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Wee1 inhibition disturbs cell-cycle progression and triggers DNA damage. (a) B16 cells transfected with the RRCPP/siWee1 complex and cell-cycle profiles were determined by flow cytometry after staining with PI. (b) Percentage of total G2/M cells included in each group. (c) For analysis by western blotting with the indicated antibodies. The results show that treatment with the RRCPP/siWee1 complex abrogates the G2/M checkpoint of B16 cells by attenuating the phosphorylation of CDK1 and upregulating the γ-H2AX protein, which is an important DNA damagerelated protein. (d) Fold expression of cyclinD1 mRNA in each treatment. (e) Cell immunofluorescence analysis of the target transfected with RRCPP/siWee1 was performed for 48 h by staining with Alexa Fluor 488-blocked γ-H2AX and DAPI.
Source publication
Small interfering RNA (siRNA)-based drugs have shown tremendous potential to date in cancer gene therapy. Despite the considerable efforts in siRNA design and manufacturing, unsatisfactory delivery systems persist as a limitation for the application of siRNA-based drugs. In this work, the cholesterol, cell-penetrating peptide conjugate cRGD (R8–cRG...
Contexts in source publication
Context 1
... clarify the mechanism of cell death induced by RRCPP/siWee1 treatment, cell-cycle assay and western blotting assay were performed on B16 cells. As shown in Figure 4a,b, the cells of the G2/M phase in RRCPP/siWee1 treatment were significantly higher (p < 0.05), ...
Context 2
... data demonstrated that the RRCPP/siWee1 complex abrogating the G2/M checkpoint may work through prolonging the G2/M period. With the reduction in Wee1 gene expression, a significant downregulation of phosphorylated CDK1 (Tyr15) was observed, following RRCPP/siWee1 treatment (Figure 4c), suggesting that RRCPP/siWee1 may abrogate the G2 checkpoint through the phosphorylation of CDK1. It has been reported that deregulation of CDK1 activity may trigger the activation of caspase and apoptosis, 45,46 which was consistent with our results in this study. ...
Context 3
... has been reported that deregulation of CDK1 activity may trigger the activation of caspase and apoptosis, 45,46 which was consistent with our results in this study. Meanwhile, the cyclinD1 mRNA was extremely upregulated in the RRCPP/ siWee1-treated cells (Figure 4d). In the RRCPP/siWee1 treatment groups, a high level of the DNA damage-associated protein γ-H2AX was also detected (Figure 4c). ...
Context 4
... the cyclinD1 mRNA was extremely upregulated in the RRCPP/ siWee1-treated cells (Figure 4d). In the RRCPP/siWee1 treatment groups, a high level of the DNA damage-associated protein γ-H2AX was also detected (Figure 4c). We further searched for DNA damage by staining with an antibody against γ-H2AX and co-staining nuclei with DAPI. ...
Citations
... Research demonstrated that caveolin-mediated endocytosis, micropinocytosis, lipid raft-mediated endocytosis, and clathrin-mediated endocytosis the currently endocytosis internalization mechanisms. 22,23 Before transfection, the cells were stimulated by series inhibitors, including genistein (GT) and filipin III (Fil) for caveolin-mediated endocytosis, wortmannin (WM) for micropinocytosis, methyl-β-cyclodextrin (M-β-CD) for lipid raft-mediated endocytosis, and chlorpromazine (CPZ) for clathrin-mediated endocytosis. ...
Background
Messenger ribonucleic acid (mRNA)-based gene therapy has great potential in cancer treatment. However, the application of mRNA-based cancer treatment could be further developed. Elevated delivery ability and enhanced immune response are advantages for expanding the application of mRNA-based cancer therapy. It is crucial that the prepared carrier can cause an immune reaction based on the efficient delivery of mRNA.
Methods
We reported DMP nanoparticle previously, which was obtained by the self-assembly of 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) and (ethylene glycol)-b-poly (ε-caprolactone) (mPEG-PCL). Research demonstrated that DMP can deliver mRNA, siRNA, and plasmid. And it is applied to various tumor types. In our work, the tumor cell lysate was introduced to the internal DMP chain, fusing cell-penetrating peptides (CPPs) modification on the surface forming the CLSV system. And then mixed encoded IL-22BP (interleukin-22 binding protein) mRNA and CLSV to form CLSV/IL-22BP complex.
Results
The size of the CLSV system was 213.2 nm, and the potential was 45.7 mV. The transfection efficiency of the CLSV system is up to 76.45% in C26 cells via the micropinocytosis pathway. The CLSV system also could induce an immune response and significantly elevate the expression of CD80, CD86, and MHC-II in vivo. Then, by binding with IL-22BP (Interleukin-22 binding protein) mRNA, the CLSV/IL-22BP complex inhibited tumor cell growth, with an inhibition rate of up to 82.3% in vitro. The CLSV/IL-22BP complex also inhibited tumor growth in vivo, the tumor cell growth inhibition up to 75.0% in the subcutaneous tumor model, and 84.9% in the abdominal cavity metastasis tumor model.
Conclusion
Our work demonstrates that the CLSV system represents a potent potential for mRNA delivery.
... What G2M checkpoints, E2F targets, and MYC targets have in common is that they all play a pivotal role in cell cycle progression and cell division. The overexpression of oncogenic G2/M checkpoint may lead to poor prognosis (18,19). The cyclin-dependent kinase (CDK)-retinoblastoma gene (RB)-E2F axis plays an important role in cell cycle progression, which controls genome replication and accurate cell division cycle (20). ...
Background
Metabolic reprogramming is involved in different stages of tumorigenesis. There are six widely recognized tumor-associated metabolic pathways, including cholesterol catabolism process, fatty acid metabolism, glutamine metabolic process, glycolysis, one carbon metabolic process, and pentose phosphate process. This study aimed to classify gastric cancer patients into different metabolic bio-similar clusters.
Method
We analyzed six tumor-associated metabolic pathways and calculated the metabolic pathway score through RNA-seq data using single sample gene set enrichment analysis. The consensus clustering analysis was performed to classify patients into different bio-similar clusters by multi-dimensional scaling. Kaplan–Meier curves were presented between different metabolic bio-similar groups for OS analysis.
Results
A training set of 370 patients from the Cancer Genome Atlas database with primary gastric cancer was chosen. Patients were classified into four metabolic bio-similar clusters, which were identified as metabolic non-specificity, metabolic-active, cholesterol-silence, and metabolic-silence clusters. Survival analysis showed that patients in metabolic-active cluster and metabolic-silence cluster have significantly poor prognosis than other patients ( p =0.031). Patients in metabolic-active cluster and metabolic-silence cluster had significantly higher intra-tumor heterogeneity than other patients ( p =0.032). Further analysis was performed in metabolic-active cluster and cholesterol-silence cluster. Three cell-cycle-related pathways, including G2M checkpoints, E2F targets, and MYC targets, were significantly upregulated in metabolic-active cluster than in cholesterol-silence cluster. A validation set of 192 gastric cancer patients from the Gene Expression Omnibus data portal verified that metabolic bio-similar cluster can predict prognosis in gastric cancer.
Conclusion
Our study established a multi-dimension metabolic prognostic model in gastric cancer, which may be feasible for predicting clinical outcome.
... They exert membrane translocation via mechanisms that remain under investigation [13,14]. The cellular uptake and subsequent gene silencing efficiency of siRNA can be greatly improved by forming non-covalent complexes containing CPPs and siRNA [15][16][17][18], by covalently conjugating them [19][20][21] or by encapsulating siRNA within CPP-functionalized nanoparticles. Some encouraging results have also been seen in vitro [22][23][24] and in vivo [25,26] after complexing siRNA to endosomal escape-facilitating CPPs. ...
The therapeutic potential of short interfering RNA (siRNA) to treat many diseases that are incurable with traditional preparations is limited by the extensive metabolism of serum nucleases, low permeability through biological membrane barriers because of a negative charge, and endosomal trapping. Effective delivery vectors are required to overcome these challenges without causing unwanted side effects. Here, we present a relatively simple synthetic protocol to obtain positively charged gold nanoparticles (AuNPs) with narrow size distribution and the surface modified with Tat-related cell-penetrating peptide. The AuNPs were characterized using TEM and the localized surface plasmon resonance technique. The synthesized AuNPs showed low toxicity in experiments in vitro and were able to effectively form complexes with double-stranded siRNA. The obtained delivery vehicles were used for intracellular delivery of siRNA in an ARPE-19 cell line transfected with secreted embryonic alkaline phosphatase (SEAP). The delivered oligonucleotide remained intact and caused a significant knockdown effect on SEAP cell production. The developed material could be useful for delivery of negatively charged macromolecules, such as antisense oligonucleotides and various RNAs, particularly for retinal pigment epithelial cell drug delivery.
... By analyzing the sequencing profiles, we identified lipocalin 2 (LCN2) as a factor involved in OSCC metastasis and EGFR resistance, indicating that it may work as a potential target; thus, we performed in-depth research on LCN2. Moreover, the use of nanotechnology for nucleic acid-based drug delivery has opened new doors in the field of cancer treatment [11][12][13][14]. Encapsulation of siRNA into nanoparticles (NPs) not only enhances siRNA pharmacokinetics to increase the siRNA concentration in cancer cells but also directly inhibits target gene expression, thus limiting cancer malignancy. ...
Background
EGFR is an important signal involved in tumor growth that can induce tumor metastasis and drug resistance. Exploring targets for effective EGFR regulation is an important topic in current research and drug development. Inhibiting EGFR can effectively inhibit the progression and lymph node metastasis of oral squamous cell carcinoma (OSCC) because OSCC is a type of cancer with high EGFR expression. However, the problem of EGFR drug resistance is particularly prominent, and identifying a new target for EGFR regulation could reveal an effective strategy.
Methods
We sequenced wild type or EGFR-resistant OSCC cells and samples from OSCC patients with or without lymph node metastasis to find new targets for EGFR regulation to effectively replace the strategy of directly inhibiting EGFR and exert an antitumor effect. We then investigated the effect of LCN2 on OSCC biological abilities in vitro and in vivo through protein expression regulation. Subsequently, we elucidated the regulatory mechanism of LCN2 through mass spectrometry, protein interaction, immunoblotting, and immunofluorescence analyses. As a proof of concept, a reduction-responsive nanoparticle (NP) platform was engineered for effective LCN2 siRNA (siLCN2) delivery, and a tongue orthotopic xenograft model as well as an EGFR-positive patient-derived xenograft (PDX) model were applied to investigate the curative effect of siLCN2.
Results
We identified lipocalin-2 (LCN2), which is upregulated in OSCC metastasis and EGFR resistance. Inhibition of LCN2 expression can effectively inhibit the proliferation and metastasis of OSCC in vitro and in vivo by inhibiting EGFR phosphorylation and downstream signal activation. Mechanistically, LCN2 binds EGFR and enhances the recycling of EGFR, thereby activating the EGFR-MEK-ERK cascade. Inhibition of LCN2 effectively inhibited the activation of EGFR. We translated this finding by systemic delivery of siLCN2 by NPs, which effectively downregulated LCN2 in the tumor tissues, thereby leading to a significant inhibition of the growth and metastasis of xenografts.
Conclusions
This research indicated that targeting LCN2 could be a promising strategy for the treatment of OSCC.
... A variant of cRGD fused to a cell-penetrating peptide (Arg 8 -cRGD) has been included in a cholesterol-PEG-PEI nanoparticle for the delivery of a siRNA against Wee1, a nuclear kinase overexpressed in melanoma [95]. This formulation inhibited both primary tumor growth (by 85%) and secondary dissemination (by 66%) in a mouse model of metastatic melanoma. ...
... The described peptide-targeted nanoparticles are summarized in Table 6. Albumin/red cell membranes Gefitinib cRGD α v β 3 /α v β 5 integrins Lung cancer (in vitro and in vivo) [86] Lipid nanoparticle GNA002 cRGD-Arg 6 α v β 3 /α v β 5 integrins + cell penetration Squamous cell Carcinoma-tongue (in vitro and in vivo) [87] Silk fibroin nanoparticle Curcumin cRGD α v β 3 /α v β 5 integrins Different cell models (in vitro) [88] Liposome Doxorubicin cRGD α v β 3 /α v β 5 integrins Colon cancer (in vitro and in vivo) [89] DSPE/PEG nanoparticle TQs-PEG4 cRGD α v β 3 /α v β 5 integrins Breast cancer (in vitro and in vivo) [90] Silver sulfide nanoparticle Doxorubicin cRGD α v β 3 /α v β 5 integrins Breast cancer (in vitro and in vivo) [91] Gold-iron oxide nanoparticle Multimodal cRGD α v β 3 /α v β 5 integrins Breast cancer (in vitro and in vivo) [92] Gallic acid/Fe 3+ nanoparticle Doxorubicin, multimodal cRGD-Platinum prodrug α v β 3 /α v β 5 integrins Glioblastoma (in vitro and in vivo) [93] DSPE nanoparticle Siponimod cRGD-PEG α v β 3 /α v β 5 integrins Breast cancer (in vitro and in vivo) [94] PEG-lipid nanoparticle Wee1 siRNA cRGD-Arg 8 α v β 3 /α v β 5 integrins + cell penetration Melanoma (in vitro and in vivo) [95] Chitosan nanoparticle VEGF siRNA cRGD-albumin ...
Simple Summary
Conventional antitumor drugs have limitations, including poor water solubility and lack of targeting capability, with consequent non-specific distribution, systemic toxicity, and low therapeutic index. Nanotechnology promises to overcome these drawbacks by exploiting the physical properties of diverse nanocarriers that can be linked to moieties with binding selectivity for cancer cells. The use of nanoparticles as therapeutic formulations allows a targeted delivery and a slow, controlled release of the drug(s), making them tunable modules for applications in precision medicine. In addition, nanoparticles are also being developed as cancer vaccines, offering an opportunity to increase both cellular and humoral immunity, thus providing a new weapon to beat cancer.
Abstract
Malignant tumors originate from a combination of genetic alterations, which induce activation of oncogenes and inactivation of oncosuppressor genes, ultimately resulting in uncontrolled growth and neoplastic transformation. Chemotherapy prevents the abnormal proliferation of cancer cells, but it also affects the entire cellular network in the human body with heavy side effects. For this reason, the ultimate aim of cancer therapy remains to selectively kill cancer cells while sparing their normal counterparts. Nanoparticle formulations have the potential to achieve this aim by providing optimized drug delivery to a pathological site with minimal accumulation in healthy tissues. In this review, we will first describe the characteristics of recently developed nanoparticles and how their physical properties and targeting functionalization are exploited depending on their therapeutic payload, route of delivery, and tumor type. Second, we will analyze how nanoparticles can overcome multidrug resistance based on their ability to combine different therapies and targeting moieties within a single formulation. Finally, we will discuss how the implementation of these strategies has led to the generation of nanoparticle-based cancer vaccines as cutting-edge instruments for cancer immunotherapy.
... A recent study showed the successful use of a new siRNA delivery system with nanoparticles displaying highly penetrating abilities. They were recently used as a carrier to deliver WEE1 siRNA in melanoma preclinical models [128]. The nanoparticle/siWEE1 complex displayed a strong anti-cancer activity in vitro in B16 cells and an anti-tumor effect in vivo in subcutaneous xenograft and lung metastasis of B16 tumor models, as compared with the negative control group [128]. ...
... They were recently used as a carrier to deliver WEE1 siRNA in melanoma preclinical models [128]. The nanoparticle/siWEE1 complex displayed a strong anti-cancer activity in vitro in B16 cells and an anti-tumor effect in vivo in subcutaneous xenograft and lung metastasis of B16 tumor models, as compared with the negative control group [128]. ...
Targeted therapies against components of the mitogen-activated protein kinase (MAPK) pathway and immunotherapies, which block immune checkpoints, have shown important clinical benefits in melanoma patients. However, most patients develop resistance, with consequent disease relapse. Therefore, there is a need to identify novel therapeutic approaches for patients who are resistant or do not respond to the current targeted and immune therapies. Melanoma is characterized by homologous recombination (HR) and DNA damage response (DDR) gene mutations and by high replicative stress, which increase the endogenous DNA damage, leading to the activation of DDR. In this review, we will discuss the current experimental evidence on how DDR can be exploited therapeutically in melanoma. Specifically, we will focus on PARP, ATM, CHK1, WEE1 and ATR inhibitors, for which preclinical data as single agents, taking advantage of synthetic lethal interactions, and in combination with chemo-targeted-immunotherapy, have been growing in melanoma, encouraging the ongoing clinical trials. The overviewed data are suggestive of considering DDR inhibitors as a valid therapeutic approach, which may positively impact the future of melanoma treatment.
... It was theorized in this study that the nature of the sturdy membrane of lipopepsomes prevented RNA degradation, leading to high levels of gene silencing 96 . In studying the treatment of melanoma, it was found that a CPP/cholesterol/PEG conjugate was able to form nanoparticles to facilitate delivery of Wee1 siRNA, leading to significant silencing of the WEE1 gene and melanoma tumor cell apoptosis in vitro as well as inhibition of tumor growth in vivo 97 . Other studies on the uses of CPPs to target melanomas use different CPP-based delivery methods, such as microneedles 98 . ...
Cell penetrating peptides (CPPs) offer unique and promising solutions to overcome barriers to intracellular delivery of potential therapeutics for a variety of diseases, particularly when used as a delivery method for RNA interference agents such as siRNA. CPP mediated siRNA delivery provides promising therapeutic potential for various pathologies including many different types of cancer as well as other pathologies. Both cell-specific and non-cell-specific CPPs can play roles in transporting several types of cargo into cells, including but not limited to drugs, viral vectors, peptide nucleic acids, nanoparticles, liposomes, and siRNA. CPPs mediate siRNA delivery through a variety of delivery methods, including covalent conjugation and nanoparticle formation, and can be fashioned to facilitate endosomal escape. While no therapeutics currently utilize CPP-mediated siRNA delivery, two major approved therapeutics use RNA interference as a treatment modality, and many clinical trials are in progress testing the use of CPPs, again with an emphasis on the treatment of cancer. Further research is needed before the clinical use of CPP/siRNA complexes is commonplace, but advances in both CPP and siRNA technology appear promising for this method of treatment.
In this chapter, the delivery of biomolecules such as oligonucleotides, proteins, peptides and others by connection of the power of modern nanoparticles with the flexibility and efficacy of CPPs is summarized. This will increase the potential of therapeutic agents, requiring protection from degradation and release in a controlled manner.
