Porcupine quills keratin peptides induces G0/G1 cell cycle arrest and apoptosis via p53/p21 pathway and caspase cascade reaction in MCF-7 breast cancer cells.

BACKGROUND: Porcupine quills, a by-product of porcupine pork, are rich in keratin, which is an excellent source of bioactive peptides. The objective of this study was to investigate the underlying mechanism of anti-proliferation effect of porcupine quills keratin peptides (PQKPs) on MCF-7 cells. RESULTS: Results showed that PQKPs induced MCF-7 cells apoptosis by significantly decreasing the secretion level of anti-apoptosis protein Bcl-2 and increasing the secretion levels of pro-apoptosis proteins Bax, cytochrome c, caspase 9, caspase 3 and PARP. PQKPs also arrested the cell cycle at G0/G1 phase via remarkably reducing the protein levels of CDK4 and enhancing the protein levels of p53 and p21. High-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) analysis identified nine peptides with molecular weights less than 1000 Da in PQKPs. Molecular docking results showed that TPGPPT and KGPAC identified from PQKPs could bind with p53 mutant and Bcl-2 protein by conventional hydrogen bonds, carbon hydrogen bonds and van der Waals force. Furthermore, the anti-proliferation impact of synthesized peptides (TPGPPT and KGPAC) was shown in MCF-7 cells. CONCLUSION: These findings indicated that PQKPs suppressed the proliferation of MCF-7 breast cancer cells by triggering apoptosis and G0/G1 cell cycle arrest. Moreover, the outcome of this study will bring fresh insights into the production and application of animal byproducts. © 2023 Society of Chemical Industry.

Spatial and temporal expression of PORCN is highly dynamic in the developing mouse cochlea.

The mammalian organ of Corti is a highly specialized sensory organ of the cochlea with a fine-grained pattern that is essential for auditory function. The sensory epithelium, the organ of Corti consists of a single row of inner hair cells and three rows of outer hair cells that are intercalated by support cells in a mosaic pattern. Previous studies show that the Wnt pathway regulates proliferation, promotes medial compartment formation in the cochlea, differentiation of the mechanosensory hair cells and axon guidance of Type II afferent neurons. WNT ligand expressions are highly dynamic throughout development but are insufficient to explain the roles of the Wnt pathway. We address a potential way for how WNTs specify the medial compartment by characterizing the expression of Porcupine (PORCN), an O-acyltransferase that is required for WNT secretion. We show PORCN expression across embryonic ages (E)12.5 - E14.5, E16.5, and postnatal day (P)1. Our results showed enriched PORCN in the medial domains during early stages of development, indicating that WNTs have a stronger influence on patterning of the medial compartment. PORCN was rapidly downregulated after E14.5, following the onset of sensory cell differentiation; residual expression remained in some hair cells and supporting cells. On E14.5 and E16.5, we also examined the spatial expression of Gsk3ß, an inhibitor of canonical Wnt signaling to determine its potential role in radial patterning of the cochlea. Gsk3ß was broadly expressed across the radial axis of the epithelium; therefore, unlikely to control WNT-mediated medial specification. In conclusion, the spatial expression of PORCN enriches WNT secretion from the medial domains of the cochlea to influence the specification of cell fates in the medial sensory domain.

Morphological evaluation of polysaccharide content and collagen composition during cutaneous wound healing in the Sunda porcupine (Hystrix javanica).

Wound healing in the Sunda porcupine is believed to occur quickly, although the wound is large and severe. Wound enclosure involves many processes to restore the lost or damaged skin structure where conjugated polysaccharide-protein and collagen, as the main components deposited in wound tissue to restore it. The aim of this study was to evaluate alteration of polysaccharide contents and collagen in untreated full-thickness wound healing in the thoracodorsal and lumbosacral regions in the Sunda porcupines. Histological analysis was performed by periodic acid Schiff, alcian blue pH 2.5, picrosirius red staining method and Low Vacuum Scanning Electron Microscope (LV-SEM) imaging to obtain the fundamental data of healing process. Wound healing began with re-epithelization followed by progressive wound contraction with 4 overlapping stages in about 30-50 days until the wound closed (21-30 days in thoracodorsal and 30-50 days in lumbosacral). Neutral polysaccharide was more widely distributed compared to the acid polysaccharide in almost all stages of wound healing. The ratio of collagen I to III appeared to be higher in the thoracodorsal region than the lumbosacral region during healing process. LV-SEM imaging showed changes in connective tissue structure in the wound border and granulation tissue which appeared abundant and mixed of thin and thick fiber. In conclusion, cutaneous full thickness wound healing in the Sunda porcupine occurred faster in the thoracodorsal region, which might be correlated to the role of neutral polysaccharide and a high ratio of collagen I to III.

Anticancer activity of grassy Hystrix brachyura bezoar and its mechanisms of action: An in vitro and in vivo based study.

Porcupine bezoar (PB) is a calcified undigested material generally found in porcupine's (Hystrix brachyura) gastrointestinal tract. The bezoar is traditionally used in South East Asia and Europe for the treatment of cancer, poisoning, dengue, typhoid, etc. However, limited scientific studies have been performed to verify its anticancer potential to substantiate its traditional claims in the treatment of cancers. Hence, this study was aimed at investigating the in vitro and in vivo anticancer properties of two grassy PB aqueous extract (PB-A and PB-B) using A375 cancer cell line and zebrafish model, respectively. This paper presents the first report on in vitro A375 cell viability assay, apoptosis assay, cell cycle arrest assay, migration assay, invasion assay, qPCR experimental assay and in vivo anti-angiogenesis assay using the grassy PBs. Experimental findings revealed IC50 value are 26.59 ± 1.37 µg/mL and 30.12 ± 3.25 µg/mL for PB-A and PB-B respectively. PBs showed anti-proliferative activity with no significant cytotoxic effect on normal human dermal fibroblast (NHDF). PBs were also found to induce apoptosis via intrinsic pathway and arrest cell cycle at G2/M phase. Additionally, the findings indicated its ability to debilitate migration and invasion of A375 cells. Further evaluation using embryo zebrafish model revealed LC50 = 450.0 ± 2.50 µg/mL and 58.7 ± 5.0 µg/mL for PB-A and PB-B which also exerted anti-angiogenesis effect in zebrafish. Moreover, stearic acid, ursodeoxycholic acid and pregnenolone were identified as possible metabolites that might contribute to the anticancer effect of the both PBs. Overall, this study demonstrated that PB-A and PB-B possess potential in vitro and in vivo anticancer effects which are elicited through selective cytotoxic effect, induction of apoptosis, inhibition of migration and invasion and anti-angiogenesis. This study provides scientific evidence that the porcupine bezoar do possess anti-cancer efficacy and further justifies its traditional utility. However, more experiments with higher vertebrae models are still warranted to validate its traditional claims as an anticancer agent.

Characterization of novel lignocellulose-degrading enzymes from the porcupine microbiome using synthetic metagenomics.

Plant cell walls are composed of cellulose, hemicellulose, and lignin, collectively known as lignocellulose. Microorganisms degrade lignocellulose to liberate sugars to meet metabolic demands. Using a metagenomic sequencing approach, we previously demonstrated that the microbiome of the North American porcupine (Erethizon dorsatum) is replete with genes that could encode lignocellulose-degrading enzymes. Here, we report the identification, synthesis and partial characterization of four novel genes from the porcupine microbiome encoding putative lignocellulose-degrading enzymes: ß-glucosidase, α-L-arabinofuranosidase, ß-xylosidase, and endo-1,4-ß-xylanase. These genes were identified via conserved catalytic domains associated with cellulose- and hemicellulose-degradation. Phylogenetic trees were created for each of these putative enzymes to depict genetic relatedness to known enzymes. Candidate genes were synthesized and cloned into plasmid expression vectors for inducible protein expression and secretion. The putative ß-glucosidase fusion protein was efficiently secreted but did not permit Escherichia coli (E. coli) to use cellobiose as a sole carbon source, nor did the affinity purified enzyme cleave p-Nitrophenyl ß-D-glucopyranoside (p-NPG) substrate in vitro over a range of physiological pH levels (pH 5-7). The putative hemicellulose-degrading ß-xylosidase and α-L-arabinofuranosidase enzymes also lacked in vitro enzyme activity, but the affinity purified endo-1,4-ß-xylanase protein cleaved a 6-chloro-4-methylumbelliferyl xylobioside substrate in acidic and neutral conditions, with maximal activity at pH 7. At this optimal pH, KM, Vmax, and kcat were determined to be 32.005 ± 4.72 µM, 1.16x10-5 ± 3.55x10-7 M/s, and 94.72 s-1, respectively. Thus, our pipeline enabled successful identification and characterization of a novel hemicellulose-degrading enzyme from the porcupine microbiome. Progress towards the goal of introducing a complete lignocellulose-degradation pathway into E. coli will be accelerated by combining synthetic metagenomic approaches with functional metagenomic library screening, which can identify novel enzymes unrelated to those found in available databases.

Direct evidence supporting the existence of a helical dislocation in protofilament packing in the intermediate filaments of oxidized trichocyte keratin.

The X-ray diffraction patterns of quill and hair, as well as other trichocyte keratin appendages, contain meridional reflections that can be indexed on an axial repeat of 470 Å. Unusually, however, many of the expected orders are not observed. A possible explanation, proposed by Fraser and MacRae (1983), was that the intermediate filaments (IF) that constitute the fibrillar component of the filament/matrix texture consist of 4-chain protofilaments arranged on a surface lattice subject to a helical dislocation. The radial projection of the resulting 8-protofilament ribbon was defined in terms of a two-dimensional unit cell characterized by vectors (a, b) with axial projections za ∼ 74 Šand zb ∼ 198 Å. This situation resembles that found in microtubules, where helical dislocations in subunit packing are also encountered, leading to a so-called "seam" along their length (Metoz and Wade, 1997). In keratin, however, the protofilaments are helical so the seam is inclined to the axis of the IF. Here we report details of the Patterson function that provides independent evidence for both the helical dislocation and the dimensions of the surface lattice. In addition, the observed meridional X-ray amplitudes have been compared with those predicted by various models of the axial distribution of electron density. A new model, adapted from one previously proposed, fits the data significantly better than has heretofore proved possible. An interpretation of the model in terms of either specific keratin-associated-protein (KAP) binding or the retention of IF symmetry by a portion of the head and/or tail domains is suggested.