In vitro gastrointestinal stability and intestinal absorption of ACE-1 and DPP4 inhibitory peptides from poultry by-product hydrolysates.

Bioactive peptides derived from food are promising health-promoting ingredients that can be used in functional foods and nutraceutical formulations. In addition to the potency towards the selected therapeutic target, the bioavailability of bioactive peptides is a major factor regarding clinical efficacy. We have previously shown that a low molecular weight peptide fraction (LMWPF) from poultry by-product hydrolysates possesses angiotensin-1-converting enzyme (ACE-1) and dipeptidyl-peptidase 4 (DPP4) inhibitory activities. The present study aimed to investigate the bioavailability of the bioactive peptides in the LMWPF. Prior to the investigation of bioavailability, a dipeptide YA was identified from this fraction as a dual inhibitor of ACE-1 and DPP4. Gastrointestinal (GI) stability and intestinal absorption of the bioactive peptides (i.e., YA as well as two previously reported bioactive dipeptides (VL and IY)) in the LMWPF were evaluated using the INFOGEST static in vitro digestion model and intestinal Caco-2 cell monolayer, respectively. Analysis of peptides after in vitro digestion confirmed that the dipeptides were resistant to the simulated GI conditions. After 4 hours of incubation, the concentration of the peptide from the apical side of the Caco-2 cell monolayer showed a significant decrease. However, the corresponding absorbed peptides were not detected on the basolateral side, suggesting that the peptides were not transported across the intestinal monolayer but rather taken up or metabolized by the Caco2 cells. Furthermore, when analyzing the gene expression of the Caco-2 cells upon peptide stimulation, a down-regulation of peptide transporters, the transcription factor CDX2, and the tight junction protein-1 (TJP1) was observed, suggesting the specific effects of the peptides on the Caco-2 cells. The study demonstrated that bioactive dipeptides found in the LMWPF were stable through in vitro GI digestion; however, the overall bioavailability may be hindered by inadequate uptake across the intestinal barrier.

Expansion of bovine skeletal muscle stem cells from spinner flasks to benchtop stirred-tank bioreactors for up to 38 days.

Introduction: Successful long-term expansion of skeletal muscle satellite cells (MuSCs) on a large scale is fundamental for cultivating animal cells for protein production. Prerequisites for efficient cell expansion include maintaining essential native cell activities such as cell adhesion, migration, proliferation, and differentiation while ensuring consistent reproducibility. Method: This study investigated the growth of bovine MuSC culture using low-volume spinner flasks and a benchtop stirred-tank bioreactor (STR). Results and discussion: Our results showed for the first time the expansion of primary MuSCs for 38 days in a bench-top STR run with low initial seeding density and FBS reduction, supported by increased expression of the satellite cell marker PAX7 and reduced expression of differentiation-inducing genes like MYOG, even without adding p38-MAPK inhibitors. Moreover, the cells retained their ability to proliferate, migrate, and differentiate after enzymatic dissociation from the microcarriers. We also showed reproducible results in a separate biological benchtop STR run.

Characterization of wooden breast myopathy: a focus on syndecans and ECM remodeling.

Introduction: The skeletal muscle deformity of commercial chickens (Gallus gallus), known as the wooden breast (WB), is associated with fibrotic myopathy of unknown etiology. For future breeding strategies and genetic improvements, it is essential to identify the molecular mechanisms underlying the phenotype. The pathophysiological hallmarks of WB include severe skeletal muscle fibrosis, inflammation, myofiber necrosis, and multifocal degeneration of muscle tissue. The transmembrane proteoglycans syndecans have a wide spectrum of biological functions and are master regulators of tissue homeostasis. They are upregulated and shed (cleaved) as a regulatory mechanism during tissue repair and regeneration. During the last decades, it has become clear that the syndecan family also has critical functions in skeletal muscle growth, however, their potential involvement in WB pathogenesis is unknown. Methods: In this study, we have categorized four groups of WB myopathy in broiler chickens and performed a comprehensive characterization of the molecular and histological profiles of two of them, with a special focus on the role of the syndecans and remodeling of the extracellular matrix (ECM). Results and discussion: Our findings reveal differential expression and shedding of the four syndecan family members and increased matrix metalloproteinase activity. Additionally, we identified alterations in key signaling pathways such as MAPK, AKT, and Wnt. Our work provides novel insights into a deeper understanding of WB pathogenesis and suggests potential therapeutic targets for this condition.

Production of food-grade microcarriers based on by-products from the food industry to facilitate the expansion of bovine skeletal muscle satellite cells for cultured meat production.

A major challenge for successful cultured meat production is the requirement for large quantities of skeletal muscle satellite cells (MuSCs). Commercial microcarriers (MCs), such as Cytodex®1, enable extensive cell expansion by offering a large surface-to-volume ratio. However, the cell-dissociation step post cell expansion makes the cell expansion less efficient. A solution is using food-grade MCs made of sustainable raw materials that do not require a dissociation step and can be included in the final meat product. This study aimed to produce food-grade MCs from food industry by-products (i.e., turkey collagen and eggshell membrane) and testing their ability to expand bovine MuSCs in spinner flask systems for eight days. The MCs' physical properties were characterized, followed by analyzing the cell adhesion, growth, and metabolic activity. All MCs had an interconnected porous structure. Hybrid MCs composed of eggshell membrane and collagen increased the mechanical hardness and stabilized the buoyancy compared to pure collagen MCs. The MuSCs successively attached and covered the entire surface of all MCs while expressing high cell proliferation, metabolic activity, and low cell cytotoxicity. Cytodex®1 MCs were included in the study. Relative gene expression of skeletal muscle markers showed reduced PAX7 and increased MYF5, which together with augmented proliferation marker MKI67 indicated activated and proliferating MuSCs on all MCs. Furthermore, the expression pattern of cell adhesion receptors (ITGb5 and SDC4) and focal adhesion marker VCL varied between the distinct MCs, indicating different specific cell receptor interactions with the various biomaterials. Altogether, our results demonstrate that these biomaterials are promising prospects to produce custom-fabricated food-grade MCs intended to expand MuSCs.

Identification of response signatures for tankyrase inhibitor treatment in tumor cell lines.

Small-molecule tankyrase 1 and tankyrase 2 (TNKS1/2) inhibitors are effective antitumor agents in selected tumor cell lines and mouse models. Here, we characterized the response signatures and the in-depth mechanisms for the antiproliferative effect of tankyrase inhibition (TNKSi). The TNKS1/2-specific inhibitor G007-LK was used to screen 537 human tumor cell lines and a panel of particularly TNKSi-sensitive tumor cell lines was identified. Transcriptome, proteome, and bioinformatic analyses revealed the overall TNKSi-induced response signatures in the selected panel. TNKSi-mediated inhibition of wingless-type mammary tumor virus integration site/ß-catenin, yes-associated protein 1 (YAP), and phosphatidylinositol-4,5-bisphosphate 3-kinase/AKT signaling was validated and correlated with lost expression of the key oncogene MYC and impaired cell growth. Moreover, we show that TNKSi induces accumulation of TNKS1/2-containing ß-catenin degradasomes functioning as core complexes interacting with YAP and angiomotin proteins during attenuation of YAP signaling. These findings provide a contextual and mechanistic framework for using TNKSi in anticancer treatment that warrants further comprehensive preclinical and clinical evaluations.

Tankyrase inhibition sensitizes melanoma to PD-1 immune checkpoint blockade in syngeneic mouse models.

The development of immune checkpoint inhibitors represents a major breakthrough in cancer therapy. Nevertheless, a substantial number of patients fail to respond to checkpoint pathway blockade. Evidence for WNT/ß-catenin signaling-mediated immune evasion is found in a subset of cancers including melanoma. Currently, there are no therapeutic strategies available for targeting WNT/ß-catenin signaling. Here we show that a specific small-molecule tankyrase inhibitor, G007-LK, decreases WNT/ß-catenin and YAP signaling in the syngeneic murine B16-F10 and Clone M-3 melanoma models and sensitizes the tumors to anti-PD-1 immune checkpoint therapy. Mechanistically, we demonstrate that the synergistic effect of tankyrase and checkpoint inhibitor treatment is dependent on loss of ß-catenin in the tumor cells, anti-PD-1-stimulated infiltration of T cells into the tumor and induction of an IFNγ- and CD8+ T cell-mediated anti-tumor immune response. Our study uncovers a combinatorial therapeutical strategy using tankyrase inhibition to overcome ß-catenin-mediated resistance to immune checkpoint blockade in melanoma.

MEK Inhibition Induces Canonical WNT Signaling through YAP in KRAS Mutated HCT-15 Cells, and a Cancer Preventive FOXO3/FOXM1 Ratio in Combination with TNKS Inhibition.

The majority of colorectal cancers are induced by subsequent mutations in APC and KRAS genes leading to aberrant activation of both canonical WNT and RAS signaling. However, due to induction of feedback rescue mechanisms some cancers do not respond well to targeted inhibitor treatments. In this study we show that the APC and KRAS mutant human colorectal cancer cell line HCT-15 induces canonical WNT signaling through YAP in a MEK dependent mechanism. This inductive loop is disrupted with combined tankyrase (TNKS) and MEK inhibition. RNA sequencing analysis suggests that combined TNKS/MEK inhibition induces metabolic stress responses in HCT-15 cells promoting a positive FOXO3/FOXM1 ratio to reduce antioxidative and cryoprotective systems.

5-FU resistant EMT-like pancreatic cancer cells are hypersensitive to photochemical internalization of the novel endoglin-targeting immunotoxin CD105-saporin.

BACKGROUND: Development of resistance to 5-fluorouracil (5-FU) is a major problem in treatment of various cancers including pancreatic cancer. In this study, we reveal important resistance mechanisms and photochemical strategies to overcome 5-FU resistance in pancreatic adenocarcinoma. METHODS: 5-FU resistant (5-FUR), epithelial-to-mesenchymal-like sub-clones of the wild type pancreatic cancer cell line Panc03.27 were previously generated in our lab. We investigated the cytotoxic effect of the endosomal/lysosomal-localizing photosensitizer TPCS2a (fimaporfin) combined with light (photochemical treatment, PCT) using MTS viability assay, and used fluorescence microscopy to show localization of TPCS2a and to investigate the effect of photodamage of lysosomes. Flow cytometric analysis was performed to investigate uptake of photosensitizer and to assess intracellular ROS levels. Expression and localization of LAMP1 was assessed using RT-qPCR, western blotting, and structured illumination microscopy. MTS viability assay was used to assess the effect of combinations of 5-FU, chloroquine (CQ), and photochemical treatment. Expression of CD105 was investigated using RT-qPCR, western blotting, flow cytometry, and fluorescence microscopy, and co-localization of TPCS2a and anti-CD105-saporin was assessed using microscopy. Lastly, the MTS assay was used to investigate cytotoxic effects of photochemical internalization (PCI) of the anti-CD105-immunotoxin. RESULTS: The 5-FUR cell lines display hypersensitivity to PCT, which was linked to increased uptake of TPCS2a, altered lysosomal distribution, lysosomal photodamage and increased expression of the lysosomal marker LAMP-1 in the 5-FUR cells. We show that inhibition of autophagy induced by either chloroquine or lysosomal photodamage increases the sensitivity to 5-FU in the resistant cells. The three 5-FUR sub-clones overexpress Endoglin (CD105). Treatment with the immunotoxin anti-CD105-saporin alone significantly reduced the viability of the CD105-expressing 5-FUR cells, whereas little effect was seen in the CD105-negative non-resistant parental cancer cell lines. Strikingly, using the intracellular drug delivery method photochemical internalization (PCI) by combining light-controlled activation of the TPCS2a with nanomolar levels of CD105-saporin resulted in strong cytotoxic effects in the 5-FUR cell population. CONCLUSION: Our findings suggested that autophagy is an important resistance mechanism against the chemotherapeutic drug 5-FU in pancreatic cancer cells, and that inhibition of the autophagy process, either by CQ or lysosomal photodamage, can contribute to increased sensitivity to 5-FU. For the first time, we demonstrate the promise of PCI-based targeting of CD105 in site-specific elimination of 5-FU resistant pancreatic cancer cells in vitro. In conclusion, PCI-based targeting of CD105 may represent a potent anticancer strategy and should be further evaluated in pre-clinical models.

Implications of targeted genomic disruption of ß-catenin in BxPC-3 pancreatic adenocarcinoma cells.

Pancreatic adenocarcinoma (PA) is among the most aggressive human tumors with an overall 5-year survival rate of <5% and available treatments are only minimal effective. WNT/ß-catenin signaling has been identified as one of 12 core signaling pathways that are commonly mutated in PA. To obtain more insight into the role of WNT/ß-catenin signaling in PA we established human PA cell lines that are deficient of the central canonical WNT signaling protein ß-catenin by using zinc-finger nuclease (ZFN) mediated targeted genomic disruption in the ß-catenin gene (CTNNB1). Five individual CTNNB1 gene disrupted clones (BxPC3ΔCTNNB1) were established from a BxPC-3 founder cell line. Despite the complete absence of ß-catenin, all clones displayed normal cell cycle distribution profiles, overall normal morphology and no elevated levels of apoptosis although increased doubling times were observed in three of the five BxPC3ΔCTNNB1 clones. This confirms that WNT/ß-catenin signaling is not mandatory for long term cell growth and survival in BxPC-3 cells. Despite a normal morphology of the ß-catenin deficient cell lines, quantitative proteomic analysis combined with pathway analysis showed a significant down regulation of proteins implied in cell adhesion combined with an up-regulation of plakoglobin. Treatment of BxPC3ΔCTNNB1 cell lines with siRNA for plakoglobin induced morphological changes compatible with a deficiency in the formation of functional cell to cell contacts. In addition, a re-localization of E-cadherin from membranous in untreated to accumulation in cytoplasmatic puncta in plakoglobin siRNA treated BxPC3ΔCTNNB1 cells was observed. In conclusion we describe in ß-catenin deficient BxPC-3 cells a rescue function for plakoglobin on cell to cell contacts and maintaining the localization of E-cadherin at the cellular surface, but not on canonical WNT signaling as measured by TFC/LEF mediated transcription.