Investigating mitochondrial fission, fusion, and autophagy in retinal pigment epithelium from donors with age-related macular degeneration.

Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in developed countries, characterized by the death of retinal pigment epithelial (RPE) cells and photoreceptors. Previous studies report an accumulation of damaged and dysfunctional mitochondria in RPE of human donors with AMD. Understanding how damaged mitochondria accumulate in AMD is an important step in discovering disease mechanisms and identifying therapeutic targets. In this report, we assessed mitochondrial fission and fusion by quantifying proteins and measured mitochondrial autophagy (mitophagy) via protein analysis and advanced imaging techniques using mitochondrial targeted mKeima in primary human RPE from donors with or without AMD. We report disease-specific differences in mitochondrial proteins that regulate fission, fusion, and mitophagy that were present at baseline and with treatments to stimulate these pathways. Data suggest AMD RPE utilize receptor-mediated mitophagy as a compensatory mechanism for deficits in the ubiquitin-mediated mitophagy pathway. These changes in mitochondrial homeostasis could lead to the buildup of damaged and dysfunctional mitochondria observed in the RPE of AMD donors.

Inflammasome Activation in Retinal Pigment Epithelium from Human Donors with Age-Related Macular Degeneration.

Age-related macular degeneration (AMD), the leading cause of blindness in the elderly, is characterized by the death of retinal pigment epithelium (RPE) and photoreceptors. One of the risk factors associated with developing AMD is the single nucleotide polymorphism (SNP) found within the gene encoding complement factor H (CFH). Part of the innate immune system, CFH inhibits alternative complement pathway activation. Multi-protein complexes called inflammasomes also play a role in the innate immune response. Previous studies reported that inflammasome activation may contribute to AMD pathology. In this study, we used primary human adult RPE cell cultures from multiple donors, with and without AMD, that were genotyped for the Y402H CFH risk allele. We found complement and inflammasome-related genes and proteins at basal levels in RPE tissue and cell cultures. Additionally, treatment with rotenone, bafilomycin A, and ATP led to inflammasome activation. Overall, the response to priming and activation was similar, irrespective of disease state or CFH genotype. While these data show that the inflammasome is present and active in RPE, our results suggest that inflammasome activation may not contribute to early AMD pathology.

Human iPSC- and Primary-Retinal Pigment Epithelial Cells for Modeling Age-Related Macular Degeneration.

Primary cultures of retinal pigment epithelium (RPE) from human adult donors (haRPE) and induced pluripotent stem cell derived-RPE (iPSC-RPE) are valuable model systems for gaining mechanistic insight and for testing potential therapies for age-related macular degeneration (AMD). This study evaluated the treatment response of haRPE and iPSC-RPE to oxidative stress and potential therapeutics addressing mitochondrial defects. haRPE and iSPC-RPE were derived from donors with or without AMD. Mitochondrial function was measured after treatment with menadione, AICAR, or trehalose and the response to treatment was compared between cell models and by disease status. In a subset of samples, haRPE and iPSC-RPE were generated from the same human donor to make a side-by-side comparison of the two cell models' response to treatment. Disease-specific responses to all three treatments was observed in the haRPE. In contrast, iPSC-RPE had a similar response to all treatments irrespective of disease status. Analysis of haRPE and iPSC-RPE generated from the same human donor showed a similar response for donors without AMD, but there were significant differences in treatment response between cell models generated from AMD donors. These results support the use of iPSC-RPE and haRPE when investigating AMD mechanisms and new therapeutics but indicates that attention to experimental conditions is required.

Quantification of mitophagy using mKeima-mito in cultured human primary retinal pigment epithelial cells.

The retinal pigment epithelium is a pigmented monolayer of cells that help maintain a healthy retina. Loss of this essential cell layer is implicated in a number of visual disorders, including age-related macular degeneration (AMD). Utilizing primary RPE cultures to investigate disease is an important step in understanding disease mechanisms. However, the use of primary RPE cultures presents a number of challenges, including the limited number of cells available and the presence of auto-fluorescent pigment that interferes with quantifying fluorescent probes. Additionally, primary RPE are difficult to transfect with exogenous nucleic acids traditionally used for fluorescent imaging. To overcome these challenges, we used an adeno-associated viral (AAV) vector to express a pH sensitive fluorescent protein, mKeima, fused to the mitochondrial targeting sequence of cytochrome oxidase subunit 8A (mKeima-mito). mKeima-mito allows for quantification of mitochondrial autophagy (mitophagy) in live-cell time-lapse imaging experiments. We also developed an image analysis pipeline to selectively quantify mKeima-mito while removing the signal of auto-fluorescent pigment from the dataset by utilizing information from the mKeima fluorescent channels. These techniques are demonstrated in primary RPE cultures expressing mKeima-mito treated with 2-[2-[4-(trifluoromethoxy)phenyl]hydrazinylidene]-propanedinitrile (FCCP), an uncoupler that depolarizes the mitochondrial membrane and leads to mitochondrial fragmentation and mitophagy. The techniques outlined provide a roadmap for investigating disease mechanisms or the effect of treatments utilizing fluorescent probes in an important cell culture model.

Testing Mitochondrial-Targeted Drugs in iPSC-RPE from Patients with Age-Related Macular Degeneration.

Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly. No universally effective treatments exist for atrophic or "dry" AMD, which results from loss of the retinal pigment epithelium (RPE) and photoreceptors and accounts for ≈80% of all AMD patients. Prior studies provide evidence for the involvement of mitochondrial dysfunction in AMD pathology. This study used induced pluripotent stem cell (iPSC) RPE derived from five AMD patients to test the efficacy of three drugs (AICAR (5-Aminoimidazole-4-carboxamide ribonucleotide), Metformin, trehalose) that target key processes in maintaining optimal mitochondrial function. The patient iPSC-RPE lines were used in a proof-of-concept drug screen, utilizing an analysis of RPE mitochondrial function following acute and extended drug exposure. Results show considerable variability in drug response across patient cell lines, supporting the need for a personalized medicine approach for treating AMD. Furthermore, our results demonstrate the feasibility of using iPSC-RPE from AMD patients to develop a personalized drug treatment regime and provide a roadmap for the future clinical management of AMD.

Impaired Mitochondrial Function in iPSC-Retinal Pigment Epithelium with the Complement Factor H Polymorphism for Age-Related Macular Degeneration.

Age-related macular degeneration (AMD), the leading cause of vision loss in the elderly, is characterized by loss of the retinal pigment epithelium (RPE). While the disease mechanism remains unclear, prior studies have linked AMD with RPE mitochondrial defects and genetic polymorphisms in the complement pathway. This study used RPE generated from induced pluripotent stem cells (iPSC-RPE), which were derived from human donors with or without AMD and genotyped for the complement factor H (CFH) AMD high-risk allele (rs1061170, Y402H) to investigate whether donor disease state or genotype had a detrimental effect on mitochondrial function and inflammation. Results show that cells derived from donors with AMD display decreased mitochondrial function under conditions of stress and elevated expression of inflammatory markers compared to iPSC-RPE from individuals without AMD. A more pronounced reduction in mitochondrial function and increased inflammatory markers was observed in CFH high-risk cells, irrespective of disease state. These results provide evidence for a previously unrecognized link between CFH and mitochondrial function that could contribute to RPE loss in AMD patients harboring the CFH high-risk genotype.

Automating Human Induced Pluripotent Stem Cell Culture and Differentiation of iPSC-Derived Retinal Pigment Epithelium for Personalized Drug Testing.

Derivation and differentiation of human induced pluripotent stem cells (hiPSCs) provide the opportunity to generate medically important cell types from individual patients and patient populations for research and the development of potential cell therapies. This technology allows disease modeling and drug screening to be carried out using diverse population cohorts and with more relevant cell phenotypes than can be accommodated using traditional immortalized cell lines. However, technical complexities in the culture and differentiation of hiPSCs, including lack of scale and standardization and prolonged experimental timelines, limit the adoption of this technology for many large-scale studies, including personalized drug screening. The entry of reproducible end-to-end automated workflows for hiPSC culture and differentiation, demonstrated on commercially available platforms, provides enhanced accessibility of this technology for both research laboratories and commercial pharmaceutical testing. Here we have utilized TECAN Fluent automated cell culture workstations to perform hiPSC culture and differentiation in a reproducible and scalable process to generate patient-derived retinal pigment epithelial cells for downstream use, including drug testing. hiPSCs derived from multiple donors with age-related macular degeneration (AMD) were introduced into our automated workflow, and cell lines were cultured and differentiated into retinal pigment epithelium (RPE). Donor hiPSC-RPE lines were subsequently entered in an automated drug testing workflow to measure mitochondrial function after exposure to "mitoactive" compounds. This work demonstrates scalable, reproducible culture and differentiation of hiPSC lines from individuals on the TECAN Fluent platform and illustrates the potential for end-to-end automation of hiPSC-based personalized drug testing.

Improving retinal mitochondrial function as a treatment for age-related macular degeneration.

Age-related macular degeneration (AMD) is the leading cause of blindness among the elderly. Currently, there are no treatments for dry AMD, which is characterized by the death of retinal pigment epithelium (RPE) and photoreceptors. Reports from human donors with AMD suggest that RPE mitochondrial defects are a key event in AMD pathology. Thus, the most effective strategy for treating dry AMD is to identify compounds that enhance mitochondrial function and subsequently, preserve the RPE. In this study, primary cultures of RPE from human donors with (n = 20) or without (n = 8) AMD were used to evaluate compounds that are designed to protect mitochondria from oxidative damage (N-acetyl-l-cysteine; NAC), remove damaged mitochondria (Rapamycin), increase mitochondrial biogenesis (Pyrroloquinoline quinone; PQQ), and improve oxidative phosphorylation (Nicotinamide mononucleotide, NMN). Mitochondrial function measured after drug treatments showed an AMD-dependent response; only RPE from donors with AMD showed improvements. All four drugs caused a significant increase in maximal respiration (p < 0.05) compared to untreated controls. Treatment with Rapamycin, PQQ, or NMN significantly increased ATP production (p < 0.05). Only Rapamycin increased basal respiration (p < 0.05). Notably, robust responses were observed in only about 50% of AMD donors, with attenuated responses observed in the remaining AMD donors. Further, within the responders, individual donors exhibited a distinct reaction to each drug. Our results suggest drugs targeting pathways involved in maintaining healthy mitochondria can improve mitochondrial function in a select population of RPE from AMD donors. The unique response of individual donors to specific drugs supports the need for personalized medicine when treating AMD.

N-Acetyl-L-cysteine Protects Human Retinal Pigment Epithelial Cells from Oxidative Damage: Implications for Age-Related Macular Degeneration.

Age-related macular degeneration (AMD) involves the loss of retinal pigment epithelium (RPE) and photoreceptors and is one of the leading causes of blindness in the elderly. Oxidative damage to proteins, lipids, and DNA has been associated with RPE dysfunction and AMD. In this study, we evaluated oxidative stress in AMD and the efficacy of antioxidant, N-acetyl-L-cysteine (NAC), in protecting RPE from oxidative damage. To test this idea, primary cultures of RPE from human donors with AMD (n = 32) or without AMD (No AMD, n = 21) were examined for expression of NADPH oxidase (NOX) genes, a source of reactive oxygen species (ROS). Additionally, the cells were pretreated with NAC for 2 hours and then treated with either hydrogen peroxide (H2O2) or tert-butyl hydroperoxide (t-BHP) to induce cellular oxidation. Twenty-four hours after treatment, ROS production, cell survival, the content of glutathione (GSH) and adenosine triphosphate (ATP), and cellular bioenergetics were measured. We found increased expression of p22phox, a NOX regulator, in AMD cells compared to No AMD cells (p = 0.02). In both AMD and No AMD cells, NAC pretreatment reduced t-BHP-induced ROS production and protected from H2O2-induced cell death and ATP depletion. In the absence of oxidation, NAC treatment improved mitochondrial function in both groups (p < 0.01). Conversely, the protective response exhibited by NAC was disease-dependent for some parameters. In the absence of oxidation, NAC significantly reduced ROS production (p < 0.001) and increased GSH content (p = 0.02) only in RPE from AMD donors. Additionally, NAC-mediated protection from H2O2-induced GSH depletion (p = 0.04) and mitochondrial dysfunction (p < 0.05) was more pronounced in AMD cells compared with No AMD cells. These results demonstrate the therapeutic benefit of NAC by mitigating oxidative damage in RPE. Additionally, the favorable outcomes observed for AMD RPE support NAC's relevance and the potential therapeutic value in treating AMD.

Altered bioenergetics and enhanced resistance to oxidative stress in human retinal pigment epithelial cells from donors with age-related macular degeneration.

Age-related macular degeneration (AMD) is the leading cause of blindness among older adults. It has been suggested that mitochondrial defects in the retinal pigment epithelium (RPE) underlies AMD pathology. To test this idea, we developed primary cultures of RPE to ask whether RPE from donors with AMD differ in their metabolic profile compared with healthy age-matched donors. Analysis of gene expression, protein content, and RPE function showed that these cultured cells replicated many of the cardinal features of RPE in vivo. Using the Seahorse Extracellular Flux Analyzer to measure bioenergetics, we observed RPE from donors with AMD exhibited reduced mitochondrial and glycolytic function compared with healthy donors. RPE from AMD donors were also more resistant to oxidative inactivation of these two energy-producing pathways and were less susceptible to oxidation-induced cell death compared with cells from healthy donors. Investigation of the potential mechanism responsible for differences in bioenergetics and resistance to oxidative stress showed RPE from AMD donors had increased PGC1α protein as well as differential expression of multiple genes in response to an oxidative challenge. Based on our data, we propose that cultured RPE from donors phenotyped for the presence or absence of AMD provides an excellent model system for studying "AMD in a dish". Our results are consistent with the ideas that (i) a bioenergetics crisis in the RPE contributes to AMD pathology, and (ii) the diseased environment in vivo causes changes in the cellular profile that are retained in vitro.

Generation of retinal pigmented epithelium from iPSCs derived from the conjunctiva of donors with and without age related macular degeneration.

Fidelity in pluripotent stem cell differentiation protocols is necessary for the therapeutic and commercial use of cells derived from embryonic and induced pluripotent stem cells. Recent advances in stem cell technology, especially the widespread availability of a range of chemically defined media, substrates and differentiation components, now allow the design and implementation of fully defined derivation and differentiation protocols intended for replication across multiple research and manufacturing locations. In this report we present an application of these criteria to the generation of retinal pigmented epithelium from iPSCs derived from the conjunctiva of donors with and without age related macular degeneration. Primary conjunctival cells from human donors aged 70-85 years were reprogrammed to derive multiple iPSC lines that were differentiated into functional RPE using a rapid and defined differentiation protocol. The combination of defined iPSC derivation and culture with a defined RPE differentiation protocol, reproducibly generated functional RPE from each donor without requiring protocol adjustments for each individual. This successful validation of a standardized, iPSC derivation and RPE differentiation process demonstrates a practical approach for applications requiring the cost-effective generation of RPE from multiple individuals such as drug testing, population studies or for therapies requiring patient-specific RPE derivations. In addition, conjunctival cells are identified as a practical source of somatic cells for deriving iPSCs from elderly individuals.

Nanoparticle formulation of ormeloxifene for pancreatic cancer.

Pancreatic cancer is the fourth most prevalent cancer with about an 85% mortality rate; thus, an utmost need exists to discover new therapeutic modalities that would enhance therapy outcomes of this disease with minimal or no side effects. Ormeloxifene (ORM), a synthetic molecule, has exhibited potent anti-cancer effects through inhibition of important oncogenic and proliferation signaling pathways. However, the anti-cancer efficacy of ORM can be further improved by developing its nanoformulation, which will also offer tumor specific targeted delivery. Therefore, we have developed a novel ORM encapsulated poly(lactic-co-glycolic acid) nanoparticle (NP) formulation (PLGA-ORM NP). This formulation was characterized for particle size, chemical composition, and drug loading efficiency, using various physico-chemical methods (TEM, FT-IR, DSC, TGA, and HPLC). Because of its facile composition, this novel formulation is compatible with antibody/aptamer conjugation to achieve tumor specific targeting. The particle size analysis of this PLGA-ORM formulation (∼100 nm) indicates that this formulation can preferentially reach and accumulate in tumors by the Enhanced Permeability and Retention (EPR) effect. Cellular uptake and internalization studies demonstrate that PLGA-ORM NPs escape lysosomal degradation, providing efficient endosomal release to cytosol. PLGA-ORM NPs showed remarkable anti-cancer potential in various pancreatic cancer cells (HPAF-II, AsPC-1, BxPC-3, Panc-1, and MiaPaca) and a BxPC-3 xenograft mice model resulting in increased animal survival. PLGA-ORM NPs suppressed pancreatic tumor growth via suppression of Akt phosphorylation and expression of MUC1, HER2, PCNA, CK19 and CD31. This study suggests that the PLGA-ORM formulation is highly efficient for the inhibition of pancreatic tumor growth and thus can be valuable for the treatment of pancreatic cancer in the future.

Ormeloxifene suppresses desmoplasia and enhances sensitivity of gemcitabine in pancreatic cancer.

The management of pancreatic ductal adenocarcinoma (PDAC) is extremely poor due to lack of an efficient therapy and development of chemoresistance to the current standard therapy, gemcitabine. Recent studies implicate the intimate reciprocal interactions between epithelia and underlying stroma due to paracrine Sonic hedgehog (SHH) signaling in producing desmoplasia and chemoresistance in PDAC. Herein, we report for the first time that a nonsteroidal drug, ormeloxifene, has potent anticancer properties and depletes tumor-associated stromal tissue by inhibiting the SHH signaling pathway in PDAC. We found that ormeloxifene inhibited cell proliferation and induced death in PDAC cells, which provoked us to investigate the combinatorial effects of ormeloxifene with gemcitabine at the molecular level. Ormeloxifene caused potent inhibition of the SHH signaling pathway via downregulation of SHH and its related important downstream targets such as Gli-1, SMO, PTCH1/2, NF-κB, p-AKT, and cyclin D1. Ormeloxifene potentiated the antitumorigenic effect of gemcitabine by 75% in PDAC xenograft mice. Furthermore, ormeloxifene depleted tumor-associated stroma in xenograft tumor tissues by inhibiting the SHH cellular signaling pathway and mouse/human collagen I expression. Xenograft tumors treated with ormeloxifene in combination with gemcitabine restored the tumor-suppressor miR-132 and inhibited stromal cell infiltration into the tumor tissues. In addition, invasiveness of tumor cells cocultivated with TGFß-stimulated human pancreatic stromal cells was effectively inhibited by ormeloxifene treatment alone or in combination with gemcitabine. We propose that ormeloxifene has high therapeutic index and in a combination therapy with gemcitabine, it possesses great promise as a treatment of choice for PDAC/pancreatic cancer.

Implications of protein corona on physico-chemical and biological properties of magnetic nanoparticles.

Interaction of serum proteins and nanoparticles leads to a nanoparticle-protein complex formation that defines the rational strategy for a clinically relevant formulation for drug delivery, hyperthermia, and magnetic resonance imaging (MRI) applications in cancer nanomedicine. Given this perspective, we have examined the pattern of human serum protein corona formation with our recently engineered magnetic nanoparticles (MNPs). The alteration in particle size, zeta potential, hemotoxicity, cellular uptake/cancer cells targeting potential, and MRI properties of the MNPs after formation of human serum (HS) protein corona were studied. Our results indicated no significant change in particle size of our MNPs upon incubation with 0.5-50 wt/v% human serum, while zeta potential of MNPs turned negative due to human serum adsorption. When incubated with an increased serum and particle concentration, apolipoprotein E was adsorbed on the surface of MNPs apart from serum albumin and transferrin. However, there was no significant primary or secondary structural alterations observed in serum proteins through Fourier transform infrared spectroscopy, X-ray diffraction, and circular dichroism. Hemolysis assay suggests almost no hemolysis at the tested concentrations (up to 1 mg/mL) for MNPs compared to the sodium dodecyl sulfate (positive control). Additionally, improved internalization and uptake of MNPs by C4-2B and Panc-1 cancer cells were observed upon incubation with human serum (HS). After serum protein adsorption to the surface of MNPs, the close vicinity within T1 (∼1.33-1.73 s) and T2 (∼12.35-13.43 ms) relaxation times suggest our MNPs retained inherent MRI potential even after biomolecular protein adsorption. All these superior clinical parameters potentially enable clinical translation and use of this formulation for next generation nanomedicine for drug delivery, cancer-targeting, imaging and theranostic applications.

Ormeloxifene efficiently inhibits ovarian cancer growth.

Ovarian cancer continues to be a leading cause of cancer related deaths for women. Anticancer agents effective against chemo-resistant cells are greatly needed for ovarian cancer treatment. Repurposing drugs currently in human use is an attractive strategy for developing novel cancer treatments with expedited translation into clinical trials. Therefore, we examined whether ormeloxifene (ORM), a non-steroidal Selective Estrogen Receptor Modulator (SERM) currently used for contraception, is therapeutically effective at inhibiting ovarian cancer growth. We report that ORM treatment inhibits cell growth and induces apoptosis in ovarian cancer cell lines, including cell lines resistant to cisplatin. Furthermore, ORM treatment decreases Akt phosphorylation, increases p53 phosphorylation, and modulates the expression and localization patterns of p27, cyclin E, cyclin D1, and CDK2. In a pre-clinical xenograft mouse ORM treatment significantly reduces tumorigenesis and metastasis. These results indicate that ORM effectively inhibits the growth of cisplatin resistant ovarian cancer cells. ORM is currently in human use and has an established record of patient safety. Our encouraging in vitro and pre-clinical in vivo findings indicate that ORM is a promising candidate for the treatment of ovarian cancer.

MicroRNA-145 targets MUC13 and suppresses growth and invasion of pancreatic cancer.

Pancreatic cancer has a poor prognosis due to late diagnosis and ineffective therapeutic multimodality. MUC13, a transmembrane mucin is highly involved in pancreatic cancer progression. Thus, understanding its regulatory molecular mechanisms may offer new avenue of therapy for prevention/treatment of pancreatic cancer. Herein, we report a novel microRNA (miR-145)-mediated mechanism regulating aberrant MUC13 expression in pancreatic cancer. We report that miR-145 expression inversely correlates with MUC13 expression in pancreatic cancer cells and human tumor tissues. miR-145 is predominantly present in normal pancreatic tissues and early Pancreatic Ductal Adenocarcinoma (PDAC) precursor lesions (PanIN I) and is progressively suppressed over the course of development from PanIN II/III to late stage poorly differentiated PDAC. We demonstrate that miR-145 targets 3' untranslated region of MUC13 and thus downregulates MUC13 protein expression in cells. Interestingly, transfection of miR-145 inhibits cell proliferation, invasion and enhances gemcitabine sensitivity. It causes reduction of HER2, P-AKT, PAK1 and an increase in p53. Similar results were found when MUC13 was specifically inhibited by shRNA directed at MUC13. Additionally, intratumoral injections of miR-145 in xenograft mice inhibited tumor growth via suppression of MUC13 and its downstream target, HER2. These results suggest miR-145 as a novel regulator of MUC13 in pancreatic cancer.

Anti-cancer activity of curcumin loaded nanoparticles in prostate cancer.

Prostate cancer is the most commonly diagnosed cancer disease in men in the Unites States and its management remains a challenge in everyday oncology practice. Thus, advanced therapeutic strategies are required to treat prostate cancer patients. Curcumin (CUR) is a promising anticancer agent for various cancer types. The objective of this study was to evaluate therapeutic potential of novel poly(lactic-co-glycolic acid)- CUR nanoparticles (PLGA-CUR NPs) for prostate cancer treatment. Our results indicate that PLGA-CUR NPs efficiently internalize in prostate cancer cells and release biologically active CUR in cytosolic compartment of cells for effective therapeutic activity. Cell proliferation (MTS), clonogenic, and Western blot analyses reveal that PLGA-CUR NPs can effectively inhibit proliferation and colony formation ability of prostate cancer cells than free CUR. PLGA-CUR NPs showed superior tumor regression compared to CUR in xenograft mice. Further investigations reveal that PLGA-CUR NPs inhibit nuclear ß-catenin and AR expression in cells and in tumor xenograft tissues. It also suppresses STAT3 and AKT phosphorylation and leads to apoptosis via inhibition of key anti-apoptotic proteins, Mcl-1, Bcl-xL and caused induction of PARP cleavage. Additionally, significant downregulation of oncogenic miR21 and up-regulation of miR-205 was observed with PLGA-CUR NPs treatment as determined by RT-PCR and in situ hybridization analyses. A superior anti-cancer potential was attained with PSMA antibody conjugated PLGA-CUR NPs in prostate cancer cells and a significant tumor targeting of (131)I labeled PSMA antibody was achieved with PLGA-CUR NPs in prostate cancer xenograft mice model. In conclusion, PLGA-CUR NPs can significantly accumulate and exhibit superior anticancer activity in prostate cancer.

Functions and regulation of MUC13 mucin in colon cancer cells.

BACKGROUND: MUC13 is overexpressed and aberrantly localized in colon cancer tissue; however, the specific functions and regulation of MUC13 expression are unknown. METHODS: Stable cell lines with either overexpressed or suppressed MUC13 levels were analyzed to determine cell growth, colony formation, cell migration, and cell invasion assays. The molecular mechanisms involved in MUC13 regulation were elucidated via chromatin immunoprecipitation (ChIP) and analysis of interleukin 6 (IL6) treatments. Colon cancer tissues were analyzed by immunohistochemistry (IHC) for the protein levels of MUC13 and P-STAT5 in colon cancer cells. RESULTS: Overexpression of MUC13 increased cell growth, colony formation, cell migration, and invasion. In concordance, MUC13 silencing decreased these tumorigenic features. Overexpression of MUC13 also modulated various cancer-associated proteins, including telomerase reverse transcriptase, sonic hedgehog, B cell lymphoma murine like site 1, and GATA like transcription factor 1. Additionally, MUC13-overexpressing cells showed increased HER2 and P-ERK expression. ChIP analysis revealed binding of STAT5 to the predicted MUC13 promoter. IL6 treatment of colon cancer cells increased the expression of MUC13 via activation of the JAK2/STAT5 signaling pathway. Suppression of JAK2 and STAT5 signaling by chemical inhibitors abolished IL6-induced MUC13 expression. IHC analysis showed increased expression of both P-STAT5 and MUC13 in colon cancer as compared to adjacent normal tissue. CONCLUSIONS: The results of this study, for the first time, suggest functional roles of MUC13 in colon cancer progression and provide information regarding the regulation of MUC13 expression via JAK2/STAT5 which may reveal promising therapeutic approaches for colon cancer treatment.

Novel curcumin-loaded magnetic nanoparticles for pancreatic cancer treatment.

Curcumin (CUR), a naturally occurring polyphenol derived from the root of Curcuma longa, has showed potent anticancer and cancer prevention activity in a variety of cancers. However, the clinical translation of CUR has been significantly hampered due to its extensive degradation, suboptimal pharmacokinetics, and poor bioavailability. To address these clinically relevant issues, we have developed a novel CUR-loaded magnetic nanoparticle (MNP-CUR) formulation. Herein, we have evaluated the in vitro and in vivo therapeutic efficacy of this novel MNP-CUR formulation in pancreatic cancer. Human pancreatic cancer cells (HPAF-II and Panc-1) exhibited efficient internalization of the MNP-CUR formulation in a dose-dependent manner. As a result, the MNP-CUR formulation effectively inhibited growth of HPAF-II and Panc-1 cells in cell proliferation and colony formation assays. The MNP-CUR formulation suppressed pancreatic tumor growth in an HPAF-II xenograft mouse model and improved the survival of mice by delaying tumor growth. The growth-inhibitory effect of MNP-CUR formulation correlated with the suppression of proliferating cell nuclear antigen (PCNA), B-cell lymphoma-extra large (Bcl-xL), induced myeloid leukemia cell differentiation protein (Mcl-1), cell surface-associated Mucin 1 (MUC1), collagen I, and enhanced membrane ß-catenin expression. MNP-CUR formulation did not show any sign of hemotoxicity and was stable after incubation with human serum proteins. In addition, the MNP-CUR formulation improved serum bioavailability of CUR in mice up to 2.5-fold as compared with free CUR. Biodistribution studies show that a significant amount of MNP-CUR formulation was able to reach the pancreatic xenograft tumor(s), which suggests its clinical translational potential. In conclusion, this study suggests that our novel MNP-CUR formulation can be valuable for the treatment of pancreatic cancer.

Plasma proteins interaction with curcumin nanoparticles: implications in cancer therapeutics.

Curcumin, a natural bioactive polyphenol, has been widely investigated as a conventional medicine for centuries. Over the past two decades, major pre-clinical and clinical trials have demonstrated its safe therapeutic profile but clinical translation has been hampered due to rapid degradation, poor water solubility, bioavailability and pharmaco-kinetics. To overcome such translational issues, many laboratories have focused on developing curcumin nanoformulations for cancer therapeutics. In this review, we discuss the evolution of curcumin nanomedicine in cancer therapeutics, the possible interactions between the surface of curcumin nanoparticles and plasma proteins, the role of nanoparticle-protein complex architecture parameters, and the rational design of clinically useful curcumin nanoformulations. Considering all the biologically relevant phenomena, curcumin nanoformulations can be developed as a new neutraceutical or pharmaceutical agent.