MicroRNA Delivery by Graphene-Based Complexes into Glioblastoma Cells.

Glioblastoma (GBM) is the most common primary and aggressive tumour in brain cancer. Novel therapies, despite achievements in chemotherapy, radiation and surgical techniques, are needed to improve the treatment of GBM tumours and extend patients' survival. Gene delivery therapy mostly uses the viral vector, which causes serious adverse events in gene therapy. Graphene-based complexes can reduce the potential side effect of viral carries, with high efficiency of microRNA (miRNA) or antisense miRNA delivery to GBM cells. The objective of this study was to use graphene-based complexes to induce deregulation of miRNA level in GBM cancer cells and to regulate the selected gene expression involved in apoptosis. The complexes were characterised by Fourier transform infrared spectroscopy (FTIR), scanning transmission electron microscopy and zeta potential. The efficiency of miRNA delivery to the cancer cells was analysed by flow cytometry. The effect of the anticancer activity of graphene-based complexes functionalised by the miRNA sequence was analysed using 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxyanilide salt (XTT) assays at the gene expression level. The results partly explain the mechanisms of miRNA deregulation stress, which is affected by graphene-based complexes together with the forced transport of mimic miR-124, miR-137 and antisense miR-21, -221 and -222 as an anticancer supportive therapy.

Up-regulation of hypoxia-inducible factor antisense as a novel approach to treat ovarian cancer.

Ovarian cancer (OC) is estimated to kill ~14,000 women in the United States in 2019. Current chemotherapies to treat OC initially show therapeutic efficacy but frequently drug resistance develops, at which point therapies with alternative targets are needed. Herein, we are describing a novel approach to sensitize these tumors to standard chemotherapies by increasing the transcription of hypoxia-inducible factor antisense. Methods: Genome-wide Bru-seq analysis was performed to fully capture the nascent transcriptional signature of OC cells treated with the gp130 inhibitor, SC144. In vitro and in vivo analysis, including characterization of hypoxia and select protein expression, combination with standard of care chemotherapy and antitumor efficacy were performed to assess the biological activity of SC144 on induction of hypoxia in OC cells. Results: Bru-seq analysis of OVCAR8 cells treated with SC144 shows upregulation of hypoxia related genes. In addition, transcription of hypoxia-inducible factor antisense (HIF1A-AS2) was induced that in turn reduced expression of HIF-1α and simultaneously increased expression of NDRG1. Furthermore, we observed decreased protein levels of EGFR, Met, c-Myc, cyclin D1, MMP-2, MMP-9 and TF, and phosphorylation of Src and P130-cas. SC144-induced alterations of HIF-1α and NDRG1 were also confirmed in prostate cancer cells. Ciclopirox olamine (CPX) induces a cellular transcriptional profile comparable to SC144, suggesting a similar cellular mechanism of action between these two compounds. In addition, SC144 sensitized OC cells to olaparib, carboplatin and cisplatin, and shows better in vivo efficacy than CPX. Conclusion: Induction of hypoxic stress responses through inhibition of gp130 represents a novel approach to design effective anticancer treatments in combination with standard-of-care chemotherapy in OC and the efficacy reported here strongly supports their clinical development.

A Self-Assembled Platform Based on Branched DNA for sgRNA/Cas9/Antisense Delivery.

Precisely assembled DNA nanostructures are promising candidates for the delivery of biomolecule-based therapeutics. Herein, we introduce a facile strategy for the construction of a branched DNA-based nanoplatform for codelivery of gene editing (sgRNA/Cas9, targeting DNA in the nucleus) and gene silencing (antisense, targeting mRNA in the cytoplasm) components for synergistic tumor therapy in vitro and in vivo. In our design, the branched DNA structure can efficiently load a sgRNA/Cas9/antisense complex targeting a tumor-associated gene, PLK1, through DNA self-assembly. With the incorporation of an active targeting aptamer and an endosomal escape peptide by host-guest interaction, the biocompatible DNA nanoplatform demonstrates efficient inhibition of tumor growth without apparent systemic toxicity. This multifunctional DNA nanocarrier provides a new strategy for the development of gene therapeutics.

Ultrasound/microbubble-mediated targeted delivery of anticancer microRNA-loaded nanoparticles to deep tissues in pigs.

In this study, we designed and validated a platform for ultrasound and microbubble-mediated delivery of FDA-approved pegylated poly lactic-co-glycolic acid (PLGA) nanoparticles loaded with anticancer microRNAs (miRNAs) to deep tissues in a pig model. Small RNAs have been shown to reprogram tumor cells and sensitize them to clinically used chemotherapy. To overcome their short intravascular circulation half-life and achieve controlled and sustained release into tumor cells, anticancer miRNAs need to be encapsulated into nanocarriers. Focused ultrasound combined with gas-filled microbubbles provides a noninvasive way to improve the permeability of tumor vasculature and increase the delivery efficiency of drug-loaded particles. A single handheld, curvilinear ultrasound array was used in this study for image-guided therapy with clinical-grade SonoVue contrast agent. First, we validated the platform on phantoms to optimize the microbubble cavitation dose based on acoustic parameters, including peak negative pressure, pulse length, and pulse repetition frequency. We then tested the system in vivo by delivering PLGA nanoparticles co-loaded with antisense-miRNA-21 and antisense-miRNA-10b to pig liver and kidney. Enhanced miRNA delivery was observed (1.9- to 3.7-fold increase) as a result of the ultrasound treatment compared to untreated control regions. Additionally, we used highly fluorescent semiconducting polymer nanoparticles to visually assess nanoparticle extravasation. Fluorescent microscopy suggested the presence of nanoparticles in the extravascular compartment. Hematoxylin and eosin staining of treated tissues did not reveal tissue damage. The results presented in this manuscript suggest that the proposed platform may be used to safely and noninvasively enhance the delivery of miRNA-loaded nanoparticles to target regions in deep organs in large animal models.

A miR-29b Byproduct Sequence Exhibits Potent Tumor-Suppressive Activities via Inhibition of NF-κB Signaling in KRAS-Mutant Colon Cancer Cells.

We previously demonstrated that miR-29b-3p is a hopeful miRNA-based therapy against colorectal cancer. In this study, we aimed to clarify a value of miR-29b-1-5p as a next-generation treatment, especially for KRAS-mutant colorectal cancer. RT-PCR assay showed that the expression of miR-29b-3p was high, and its partner strand, miR-29b-1-5p, level was only negligible in clinical colorectal cancer samples. Mimic-miR-29b-1-5p significantly inhibited proliferation of KRAS-mutant colorectal cancer cell lines DLD1 and SW480 and KRAS wild-type HT29 cells. Proliferative activity was further examined by either miR-29b-1-5p strand or its opposite complementary sequence because miR-29b-1-5p is a passenger miRNA and may have no physiologic function. We found that completely opposite complementary strand to miR-29b-1-5p, but not miR-29b-1-5p, possessed a potent antitumor effect and named this byproduct miRNA sequence "MIRTX." MIRTX directly targeted the 3'-UTR of CXCR2 and PIK3R1 mRNA and suppressed the NF-κB signaling pathway in KRAS-mutated colorectal cancer cells. MIRTX induced apoptosis in DLD1 with downregulation of antiapoptotic BCL2, BCL-xL, and MCL1 and upregulation of cleaved caspase-3 and cleaved PARP. In mouse xenograft models, systemic administration of MIRTX using a super carbonate apatite as a delivery vehicle significantly inhibited tumor growth of DLD1 and HT29 cells without any particular toxicities. In conclusion, these findings indicate that inhibition of NF-κB signaling by this novel miRNA-based therapeutic could be a promising treatment against refractory KRAS-mutant colorectal cancer and KRAS wild-type colorectal cancer. Mol Cancer Ther; 17(5); 977-87. ©2018 AACR.

Structurally modulated codelivery of siRNA and Argonaute 2 for enhanced RNA interference.

Small interfering RNA (siRNA) represents a promising class of inhibitors in both fundamental research and the clinic. Numerous delivery vehicles have been developed to facilitate siRNA delivery. Nevertheless, achieving highly potent RNA interference (RNAi) toward clinical translation requires efficient formation of RNA-induced gene-silencing complex (RISC) in the cytoplasm. Here we coencapsulate siRNA and the central RNAi effector protein Argonaute 2 (Ago2) via different delivery carriers as a platform to augment RNAi. The physical clustering between siRNA and Ago2 is found to be indispensable for enhanced RNAi. Moreover, by utilizing polyamines bearing the same backbone but distinct cationic side-group arrangements of ethylene diamine repeats as the delivery vehicles, we find that the molecular structure of these polyamines modulates the degree of siRNA/Ago2-mediated improvement of RNAi. We apply this strategy to silence the oncogene STAT3 and significantly prolong survival in mice challenged with melanoma. Our findings suggest a paradigm for RNAi via the synergistic coassembly of RNA with helper proteins.

Orlistat and antisense-miRNA-loaded PLGA-PEG nanoparticles for enhanced triple negative breast cancer therapy.

BACKGROUND: This study explores the use of hydrophilic poly(ethylene glycol)-conjugated poly(lactic-co-glycolic acid) nanoparticles (PLGA-PEG-NPs) as delivery system to improve the antitumor effect of antiobesity drug orlistat for triple-negative breast cancer (TNBC) therapy by improving its bioavailability. MATERIALS & METHODS: PLGA-PEG-NPs were synthesized by emulsion-diffusion-evaporation method, and the experiments were conducted in vitro in MDA-MB-231 and SKBr3 TNBC and normal breast fibroblast cells. RESULTS: Delivery of orlistat via PLGA-PEG-NPs reduced its IC50 compared with free orlistat. Combined treatment of orlistat-loaded NPs and doxorubicin or antisense-miR-21-loaded NPs significantly enhanced apoptotic effect compared with independent doxorubicin, anti-miR-21-loaded NPs, orlistat-loaded NPs or free orlistat treatments. CONCLUSION: We demonstrate that orlistat in combination with antisense-miR-21 or current chemotherapy holds great promise as a novel and versatile treatment agent for TNBC.

Abnormal Expression of miR-21 and miR-95 in Cancer Stem-Like Cells is Associated with Radioresistance of Lung Cancer.

This study demonstrated that miR-21 and miR-95 expression were significantly higher in the ALDH1(+)CD133(+)subpopulation than in the ALDH1(-)CD133(-) subpopulation of lung cancer cells. Combined delivery of anti-miR-21 and anti-miR-95 by calcium phosphate nanoparticles significantly inhibited tumor growth in a xenograft tumor model and sensitized radiotherapy. The anti-miRNAs significantly reduced miR-21 and miR-95 levels, increased PTEN, SNX1, and SGPP1 protein expression, but reduced Akt Ser(473) and Thr(308) phosphorylation. ALDH1(+)CD133(+) subpopulation of NSCLC tumor cells confers radioresistance due to high expression of miR-21 and miR-95. Targeting inhibition of miR-21 and miR-95 can inhibit tumor growth through elevating PTEN, SNX1, and SGPP1 expression and inhibiting Akt phosphorylation.

Microvesicle-delivery miR-150 promotes tumorigenesis by up-regulating VEGF, and the neutralization of miR-150 attenuate tumor development.

Tumor-associated macrophages (TAMs) mostly exhibit M2-like (alternatively activated) properties and play positive roles in angiogenesis and tumorigenesis. Vascular endothelial growth factor (VEGF) is a key angiogenic factor. During tumor development, TAMs secrete VEGF and other factors to promote angiogenesis; thus, anti-treatment against TAMs and VEGF can repress cancer development, which has been demonstrated in clinical trials and on an experimental level. In the present work, we show that miR-150 is an oncomir because of its promotional effect on VEGF. MiR-150 targets TAMs to up-regulate their secretion of VEGF in vitro. With the utilization of cell-derived vesicles, named microvesicles (MVs), we transferred antisense RNA targeted to miR-150 into mice and found that the neutralization of miR-150 down-regulates miR-150 and VEGF levels in vivo and attenuates angiogenesis. Therefore, we proposed the therapeutic potential of neutralizing miR-150 to treat cancer and demonstrated a novel, natural, microvesicle-based method for the transfer of nucleic acids.

Phospholamban antisense RNA improves SR Ca2+-ATPase activity and left ventricular function in STZ-induced diabetic rats.

OBJECTIVE: To study the effect of phospholamban antisense RNA (asPLB) on sarcoplasmic reticulum Ca2+-ATPase activity and cardiac function in rats with diabetes mellitus (DM) mediated by recombinant adeno-associated virus (rAAV) vector. METHODS: Six weeks after the induction of DM by streptozotocin injected intraperitoneally, the rats were divided into three groups, namely: DM-rAAV-asPLB group, DM-saline group and DM group (control group). The rats in the DM-rAAV-asPLB group were intramyocardially injected with rAAV-asPLB, the rats in the DM-saline group were injected with saline, and those in the control group did not receive any treatment. Six weeks after gene transfer, the expressions of PLB protein and PLB phosphorylation were detected by Western-blot, while the activity of sarcoplasmic reticulum (SR) Ca2+-ATPase and left ventricular function were measured. RESULTS: The PLB protein expression level was significantly higher whereas the PLB phosphorylation, SR Ca2+-ATPase activity and left ventricular function were significantly lower in the DM-saline group than in the control group. No significant difference was found in PLB protein expression level, PLB phosphorylation or SR Ca2+-ATPase activity between the DM-rAAV-asPLB group and the control group. The left ventricular function in the DM-rAAV-asPLB group was poorer than in the control group and was better than in the DM-saline group. CONCLUSION: rAAV-asPLB can down-regulate PLB protein expression and up-regulate PLB phosphorylation and SR Ca2+-ATPase activity, thus contributing to the improvement of in vivo left ventricular function.

Targeting miR-21 inhibits in vitro and in vivo multiple myeloma cell growth.

PURPOSE: Deregulated expression of miRNAs plays a role in the pathogenesis and progression of multiple myeloma. Among upregulated miRNAs, miR-21 has oncogenic potential and therefore represents an attractive target for the treatment of multiple myeloma. EXPERIMENTAL DESIGN: Here, we investigated the in vitro and in vivo anti-multiple myeloma activity of miR-21 inhibitors. RESULTS: Either transient-enforced expression or lentivirus-based constitutive expression of miR-21 inhibitors triggered significant growth inhibition of primary patient multiple myeloma cells or interleukin-6-dependent/independent multiple myeloma cell lines and overcame the protective activity of human bone marrow stromal cells. Conversely, transfection of miR-21 mimics significantly increased proliferation of multiple myeloma cells, showing its tumor-promoting potential in multiple myeloma. Importantly, upregulation of miR-21 canonical validated targets (PTEN, Rho-B, and BTG2), together with functional impairment of both AKT and extracellular signal-regulated kinase signaling, were achieved by transfection of miR-21 inhibitors into multiple myeloma cells. In vivo delivery of miR-21 inhibitors in severe combined immunodeficient mice bearing human multiple myeloma xenografts expressing miR-21 induced significant antitumor activity. Upregulation of PTEN and downregulation of p-AKT were observed in retrieved xenografts following treatment with miR-21 inhibitors. CONCLUSION: Our findings show the first evidence that in vivo antagonism of miR-21 exerts anti-multiple myeloma activity, providing the rationale for clinical development of miR-21 inhibitors in this still incurable disease.

Pulmonary delivery and tissue distribution of aerosolized antisense 2'-O-Methyl RNA containing nanoplexes in the isolated perfused and ventilated rat lung.

Pulmonary delivery of drugs, particularly in the treatment of lung cancer, is an attractive strategy for future targeted therapy. In this context, inhalation of nanoplexes might offer a new mode for drug delivery in gene therapy. However, limited data are currently available demonstrating pulmonary delivery, cellular uptake as well as local tolerability in lung tissue. The aim of this study was to elucidate the pulmonary delivery, tissue distribution and local tolerability of aerosolized chitosan-coated poly(lactide-co-glycolide) based nanoplexes containing antisense 2'-O-Methyl RNA (OMR). Therefore, an aerosol of OMR-nanoplexes or OMR alone was administered intra-tracheally using the model of the isolated perfused and ventilated rat lung. Localization of OMR in rat lung tissue was examined by immunohistochemistry. Administration of the OMR-nanoplex formulation resulted in significantly higher cellular OMR uptake of the respiratory epithelium in contrast to the administration of OMR alone, indicating that drug administration via aerosolized nanoplexes is able to target lung tissue. No prominent changes in lung physiology parameters were observed following inhalation, suggesting good local tolerability of OMR-nanoplex formulation.

Anti-tumor efficacy of naked siRNAs for ERBB3 or AKT2 against lung adenocarcinoma cell xenografts.

The use of siRNAs against specific molecular targets has potential for cancer therapy but has been thought to be limited by the need for formulation to improve cellular uptake. Lung adenocarcinoma cells are markedly suppressed in culture by siRNAs to the receptor ERBB3 or its downstream signaling partner AKT2. We now demonstrate that naked, unformulated siRNAs to ERBB3 or AKT2, administered i.v. as saline solutions, 2 µg/g five times per week for 3 weeks (total dose 30 µg/g), were effective suppressors of growth of A549 human lung adenocarcinoma cell xenografts in athymic mice, 12 mice per group, in four different experiments. ERBB3 and AKT2 siRNAs each inhibited growth by 70-90% on average, compared to saline-treated or untreated controls; a nonsilencing siRNA was without significant effect. Lesser but significant effects were noted with a total dose of 12 µg/g. With the higher dose, effects persisted for several weeks after the end of treatment. Expected reductions of ERBB3 and AKT2 mRNAs and proteins occurred and correlated with decrease in tumor volume. There were no significant changes in serum cytokines. These results show that naked siRNAs to ERBB3 or AKT2 may have potential for lung cancer therapy.

Tissue slice model of human lung cancer to investigate telomerase inhibition by nanoparticle delivery of antisense 2'-O-methyl-RNA.

Nanoparticles delivery of oligonucleotides represents a potential approach for cancer treatment. However, most of the experiments were based on established cancer cell lines and may not reflect the original solid tumor in vivo. Both, tumor microenvironment and tumor cell biological properties in the tumor can influence the delivery efficiency of oligonucleotides. Therefore, it is important to understand the effect of nanoparticles delivery of oligonucleotides on tumor response in intact tissue architecture of individual tumors. We used freshly isolated human tumor tissue slices and primary lung cancer cells from non-small cell lung cancer patients to evaluate this nanocarrier system. Chitosan-coated poly(lactide-co-glycolide) (PLGA) nanoparticles were used to form oligonucleotide-nanoparticle-complexes (nanoplexes) with antisense 2'-O-methyl-RNA (OMR) that can inhibit telomerase activity by binding to the RNA component of telomerase. OMR cellular uptake was strongly enhanced by nanoplexes mediated delivery in both, primary cells and tissue slices. More than 80% of primary cancer cells and 50% of cells in tissue slices showed OMR uptake. Telomerase activity was inhibited by approximately 45% in primary cancer cells and about 40% in tissue slices. Nanoplexes could penetrate into tumor tissue without influencing tissue architecture and the delivered OMR was able to inhibit telomerase activity with relatively low cytotoxicity.

Placenta growth factor induces melanoma resistance to temozolomide through a mechanism that involves the activation of the transcription factor NF-κB.

Placenta growth factor (PlGF) and its receptor vascular endothelial growth factor receptor-1 (VEGFR-1) are co-expressed in a large number of human melanoma cell lines. Moreover, a correlation between in vivo PlGF production and melanoma progression has been suggested. To investigate whether PlGF might have a role in protecting melanoma cells from the cytotoxic effects of the anticancer agent temozolomide (TMZ), which is used for the treatment of this malignancy, we stably transfected a doxycycline-inducible PlGF antisense mRNA into a human melanoma cell clone that secretes VEGF-A and PlGF and expresses receptors for both growth factors. Induction of PlGF antisense mRNA in the transfected cells (13443/ASP3 subclone) halved TMZ IC(50), and exogenous addition of PlGF to the culture medium 24 h before TMZ treatment, partially restored IC(50) values to that of control cells. The increased sensitivity of 13443/ASP3 cells upon PlGF antisense mRNA expression was not due to down-regulation of O6-methylguanine-DNA methyltransferase, a DNA repair protein that represents the main mechanism of resistance to TMZ. Since the activity of the transcription factor nuclear factor-κB (NF-κB) has been correlated to melanoma chemoresistance, we investigated whether NF-κB was involved in PlGF-induced melanoma cell resistance to TMZ. Induction of PlGF antisense mRNA in 13443/ASP3 cells halved the levels of active NF-κB and the specific inhibition of this transcription factor increased sensitivity of 13443/ASP3 cells to TMZ. In conclusion, our data strongly suggest that PlGF plays a role in melanoma cell resistance to TMZ through a pathway that involves NF-κB activation.

Antisense treatment in human prostate cancer and melanoma.

Antisense reagents and technology have developed as extraordinarily useful tools for analysis of gene function. The capacity of antisense to reduce expression of RNA (including protein-encoding mRNA and non-coding RNA) important in a multitude of diseases (including cancer) has led to the concept of using antisense molecules as drugs to treat those diseases. Both antisense RNA (RNAi) and antisense oligonucleotides (ASOs) are being developed for this purpose, with ASOs currently the most advanced in clinical testing. ASOs inhibit translation or induce degradation of complementary target RNA, and both Phase I and Phase II trials are either completed or in progress for a number of diseases. In this review, we focus on antisense approaches to treatment of two cancers (melanoma and hormone-resistant prostate cancer) where the early application of ASOs has provided important information revealing both potential for success and lessons for future preclinical and clinical investigation of ASOs as anti-cancer drugs. The progress of clinical application of two ASOs showing promise in treatment of human cancers--Oblimersen (G3139), targeting BCL2 for the treatment of metastatic melanoma, and Custirsen (OGX-11), targeting clusterin for the treatment of hormone refractory prostate cancer (HRPC)--is examined.

Peptide nucleic acids conjugated to short basic peptides show improved pharmacokinetics and antisense activity in adipose tissue.

A peptide nucleic acid (PNA) targeting a splice junction of the murine PTEN primary transcript was covalently conjugated to various basic peptides. When systemically administered to healthy mice, the conjugates displayed sequence-specific alteration of PTEN mRNA splicing as well as inhibition of full length PTEN protein expression. Correlating activity with drug concentration in various tissues indicated strong tissue-dependence, with highest levels of activity observed in adipose tissue. While the presence of a peptide carrier was found to be crucial for efficient delivery to tissue, little difference was observed between the various peptides evaluated. A second PNA-conjugate targeting the murine insulin receptor primary transcript showed a similar activity profile, suggesting that short basic peptides can generally be used to effectively deliver peptide nucleic acids to adipose tissue.

Treatment of prostate cancer cells with adenoviral vector-mediated antisense RNA using androgen-dependent and androgen-independent promoters.

The present study was designed to develop a novel antisense approach to prostate cancer therapy. We constructed ODC/AdoMetDC double antisense RNA recombinant adenovirus mediated by a prostate-specific androgen-dependent promoter (pADxsi-P2-AdoMetDC-ODC-PolyA) or a prostate-specific androgen-independent promoter (pADxsi-P1-AdoMetDC-ODC-PolyA). Western blot analysis was performed to detect the ODC and AdoMetDC protein levels. The growth curves for each group of cells were determined by MTT assay. Flow cytometry was conducted to detect cell apoptosis, proliferation, and cell cycle distribution in order to demonstrate the effects of the recombinant adenoviruses on the prostate cancer cells. RT-PCR, Western blotting, MTT, and tumor growth inhibition assay in nude mice demonstrated that pADxsi-P1-AdoMetDC-ODC-PolyA and pADxsi-P2-AdoMetDC-ODC-PolyA exhibited inhibitory effects on cell proliferation at both the gene and protein levels. Meanwhile, inhibiting effects of the pADxsi-P2-AdoMetDC-ODC-PolyA is more profound than those of the pADxsi-P1-AdoMetDC-ODC-PolyA vector. Both of the adenoviral vectors exhibited significant inhibitory effects on the growth of prostate tumors, and the inhibitory effects of the pADxsi-P2-AdoMetDC-ODC-PolyA vector were stronger than those of the pADxsi-P1-AdoMetDC-ODC-PolyA vector.

Regression of murine lung tumors by the let-7 microRNA.

MicroRNAs (miRNAs) have recently emerged as an important new class of cellular regulators that control various cellular processes and are implicated in human diseases, including cancer. Here, we show that loss of let-7 function enhances lung tumor formation in vivo, strongly supporting the hypothesis that let-7 is a tumor suppressor. Moreover, we report that exogenous delivery of let-7 to established tumors in mouse models of non-small-cell lung cancer (NSCLC) significantly reduces the tumor burden. These results demonstrate the therapeutic potential of let-7 in NSCLC and point to miRNA replacement therapy as a promising approach in cancer treatment.