RNA Nanotechnology for Codelivering High-Payload Nucleoside Analogs to Cancer with a Synergetic Effect.

Nucleoside analogs are potent inhibitors for cancer treatment, but the main obstacles to their application in humans are their toxicity, nonspecificity, and lack of targeted delivery tools. Here, we report the use of RNA four-way junctions (4WJs) to deliver two nucleoside analogs, floxuridine (FUDR) and gemcitabine (GEM), with high payloads through routine and simple solid-state RNA synthesis and nanoparticle assembly. The design of RNA nanotechnology for the co-delivery of nucleoside analogs and the chemotherapeutic drug paclitaxel (PTX) resulted in synergistic effects and high efficacy in the treatment of Triple-Negative Breast Cancer (TNBC). The 4WJ-drug complexes were confirmed to have efficient tumor spontaneous targeting and no toxicity because the motility of RNA nanoparticles has been previously shown to enable these RNA-drug complexes to spontaneously accumulate in tumor blood vessels. The negative charge of RNA enables those RNA complexes that are not targeted to tumor vasculature to circulate in the blood and enter the urine through the kidney glomerulus, without accumulating in organs, therefore being nontoxic. Drug incorporation into RNA 4WJ can be precisely controlled with a defined loading amount, location, and ratio. The incorporation of nucleoside analogs into 4WJ only requires one step using nucleoside analogue phosphoramidites during solid-phase RNA synthesis, without the need for additional conjugation and purification processes.

In Vitro and In Vivo Evaluation of the Pathology and Safety Aspects of Three- and Four-Way Junction RNA Nanoparticles.

RNA therapeutics has advanced into the third milestone in pharmaceutical drug development, following chemical and protein therapeutics. RNA itself can serve as therapeutics, carriers, regulators, or substrates in drug development. Due to RNA's motile, dynamic, and deformable properties, RNA nanoparticles have demonstrated spontaneous targeting and accumulation in cancer vasculature and fast excretion through the kidney glomerulus to urine to prevent possible interactions with healthy organs. Furthermore, the negatively charged phosphate backbone of RNA results in general repulsion from negatively charged lipid cell membranes for further avoidance of vital organs. Thus, RNA nanoparticles can spontaneously enrich tumor vasculature and efficiently enter tumor cells via specific targeting, while those not entering the tumor tissue will clear from the body quickly. These favorable parameters have led to the expectation that RNA has low or little toxicity. RNA nanoparticles have been well characterized for their anticancer efficacy; however, little detail on RNA nanoparticle pathology and safety is known. Here, we report the in vitro and in vivo assessment of the pathology and safety aspects of different RNA nanoparticles including RNA three-way junction (3WJ) harboring 2'-F modified pyrimidine, folic acid, and Survivin siRNA, as well as the RNA four-way junction (4WJ) harboring 2'-F modified pyrimidine and 24 copies of SN38. Both animal models and patient serum were investigated. In vitro studies include hemolysis, platelet aggregation, complement activation, plasma coagulation, and interferon induction. In vivo studies include hematoxylin and eosin (H&E) staining, hematological and biochemical analysis as the serum profiling, and animal organ weight study. No significant toxicity, side effect, or immune responses were detected during the extensive safety evaluations of RNA nanoparticles. These results further complement previous cancer inhibition studies and demonstrate RNA nanoparticles as an effective and safe drug delivery vehicle for future clinical translations.

RNA four-way junction (4WJ) for spontaneous cancer-targeting, effective tumor-regression, metastasis suppression, fast renal excretion and undetectable toxicity.

The field of RNA therapeutics has been emerging as the third milestone in pharmaceutical drug development. RNA nanoparticles have displayed motile and deformable properties to allow for high tumor accumulation with undetectable healthy organ accumulation. Therefore, RNA nanoparticles have the potential to serve as potent drug delivery vehicles with strong anti-cancer responses. Herein, we report the physicochemical basis for the rational design of a branched RNA four-way junction (4WJ) nanoparticle that results in advantageous high-thermostability and -drug payload for cancer therapy, including metastatic tumors in the lung. The 4WJ nanostructure displayed versatility through functionalization with an anti-cancer chemical drug, SN38, for the treatment of two different cancer models including colorectal cancer xenograft and orthotopic lung metastases of colon cancer. The resulting 4WJ RNA drug complex spontaneously targeted cancers effectively for cancer inhibition with and without ligands. The 4WJ displayed fast renal excretion, rapid body clearance, and little organ accumulation with undetectable toxicity and immunogenicity. The safety parameters were documented by organ histology, blood biochemistry, and pathological analysis. The highly efficient cancer inhibition, undetectable drug toxicity, and favorable Chemical, Manufacturing, and Control (CMC) production of RNA nanoparticles document a candidate with high potential for translation in cancer therapy.

Type-3 Hyaluronan Synthase Attenuates Tumor Cells Invasion in Human Mammary Parenchymal Tissues.

The microenvironment for tumor growth and developing metastasis should be essential. This study demonstrated that the hyaluronic acid synthase 3 (HAS3) protein and its enzymatic product hyaluronic acid (HA) encompassed in the subcutaneous extracellular matrix can attenuate the invasion of human breast tumor cells. Decreased HA levels in subcutaneous Has3-KO mouse tissues promoted orthotopic breast cancer (E0771) cell-derived allograft tumor growth. MDA-MB-231 cells premixed with higher concentration HA attenuate tumor growth in xenografted nude mice. Human patient-derived xenotransplantation (PDX) experiments found that HA selected the highly migratory breast cancer cells with CD44 expression accumulated in the tumor/stroma junction. In conclusion, HAS3 and HA were detected in the stroma breast tissues at a high level attenuates effects for induced breast cancer cell death, and inhibit the cancer cells invasion at the initial stage. However, the highly migratory cancer cells were resistant to the HA-mediated effects with unknown mechanisms.

Thermostability, Tunability, and Tenacity of RNA as Rubbery Anionic Polymeric Materials in Nanotechnology and Nanomedicine-Specific Cancer Targeting with Undetectable Toxicity.

RNA nanotechnology is the bottom-up self-assembly of nanometer-scale architectures, resembling LEGOs, composed mainly of RNA. The ideal building material should be (1) versatile and controllable in shape and stoichiometry, (2) spontaneously self-assemble, and (3) thermodynamically, chemically, and enzymatically stable with a long shelf life. RNA building blocks exhibit each of the above. RNA is a polynucleic acid, making it a polymer, and its negative-charge prevents nonspecific binding to negatively charged cell membranes. The thermostability makes it suitable for logic gates, resistive memory, sensor set-ups, and NEM devices. RNA can be designed and manipulated with a level of simplicity of DNA while displaying versatile structure and enzyme activity of proteins. RNA can fold into single-stranded loops or bulges to serve as mounting dovetails for intermolecular or domain interactions without external linking dowels. RNA nanoparticles display rubber- and amoeba-like properties and are stretchable and shrinkable through multiple repeats, leading to enhanced tumor targeting and fast renal excretion to reduce toxicities. It was predicted in 2014 that RNA would be the third milestone in pharmaceutical drug development. The recent approval of several RNA drugs and COVID-19 mRNA vaccines by FDA suggests that this milestone is being realized. Here, we review the unique properties of RNA nanotechnology, summarize its recent advancements, describe its distinct attributes inside or outside the body and discuss potential applications in nanotechnology, medicine, and material science.

Tea polyphenol epigallocatechin-3-gallate inhibits cell proliferation in a patient-derived triple-negative breast cancer xenograft mouse model via inhibition of proline-dehydrogenase-induced effects.

Triple-negative breast cancers (TNBCs) lack specific targeted therapy options and have evolved into highly chemo-resistant tumors that metastasize to multiple organs. The present study demonstrated that the proline dehydrogenase (PRODH) mRNA level in paired (tumor vs. normal) human breast tissue samples (n=234) was 6.6-fold greater than normal cells (*p=0.021). We established stable PRODH-overexpressing TNBC (HS578T) cells, and the malignant phenotypes were evaluated using soft agar colony formation and Transwell migration assays. The results demonstrated that PRODH induced epithelial-mesenchymal transition in cancer cells and increased cell proliferation. The present study found that the tea polyphenol epigallocatechin-3-gallate (EGCG) significantly inhibited PRODH and its regulated proteins, such as alpha-smooth muscle actin (alpha-SMA) expression in TNBC cells. These findings support the targeting of the PRODH signaling pathway as a potential therapeutic strategy in preventing cancer cell metastasis. The patient-derived xenograft (PDX) mouse model is highly relevant to real human tumor growth. We established a TNBC-PDX (F4, n=4 in each group)mouse model. The PDX mice were treated with EGCG (50 mg/kg), and the results indicated that EGCG significantly inhibited PDX tumor growth (*p = 0.013). These experiments provide additional evidence to evaluate the antitumor effects of EGCG-induced PRODH inhibition for clinical therapeutic application, especially in TNBC patients.

Multivalent rubber-like RNA nanoparticles for targeted co-delivery of paclitaxel and MiRNA to silence the drug efflux transporter and liver cancer drug resistance.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide. Analogous to the border customs, liver mainly functions as a filter to detoxify chemicals and metabolite administered orally or intravenously. Besides, the liver cancer cells overexpress the drug exporters which cause high drug effluxion from liver cancer cells, leading to chemoresistance and a diminished chemotherapeutic effect on liver cancer. Recently, we found that RNA nanoparticles display rubber-like property that can rapidly deliver therapeutics to tumor site efficiently and the rest of the RNA nanoparticle were cleared by renal excretion within half hour after systemic injection. Therefore, we designed a new multivalent RNA nanoparticle harboring three copies of hepatocyte targeting-ligands, one copy of miR122, and 24 copies of Paclitaxel to overcome the drug effluxion and chemoresistance thus, synergistically treating HCC. The hepatocyte targeting ligands introduce tumor specificity to the RNA nanoparticles as they selectively bind and internalize into liver cancer cells. The rubber-like RNA nanoparticles allow for enhanced targeting ability to the HCC tumors. The RNA nanoparticles carrying miR122 and PTX were delivered to the liver cancer cells efficiently due to their rubber-like property to enhance their EPR as well as the receptor-mediated endocytosis by hepatocyte targeting-ligands. The miR122 efficiently silenced the drug exporters and the oncogenic proteins. The synergistic effect between miR122 and PTX was confirmed by HSA (Highest Single Agent) synergy model. IC50 was determined to be 460 nM. In vivo studies on mice xenografts revealed that the RNA nanoparticle predominantly accumulated in HCC tumor sites and efficiently inhibited the tumor growth after multiple IV injection. This demonstrates the potential of the rubber-like multivalent RNA nanoparticles to conquest the liver cancer, a currently incurable lethal disease.

Melatonin promotes neuroblastoma cell differentiation by activating hyaluronan synthase 3-induced mitophagy.

Neuroblastoma is the second most common pediatric malignancy and has a high rate of spontaneous remission. Uncovering the mechanisms underlying neuroblastoma cell differentiation is critical for therapeutic purposes. A neuroblastoma cell line (N2a) treated with either serum withdrawal (<2.5%) or melatonin (>0.1 nmol/L) for 24 hours was used as a cell differentiation research model. Interestingly, the hyaluronan synthase 3 (HAS3) protein was induced in differentiated N2a cells. N2a-allografted nude mice received an intraperitoneal injection of melatonin (40 or 80 mg/kg/day for 3 weeks). The mean tumor volume in mice treated with 80 mg/kg melatonin was smaller than that in PBS-treated mice (1416.3 and 3041.3 mm3 , respectively, difference = 1625 mm3 , *P = 0.0003, n = 7 per group). Compared with the vector control group, N2a cells with forced HAS3 overexpression showed significantly increased neuron length (*P = 0.00082) and neurite outgrowth (*P = 0.00059). Intracellular changes in autophagy, including distorted mitochondria with abnormal circular inner membranes, were detected by transmission electron microscopy (TEM). Our study demonstrated that HAS3-mediated signaling activated by physiological concentrations of melatonin (>0.1 nmol/L) triggered significant N2a cell differentiation. These results provide molecular data with potential clinical relevance for therapeutic drug development.

Study of the antitumor mechanisms of apiole derivatives (AP-02) from Petroselinum crispum through induction of G0/G1 phase cell cycle arrest in human COLO 205 cancer cells.

BACKGROUND: Apiole was isolated from the leaves of various plants and vegetables and has been demonstrated to inhibit human colon cancer cell (COLO 205 cells) growth through induction of G0/G1 cell cycle arrest and apoptotic cell death. This study further explored the antitumor effects of apiole derivatives AP-02, 04, and 05 in COLO 205 cancer cells. METHODS: Human breast (MDA-MB-231, ZR75), lung (A549, PE089), colon (COLO 205, HT 29), and hepatocellular (Hep G2, Hep 3B) cancer cells were treated with apiole and its derivatives in a dose-dependent manner. Flow cytometry analysis was subsequently performed to determine the mechanism of AP-02-induced G0/G1 cell cycle arrest. The in vivo antitumor effect of AP-02 (1 and 5 mg/kg, administered twice per week) was examined by treating athymic nude mice bearing COLO 205 tumor xenografts. The molecular mechanisms of AP-02-induced antitumor effects were determined using western blot analysis. RESULTS: AP-02 was the most effective compound, especially for inhibition of COLO 205 colon cancer cell growth. The cytotoxicity of AP-02 in normal colon epithelial (FHC) cells was significantly lower than that in other normal cells derived from the breast, lung or liver. Flow cytometry analysis indicated that AP-02-induced G0/G1 cell cycle arrest in COLO 205 cells but not in HT 29 cells (< 5 µM for 24 h, **p < 0.01). Tumor growth volume was also significantly inhibited in AP-02 (> 1 mg/kg)-treated athymic nude mice bearing COLO 205 tumor xenografts compared to control mice (*p < 0.05). Furthermore, G0/G1 phase regulatory proteins (p53 and p21/Cip1) and an invasion suppressor protein (E-cadherin) were significantly upregulated, while cyclin D1 was significantly downregulated, in AP-02-treated tumor tissues compared to the control group (> 1 mg/kg, *p < 0.05). CONCLUSIONS: Our results provide in vitro and in vivo molecular evidence of AP-02-induced anti-proliferative effects on colon cancer, indicating that this compound might have potential clinical applications.

Small G protein signalling modulator 2 (SGSM2) is involved in oestrogen receptor-positive breast cancer metastasis through enhancement of migratory cell adhesion via interaction with E-cadherin.

The function of small G protein signalling modulators (SGSM1/2/3) in cancer remains unknown. Our findings demonstrated that SGSM2 is a plasma membrane protein that strongly interacted with E-cadherin/ß-catenin. SGSM2 downregulation enhanced the phosphorylation of focal adhesion kinase (FAK; Y576/577), decreased the expression of epithelial markers such as E-cadherin, ß-catenin, and Paxillin, and increased the expression of Snail and Twist-1, which reduced cell adhesion and promoted cancer cell migration. Oestrogen and fibronectin treatment was found to promote the colocalization of SGSM2 at the leading edge with phospho-FAK (Y397). The BioGRID database showed that SGSM2 potentially interacts with cytoskeleton remodelling and cell-cell junction proteins. These evidences suggest that SGSM2 plays a role in modulating cell adhesion and cytoskeleton dynamics during cancer migration.

α-Tocopherol succinate enhances pterostilbene anti-tumor activity in human breast cancer cells in vivo and in vitro.

Vitamin E (Vit. E) is considered an essential dietary nutrient for humans and animals. An enormous body of evidence indicates the biological and protective effects of Vit. E consumption. Tocopherol-associated protein (TAP) is a major tocopherol-binding protein affecting Vit. E stimulation and downstream signaling transduction. However, how Vit. E utilizes TAP as an anti-cancer mechanism remains unclear. Microarray analysis of signature gene profiles in breast cancer cells treated with α-tocopheryl succinate (α-TOS, a Vit. E isoform) resulted in cell cycle arrest and anti-cancer activity in breast cancer cells. Pterostilbene (PS), a natural dietary antioxidant found in blueberries, in combination with α-TOS synergistically maximized breast cancer cell growth inhibition by disrupting signal transduction, transcription factors and cell cycle proteins. In a xenograft mouse model, PS treatment with Vit. E inhibited breast tumor growth and cell invasion, which were evaluated using our recently developed circulating tumor cell (CTC) detection assay. Because dietary Vit. E and PS supplementation contributed to preventative and therapeutic effects in vitro and in vivo, this combination may benefit breast cancer therapy in the clinic.

Pu-erh Tea Extract Attenuates Nicotine-Induced Foam Cell Formation in Primary Cultured Monocytes: An in Vitro Mechanistic Study.

In this study, the mechanisms by which pu-erh tea extract (PETE) attenuates nicotine-induced foam cell formation were investigated. Monocytes were purified from healthy individuals using commercial antibodies coated with magnetic beads. We found that the nicotine-induced (1-10 µM) expression of oxidized low-density lipoprotein receptors (ox-LDLRs) and α9-nAchRs in monocytes was significantly attenuated by 24 h of PETE (10 µg/mL; ∗, p < 0.05) cotreatment. Nicotine (1 µM for 24 h) significantly induced the expression of the surface adhesion molecule ICAM-1 and the monocyte integrin adhesion molecule (CD11b) by human umbilical vein endothelial cells (HUVECs) and triggered monocytes to differentiate into macrophages via interactions with the endothelium. After treatment with nicotine (0.1-10 µM for 24 h), the HUVECs released chemotactic factors (IL-8) to attract monocytes into the tunica intima of the artery, and the monocytes then transformed into foam cells. We demonstrated that PETE treatment (>1 µg/mL for 24 h; ∗, p < 0.05) significantly attenuates nicotine-induced (1 µM) monocyte migration toward HUVECs and foam cell formation. This study suggests that tea components effectively attenuate the initial step (foam cell formation) of nicotine-induced atherosclerosis in circulating monocytes.

Alteration of α-tocopherol-associated protein (TAP) expression in human breast epithelial cells during breast cancer development.

Breast cancer is the most common malignancy among women and has an age-specific incidence profile. Over the last decade, many studies have demonstrated the anticancer activity of α-tocopherol, the main and most active form of natural vitamin E. α-Tocopherol-associated protein (TAP) was found to be one of the major α-tocopherol binding proteins in human serum and in liver, brain, and prostate tissues. In this study, we found that reduced TAP expression was significantly correlated with Her2/neu receptor expression, breast cancer stage and nodal stage in paired normal and cancerous breast tissue samples from 93 patients using real-time PCR analysis. A cell viability assay showed that α-tocopheryl succinate (α-TOS), a synthetic derivative of α-tocopherol, enhanced the cells' sensitivity to doxorubicin and resulted in a reduction in cell viability in breast cancers. Taken together, these data suggest that the use of vitamin E or its analogue as a dietary supplement may be beneficial for the treatment of cancer.