral administration of the PEG-PCL nanoparticles with encapsulated Man-PEI/plasmid complexes transfected intestinal cells with the plasmid in vivo, while presenting a time-dependent progression through the intestines. Conclusively, our carrier system can deliver genetic material to the GI tract and actively target mannose receptor overexpressing cells.

Advanced bioinformatic analysis and pathway prediction of NSCLC cells upon cisplatin resistance.

This study aims to identify pathway involvement in the development of cisplatin (cis-diamminedichloroplatinum (II); CDDP) resistance in A549 lung cancer (LC) cells by utilizing advanced bioinformatics software. We developed CDDP-resistant A549 (A549/DDP) cells through prolonged incubation with the drug and performed RNA-seq on RNA extracts to determine differential mRNA and miRNA expression between A549/DDP and A549 cells. We analyzed the gene dysregulation with Ingenuity Pathway Analysis (IPA; QIAGEN) software. In contrast to prior research, which relied on the clustering of dysregulated genes to pathways as an indication of pathway activity, we utilized the IPA software for the dynamic evaluation of pathway activity depending on the gene dysregulation levels. We predicted 15 pathways significantly contributing to the chemoresistance, with several of them to have not been previously reported or analyzed in detail. Among them, the PKR signaling, cholesterol biosynthesis, and TEC signaling pathways are included, as well as genes, such as PIK3R3, miR-34c-5p, and MDM2, among others. We also provide a preliminary analysis of SNPs and indels, present exclusively in A549/DDP cells. This study's results provide novel potential mechanisms and molecular targets that can be explored in future studies and assist in improving the understanding of the chemoresistance phenotype.

A grafted peptidomimetic for EGFR heterodimerization inhibition: Implications in NSCLC models.

Among the lung cancers, approximately 85% are histologically classified as non-small-cell lung cancer (NSCLC), a leading cause of cancer deaths worldwide. Epidermal growth factor receptors (EGFRs) are known to play a crucial role in lung cancer. HER2 overexpression is detected by immunohistochemistry in 2.4%-38% of NSCLC samples. EGFRs have been targeted with three generations of tyrosine kinase inhibitors (TKIs), and drug resistance has become a major issue; HER2 dimerization with EGFR also plays a major role in the development of resistance to TKI therapy. We have designed grafted peptides to bind to the HER2 extracellular domain (ECD) and inhibit protein-protein interactions of EGFR:HER2 and HER2:HER3. A sunflower trypsin inhibitor (SFTI-1) template was used to graft a peptidomimetic compound. Among several grafted peptides, SFTI-G5 exhibited antiproliferative activity in HER2-positive NSCLC cell lines such as Calu-3 cells with an IC50 value of 0.073 µM. SFTI-G5 was shown to bind to ECD of HER2 and inhibit EGFR:HER2 and HER2:HER3 dimerization and inhibit the phosphorylation of HER2 and downstream signaling proteins. As a proof-of-concept, the in vivo activity of SFTI-G5 was evaluated in two NSCLC mouse models. SFTI-G5 was able to inhibit tumor growth in both models. Furthermore, SFTI-G5 was shown to inhibit EGFR dimerization in tissue samples obtained from in vivo models. These grafted peptides can be used as novel dual inhibitors of EGFR dimerization in NSCLC.

Combination of miR­143 and miR­506 reduces lung and pancreatic cancer cell growth through the downregulation of cyclin­dependent kinases.

Lung cancer (LC) and pancreatic cancer (PC) are the first and fourth leading causes of cancer­related deaths in the US. Deregulated cell cycle progression is the cornerstone for rapid cell proliferation, tumor development, and progression. Here, we provide evidence that a novel combinatorial miR treatment inhibits cell cycle progression at two phase transitions, through their activity on the CDK4 and CDK1 genes. Following transfection with miR­143 and miR­506, we analyzed the differential gene expression of CDK4 and CDK1, using qPCR or western blot analysis, and evaluated cell cycle inhibition, apoptosis and cytotoxicity. The combinatorial miR­143/506 treatment downregulated CDK4 and CDK1 levels, and induced apoptosis in LC cells, while sparing normal lung fibroblasts. Moreover, the combinatorial miR treatment demonstrated a comparable activity to clinically tested cell cycle inhibitors in inhibiting cell cycle progression, by presenting substantial inhibition at the G1/S and G2/M cell cycle transitions. More importantly, the miR­143/506 treatment presented a broader application, effectively downregulating CDK1 and CDK4 levels, and reducing cell growth in PC cells. These findings suggest that the miR­143/506 combination acts as a promising approach to inhibit cell cycle progression for cancer treatment with minimal toxicity to normal cells.

Cellular Migration Assay: An In Vitro Technique to Simulate the Wound Repair Mechanism.

Wound regeneration is a complex process, which necessitates proper coordination among the inflammatory response, vascularization, matrix formation, and reformation of epithelial tissue. It is a unique process, where healing and regeneration take place simultaneously. Matrix formation is the first critical stage that starts the communication between the keratinocytes, fibroblasts, and integrins. This, in turn, stimulates the differentiation of monocytes into macrophages, to produce cytokines for fibroblasts. This phenomenon is the crucial part for the keratinocyte migration and epithelialization to fill the wound. To understand the complex procedure of wound regeneration, there is a need for easy, convenient, and low-cost approaches that will simulate the wound-repairing process. Scratch assay or cellular migration assay is one of the most convenient and affordable approaches, commonly used by the scientific community. In this chapter, we present the fundamental principles of the experimental procedures required for the Scratch assay.

Nucleic Acid Delivery with α-Tocopherol-Polyethyleneimine-Polyethylene Glycol Nanocarrier System.

PURPOSE: Nucleic acid-based therapies are a promising therapeutic tool. The major obstacle in their clinical translation is their efficient delivery to the desired tissue. We developed a novel nanosized delivery system composed of conjugates of α-tocopherol, polyethyleneimine, and polyethylene glycol (TPP) to deliver nucleic acids. METHODS: We synthesized a panel of TPP molecules using different molecular weights of PEG and PEI and analyzed with various analytical approaches. The optimized version of TPP (TPP111 - the 1:1:1 molecular ratio) was self-assembled in water to produce nanostructures and then evaluated in diversified in vitro and in vivo studies. RESULTS: Through a panel of synthesized molecules, TPP111 conjugate components self-assembled in water, forming globular shaped nanostructures of ~90 nm, with high nucleic acid entrapment efficiency. The polymer had low cytotoxicity in vitro and protected nucleic acids from nucleases. Using a luciferase-expressing plasmid, TPP111-plasmid nano-complexes were rapidly up-taken by cancer cells in vitro and induced strong transfection, comparable to PEI. Colocalization of the nano-complexes and endosomes/lysosomes suggested an endosome-mediated uptake. Using a subcutaneous tumor model, intravenously injected nano-complexes preferentially accumulated to the tumor area over 24 h. CONCLUSION: These results indicate that we successfully synthesized the TPP111 nanocarrier system, which can deliver nucleic acids in vitro and in vivo and merits further evaluation.

In vivo studies of a peptidomimetic that targets EGFR dimerization in NSCLC.

Studies related to lung cancer have shown a link between human epidermal growth factor receptor-2 (HER2) expression and poor prognosis in patients with non-small cell lung cancer (NSCLC). HER2 overexpression has been observed in 3-38% of NSCLC, while strong HER2 protein overexpression is found in 2.5% of NSCLC. However, HER2 dimerization is important in lung cancer, including EGFR mutated NSCLC. Since HER2 dimerization leads to cell proliferation, targeting the dimerization of HER2 will have a significant impact on cancer therapies. A peptidomimetic has been designed that can be used as a therapeutic agent for a subset of NSCLC patients overexpressing HER2 or possessing HER2 as well as EGFR mutation. A cyclic peptidomimetic (18) has been designed to inhibit protein-protein interactions of HER2 with its dimerization partners EGFR and HER3. Compound 18 exhibited antiproliferative activity in HER2-positive NSCLC cell lines at nanomolar concentrations. Western blot analysis showed that 18 inhibited phosphorylation of HER2 and Akt in vitro and in vivo. Stability studies of 18 at various temperature and pH (pH 1 and pH 7.6), and in the presence of liver microsomes indicated that 18 was stable against thermal and chemical degradation. Pharmacokinetic parameters were evaluated in nude mice by administrating single doses of 4 mg/kg and 6 mg/kg of 18 via IV. The anticancer activity of 18 was evaluated using an experimental metastasis lung cancer model in mice. Compound 18 suppressed the tumor growth in mice when compared to control. A proximity ligation assay further proved that 18 inhibits HER2:HER3 and EGFR: HER2 dimerization. Overall, these results suggest that 18 can be a potential treatment for HER2-dimerization related NSCLC.

Nanoparticle Delivery and Tumor Vascular Normalization: The Chicken or The Egg?

Tumor-induced angiogenesis has been a significant focus of anti-cancer therapies for several decades. The immature and "leaky" tumor vasculature leads to significant cancer cell intravasation, increasing the metastatic potential, while the disoriented and hypo-perfused tumor vessels hamper the anti-tumor efficacy of immune cells and prevent the efficient diffusion of chemotherapeutic drugs. Therefore, tumor vascular normalization has emerged as a new treatment goal, aiming to provide a mature tumor vasculature, with higher perfusion, decreased cancer cell extravasation, and higher efficacy for anti-cancer therapies. Here we propose an overview of the nanodelivery approaches that target tumor vasculature, aiming to achieve vascular normalization. At the same time, abnormal vascular architecture and leaky tumor vessels have been the cornerstone for nanodelivery approaches through the enhanced permeability and retention (EPR) effect. Vascular normalization presents new opportunities and requirements for efficient nanoparticle delivery against the tumor cells and overall improved anti-cancer therapies.

miRNAs in gastrointestinal diseases: can we effectively deliver RNA-based therapeutics orally?

Nucleic acid-based therapeutics are evaluated for their potential of treating a plethora of diseases, including cancer and inflammation. Short nucleic acids, such as miRNAs, have emerged as versatile regulators for gene expression and are studied for therapeutic purposes. However, their inherent instability in vivo following enteral and parenteral administration has prompted the development of novel methodologies for their delivery. Although research on the oral delivery of siRNAs is progressing, with the development and utilization of promising carrier-based methodologies for the treatment of a plethora of gastrointestinal diseases, research on miRNA-based oral therapeutics is lagging behind. In this review, we present the potential role of miRNAs in diseases of the GI tract, and analyze current research and the cardinal features of the novel carrier systems used for nucleic acid oral delivery that can be expanded for oral miRNA administration.

Evaluation of the feasibility of intrapancreatic delivery of drug-loaded microparticles via EUS-guided fine needle injection using a swine model.

Background and study aims Patients with pancreatic cancer often have locally advanced or metastatic disease and are not candidates for curative surgery. Polymer-based microparticles (MPs) represent a drug delivery system that offers sustained release of a chemotherapeutic drug after intralesional injection for local tumor management. The aim of this study was to determine the feasibility of endoscopic ultrasound-guided fine-needle injection (EUS-FNI) of drug-loaded MPs tagged with a fluorophore and fiducial markers for locating the injection site. Secondary aims were to determine the tissue-specific effects of MPs. Methods Five pigs underwent EUS with selection of an injection site within the pancreas that was marked by placing fiducial markers prior to the MPs injection. EUS-FNI of either blank microparticles (BMPs), containing no drug, or gemcitabine-loaded microparticles (GMPs) was performed. A saline flush containing Spot Endoscopic Marker was used to expel any residual MPs in the needle shaft and tattoo the injection site. Results A green fluorescent protein flashlight was used to successfully identify the site of MP injection sites in the dissected pancreas. Frozen sections of pig pancreas demonstrated a defined deposit, confirming the delivery of the MPs. Finally, fluorescence microscopy showed activation of caspase-mediated cell death in pancreases of animals that received injections of GMPs. Conclusions This pilot study demonstrated that fiducial marker placement and pancreatic EUS-FNI of MPs was successful in all pigs with no animals demonstrating pancreatitis. Further studies are needed to determine the role for intralesional injection of drug-loaded MPs in borderline resectable or unresectable pancreatic cancer.

[Corrigendum] Phospho­valproic acid inhibits pancreatic cancer growth in mice: Enhanced efficacy by its formulation in poly­(L)­lactic acid­poly(ethylene glycol) nanoparticles.

Subsequently to the publication of the aobve article, the authors have realized that they overlooked including a 'Competing Interests' section. This should have been included in the paper as follows: 'GM, RW and GGM declare they have no competing interests. BR has an equity position in Medicon Pharmaceuticals, Inc., the company that provided the test agent'. The authors regret that this statement was not included in the paper, and apologize for any inconvenience caused. [the original article was published in the International Journal of Oncology 51: 1035-1044, 2017; DOI: 10.3892/ijo.2017.4103].

Multipronged activity of combinatorial miR-143 and miR-506 inhibits Lung Cancer cell cycle progression and angiogenesis in vitro.

Lung cancer (LC) is the leading cause of cancer-related deaths. Downregulation of CDK1, 4 and 6, key regulators of cell cycle progression, correlates with decreased LC cell proliferation. Enforced expression of miRNAs (miRs) is a promising approach to regulate genes. Here, we study the combinatorial treatment of miR-143 and miR-506 to target the CDK1, 4/6 genes, respectively. We analyzed the differential expression of CDK genes by qPCR, and western blot, and evaluated changes in the cell cycle distribution upon combinatorial treatment. We used an antibody microarray analysis to evaluate protein expression, focusing on the cell cycle pathway, and performed RNA-sequencing for pathway analysis. The combinatorial miR treatment significantly downregulated CDK1, 4 and 6 expression, and induced a shift of the cell cycle populations, indicating a G1 and G2 cell cycle block. The two miRs induces strong cytotoxic activity, with potential synergism, and a significant Caspase 3/7 activation. We identified a strong inhibition of tube formation in the presence or absence VEGF in an in vitro angiogenesis model. Together with the pathways analysis of the RNA-sequencing data, our findings establish the combinatorial miR transfection as a viable strategy for lung cancer treatment that merits further investigation.