analysis-combinatorial-mirna-treatments-to-regulate-cell-cycle

Analysis of Combinatorial miRNA Treatments to Regulate Cell Cycle and Angiogenesis

Lung cancer (LC) is the leading cause of cancer-related deaths worldwide. Similar to other cancer cells, a fundamental characteristic of LC cells is ...

murine-dermal-lymphatic-endothelial-cell-isolation

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 ...

University of Louisiana Monroe

University of Louisiana at Monroe

Pancreatic cancer (PC) is one of the most difficult cancers to treat. Since the current chemotherapy is inadequate and various biological approaches have failed, the need for agents that have a potential to treat PC is pressing. Phospho-valproic acid (P-V), a novel anticancer agent, is efficacious in xenograft models of human PC and is apparently safe. In the present study, we evaluated whether formulating P-V in nanoparticles could enhance its anticancer efficacy. In a mouse model of Kras/pancreatitis-associated PC, P-V, orally administered, inhibited the incidence of acinar-to-ductal metaplasia by 60%. To improve its efficacy, we formulated P-V in five different polymeric nanoparticles. Poly-(L)-lactic acid- poly(ethylene glycol) (PLLA-PEG) nanoparticles proved the optimal formulation. PLLA-PEG improved P-V's pharmacokinetics in mice enhancing the levels of P-V in blood. Compared to control, P-V formulated in PLLA-PEG suppressed the growth of MIA PaCa-2 xenografts by 81%, whereas P-V alone reduced it by 51% (p<0.01). Furthermore, P-V formulated in PLLA-PEG inhibited acinar-to-ductal metaplasia in mice with activated Kras, reducing it by 87% (p<0.02). In both disease models, P-V suppressed STAT3 phosphorylation at the Ser727 and Tyr705 residues; STAT3 is the pivotal molecular target of P-V. In conclusion, P-V is a promising agent against PC, and its formulation in PLLA-PEG nanoparticles enhances its efficacy by improving its pharmacokinetics.

Phospho-valproic acid inhibits pancreatic cancer growth in mice: enhanced efficacy by its formulation in poly-(L)-lactic acid-poly(ethylene glycol) nanoparticles.

Curcumin reduces Hodgkin's lymphoma (HL) cell growth in vitro, but its unfavorable pharmacokinetics highlight the need for novel in vivo delivery systems. Thus, we explored whether formulation of curcumin in solid lipid nanoparticles (SLN-curc) or d-α-Tocopheryl polyethylene glycol 1000 succinate (TPGS) nanoparticles (TPGS-curc) could enhance its efficacy in mice. Curcumin formulated in SLN and in TPGS resulted in higher curcumin plasma levels in mice. Compared to vehicle-treated controls, SLN-curc and TPGS-curc reduced HL xenograft growth by 50.5% (p < 0.02) and 43.0% (p < 0.04), respectively, while curcumin reduced it by 35.8% (p < 0.05). In addition, SLN-curc reduced the expression of proteins involved in cell proliferation and apoptosis (XIAP and Mcl-1) in HL tumor extracts. In HL cells in culture, curcumin decreased the expression of relevant anti-inflammatory cytokines (IL-6 and TNF-α) in a concentration-dependent manner. Moreover, when given in combination with bleomycin, doxorubicin and vinblastine, curcumin showed an additive growth inhibitory effect. In conclusion, SLNs appear as an appropriate and effective drug delivery system for curcumin. Given the efficacy of SLN-curc and the enhanced growth inhibitory effect when combined with chemotherapeutic drugs, we speculate that curcumin, when appropriately formulated, is a promising adjuvant agent for the treatment of HL and merits further evaluation.

Curcumin formulated in solid lipid nanoparticles has enhanced efficacy in Hodgkin's lymphoma in mice.

miRNAs are promising therapeutic targets or tools for the treatment of numerous diseases, with most prominently, cancer. The inherent capacity of these short nucleic acids to regulate multiple cancer-related pathways simultaneously has prompted strong research on understanding miR functions and their potential use for therapeutic purposes. A key determinant of miR therapeutics' potential for treatment is their delivery. Viral and non-viral vectors attempt to address the major limitations associated with miR delivery, but several hurdles have been identified. Here, we present an overview on the general limitations of miR delivery, and the delivery strategies exploited to overcome them. We provide an introduction on the advantages and disadvantages of viral and non-viral vectors, and we go into detail to analyze the most prominently used non-viral systems. We provide with an update on the most recent research on this topic and we describe the mechanism and limitations of the lipid-, polymer- and inorganic material- based miR delivery systems.

Non-viral based miR delivery and recent developments.

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.

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

 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.  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.  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.  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.

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

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.

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

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 IC 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.

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

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.

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

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.

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

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.

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

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.

ArticleTotal : 2	Year
Publication title Cited by 9	2019
Publication title Cited by 1	2023

Article	Year
Phospho-valproic acid inhibits pancreatic cancer growth in mice: enhanced efficacy by its formulation in poly-(L)-lactic acid-poly(ethylene glycol) nanoparticles. International journal of oncology\| PubMed ID: 28849098	2017
Curcumin formulated in solid lipid nanoparticles has enhanced efficacy in Hodgkin's lymphoma in mice. Archives of biochemistry and biophysics\| PubMed ID: 29679536	2018
Non-viral based miR delivery and recent developments. European journal of pharmaceutics and biopharmaceutics : official journal of Arbeitsgemeinschaft fur Pharmazeutische Verfahrenstechnik e.V\| PubMed ID: 29679644	2018
Multipronged activity of combinatorial miR-143 and miR-506 inhibits Lung Cancer cell cycle progression and angiogenesis in vitro. Scientific reports\| PubMed ID: 30002440	2018
Evaluation of the feasibility of intrapancreatic delivery of drug-loaded microparticles via EUS-guided fine needle injection using a swine model. Endoscopy international open\| PubMed ID: 31404394	2019
Cellular Migration Assay: An In Vitro Technique to Simulate the Wound Repair Mechanism. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 32808260	2021
A grafted peptidomimetic for EGFR heterodimerization inhibition: Implications in NSCLC models. European journal of medicinal chemistry\| PubMed ID: 33667849	2021
Advanced bioinformatic analysis and pathway prediction of NSCLC cells upon cisplatin resistance. Scientific reports\| PubMed ID: 33753779	2021
Chlorotoxin and Lung Cancer: A Targeting Perspective for Drug Delivery. Pharmaceutics\| PubMed ID: 36559106	2022
Endothelial-Specific Targeting of RhoA Signaling via CD31 Antibody-Conjugated Nanoparticles. The Journal of pharmacology and experimental therapeutics\| PubMed ID: 36746610	2023
Lyophilized liposomal formulation of a peptidomimetic-Dox conjugate for HER2 positive breast and lung cancer. International journal of pharmaceutics\| PubMed ID: 37059241	2023

George Mattheolabakis

Biography

JoVE Journal Publications

Other Publications