Selectin-targeting glycosaminoglycan-peptide conjugate limits neutrophil-mediated cardiac reperfusion injury.

AIMS: One of the hallmarks of myocardial infarction (MI) is excessive inflammation. During an inflammatory insult, damaged endothelial cells shed their glycocalyx, a carbohydrate-rich layer on the cell surface which provides a regulatory interface to immune cell adhesion. Selectin-mediated neutrophilia occurs as a result of endothelial injury and inflammation. We recently designed a novel selectin-targeting glycocalyx mimetic (termed DS-IkL) capable of binding inflamed endothelial cells. This study examines the capacity of DS-IkL to limit neutrophil binding and platelet activation on inflamed endothelial cells, as well as the cardioprotective effects of DS-IkL after acute myocardial infarction. METHODS AND RESULTS: In vitro, DS-IkL diminished neutrophil interactions with both recombinant selectin and inflamed endothelial cells, and limited platelet activation on inflamed endothelial cells. Our data demonstrated that DS-IkL localized to regions of vascular inflammation in vivo after 45 min of left anterior descending coronary artery ligation-induced MI. Further, findings from this study show DS-IkL treatment had short- and long-term cardioprotective effects after ischaemia/reperfusion of the left anterior descending coronary artery. Mice treated with DS-IkL immediately after ischaemia/reperfusion and 24 h later exhibited reduced neutrophil extravasation, macrophage accumulation, fibroblast and endothelial cell proliferation, and fibrosis compared to saline controls. CONCLUSIONS: Our findings suggest that DS-IkL has great therapeutic potential after MI by limiting reperfusion injury induced by the immune response.

Development and Characterization of a Novel Peptide-Drug Conjugate with DM1 for Treatment of FGFR2-Positive Tumors.

A maytansin derivative, DM1, is a promising therapeutic compound for treating tumors, but is also a highly poisonous substance with various side effects. For clinical expansion, we tried to develop novel peptide-drug conjugates (PDCs) with DM1. In the study, a one-bead one-compound (OBOC) platform was used to screen and identify a novel, highly stable, non-natural amino acid peptide targeting the tyrosine receptor FGFR2. Then, the identified peptide, named LLC2B, was conjugated with the cytotoxin DM1. Our results show that LLC2B has high affinity for the FGFR2 protein according to an isothermal titration calorimetry (ITC) test. LLC2B-Cy5.5 binding to FGFR2-positive cancer cells was confirmed by fluorescent microscopic imaging and flow cytometry in vitro. Using xenografted nude mouse models established with breast cancer MCF-7 cells and esophageal squamous cell carcinoma KYSE180 cells, respectively, LLC2B-Cy5.5 was observed to specifically target tumor tissues 24 h after tail vein injection. Incubation assays, both in aqueous solution at room temperature and in human plasma at 37 °C, suggested that LLC2B has high stability and strong anti-proteolytic ability. Then, we used two different linkers, one of molecular disulfide bonds and another of a maleimide group, to couple LLC2B to the toxin DM1. The novel peptide-drug conjugates (PDCs) inhibited tumor growth and significantly increased the maximum tolerated dose of DM1 in xenografted mice. In brief, our results suggest that LLC2B-DM1 can be developed into a potential PDC for tumor treatment in the future.

CircASH2L Promotes Ovarian Cancer Tumorigenesis, Angiogenesis, and Lymphangiogenesis by Regulating the miR-665/VEGFA Axis as a Competing Endogenous RNA.

Ovarian cancer is the leading cause of gynecologic cancer-related deaths. Emerging research has revealed a close relationship between circular RNAs (circRNAs) and ovarian cancer development, metastasis, and prognosis. The objective of our research was to further explore the relationship between circASH2L and ovarian cancer. Quantitative real-time polymerase chain reaction was used to detect the differential expression of circRNAs between normal ovaries and ovarian cancer tissues. The impact of circASH2L on the proliferation, invasion, and tumorigenicity of ovarian cancer cells was evaluated using gain- and loss-of-function experiments. The molecular mechanisms of circASH2L function were investigated using bioinformatics analysis, RNA fluorescence in situ hybridization, western blots, and dual-luciferase reporter assays. The results showed that circASH2L was remarkably upregulated in ovarian cancer. The invasion and growth of ovarian cancer cells were suppressed by circASH2L knockdown in vitro, and downregulation of circASH2L restrained both angiogenesis and lymphangiogenesis of tumor xenografts in vivo. Furthermore, circASH2L was mostly distributed in the cytoplasm, where it competes with vascular endothelial growth factor A (VEGFA) for binding to miR-665. These findings indicate that circASH2L has an oncogenic function in ovarian cancer. In conclusion, circASH2L plays a critical role in regulating ovarian cancer cell tumorigenesis, angiogenesis, and lymphangiogenesis through the miR-665/VEGFA axis and, therefore, is a possible candidate target for ovarian cancer treatment.

Novel nanococktail of a dual PI3K/mTOR inhibitor and cabazitaxel for castration-resistant prostate cancer.

Prognosis of castration-resistant prostate cancer (CRPC) carries is poor, and no effective therapeutic regimen is yet known. The phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) pathway played a predominant role and may be a promising molecular target for CRPC. However, the toxicity of the dual PI3K inhibitors in clinical trials limits their clinical efficacy for CRPC. To solve this problem, we employed a highly integrated precision nanomedicine strategy to molecularly and physically target CRPC through synergistic effects, enhanced targeted drug delivery efficiency, and reduced unwanted side-effects. Gedatolisib (Ge), a potent inhibitor of PI3K/mTOR, was formulated into our disulfied-crosslinked micelle plateform (NanoGe), which exhibits excellent water solubility, small size (23.25±2 nm), excellent stability with redox stimulus-responsive disintegration, and preferential uptake at tumor sites. NanoGe improved the anti-neoplastic effect of free Ge by 53 times in PC-3M cells and 13 times in C4-2B cells though its enhanced uptake via caveolae- and clathrin-mediated endocytic pathways and the subsequent inhibition of the PI3K/mTOR pathway, resulting in Bax/Bcl-2 dependent apoptosis. In an animal xenograft model, NanoGe showed superior efficacy than free Ge, and synergized with nanoformulated cabazitaxel (NanoCa) as a nanococktail format to achieve a cure rate of 83%. Taken together, our results demonstrate the potency of NanoGe in combination with NanoCa is potent against prostate cancer.

A cyclic peptide retards the proliferation of DU145 prostate cancer cells in vitro and in vivo through inhibition of FGFR2.

In malignancies, fibroblast growth factor receptors (FGFRs) signaling is reinforced through overexpression of fibroblast growth factors (FGFs) or their receptors. FGFR2 has been proposed as a target for cancer therapy, because both the expression and activation of FGFR2 are boosted in various malignant carcinomas. Although several chemicals have been designed against FGFR2, they did not exhibit enough specificity and might bring potential accumulated toxicity. In this study, we developed an epitope peptide (P5) and its cyclic derivative (DcP5) based on the structure of FGF2 to limit the activation of FGFR2. The anticancer activities of P5 and DcP5 were examined in vitro and in vivo. Our results demonstrated that P5 significantly inhibited the cell proliferation in FGFR2-dependent manner in DU145 cells and retarded tumor growth in DU145 xenograft model with negligible toxicity toward normal organs. Further investigations found that the Gln4 and Glu6 residues of P5 bind to FGFR2 to abolish its activation. Moreover, we developed the P5 cyclic derivative, DcP5, which achieved reinforced stability and anticancer activity in vivo. Our findings suggest P5 and its cyclic derivative DcP5 as potential candidates for anticancer therapy.

Long Non-coding RNA LINC01969 Promotes Ovarian Cancer by Regulating the miR-144-5p/LARP1 Axis as a Competing Endogenous RNA.

Accumulating evidence has shown that long non-coding RNAs (lncRNAs) can be used as biological markers and treatment targets in cancer and play various roles in cancer-related biological processes. However, the lncRNA expression profiles and their roles and action mechanisms in ovarian cancer (OC) are largely unknown. Here, we assessed the lncRNA expression profiles in OC tissues from The Cancer Genome Atlas (TCGA) database, and one upregulated lncRNA, LINC01969, was selected for further study. LINC01969 expression levels in 41 patients were verified using quantitative real-time polymerase chain reaction (qRT-PCR). The in vitro effects of LINC01969 on OC cell migration, invasion, and proliferation were determined by the CCK-8, ethynyl-2-deoxyuridine (EdU), wound healing, and Transwell assays. Epithelial-mesenchymal transition (EMT) was evaluated using qRT-PCR and Western blotting. The molecular mechanisms of LINC01969 in OC were assessed through bioinformatics analysis, RNA-binding protein immunoprecipitation (RIP), dual luciferase reporter gene assays, and a rescue experiment. Finally, in vivo experiments were conducted to evaluate the functions of LINC01969. The results of the current study showed that LINC01969 was dramatically upregulated in OC, and patients with lower LINC01969 expression levels tended to have better overall survival. Further experiments demonstrated that LINC01969 promoted the migration, invasion, and proliferation of OC cells in vitro and sped up tumor growth in vivo. Additionally, LINC01969, which primarily exists in the cytoplasm, boosted LARP1 expression by sponging miR-144-5p and promoted the malignant phenotypes of OC cells. In conclusion, the LINC01969/miR-144-5p/LARP1 axis is a newly identified regulatory signaling pathway involved in OC progression.

One-bead one-compound combinatorial library derived targeting ligands for detection and treatment of oral squamous cancer.

Oral squamous cancers (OSC) are hallmarked by poor prognosis, delayed clinical detection, and a lack of defined, characteristic biomarkers. By screening combinatorial one-bead one-compound (OBOC) peptide libraries against oral squamous cancer cell lines, two cyclic peptide ligands, LLY12 and LLY13 were previously identified. These ligands are capable of specific binding to the oral cancer cell lines (MOK-101, HSC-3, SCC-4 and SCC-10a) but not non-cancerous keratinocytes, leukocytes, fibroblast, and endothelial cells. These two peptides were synthesized and evaluated for their binding property, cytotoxicity and cell permeability. In vitro studies indicate that both LLY12 and LLY13 were able to bind to oral cancer cells with high specificity but did not show any cytotoxicity against human keratinocytes. Biotinylated LLY13, in complex with streptavidin-alexa488 was taken up by live oral cancer cells, thus rendering it as an excellent candidate vehicle for efficient delivery of drug loaded-nanoparticles. In vivo and ex vivo near infra-red fluorescence imaging studies confirmed the in vivo targeting efficiency and specificity of LLY13 in oral cancer orthotopic murine xenograft model. In vivo studies also showed that LLY13 was able to accumulate in the OSC tumors and demarcate the tumor margins in orthotopic xenograft model. Together, our data supports LLY13 as a promising theranostic agent against OSC.

Galectin-1 inhibition induces cell apoptosis through dual suppression of CXCR4 and Ras pathways in human malignant peripheral nerve sheath tumors.

BACKGROUND: The Ras signaling pathway is commonly dysregulated in human malignant peripheral nerve sheath tumors (MPNSTs). It is well known that galectin-1 (Gal-1) is essential to stabilize membrane Ras and thereby induce the activation of Ras. However, the role of Gal-1 in MPNST progression remains unknown. The aim of this study was to examine whether Gal-1 knockdown could have an effect on the Ras signaling pathway. METHODS: Cell viability, apoptosis assay, and colony formation were performed to examine the effects of inhibition of Gal-1 in MPNST cells. We used a human MPNST xenograft model to assess growth and metastasis inhibitory effects of Gal-1 inhibitor LLS2. RESULTS: Gal-1 was upregulated in MPNST patients and was highly expressed in MPNST cells. Knockdown of Gal-1 by small interfering (si)RNA in Gal-1 expressing MPNST cells significantly reduces cell proliferation through the suppression of C-X-C chemokine receptor type 4 (CXCR4) and the rat sarcoma viral oncogene homolog (RAS)/extracellular signal-regulated kinase (ERK) pathway, which are important oncogenic signaling in MPNST development. Moreover, Gal-1 knockdown induces apoptosis and inhibits colony formation. LLS2, a novel Gal-1 allosteric small molecule inhibitor, is cytotoxic against MPNST cells and was able to induce apoptosis and suppress colony formation in MPNST cells. LLS2 treatment and Gal-1 knockdown exhibited similar effects on the suppression of CXCR4 and RAS/ERK pathways. More importantly, inhibition of Gal-1 expression or function by treatment with either siRNA or LLS2 resulted in significant tumor responses in an MPNST xenograft model. CONCLUSION: Our results identified an oncogenic role of Gal-1 in MPNST and that its inhibitor, LLS2, is a potential therapeutic agent, applied topically or systemically, against MPNST.

Preventive effects of combinative natural foods produced by elite crop varieties rich in anticancer effects on N-nitrosodiethylamine-induced hepatocellular carcinoma in rats.

The World Cancer Research Fund International has released 32 anticancer effects (ACEs) that targeted every stage of cancer processes. Thus, we designed two formulas of natural food combination Diet I and Diet II, mainly produced by elite crop varieties rich in ACEs with different mixture ratios, and evaluated their cancer preventive effects on N-nitrosodiethylamine (NDEA)-induced hepatocarcinogenesis. After 20 weeks of dietary intervention, Diet I and Diet II reduced incidence, size, and number of hepatic nodules (p < 0.01) and prevented hepatic tumor formation in NDEA-induced hepatocarcinogenesis rats. Low-grade hepatic dysplasia incidence was 20% for Diet II and 40% for Diet I, and apparent hepatocellular carcinomas (HCC) rates were both 0, while 90% HCC in control diet treatment group (p < 0.01). Diet I and Diet II ameliorated abnormal liver function enzymes, reduced serum alpha fetal protein, tumor-specific growth factor, dickkopf-related protein 1, tumor necrosis factor-alpha and interleukin-6 levels, regulated hepatic phase I and II xenobiotic-metabolizing enzymes, enhanced antioxidant capacity, suppressed NDEA-initiated oxidative DNA damage, and induced apoptosis coupled to down-regulation of proinflammatory, invasion, and angiogenesis markers. Daily intake of combination diet produced from ACEs-rich elite crop varieties can effectively prevent or delay occurrence and development of NDEA-induced hepatocarcinogenesis in rats.

A highly integrated precision nanomedicine strategy to target esophageal squamous cell cancer molecularly and physically.

The prognosis of esophageal squamous cell carcinoma is poor. We hereby presented a highly integrated and clinically relevant precision nanomedicine strategy to target ESCC molecularly and physically for significant improvement of the treatment efficacy. We firstly identified PI3K overexpression in patient samples and its relation to poor patient survival. With our highly versatile tumor-targeted drug delivery platform (DCM), we were able to load a potent but toxic docetaxel (DTX) and a PI3K inhibitor (AZD8186) with favorable physical properties. The combination of the DTX-DCM and AZD8186-DCM showed a highly efficacious and synergistic anti-tumor effect and decreased hematotoxicity. A pro-apoptotic protein, Bax was significantly upregulated in ESCC cells treated with combination therapy compared to that with monotherapy. This study utilized a highly integrated precision nano-medicine strategy that combines the identification of cancer molecular target from human patients, precision drug delivery and effective combination therapy for the development of better ESCC treatment.

Nanoformulated paclitaxel and AZD9291 synergistically eradicate non-small-cell lung cancers in vivo.

AIM: This study aims to develop new nanoformulations of EGFR T790M targeted inhibitor AZD9291 and paclitaxel (PTX) for combination therapy of lung cancer. MATERIALS & METHODS: We prepared and characterized PTX- and AZD9291-loaded disulfide cross-linking micelles (DCMs), and evaluate their combination effect and toxicity in vitro and in lung cancer-bearing mice. RESULTS: Drug-loaded DCMs were relatively small in size, and possessed glutathione-responsive drug release. The combination of PTX-DCMs and AZD92921-DCMs exhibited strong synergistic effects in both cell line and in vivo without additional toxicity. Molecular studies demonstrated the synergistic modification in both IKB-α/NF-κB/Bcl-2 and EGFR/Akt pathways. CONCLUSION: The combination of DCM-loaded AZD9291 and PTX could potentially offer more effective and less toxicity treatment options for lung cancer patients.

Targeting Galectin-1 Impairs Castration-Resistant Prostate Cancer Progression and Invasion.

Purpose: The majority of patients with prostate cancer who are treated with androgen-deprivation therapy (ADT) will eventually develop fatal metastatic castration-resistant prostate cancer (mCRPC). Currently, there are no effective durable therapies for patients with mCRPC. High expression of galectin-1 (Gal-1) is associated with prostate cancer progression and poor clinical outcome. The role of Gal-1 in tumor progression is largely unknown. Here, we characterized Gal-1 functions and evaluated the therapeutic effects of a newly developed Gal-1 inhibitor, LLS30, in mCRPC.Experimental Design: Cell viability, colony formation, migration, and invasion assays were performed to examine the effects of inhibition of Gal-1 in CRPC cells. We used two human CRPC xenograft models to assess growth-inhibitory effects of LLS30. Genome-wide gene expression analysis was conducted to elucidate the effects of LLS30 on metastatic PC3 cells.Results: Gal-1 was highly expressed in CRPC cells, but not in androgen-sensitive cells. Gal-1 knockdown significantly inhibited CRPC cells' growth, anchorage-independent growth, migration, and invasion through the suppression of androgen receptor (AR) and Akt signaling. LLS30 targets Gal-1 as an allosteric inhibitor and decreases Gal-1-binding affinity to its binding partners. LLS30 showed in vivo efficacy in both AR-positive and AR-negative xenograft models. LLS30 not only can potentiate the antitumor effect of docetaxel to cause complete regression of tumors, but can also effectively inhibit the invasion and metastasis of prostate cancer cells in vivoConclusions: Our study provides evidence that Gal-1 is an important target for mCRPC therapy, and LLS30 is a promising small-molecule compound that can potentially overcome mCRPC. Clin Cancer Res; 24(17); 4319-31. ©2018 AACR.

Self-indicating, fully active pharmaceutical ingredients nanoparticles (FAPIN) for multimodal imaging guided trimodality cancer therapy.

Conventional drug delivery systems contain substantial amounts of excipients such as polymers and lipids, typically with low drug loading capacity and lack of intrinsic traceability and multifunctionality. Here, we report fully active pharmaceutical ingredient nanoparticles (FAPIN) which were self-assembled by minimal materials, but seamlessly orchestrated versatile theranostic functionalities including: i) self-delivery: no additional carriers were required, all components in the formulation are active pharmaceutical ingredients; ii) self-indicating: no additional imaging tags were needed. The nanoparticle itself was composed of 100% imaging agents, so that the stability, drug release, subcellular dispositions, biodistribution and therapeutic efficacy of FAPINs can be readily visualized by ample imaging capacities, including energy transfer relay dominated, dual-color fluorogenic property, near-infrared fluorescence imaging and magnetic resonance imaging; and iii) highly effective trimodality cancer therapy, encompassing photodynamic-, photothermal- and chemo-therapies. FAPINs were fabricated with very simple material (a photosensitizer-drug conjugate), unusually achieved ∼10 times better in vitro antitumor activity than their free counterparts, and were remarkably efficacious in patient-derived xenograft (PDX) glioblastoma multiforme animal models. Only two doses of FAPINs enabled complete ablation of highly-malignant PDX tumors in 50% of the mice.

Image-guided photo-therapeutic nanoporphyrin synergized HSP90 inhibitor in patient-derived xenograft bladder cancer model.

Photodynamic therapy is a promising and effective non-invasive therapeutic approach for the treatment of bladder cancers. Therapies targeting HSP90 have the advantage of tumor cell selectivity and have shown great preclinical efficacy. In this study, we evaluated a novel multifunctional nanoporphyrin platform loaded with an HSP90 inhibitor 17AAG (NP-AAG) for use as a multi-modality therapy against bladder cancer. NP-AAG was efficiently accumulated and retained at bladder cancer patient-derived xenograft (PDX) over 7 days. PDX tumors could be synergistically eradicated with a single intravenous injection of NP-AAG followed by multiple light treatments within 7 days. NP-AAG mediated treatment could not only specifically deliver 17AAG and produce heat and reactive oxygen species, but also more effectively inhibit essential bladder cancer essential signaling molecules like Akt, Src, and Erk, as well as HIF-1α induced by photo-therapy. This multifunctional nanoplatform has high clinical relevance and could dramatically improve management for bladder cancers with minimal toxicity.

Combinatorial chemistry in drug discovery.

Several combinatorial methods have been developed to create focused or diverse chemical libraries with a wide range of linear or macrocyclic chemical molecules: peptides, non-peptide oligomers, peptidomimetics, small-molecules, and natural product-like organic molecules. Each combinatorial approach has its own unique high-throughput screening and encoding strategy. In this article, we provide a brief overview of combinatorial chemistry in drug discovery with emphasis on recently developed new technologies for design, synthesis, screening and decoding of combinatorial library. Examples of successful application of combinatorial chemistry in hit discovery and lead optimization are given. The limitations and strengths of combinatorial chemistry are also briefly discussed. We are now in a better position to truly leverage the power of combinatorial technologies for the discovery and development of next-generation drugs.

Single-Bead Quantification of Peptide Loading Distribution for One-Bead One-Compound Library Synthesis Using Confocal Raman Spectroscopy.

We report an analytical method to determine peptide loading of "one-bead one-compound" (OBOC) combinatorial peptide libraries at single-bead level. The quantification is based on a linear relationship between the amount of N-terminal amino groups on individual peptide beads and the intensity of Raman signal obtained from a specifically designed reporter labeled on amino groups. Confocal Raman spectroscopy was employed to characterize peptide loading of beads with defined peptide sequences and from OBOC combinatorial peptide libraries. Although amine loading of blank TentaGel beads was found to be uniform, peptide loading among beads of OBOC peptide libraries varied substantially, particularly for those libraries with long sequences. Construction of OBOC libraries can be monitored with this novel analytical technique so that synthetic conditions can be optimized for the preparation of high-quality OBOC peptide libraries. As the variability of peptide loading of individual library beads can significantly influence the screening results, quantitative information obtained by this method will allow us to gain insight into the complexity and challenge of OBOC library synthesis and screening.

A versatile nanoplatform for synergistic combination therapy to treat human esophageal cancer.

One of the major goals of precision oncology is to promote combination therapy to improve efficacy and reduce side effects of anti-cancer drugs based on their molecular mechanisms. In this study, we aimed to develop and validate new nanoformulations of docetaxel (DTX) and bortezomib (BTZ) for targeted combination therapy to treat human esophageal cancer. By leveraging our versatile disulfide cross-linked micelles (DCMs) platform, we developed nanoformulations of DTX and BTZ (named DTX-DCMs and BTZ-DCMs). Their physical properties were characterized; their anti-cancer efficacies and mechanisms of action were investigated in a human esophageal cancer cell line in vitro. Furthermore, the in vitro anti-tumor activities of combination therapies (concurrent drug treatment, sequential drug treatment, and treatment using different ratios of the drugs) were examined in comparison with the single drug treatment and free drug strategies. These drug-loaded nanoparticles were spherical in shape and relatively small in size of approximately 20-22 nm. The entrapment efficiencies of DTX and BTZ into nanoparticles were 82.4% and 84.1%, respectively. The drug release rates of DTX-DCMs and BTZ-DCMs were sustained, and greatly increased in the presence of GSH. These nanodrugs were effectively internalized by KYSE30 esophageal cancer cells, and dose-dependently induced cell apoptosis. We further revealed a strong synergistic effect between DTX-DCMs and BTZ-DCMs against KYSE30 esophageal cancer cells. Sequential combination therapy with DTX-DCMs followed by BTZ-DCMs exhibited the best anti-tumor efficacy in vitro. This study demonstrates that DTX and BTZ could be successfully nanoformulated into disulfide cross-linked micelles. The nanoformulations of DTX and BTZ demonstrate an immense potential for synergistic combination therapy to treat human esophageal cancer.

Cholic acid-based novel micellar nanoplatform for delivering FDA-approved taxanes.

AIM: To structurally modify our existing cholic acid (CA)-based telodendrimer (TD; PEG5K-CA8) for effective micellar nanoencapsulation and delivery of the US FDA-approved members of taxane family. MATERIALS & METHODS: Generation of hybrid TDs was achieved by replacing four of the eight CAs with biocompatible organic moieties using solution-phase peptide synthesis. Drug loading was done using the standard evaporation method. RESULTS: Hybrid TDs can generate micelles with narrow size distributions, low critical micelle concentration values (1-6 µM), better hematocompatibility and lack of in vitro cytotoxicity. CONCLUSION: Along with PEG5K-CA8, CA-based hybrid nanoplatform is the first of its kind that can stably encapsulate all three FDA-approved taxanes with nearly 100% efficiency up to 20% (w/w) loading.