Folate Receptor Expression Research Articles

The Interplay between Fe3O4 Superparamagnetic Nanoparticles, Sodium Butyrate, and Folic Acid for Intracellular Transport.

Combined treatments which use nanoparticles and drugs could be a synergistic strategy for the treatment of a variety of cancers to overcome drug resistance, low efficacy, and high-dose-induced systemic toxicity. In this study, the effects on human colon adenocarcinoma cells of surface modified Fe3O4 magnetic nanoparticles (MNPs) in combination with sodium butyrate (NaBu), added as a free formulation, were examined demonstrating that the co-delivery produced a cytotoxic effect on malignant cells. Two different MNP coatings were investigated: a simple polyethylene glycol (PEG) layer and a mixed folic acid (FA) and PEG layer. Our results demonstrated that MNPs with FA (FA-PEG@MNPs) have a better cellular uptake than the ones without FA (PEG@MNPs), probably due to the presence of folate that acts as an activator of folate receptors (FRs) expression. However, in the presence of NaBu, the difference between the two types of MNPs was reduced. These similar behaviors for both MNPs likely occurred because of the differentiation induced by butyrate that increases the uptake of ferromagnetic nanoparticles. Moreover, we observed a strong decrease of cell viability in a NaBu dose-dependent manner. Taking into account these results, the cooperation of multifunctional MNPs with NaBu, taking into consideration the particular cancer-cell properties, can be a valuable tool for future cancer treatment.

Screening of Polymer-Based Drug Delivery Vehicles Targeting Folate Receptors in Triple-Negative Breast Cancer

PurposeTo compare cellular uptake and cytotoxicity of fluorescein (FL)-labeled polyethylene glycols (PEGs) carrying 2 folate groups (targeted delivery vehicles [TDVs]) to non-PEGylated molecules with 1 or 2 folate groups. Materials and MethodsThree PEGylated TDVs and 2 non-PEGylated folic acid (FA)–fluorescein (FL) conjugates (FA-FL and FA-FL-FA) were synthesized. Two triple-negative breast cancer cell lines (MDA-MB-231and MDA-MB-468) were cultured to 70% confluency and incubated for 2 h in a folate-depleted medium. Folate receptor (FR) expression was confirmed by immunocytochemistry. Cellular uptake and cytotoxicity of compounds were measured by flow cytometry. Intracellular localization was confirmed using confocal microscopy. ResultsMDA-MB-231 demonstrated 40% more FR staining than MD-MB-468. Intracellular localization of the 2 non-PEGylated molecules (FA-FL and FA-FL-FA) and the 3 PEGylated TDVs was confirmed with confocal microscopy. Cellular uptake was independent of concentration for FA-FL, but there was 26.8% more cytotoxicity at 30 μg/mL compared with no treatment (P ≤ .05). Uptake was > 90% for FA-FL-FA at 10 μg/mL and 30 μg/mL without significant cytotoxicity (P ≤ .005). Cellular uptake was > 80% for all TDVs. The molecule containing monodispersed PEG with Mn = 1,000 g/mol had the highest uptake in both cell lines without cytotoxicity. Maximum toxicity was demonstrated by the molecule containing PEG2,000 only at the highest dose of 30 μg/mL (8.66% ± 3.94% cytotoxicity; cut-off was 20%). ConclusionsThe molecule containing monodispersed PEG with Mn = 1,000 g/mol and 2 FA targeting groups demonstrated better targetability and cellular uptake as a TDV.

Folic acid promotes proliferation and differentiation of porcine pancreatic stem cells into insulin-secreting cells through canonical Wnt and ERK signaling pathway

Porcine pancreatic stem cells (pPSCs) can be induced to differentiate into insulin-producing cells in vitro and thus serve as a major cells source for β-cell regeneration. However, this application is limited by the weak cell proliferation ability and low insulin induction efficiency.In this study, we explored the role of folic acid in the proliferation of pPSCs and the formation of insulin-secreting cells. We found that FA-treated pPSCs cells had a high EDU positive rate, and the proliferation marker molecules PCNA, CyclinD1 and c-Myc were up-regulated, while the expression of folate receptor α (FOLRα) was up-regulated. In further research, interference FOLRα or adding canonical Wnt signaling pathway or ERK signaling pathway inhibitors could significantly inhibit the effect of FA on pPSCs proliferation. Meanwhile, during the differentiation of pPSCs into insulin-secreting cells, we found that the maturation marker genes Insulin, NKX6.1, MafA, and NeuroD1 was upregulated in insulin-secreting cell masses differentiationed from pPSCs after FA treatment, and the functional molecules Insulin and C-peptide were increased, the ability to secrete insulin in response to high glucose was also increased. With the addition of Wnt and ERK signaling pathway inhibitors, the pro-differentiation effect of FA was weakened.In conclusion, FA promotes the proliferation of pPSCs by binding to folate receptor α (FOLRα) and increase the efficiency of directed differentiation of pPSCs into insulin-producing cells by regulating canonical Wnt and ERK signaling pathway. This study lays theoretical foundation for solving the bottleneck in the treatment of diabetes with stem cell transplantation in future.

Carbon dots-based nanocarrier system with intrinsic tumor targeting ability for cancer treatment

Doxorubicin (DOX) is a traditional broad-spectrum antitumor drug, which has a wide range of clinical applications, but has no tumor non-specificity. Nanoparticles have been explored as drug delivery agents to enhance the therapeutic efficacy and reduce toxic and side effects. Carbon dots (CDs), a carbon-based nanomaterial, has many unique advantages such as easy synthesis, good biocompatibility, and low toxicity. In this study, folic acid was used as raw material to prepare new CDs, and DOX was loaded on the surface of CDs through electrostatic interaction. The prepared nano-drugs CDs/DOX could effectively release DOX under mild acidic pH stimulation. Cell imaging showed that CDs/DOX could transport doxorubicin (DOX) to cancer cells and make them accumulated in nucleus freely. Flow cytometry tests and cellular toxicity assay together confirmed that CDs/DOX could target tumor cells with high expression of folate receptor and increase anti-tumor activity. The therapeutic effect on 4T1 tumor-bearing mice model indicated that CDs/DOX could alleviate DOX-induced toxicity, effectively inhibit tumor growth, and prolong the survival time. Hence, such a targeting nanocarrier is likely to be a candidate for cancer treatment.

Antitumor Activity of a Novel Double-Targeted System for Folate Receptor-Mediated Delivery of Mitomycin C.

In this study, we designed, formulated, and investigated the potential antitumor activity of a folate receptor (FR)-mediated double-targeted drug delivery system. The system comprised of the FR ligand folic acid (FA), glycine-phenylalanine-leucine-glycine (Gly-Phe-Leu-Gly, GFLG), which can be specifically cleaved by cathepsin B, and the anticancer drug mitomycin C (MMC). The antitumor effect of FA-GFLG-MMC was compared to that of MMC. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay revealed that FA-GFLG-MMC has a significantly higher inhibitory effect on HeLa, SiHa, and PC9 cells (high FR expression) than that on 16HBE and A549 cells (low FR expression). Furthermore, FA-GFLG-MMC inhibited cancer cell proliferation in a dose-dependent manner. Free MMC was toxic to both cancer and normal cells. Apoptosis of the HeLa, SiHa, and PC9 cells was higher than that of the A549 cells; however, the apoptotic effect on 16HBE cells was minimal. Proapoptotic protein bcl-2-associated X-protein (BAX) and antiapoptotic protein BCL-2 play critical roles in cellular defense and apoptotic signal transduction. BAX/BCL-2 ratio is used to determine the intensity of an apoptotic signal and assess whether a cell will survive or undergo apoptosis. BAX and BCL-2 expression in cells treated with 5 μM FA-GFLG-MMC was studied by Western blotting. FA-GFLG-MMC increased the BAX/BCL-2 ratio in HeLa, SiHa, and PC9 cells. The results show that FA-GFLG-MMC can effectively inhibit tumor cell proliferation by inducing apoptosis. Therefore, the system developed can enhance the delivery of anticancer drugs to cancer cells and thereby reduce their toxic effects on normal cells.

Detection of membrane receptors on per tumor cell by nonimmobilized cell capillary electrophoresis and a mathematic model

Folate receptors (FRs) are a class of valuable therapeutic target which is highly expressed on a variety of cancers. The accurate detection of the expression of FRs in different cells is conducive to improve the accuracy of FR targeted tumor therapy. Herein, a method based on nonimmobilized cell capillary electrophoresis (NICCE) combined with a mathematic model to quantify FRs on each single tumor cell was developed. At first, we studied the interactions between FA and A549, HT-29, HepG2, and U87MG cells by NICCE respectively, and calculated the kinetic parameters (Ka, k’, ka, and kd). Next, we established a mathematic model to accurately determine the number of moles of FRs on per A549, HT-29, HepG2, and U87MG cell for the first time, that were (10.44 ± 0.53) × 10–19 mol, (34.32 ± 1.33) × 10–19 mol, (337.14 ± 10.11) × 10–19 mol, and (37.31 ± 2.13) × 10–19 mol. Then, these re-sults were proved to be consistent with the results of enzyme-linked immunosorbent assay (ELISA). Therefore, this method is simple, rapid, sensitive, and without protein separation or purification, which is expected to achieve clinical detection of cell membrane receptor expression level of cell membrane receptors on a single cell, which may be greatly beneficial to further clinical diagnosis and therapy.

Reprogramming of profibrotic macrophages for treatment of bleomycin‐induced pulmonary fibrosis

Fibrotic diseases cause organ failure that lead to ~45% of all deaths in the United States. Activated macrophages stimulate fibrosis by secreting cytokines that induce fibroblasts to synthesize collagen and extracellular matrix proteins. Although suppression of macrophage‐derived cytokine production can halt progression of fibrosis, therapeutic agents that prevent release of these cytokines (e.g., TLR7 agonists) have proven too toxic to administer systemically. Based on the expression of folate receptor β solely on activated myeloid cells, we have created a folate‐targeted TLR7 agonist (FA‐TLR7‐54) that selectively accumulates in profibrotic macrophages and suppresses fibrosis‐inducing cytokine production. We demonstrate that FA‐TLR7‐54 reprograms M2‐like fibrosis‐inducing macrophages into fibrosis‐suppressing macrophages, resulting in dramatic declines in profibrotic cytokine release, hydroxyproline biosynthesis, and collagen deposition, with concomitant increases in alveolar airspaces. Although nontargeted TLR7‐54 is lethal at fibrosis‐suppressing doses, FA‐TLR7‐54 halts fibrosis without evidence of toxicity. Taken together, FA‐TLR7‐54 is shown to constitute a novel and potent approach for treating fibrosis without causing dose‐limiting systemic toxicities.

Impact of extracellular folic acid levels on oviductal gene expression

Folate plays a specific role as methyl donor for nucleotide synthesis and genomic methylation patterns, which in turn are important epigenetic determinants in gene expression. Previous studies have revealed the presence of folate in bovine oviductal fluid as well as the existence of a fine-tuned regulation of the gene expression of folate receptors and transporters in bovine oviduct epithelial cells (BOECs). However, the functional implications of folate in the oviduct remain unknown. The present study aimed to assess the effect of folic acid (FA) on expression levels of selected genes that potentially respond to the folate status in in vitro BOECs. To obtain an insight into the optimization of a culture system for assays, gene expression of folate receptors and transporters was compared between BOECs grown in monolayers and in suspension. The results showed that BOECs from isthmus and ampulla in suspension culture better preserved the region-dependent gene expression profile than in monolayers. Subsequently, BOECs from both anatomical regions were separately cultured in suspension for 24 h assaying different FA concentrations: I) TCM-199 (control); II) TCM-199 + 1 μM FA (similar to the oviduct concentration); III) TCM-199 + 10 μM FA and IV) TCM-199 + 100 μM FA. Expression analysis of genes related to important cellular processes including folate transport, DNA methylation, cell-cell interaction, antioxidant activity and signaling pathways was performed in BOECs using RT-qPCR. Our data demonstrated that addition of 1 μM FA did not affect mRNA levels of most genes analyzed. In contrast, BOECs cultured with 10 μM FA exhibited increased mRNA expression levels of genes involved in folate intake, DNA methylation and antioxidant protection. It is worth noting that at 100 μM FA, transcriptional response in BOECs mainly resulted in decreased mRNA levels of the majority of the genes assayed. Interestingly, cytotoxicity analysis showed a similar LDH activity in the culture media of the experimental groups, indicating that cell integrity was not affected by the FA concentrations assayed. In conclusion, our findings suggest that folate can affect BOECs, promoting changes in gene activity in a framework of functional readjustments in response to environmental conditions.

An Efficient Photodynamic Therapy Treatment for Human Pancreatic Adenocarcinoma.

To date, pancreatic adenocarcinoma (ADKP) is a devastating disease for which the incidence rate is close to the mortality rate. The survival rate has evolved only 2–5% in 45 years, highlighting the failure of current therapies. Otherwise, the use of photodynamic therapy (PDT), based on the use of an adapted photosensitizer (PS) has already proved its worth and has prompted a growing interest in the field of oncology. We have developed a new photosensitizer (PS-FOL/PS2), protected by a recently published patent (WO2019 016397-A1, 24 January 2019). This photosensitizer is associated with an addressing molecule (folic acid) targeting the folate receptor 1 (FOLR1) with a high affinity. Folate binds to FOLR1, in a specific way, expressed in 100% of ADKP or over-expressed in 30% of cases. The first objective of this study is to evaluate the effectiveness of this PS2-PDT in four ADKP cell lines: Capan-1, Capan-2, MiapaCa-2, and Panc-1. For this purpose, we first evaluated the gene and protein expression of FOLR1 on four ADKP cell lines. Subsequently, we evaluated PS2’s efficacy in our cell lines and we assessed the impact of PDT on the secretome of cancer cells and its impact on the immune system. Finally, we evaluate the PDT efficacy on a humanized SCID mouse model of pancreatic cancer. In a very interesting way, we observed a significant increase in the proliferation of activated-human PBMC when cultured with conditioned media of ADKP cancer cells subjected to PDT. Furthermore, to evaluate in vivo the impact of this new PS, we analyzed the tumor growth in a humanized SCID mice model of pancreatic cancer. Four conditions were tested: Untreated, mice (nontreated), mice with PS (PS2), mice subjected to illumination (Light only), and mice subjected to illumination in the presence of PS (PDT). We noticed that the mice subjected to PDT presented a strong decrease in the growth of the tumor over time after illumination. Our investigations have not only suggested that PS2-PDT is an effective therapy in the treatment of PDAC but also that it activates the immune system and could be considered as a real adjuvant for anti-cancer vaccination. Thus, this new study provides new treatment options for patients in a therapeutic impasse and will provide a new arsenal in the fight against PDAC.

Bioinformatics analysis of FOLR1 expression, functional enrichment, related signaling pathways and relationship with prognosis in ovarian cancer

Abstract Objective To investigate folate-receptor 1 (FOLR1) expression in ovarian cancer and its association with patient prognosis. Methods TCGA and Oncomine databases were used to collect data about FOLR1 mRNA expression in multiple carcinomas. FOLR1 mRNA expression levels in ovarian cancer samples and corresponding adjacent normal ovary tissue were compared. A protein-protein interaction (PPI) network was constructed using the STRING database of FOLR1 and relevant genes. The overall survival (OS) and progression free survival (PFS) rates of ovarian cancer patients in high- and low- FOLR1 expression groups were compared by log-rank test. Sixty-six ovarian epithelial carcinoma samples were included in the study, and tumor specimens of the 66 cases were tested for FOLR1 protein expression by an immunohistochemistry assay. Results FOLR1 mRNA was significantly elevated in ovarian cancer compared to other carcinomas. FOLR1 mRNA expression levels were significantly higher in tumor tissues than in the corresponding normal tissues (P<0.05) of ovarian cancer patients. The PPI network indicated that the local clustering coefficient was 0.898, indicating that the PPI network was enriched significantly (P<0.05). The median PFS values were 22.39 and 19.00 months for lowand high-FOLR1 expression groups, respectively, with significant statistical difference between the two (HR=1.26, 95%CI:1.09-1.45, P<0.05). FOLR1 protein expression was correlated with tumor differentiation (P<0.05) in ovarian cancer patients. However, its levels were not correlated with patient age, tumor diameter, lymph node metastasis or FIGO stage (P>0.05). Conclusion FOLR1 is upregulated in epithelial ovarian cancer, and its expression is correlated with patients’ progression free survival, making it a valuable biomarker for prognosis.

Folate Receptor-Targeting and Reactive Oxygen Species-Responsive Liposomal Formulation of Methotrexate for Treatment of Rheumatoid Arthritis.

Multifunctional nanomedicines with active targeting and stimuli-responsive drug release function utilizing pathophysiological features of the disease are regarded as an effective strategy for treatment of rheumatoid arthritis (RA). Under the inflammatory environment of RA, activated macrophages revealed increased expression of folate receptor and elevated intracellular reactive oxygen species (ROS) level. In this study, we successfully conjugated folate to polyethylene glycol 100 monostearate as film-forming material and further prepared methotrexate (MTX) and catalase (CAT) co-encapsulated liposomes, herein, shortened to FOL-MTX&CAT-L, that could actively target to activated macrophages. Thereafter, elevated intracellular hydrogen peroxide, the main source of ROS, diffused into liposomes and encapsulated CAT catalyzed the decomposition of hydrogen peroxide into oxygen and water. Continuous oxygen-generation inside liposomes would eventually disorganize its structure and release the encapsulated MTX. We characterized the in vitro drug release, cellular uptake and cytotoxicity studies as well as in vivo pharmacokinetics, biodistribution, therapeutic efficacy and safety studies of FOL-MTX&CAT-L. In vitro results revealed that FOL-MTX&CAT-L possessed sufficient ROS-sensitive drug release, displayed an improved cellular uptake through folate-mediated endocytosis and exhibited a higher cytotoxic effect on activated RAW264.7 cells. Moreover, in vivo results showed prolonged blood circulation time of PEGylated liposomes, enhanced accumulation of MTX in inflamed joints of collagen-induced arthritis (CIA) mice, reinforced therapeutic efficacy and minimal toxicity toward major organs. These results imply that FOL-MTX&CAT-L may be used as an effective nanomedicine system for RA treatment.

MicroRNA-29b inhibits cell growth and promotes sensitivity to oxaliplatin in colon cancer by targeting FOLR1.

The present study was aimed to explore the functional role of microRNA (miR)-29b in colon cancer, as well as underlying mechanisms. Expressions of miR-29b and folate receptor 1 (FOLR1) were measured in both human colon tumor samples and cell lines. Colon cancer cell lines SW480 and SW620 were transfected with miR-29b mimic, antisense oligonucleotides (ASO)-miR-29b, small interfering (siRNA) against FOLR1 (si-FOLR1), or corresponding negative controls (NCs), and then were incubated with or without oxaliplatin (L-OHP). Thereafter, cell viability, cytotoxicity, cell apoptosis, and expression of FOLR1, ATP Binding Cassette Subfamily G Member 2 (ABCG2) and p-glycoprotein (p-gp) were analyzed. We found that miR-29b was significantly decreased, while FOLR1 was statistically elevated in colon cancer samples and cell lines compared to the nontumor samples and nontumourigenic immortalized human colon epithelial cell line FHC. Overexpression of miR-29b markedly inhibited cell viability, promoted sensitivity to L-OHP, stimulated cell apoptosis (all p < .05), and decreased the levels of ABCG2 and p-gp in cancer cells, whereas suppression of miR-29b showed contrary results. Moreover, we observed that FOLR1 was a direct target of miR-29b and was negatively regulated by miR-29b. In addition, the findings revealed that the effects of FOLR1 inhibition on cell viability, sensitivity to L-OHP, cell apoptosis, and the levels of ABCG2 and p-gp were similar to overexpression of miR-29b. Taken together, our study suggests that miR-29b inhibits cell growth and promotes sensitivity to L-OHP in colon cancer by targeting FOLR1.

Selectivity Enhancement of Paclitaxel Liposome Towards Folate Receptor-Positive Tumor Cells by Ligand Number Optimization Approach.

The present work aims to develop folate-targeted paclitaxel liposome (F-PTX-LIP), which will selectively target tumor cells overexpressing folate receptor (FR) and leave normal cells. Liposomes were prepared by thin-film hydration method followed by post-insertion of synthesized ligand 1,2-distearoyl-sn-glycero-phosphoethanolamine-polyethyleneglycol 2000-folic acid (DSPE-PEG2000-FA) on the outer surface of the liposome. The synthesized ligand was evaluated for in vivo acute toxicity in Balb/c mice. Developed liposomal formulations were characterized using transmission electron microscopy (TEM) and small-angle neutron scattering (SANS). We have investigated the effect of ligand number on cell uptake and cytotoxicity by confocal laser scanning microscopy (CLSM), competitive inhibition and 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay. Compared to lung adenocarcinoma cells (A549), uptake in human ovarian carcinoma cells (SKOV3) was 2.2- and 1.2-fold higher for liposome with 480 and 240 ligand number respectively. Competitive inhibition experiment shows that prior incubation of SKOV3 cells with free folic acid significantly reduced the cell uptake of F-PTX-LIP with 480 ligand number (480 F-PTX-LIP) by 2.6-fold. 480 F-PTX-LIP displays higher cytotoxicity than free drug and PTX liposome. Moreover, it specifically targets the cells with higher folate receptor expression. Optimized 480 F-PTX-LIP formulation can be potentially useful for the treatment of folate receptor-positive tumors.

58P - IKS01, a next generation antibody drug conjugate (ADC) designed to be efficacious in tumors with low and moderate levels of folate receptor expression

BackgroundFrequent over-expression of FRA in ovarian and non-small-cell lung cancer (NSCLC), and relative lack of expression in normal tissue, make it an attractive therapeutic target. Ovarian cancer is the ninth most common cancer in women and the leading cause of death in gynaecological cancer. Lung cancer remains a leading cause of cancer-related death with NSCLC accounting for 80 % of cases. ADCs are the focus of intense interest as a means to provide selective tumor killing with increased efficacy and less off-target toxicity than standard of care chemotherapies. Clinical evaluation of mirvetuximab soravtansine in solid tumor indications demonstrated that FRA can be successfully targeted by an ADC. However, anti-tumor activity appears to be generally limited to patients whose tumors express high levels of FRA. IKS01 is an ADC comprised of an FRA-targeting antibody conjugated via Iksuda’s PermaLink conjugation technology to Femtogenix’s highly potent FGX2-62 payload. FGX2-62 is a pyrridinobenzodiazepine DNA mono-alkylating agent with pM potency in a range of tumor cell lines. IKS01 has been designed to be sufficiently potent to have target-dependent efficacy even in tumors with low or moderate expression of the target antigen. MethodsIKS01 was generated with a drug to antibody ratio of 2. Efficacy was evaluated in human cell line derived xenograft models with a range of FRA expression, including the choriocarcinoma Jeg-3 model (high expression), the ovarian adenocarcinoma OV-90 model (low expression) and the lung adenocarcinoma NCI-H2110 model (moderate expression). An FRA-targeting benchmark ADC with a format that has shown clinical efficacy was included for comparison. ResultsIKS01 is highly effective in causing tumor regressions in FRA-expressing tumor models at doses that are well tolerated. IKS01 was significantly more active than benchmark ADC in the high FRA-expressing Jeg-3 model and caused complete or near complete regressions in low/moderate FRA-expressing xenograft models where the benchmark ADC showed limited or no activity. ConclusionsIKS01 shows marked anti-tumor efficacy in pre-clinical models of ovarian and lung cancer, even in models with low or moderate FRA expression. Legal entity responsible for the studyIksuda Therapeutics Ltd. FundingIksuda Therapeutics Ltd. DisclosureJ. Thirlway: Shareholder/Stockholder/Stock options: Iksuda Therapeutics Ltd. A. Lodge: Shareholder/Stockholder/Stock options: Iksuda Therapeutics Ltd. A. Pelava: Shareholder/Stockholder/Stock options: Iksuda Therapeutics Ltd. D.J. Williamson: Shareholder/Stockholder/Stock options: Iksuda Therapeutics Ltd. D. Carta: Shareholder/Stockholder/Stock options: Iksuda Therapeutics Ltd. M. Al Nakeeb: Shareholder/Stockholder/Stock options: Iksuda Therapeutics Ltd. J. Mysliwy: Shareholder/Stockholder/Stock options: Iksuda Therapeutics Ltd. P.J.M. Jackson: Shareholder/Stockholder/Stock options: Femtogenix Ltd. D.E. Thurston: Shareholder/Stockholder/Stock options: Femtogenix Ltd. R.J. Lutz: Shareholder/Stockholder/Stock options: Iksuda Therapeutics Ltd.

DHA regulates placental expression of Folate and Vitamin B12 receptors in women with pregestational obesity

Bioavailability of nutrients is highly diverse and depends on a variety of endogenous and exogenous factors in humans. This data article reports on the plasma response of 10 human subjects (5 females, 5 males) to a single dose of a multivitamin drink within 6h (blood taken after 1, 2, 4, and 6h). Nutrients, which were considered in the assessment, were folate (Radioimmuno Assay), vitamin B12 (Radioimmuno Assay) and resveratrol and its plasma metabolites resveratrol-3-O-glucuronide (R3G), resveratrol-4′-O-glucuronide (R4G), resveratrol-3-O-sulfate (R3S) and resveratrol-3-O-4′-O-disulfate (RD, all HPLC). Biological outcome measures were malondialdehyde (MDA, HPLC) and Ferric Reducing ability potential (FRAP, Microplate reader).Mean plasma concentration increased over time significantly for folate (p < 0.05, maximum concentration (Tmax) after 2h), R3G, R4G, R3S (all p < 0.05, Tmax after 1h), RD (p < 0.05, Tmax after 2h) as well as MDA, which decreased (p < 0.05, Tmax after 2h). No significant change was observed for vitamin B12 and FRAP. Within this mean development, individual changes of participants were highly diverse such as for folate from +42 to +422%, for MDA from −49 to +30% or vitamin B12 from −4 to +33%. For R4G 4 out of 10 subjects showed even no increase in plasma at all. For R4G plasma response ranged from 0 to 36 ng/ml, for R3G from 0 to 53 ng/ml or for R4S from 62 to 265 ng/ml. There was no gender difference regarding the plasma response.

Detecting Functional and Accessible Folate Receptor Expression in Cancer and Polycystic Kidneys.

Folate-based small molecule drug conjugates (SMDCs) are currently under development and have shown promising preclinical and clinical results against various cancers and polycystic kidney disease. Two requisites for response to a folate-based SMDC are (i) folate receptor alpha (FRα) protein is expressed in the diseased tissues, and (ii) FRα in those tissues is accessible and functionally competent to bind systemically administered SMDCs. Here we report on the development of a small molecule reporter conjugate (SMRC), called EC2220, which is composed of a folate ligand for FRα binding, a multilysine containing linker that can cross-link to FRα in the presence of formaldehyde fixation, and a small hapten (fluorescein) used for immunohistochemical detection. Data show that EC2220 produces a far greater IHC signal in FRα-positive tissues over that produced with EC17, a folate-fluorescein SMRC that is released from the formaldehyde-denatured FRα protein. Furthermore, the extent of the EC2220 IHC signal was proportional to the level of FRα expression. This EC2220-based assay was qualified both in vitro and in vivo using normal tissue, cancer tissue, and polycystic kidneys. Overall, EC2220 is a sensitive and effective reagent for evaluating functional and accessible receptor expression in vitro and in vivo.

Quantitative bioimage analytics enables measurement of targeted cellular stress response induced by celastrol-loaded nanoparticles

The cellular stress response, which provides protection against proteotoxic stresses, is characterized by the activation of heat shock factor 1 and the formation of nuclear stress bodies (nSBs). In this study, we developed a computerized method to quantify the formation and size distribution of nSBs, as stress response induction is of interest in cancer research, neurodegenerative diseases, and in other pathophysiological processes. We employed an advanced bioimaging and analytics workflow to enable quantitative detailed subcellular analysis of cell populations even down to single-cell level. This type of detailed analysis requires automated single cell analysis to allow for detection of both size and distribution of nSBs. For specific induction of nSB we used mesoporous silica nanoparticles (MSNs) loaded with celastrol, a plant-derived triterpene with the ability to activate the stress response. To enable specific targeting, we employed folic acid functionalized nanoparticles, which yields targeting to folate receptor expressing cancer cells. In this way, we could assess the ability to quantitatively detect directed and spatio-temporal nSB induction using 2D and 3D confocal imaging. Our results demonstrate successful implementation of an imaging and analytics workflow based on a freely available, general-purpose software platform, BioImageXD, also compatible with other imaging modalities due to full 3D/4D and high-throughput batch processing support. The developed quantitative imaging analytics workflow opens possibilities for detailed stress response examination in cell populations, with significant potential in the analysis of targeted drug delivery systems related to cell stress and other cytoprotective cellular processes.

Expression of Folate Transport Proteins in a Mouse Model of 4NQO‐Induced Oral Cancer

IntroductionHead and neck cancer is the sixth most common cancer worldwide. Risk factors include tobacco exposure, alcohol use, male sex, and a poor diet. Administration of 4‐nitroquinoline‐1‐oxide (4NQO) in drinking water causes oral cancer in mice, and mimics the effects of tobacco use. Folate is essential for nucleotide biosynthesis as well as epigenetic regulation. Folate is imported by folate receptors (FR), proton‐coupled folate transporters (PCFT), and the reduced folate carrier (RFC). This study aimed to investigate the effects of a high saturated fat (HF) or low fat (LF) diet, sex, and 4NQO treatment on expression of folate transport proteins in mice tongues. We hypothesized that there would be a decreased expression of these proteins in the HNSCC tongue samples compared to the control tongue samples.Materials and MethodsFive week old mice (C57BI/6, 36 per sex) were provided either a low saturated fat diet (10 kcal% fat; LF) or a high fat diet (60 kcal% fat, HF). After one week, mice were then randomly assigned to 17 weeks on one of three water treatment groups: water alone (control); propylene glycol in water (1.25%, PG‐water); or 4NQO in PG‐water (50 mg/ml; 4NQO). After 17 weeks, the mice were provided water alone for six more weeks but maintained on their diet. Tongues from euthanized animals were fixed in formalin and processed for histological examination. Immunohistochemistry was performed to detect the expression of FR, PCFT, and RFC in these samples. Slides were visually graded, and the total score for the expression of each protein was acquired by adding the intensity grade (0–3) and distribution grade (0–3). Statistical analysis was performed using Graphpad Prism 7.0. All animal work was in compliance with the Institutional Animal Care and Use Committee at Midwestern University, Downers Grove, IL.ResultsCompared to the control tongues, there was decreased expression of all three folate transport proteins in tumors (p&lt;0.001). The total score (intensity + distribution) in oral cancer versus control tissue for FR, RFC, and PCFT, respectively were: 3.21 ± 0.10 vs 4.48 + 0.14; 4.38 ± 0.11 vs 5.13 ± 0.10; and 4.22 ± 0.11 vs 5.65 ± 0.69. All slides displayed a 100% distribution rate of FR, PCFT, and RFC in the epithelium. There were no significant differences in folate transport protein expression in males versus females. In the 4NQO tongues, a statistically significant decrease in FR expression was observed between the HF diet group and the LF group (3.97 ± 0.20 vs 3.05 ± 0.10, p &lt;0.0002). No differences were observed in the expression of RFC and PCFT proteins with regards to diet or sex.ConclusionWe measured decreased expression in all three folate transport proteins in oral cancers when compared to normal tongue epithelium. We speculate that mutations leading to decreased folate import can promote carcinogenesis through epigenetic mechanisms.Support or Funding InformationThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Folate-modified and curcumin-loaded dendritic magnetite nanocarriers for the targeted thermo-chemotherapy of cancer cells

A novel multifunctional nanoplatform constructed from methoxy-PEGylated poly(amidoamine) (PAMAM) generation 3 dendrimers with superparamagnetic iron oxide nanoparticles (SPIONs) entrapped in their core, containing curcumin as the payload drug and folic acid (folate) as the targeting ligand (abbreviated as FA-mPEG-PAMAM G3-CUR@SPIONs), is presented in this study. SPIONs entrapped in the core of the nanocomplex may act as a hyperthermia agent and generate localized heat upon excitation with an alternating magnetic field (AMF), thus enabling a thermo-chemotherapy strategy for cancer treatment. Accordingly, the cytotoxic effect and the mode of cell death triggered by the nanocomplex in combination with AMF were evaluated on two different cancer cell lines with various folate receptor (FR) expression levels, including KB nasopharyngeal cancer cells overexpressing FRs as the model and MCF-7 breast cancer cells with low level of FRs as the blank sample. The obtained results showed that KB cell death was greater than the cell death observed in MCF-7 cells. Moreover, a majority of cell death in both cell lines were related to apoptosis when the folate-modified nanocomplex was used instated of the non-folate-modified nanocomplex. Therefore, functionalizing the nanocomplex with folate modulated the response to thermo-chemotherapy by shifting the cell death pathways toward apoptosis.

Arsenic‑induced BRCA1 CpG promoter methylation is associated with the downregulation of ERα and resistance to tamoxifen in MCF7 breast cancer cells and mouse mammary tumor xenografts.

A significant percentage (~30%) of estrogen receptor-α (ERα)-positive tumors become refractory to endocrine therapies; however, the mechanisms responsible for this resistance remain largely unknown. Chronic exposure to arsenic through foods and contaminated water has been linked to an increased incidence of several tumors and long-term health complications. Preclinical and population studies have indicated that arsenic exposure may interfere with endocrine regulation and increase the risk of breast tumorigenesis. In this study, we examined the effects of sodium arsenite (NaAsIII) exposure in ERα-positive breast cancer cells in vitro and in mammary tumor xenografts. The results revealed that acute (within 4 days) and long-term (10 days to 7 weeks) in vitro exposure to environmentally relevant doses reduced breast cancer 1 (BRCA1) and ERα expression associated with the gain of cyclin D1 (CCND1) and folate receptor 1 (FOLR1), and the loss of methylenetetrahydrofolate reductase (MTHFR) expression. Furthermore, long-term exposure to NaAsIII induced the proliferation and compromised the response of MCF7 cells to tamoxifen (TAM). The in vitro exposure to NaAsIII induced BRCA1 CpG methylation associated with the increased recruitment of DNA methyltransferase 1 (DNMT1) and the loss of RNA polymerase II (PolII) at the BRCA1 gene. Xenografts of NaAsIII-preconditioned MCF7 cells (MCF7NaAsIII) into the mammary fat pads of nude mice produced a larger tumor volume compared to tumors from control MCF7 cells and were more refractory to TAM in association with the reduced expression of BRCA1 and ERα, CpG hypermethylation of estrogen receptor 1 (ESR1) and BRCA1, and the increased expression of FOLR1. These cumulative data support the hypothesis that exposure to AsIII may contribute to reducing the efficacy of endocrine therapy against ERα-positive breast tumors by hampering the expression of ERα and BRCA1 via CpG methylation, respectively of ESR1 and BRCA1.