Protective action by annatto-derived delta-tocotrienol (δ-TCT) and soy-derived alpha-tocopherol (α-TOC) through the regulation of PI3K/Akt-Cyclin D1 pathway against the nicotine-induced DNA damages is the focus of the present study. Nicotine, which has been widely reported to have numerous adverse effects on the reproductive system, was used as reproductive toxicant. 48 female balb/c mice (6-8 weeks) (23-25 g) were randomly divided into 8 groups (G1-G8; n = 6) and treated with either nicotine or/and annatto δ-TCT/soy α-TOC for 7 consecutive days. On Day 8, the females were superovulated and mated before euthanized for embryo collection (46 hours post-coitum). Fifty 2-cell embryos from each group were used in gene expression analysis using Affymetrix QuantiGene Plex2.0 assay. Findings indicated that nicotine (G2) significantly decreased (p < 0.05) the number of produced 2-cell embryos compared to control (G1). Intervention with mixed annatto δ-TCT (G3) and pure annatto δ-TCT (G4) significantly increased the number of produced 2-cell embryos by 127 % and 79 % respectively compared to G2, but these were lower than G1. Concurrent treatment with soy α-TOC (G5) decreased embryo production by 7 %. Supplementations with δ-TCT and α-TOC alone (G6-G8) significantly increased (p < 0.05) the number of produced 2-cell embryos by 50 %, 36 % and 41 % respectively, compared to control (G1). These results were found to be associated with the alterations in the PI3K/Akt-Cyclin D1 gene expressions, indicating the inhibitory effects of annatto δ-TCT and soy α-TOC against the nicotinic embryonic damages. To our knowledge, this is the first attempt on studying the benefits of annatto δ-TCT on murine preimplantation 2-cell embryos.
ABSTRACT
Metabolic footprinting involves the determination of metabolites excreted or secreted by the cells.
This study aimed to identify the differential extracellular metabolites in colorectal cancer (CRC)
cells for the determination of molecular changes that occur as CRC progresses. CRC cells at
different stages ie; SW 1116 (stage A), HT 29 and SW 480 (stage B), HCT 15 and DLD-1 (stage
C), and HCT 116 (stage D) were grown in culture. The media in which the cells were grown are
subjected to metabolomics profiling using Liquid Chromatography Mass SpectrometryQuadrupole Time of Flight (LC/MS Q-TOF). Statistical and metabolic pathway analysis was
performed using Metaboanalyst software and identification of metabolites was determined by the
METLIN database. A total of 27 differential extracellular metabolites were identified in CRC cells
of different stages compared to stage A cells. Data from the Partial least squares-discriminant
analysis (PLS-DA) score plot shows a clear separation between CRC cells of different stages with
a few overlaps between stage B and C. Further analysis using variable importance in projection
(VIP) revealed 14 differential extracellular metabolites that were most significant in differentiating
CRC cells of the advanced stages from stage A which are 5-hydroxy-L-tryptophan,
indoleacetaldehyde, 4,5-dimethylthiazole, 8-oxodiacetoxyscirpenol, bisnorbiotin, 5-amino-6-
(5'phosphoribosylamino) uracil, glyceryl 5-hydroxydecanoate, sphinganine, 8,8-diethoxy-2,6-
dimethyl-2-octanol, l-cystine, thiamine acetic acid, phytosphingosine, PE
(20:4(5Z,8Z,11Z,14Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), N-(2R-hydroxypentacosano-yl)-2Samino-1,3S,4R-octadecanetriol. The different expressions of metabolites may indicate altered
metabolic pathways in the more advanced CRC cells compared to stage A. This study highlights
the importance of conducting both metabolomics profiling of extracellular and intracellular to
generate a more complete understanding on the molecular changes that occur as CRC progresses
Prolonged disuse of the musculoskeletal system is associated with reduced mechanical loading and lack of anabolic stimulus. As a form of mechanical signal, the multidirectional orbital fluid shear stress transmits anabolic signal to bone forming cells in promoting cell differentiation, metabolism and proliferation. Signals are channeled through the cytoskeleton framework, directly modifying gene and protein expression. For that reason, we aimed to study the organization of Normal Human Osteoblast (NHOst) cytoskeleton with regards to orbital fluid shear (OFS) stress. Of special interest were the consequences of cytoskeletal reorganization on NHOst metabolism, proliferation, and osteogenic functional markers. Cells stimulated at 250 RPM in a shaking incubator resulted in the rearrangement of actin and tubulin fibers after 72 h. Orbital shear stress increased NHOst mitochondrial metabolism and proliferation, simultaneously preventing apoptosis. The ratio of RANKL/OPG was reduced, suggesting that orbital shear stress has the potential to inhibit osteoclastogenesis and osteoclast activity. Increase in ALP activity and OCN protein production suggests that stimulation retained osteoblast function. Shear stress possibly generated through actin seemed to hold an anabolic response as osteoblast metabolism and functional markers were enhanced. We hypothesize that by applying orbital shear stress with suitable magnitude and duration as a non-drug anabolic treatment can help improve bone regeneration in prolonged disuse cases.
Exposure of Normal Human Osteoblast cells (NHOst) to a period of hypothermia may interrupt their cellular functions, lead to changes in bone matrix and disrupt the balance between bone formation and resorption, resulting in bone loss or delayed fracture healing. To investigate this possibility, we exposed NHOst cells to moderate (35 °C) and severe (27 °C) hypothermia for 1, 12, 24 and 72 h. The effects of hypothermia with respect to cell cytoskeleton organization, metabolic activity and the expression of cold shock chaperone proteins, osteoblast transcription factors and functional markers, were examined. Our findings showed that prolonged moderate hypothermia retained the polymerization of the cytoskeletal components. NHOst cell metabolism was affected differently according to hypothermia severity. The osteoblast transcription factors Runx2 and osterix were necessary for the transcription and translation of bone matrix proteins, where alkaline phosphatase (Alp) activity and osteocalcin (OCN) bone protein were over expressed under hypothermic conditions. Consequently, bone mineralization was stimulated after exposure to moderate hypothermia for 1 week, indicating bone function was not impaired. The cold shock chaperone protein Rbm3 was significantly upregulated (p<0.001) during the cellular stress adaption under hypothermic conditions. We suggest that Rbm3 has a dual function: one as a chaperone protein that stabilizes mRNA transcripts and a second one in enhancing the transcription of Alp and Ocn genes. Our studies demonstrated that hypothermia permitted the in vitro maturation of NHOst cells probably through an osterix-dependent pathway. For that reason, we suggest that moderate hypothermia can be clinically applied to counteract heat production at the fracture site that delays fracture healing.