Short-chain fatty acid-releasing nano-prodrugs for attenuating growth and metastasis of melanoma.

Low-molecular-weight (LMW) short-chain fatty acids (SCFAs), such as propionic and butyric acids, have been reported to possess anti-neoplastic effects; however, rapid renal clearance and high dose-based side effects limit their clinical translation. Hence, in this study, we have designed a new self-assembling nano-prodrugs that can effectively supply SCFAs: endogenous enzyme-metabolizable block copolymer poly(ethylene glycol)block-poly(vinyl ester) possessing several units of SCFAs conjugated as side chains via ester linkages. These amphiphilic polymers spontaneously self-assemble into nanostructures under aqueous conditions to form orally administrable nano-prodrugs (butyric acid: NanoBA and propionic acid: NanoPA). Herein, we show the therapeutic efficacy of SCFA nanoparticles (NanoSCFA) in a mouse model of metastasis (melanoma). Ad libitum intake of our NanoSCFA markedly demonstrated a decrease in the metastatic tumor nodules in the lungs compared with the effect observed after LMW SCFA administration with no discernible toxicity to the GI tract. In contrast, LMW SCFAs, even at a lower concentration than that of the NanoSCFA, facilitated villus atrophy. Taken together, our work suggests that the use of NanoSCFA as a therapeutic intervention for metastatic cancer is preferable over typical LMW SCFAs. STATEMENT OF SIGNIFICANCE: Low-molecular-weight (LMW) short-chain fatty acids (SCFAs) have shown versatile therapeutic effects on various diseases, including anti-tumorigenesis effects. However, their clinical translation is limited due to their poor pharmacokinetic profile and adverse effects. To overcome these limitations, we have developed new amphiphilic block copolymer-based SCFA-prodrugs, which self-assemble into nanoparticles in aqueous media (NanoSCFA). SCFAs are covalently conjugated to the hydrophobic polymer segment via ester linkage, which can be enzymatically metabolized after oral administration. In the present study, we confirmed that ad libitum intake of NanoSCFAs retarded the growth and metastatic potential of B16-F10 tumors compared to the LMW SCFAs with negligible discernible toxicity, reflecting NanoSCFA as a preferable therapeutic intervention to LMW SCFA counterparts.

Design of a new self-assembling antioxidant nanomedicine to ameliorate oxidative stress in zebrafish embryos.

Oxidative stress, which is a persistent state of elevated reactive oxygen species (ROS), is implicated in the pathogeneses of several diseases, making antioxidant-based therapeutics the aptest intervention. Nevertheless, the clinical failure of conventional low-molecular-weight (LMW) antioxidants in oxidative stress-related diseases to yield favorable therapeutic outcomes and an increased mortality rate attributable to their poor pharmacokinetic characteristics, necessitates the development of alternative therapeutics. In light of this, we designed and synthesized a new amphiphilic polymer functionalized with a clinically safe base polymer of poly(styrene-co-maleic anhydride) copolymer conjugated with the LMW pleiotropic antioxidant TEMPO (a potent antioxidant) and biocompatible poly(ethylene glycol) (TEMPO-installed PSMA-g-PEG), which self-assembles into nano-sized micelles (SMAPoTN) under physiological conditions. We investigated its safety and antioxidant ability using zebrafish models. Common LMW antioxidants, such as 4-hydroxy-TEMPO (TEMPOL), vitamin C, N-acetyl-L-cysteine, and edaravone exposure induced phenotypic distortions, a manifestation of developmental toxicity, and resulted in high lethality in zebrafish larvae. LMW TEMPOL also adversely affected embryo hatchability, induced arrhythmia and cardiac edema, and failed to protect against oxidative stress. In contrast, exposure of zebrafish embryos to SMAPoTN increased the hatchability, protected embryos against various inducers of oxidative stress, and did not induce any phenotypic alterations or discernible toxicity. Taken together, we conclude that SMAPoTN surpasses LMW TEMPOL in terms of the ability to protect zebrafish, attributable to efficient ROS scavenging without perturbing normal redox homeostasis. These results imply that SMAPoTN can be used as a therapeutic intervention against various oxidative stress-induced diseases. STATEMENT OF SIGNIFICANCE: Failure of low molecular weight (LMW) antioxidants to improve therapeutic index in various oxidative stress-related pathogenesis, attributable to their poor pharmacokinetic characteristics, greatly limits their clinical translation. To overcome this limitation, we developed a self-assembling antioxidant nanoparticle (SMAPoTN) comprised of amphiphilic polymer; poly(styrene-co-maleic anhydride) conjugated with TEMPO as an antioxidant and biocompatible poly(ethylene glycol). Preliminary studies carried out in the in vivo models of zebrafish embryos confirmed that exposure of LMW antioxidant resulted in acute developmental toxicity, high lethality, and failure to rescue embryos against oxidative stress inducers. In contrast, SMAPoTN did not exert discernible toxicity and significantly improved their survival under oxidative stress. Our finding establishes antioxidant nanoparticles as more suitable therapeutic intervention for oxidative stress-induced diseases than LMW antioxidants.

Antioxidant Nanomedicine Significantly Enhances the Survival Benefit of Radiation Cancer Therapy by Mitigating Oxidative Stress-Induced Side Effects.

Oxidative stress-induced off-target effects limit the therapeutic window of radiation therapy. Although many antioxidants have been evaluated as radioprotective agents, none of them are in widespread clinical use, owing to the side effects of the antioxidants themselves and the lack of apparent benefit. Aiming for a truly effective radioprotective agent in radiation cancer therapy, the performance of a self-assembling antioxidant nanoparticle (herein denoted as redox nanoparticle; RNP) is evaluated in the local irradiation of a subcutaneous tumor-bearing mouse model. Since RNP is covered with a biocompatible shell layer and possesses a core-shell type structure of several tens of nanometers in size, its lifetime in the systemic circulation is prolonged. Moreover, since 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO), one of the most potent antioxidants, is covalently encapsulated in the core of RNP, it exerts intense antioxidant activity and induces fewer adverse effects by avoiding leakage of the TEMPO molecules. Preadministration of RNP to the mouse model effectively mitigates side effects in normal tissues and significantly extends the survival benefit of radiation cancer therapy. Moreover, RNP pretreatment noticeably increases the apoptosis/necrosis ratio of radiation-induced cell death, a highly desirable property to reduce the chronic side effects of ionizing irradiation.

Newly Developed Self-Assembling Antioxidants as Potential Therapeutics for the Cancers.

Elevated reactive oxygen species (ROS) have been implicated as significant for cancer survival by functioning as oncogene activators and secondary messengers. Hence, the attenuation of ROS-signaling pathways in cancer by antioxidants seems a suitable therapeutic regime for targeting cancers. Low molecular weight (LMW) antioxidants such as 2,2,6,6-tetramethylpyperidine-1-oxyl (TEMPO), although they are catalytically effective in vitro, exerts off-target effects in vivo due to their size, thus, limiting their clinical use. Here, we discuss the superior impacts of our TEMPO radical-conjugated self-assembling antioxidant nanoparticle (RNP) compared to the LMW counterpart in terms of pharmacokinetics, therapeutic effect, and adverse effects in various cancer models.

Management of tumor growth and angiogenesis in triple-negative breast cancer by using redox nanoparticles.

In cancer, angiogenesis is a critical phenomenon of nascent blood vessel development to facilitate the oxygen and nutrient supply prerequisite for tumor progression. Therefore, targeting tumors at the angiogenesis step may be significant to prevent their advanced progression and metastasis. Although angiogenesis inhibitors can limit the further growth of tumors, complete eradication of tumors may not be possible by monotherapy alone. Therefore, a therapeutic regimen targeting both tumor growth and its vasculature is essential. Because reactive oxygen species (ROS) are fundamental to both angiogenesis and tumor growth, the use of antioxidants may be an effective dual approach to inhibit tumors. We previously confirmed that our original antioxidant nitroxide radical-containing nanoparticles (RNPs) such as pH-sensitive RNPN, and pH-insensitive RNPO, effectively attenuates the tumorigenic and metastasis potentials of triple-negative breast cancer. In this study, we further investigated the efficacy of RNPs to limit the tumor progression by inhibiting the ROS-regulated cancer angiogenesis in a triple-negative breast cancer model. Here, we confirmed that RNPs significantly inhibited in vitro angiogenesis, attributed to the downregulation of the ROS-regulated angiogenesis inducer, vascular endothelial growth factor (VEGF) in the breast cancer cell line (MDA-MB231) and human umbilical vein endothelial cells (HUVEC), which was consistent with decreased cellular ROS. TEMPOL, a low-molecular-weight (LMW) control antioxidant, exhibited anti-angiogenic effects accompanied by cytotoxicity to the endothelial cells. In an in vivo xenograft model for breast cancer, RNPs exerted significant anti-tumor effect due to the decreased expression of tumor VEGF, which prevented accumulation of the endothelial cells. It should be noted that such efficacy of RNPs was obtained with negligible off-target effects. On the other hand, TEMPOL, because of its size, exerted anti-angiogenesis effect accompanied with injuries to the kidneys, which corroborated with previous reports. Our findings imply that RNPs are more potential antioxidants than their LMW counterparts, such as TEMPOL, for the management of breast cancers.

Fibrinolytic tissue plasminogen activator installed redox-active nanoparticles (t-PA@iRNP) for cancer therapy.

Favorable blood flow within solid tumors has become the principal strategy for drug delivery. The use of thrombolytic drugs, such as tissue plasminogen activator (t-PA), in combination with other drugs or drug carriers may increase their therapeutic effect by increasing drug delivery near the solid tumor through fibrin degradation and blood flow restoration. We, therefore, designed t-PA-installed redox-active nanoparticles (t-PA@iRNP) to improve the perfusion of antioxidant nanoparticles in tumors, via fibrin degradation to decompress tumor vessels. Additionally, antioxidant iRNP was developed for tumor inhibition by reduction of critically elevated levels of reactive oxygen species (ROS) in tumors. The t-PA@iRNP, when administered to a colon cancer model, degraded the deposited fibrin and improved the iRNP and immune cells penetration in tumor tissues via the restored blood flow, thus more effectively inhibited tumor growth. The anti-tumor effect of iRNP was attributed to ROS-reduction mediated downregulation of crucial a transcriptional factor, NF-κB. Conclusively, this study provides a new strategy to enhance the delivery of nanotherapeutics into solid tumors.

Simple and Convenient Method for the Isolation, Culture, and Re-collection of Cancer Cells from Blood by Using Glass-Bead Filters.

Circulating tumor cells (CTCs) are tumor cells that originate from primary cancer tissues, enter the bloodstream in the body, and metastasize to the other organs. Simple and convenient methods for their detection, capture, and recovery from the blood of cancer patients would be highly desirable. We report here on a simple and convenient methodology to trap, culture, and re-collect cancer cells, the sizes of which are greater than those of normal hematologic cells, by the use of glass-bead filters (GBFs). We prepared GBFs with a diameter of 24 mm and thicknesses of 0.4 mm and 1.2 mm, with well-defined pores, by sintering round-shaped glass beads (diameter: 63-106 µm). A small integrated glass-bead filter (iGBF) with a diameter of ca. 9.6 mm for the use in filtering a small volume of blood was also designed and prepared. Using GBF and iGBF, it was possible to efficiently capture mouse Lewis lung carcinoma cells expressing green fluorescent protein spiked in saline/blood by single and repeated (circulation) filtrations in in vitro experiments with very small amounts of red blood cells being captured. In addition, we successfully captured B16 CTCs from the blood of a B16 melanoma metastasis mouse model by iGBF. Cancer cells/CTCs captured on/in the GBF could be cultured and efficiently recovered from the filters. Filtration by GBF had negligible effect on the adherent and proliferative characteristics of cancer cells. Simple and convenient methods for the capture, culture, and re-collection of CTCs by GBF along with flexibility of GBF, which permits them to be molded into suitable architectures having the desired shape and size, should be useful for early and convenient diagnosis and treatment of cancer and related diseases.

Design and synthesis of a luminescent iridium complex-peptide hybrid (IPH) that detects cancer cells and induces their apoptosis.

Tumor necrosis factor related apoptosis inducing ligand (TRAIL) triggers the cell-extrinsic apoptosis pathway by complexation with its signaling receptors such as death receptors (DR4 and DR5). TRAIL is a C3-symmetric type II transmembrane protein, consists of three monomeric units. Cyclometalated iridium(III) complexes such as fac-Ir(tpy)3 (tpy = 2-(4-tolyl)pyridine) also possess a C3-symmetric structure and are known to have excellent luminescence properties. In this study, we report on the design and synthesis of a C3-symmetric and luminescent Ir complex-peptide hybrid (IPH), which contains a cyclic peptide that had been reported to bind to death receptor (DR5). The results of MTT assay of Jurkat, K562 and Molt-4 cells with IPH and co-staining experiments with IPH and an anti-DR5 antibody indicate that IPH binds to DR5 and induces apoptosis in a manner parallel to the DR5 expression level. Mechanistic studies of cell death suggest that apoptosis and necrosis-like cell death are differentiated by the position of the hydrophilic part that connects Ir complex and the peptide units. These findings suggest that IPHs could be a promising tool for controlling apoptosis and necrosis by activation of the extra-and intracellular cell death pathway and to develop new anticancer drugs that detect cancer cells and induce their cell death.

Nitroxide radical-containing nanoparticles attenuate tumorigenic potential of triple negative breast cancer.

The critical importance of reactive oxygen species (ROS) as oncogene activators and essential secondary messengers in cancer cell survival have been widely reported. Since oxidative stress has been implicated as being pivotal in various cancers, antioxidant therapy seems an apt strategy to abrogate ROS-mediated cellular processes to attenuate cancers. We therefore synthesized ROS scavenging nitroxide radical-containing nanoparticles (RNPs); pH insensitive RNPO and pH sensitive RNPN, to impede the proliferative and metastatic characteristics of the triple negative breast cancer cell line, MDA-MB-231, both in vitro and in vivo. RNPs significantly curtailed the proliferative and clonogenic potential of MDA-MB-231 and MCF-7 cell lines. Inhibition of ROS-mediated migratory and invasive characteristics of MDA-MB-231, via down regulation of NF-κB and MMP-2, was also confirmed. Furthermore, a significant anti-tumor and anti-metastatic potential of RNPs was observed in an MDA-MB-231 mouse xenograft model. Such tumoricidal effects of RNPs were attained with negligible adverse effects, compared to conventional low molecular weight antioxidants, TEMPOL. Thus, the tumoricidal effects of RNPs are suggestive of insights on precedence of nanoparticle-based therapeutics over current low molecular weight antioxidants to curtail ROS-induced tumorigenesis of various cancers.

Size-Based Differentiation of Cancer and Normal Cells by a Particle Size Analyzer Assisted by a Cell-Recognition PC Software.

Detection of anomalous cells such as cancer cells from normal blood cells has the potential to contribute greatly to cancer diagnosis and therapy. Conventional methods for the detection of cancer cells are usually tedious and cumbersome. Herein, we report on the use of a particle size analyzer for the convenient size-based differentiation of cancer cells from normal cells. Measurements made using a particle size analyzer revealed that size parameters for cancer cells are significantly greater (e.g., inner diameter and width) than the corresponding values for normal cells (white blood cells (WBC), lymphocytes and splenocytes), with no significant difference in shape parameters (e.g., circularity and convexity). The inner diameter of many cancer cell lines is greater than 10 µm, in contrast to normal cells. For the detection of WBC having similar size to that of cancer cells, we developed a PC software "Cancer Cell Finder" that differentiates them from cancer cells based on brightness stationary points on a cell surface. Furthermore, the aforementioned method was validated for cancer cell/clusters detection in spiked mouse blood samples (a B16 melanoma mouse xenograft model) and circulating tumor cell cluster-like particles in the cat and dog (diagnosed with cancer) blood samples. These results provide insights into the possible applicability of the use of a particle size analyzer in conjunction with PC software for the convenient detection of cancer cells in experimental and clinical samples for theranostics.

Application of surface enhanced Raman spectroscopy as a diagnostic system for hypersialylated metastatic cancers.

Early diagnosis of metastatic cancers could greatly limit the number of cancer-associated deaths. Aberrant surface expression of sialic acid (hypersialylation) on tumors correlating with metastatic incidence and its involvement in tumorigenesis and progression is widely reported; hence detection of hypersialylated tumors may be an effective strategy to identify metastatic cancers. We herein report on the application of phenylboronic acid-installed PEGylated gold nanoparticles coupled with Toluidine blue O (T/BA-GNPs) as SERS probes to target surface sialic acid (N-acetylneuraminic acid, Neu5Ac). Strong SERS signals from metastatic cancer cell lines (breast cancer; MDA-MB231 and colon cancer; Colon-26) were observed, contrary to non-metastatic MCF-7 cells (breast cancer). The detected SERS signals from various cancer cell lines correlated with their reported metastatic potential, implying that our T/BA-GNP based SERS system was capable of distinguishing the metastaticity of cells based on the surface Neu5Ac density. T/BA-GNP based SERS system could also significantly differentiate between hypersialylated tumor tissues and healthy tissues with high SERS signal to noise ratio, due to plasmon coupling between the specifically aggregated functionalized GNPs. Furthermore, we also confirmed reduction in SERS signals from MDA-MB231 surface upon treatment with our original reactive oxygen species (ROS)-scavenging polymeric micelle, nitroxide-radical containing nanoparticles (RNPs). The ROS-mediated abrogation of sialylation by impairing the activation of NF-κB-sialyltransferase signaling cascade upon RNP treatment was confirmed by expression studies and the T/BA-GNPs based SERS system. The aforementioned findings thus, establish T/BA-GNPs based SERS as a potential cytodiagnostic system to detect hypersialylated metastatic tumors and RNPs as anti-metastatic cancer drug candidates.

Therapeutic implications of targeting energy metabolism in breast cancer.

PPARs are ligand activated transcription factors. PPARγ agonists have been reported as a new and potentially efficacious treatment of inflammation, diabetes, obesity, cancer, AD, and schizophrenia. Since cancer cells show dysregulation of glycolysis they are potentially manageable through changes in metabolic environment. Interestingly, several of the genes involved in maintaining the metabolic environment and the central energy generation pathway are regulated or predicted to be regulated by PPARγ. The use of synthetic PPARγ ligands as drugs and their recent withdrawal/restricted usage highlight the lack of understanding of the molecular basis of these drugs, their off-target effects, and their network. These data further underscores the complexity of nuclear receptor signalling mechanisms. This paper will discuss the function and role of PPARγ in energy metabolism and cancer biology in general and its emergence as a promising therapeutic target in breast cancer.

Coffee component hydroxyl hydroquinone (HHQ) as a putative ligand for PPAR gamma and implications in breast cancer.

BACKGROUND: Coffee contains several compounds that have the potential to influence breast cancer risk and survival. However, epidemiologic data on the relation between coffee compounds and breast cancer survival are sparse and inconsistent. RESULTS: We show that coffee component HHQ has significant apoptotic effect on MDA-MB-231 and MCF-7 cells in vitro, and that ROS generation, change in mitochondrial membrane permeability, upregulation of Bax and Caspase-8 as well as down regulation of PGK1 and PKM2 expression may be important apoptosis-inducing mechanisms. The results suggest that PPARγ ligands may serve as potential therapeutic agents for breast cancer therapy. HHQ was also validated as a ligand for PPARγ by docking procedure. CONCLUSION: This is the first report on the anti-breast cancer (in vitro) activity of HHQ.

Glycolytic enzymes PGK1 and PKM2 as novel transcriptional targets of PPARγ in breast cancer pathophysiology.

Peroxisome proliferator-activated receptor γ (PPARγ) is a nuclear receptor and plays important roles in breast cancer cell proliferation. The complexity of the underlying biochemical and molecular mechanisms of breast cancer and the involvement of PPARγ in breast cancer pathophysiology are unclear. In this study, we carried out prediction of the peroxisome proliferator response element (PPRE) motifs in 2332 genes reported to be involved in breast cancer in literature. A total of 178 genes were found to have PPRE (DR1/DR2) and/or PPAR-associated conserved motif (PACM) motifs. We further constructed protein-protein interaction network, disease gene network and gene ontology (GO) analyses to identify novel key genes for experimental validation. We identified two genes in the glycolytic pathway (phosphoglycerate kinase 1 (PGK1) and pyruvate kinase M2 (PKM2)) at the ATP production steps and experimentally validated their repression by PPARγ in two breast cancer cell lines MDA-MB-231 and MCF-7. Further analysis suggested that this repression leads to decrease in ATP levels and apoptosis. These investigations will help us in understanding the molecular mechanisms by which PPARγ regulates the cellular energy pathway and the use of its ligands in human breast cancer therapeutics.