Human Colon Cancer HT29 Cell Line Treatment with High-Dose LAscorbic Acid Results to Reduced Angiogenic Proteins Expression and Elevated Pro-apoptotic Proteins Expression.

BACKGROUND: Some studies have shown anticarcinogenic effects of high dose L-Ascorbic Acid. However, there are controversies around the therapeutic administration of Ascorbic acid as an anticancer medicine. OBJECTIVE: We conducted a case-control study to investigate the role of pharmacologic concentration of Ascorbic acid on viability and angiogenesis of the human colon cancer (HT29) cell line. METHODS: The HT29 cells were cultured in DMEM-HG and treated with 10 mM ascorbic acid for 3h. The culture medium was exchanged, and after incubation at 37 ºC for 24 h, the cells were collected and utilized to evaluate viability, ROS production, gene expression and protein expression levels. The control group consisted of untreated HT29 cells. The viability of the cells was determined using the MTT method. Moreover, Nitro Blue Tetrazolium (NBT) was used to detect the ROS production capacity. The mRNA transcript's level and protein expression were evaluated by Real-time PCR and Western blotting, respectively. RESULTS: The ascorbic acid-treated group showed a significant increase in ROS production and an obvious reduction in viability compared to the control group. The treated group showed significantly increased levels of both early apoptotic markers (Bax, Cyt C, Caspase3, and Caspase 9) and late apoptotic markers (Caspase 8). Bcl2 expression showed significantly decreased levels relative to the control group. Ascorbic acid therapy substantially reduced the expression of bFGF, bFGFR, PDGF, PDGFR and PLC- γ compared to the control group. CONCLUSION: The results confirm that high-dose L-ascorbic acid reduces HT29 cell line viability in vitro.

Silver nanoparticles induce oxidative stress, apoptosis and impaired steroidogenesis in ovarian granulosa cells of cattle.

There have been increased effects of silver nanoparticle (Ag-NPs) on livestock during the past decade, but data related to adverse effects of Ag-NPs on reproductive tissues are limited. In the present study, the possible cytotoxic effects of Ag-NPs on oxidant/antioxidant balance, apoptosis and steroid hormone production in ovarian granulosa cells of cattle were studied.Cultured granulosa cells were treated with 10 nm Ag-NPs at various concentrations (1-100 µg/ml) for 24 h, and cell toxicity, oxidant/antioxidant markers, reactive oxygen species (ROS) production, abundances of apoptotic/antiapoptotic and steroidogenesis related mRNA transcripts were determined. The amount of DNA fragmentation was also determined using the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Results indicated treatment of granulosa cells with Ag-NPs induced an increase of reactive oxygen species production concomitant with increased malondialdehyde concentrations and decreased antioxidant enzyme activities. There was a maximal percentage of TUNEL+ cells (46.6%) after treatment with 50 and 100 µg/ml of Ag-NPs. The Ag-NPs could induce apoptosis in granulosa cells as indicated by the increase in caspase-3 activity, and larger abundance of BCL2 associated X (BAX) and lesser abundance of B-cell lymphoma 2 (BCL2) mRNA transcripts. The abundance of steroidogenic enzyme mRNA transcripts decreased concomitant with suppression of steroid hormone synthesis from Ag-NP-treated cells. Findings indicate silver nanoparticles (SNP) induce apoptosis and oxidative stress and change the pattern of steroid hormone synthesis in granulosa cells of cattle. The results indicate Ag-NPs may inhibit the function and viability of ovarian cells.

Betaine ameliorates impaired steroidogenesis and apoptosis in mice granulosa cells induced by high glucose concentration.

Betaine is a bioactive peptide whose beneficial effects on diabetes complications have been considered, previously. The present study aimed to investigate the possible protective effects of betaine against hyperglycemia-induced steroidogenesis impairment and apoptosis in mice granulosa cells. Ovarian granulosa cells were isolated from C57/BL6 mice and cultured in steroidogenesis medium (SM) containing 30 ng/ml FSH and 0.5 µM testosterone. The cells were cultured in SM containing low (5 mM) or high (30 mM) glucose concentrations for 24 h in the presence or absence of betaine (5 mM). At the end of the experiment, estradiol and progesterone were measured by ELISA in the culture medium. Expression of apoptosis and steroidogenesis associated genes and caspase-3 activity were determined by qRT-PCR and colorimetric assays, respectively. Exposure of mice granulosa cells to high glucose concentration inhibited the steroidogenesis by decreasing estradiol and progesterone secretion and downregulation of steroidogenesis-related genes including 3ßHSD, Cyp11a1, Cyp19a1, and StAR. Betaine treatment could ameliorate the steroidogenesis impairment at molecular and biochemical levels. High glucose concentration also enhanced apoptosis in mice granulosa cells that were characterized by elevation of caspase-3 activity, upregulation of bax gene and downregulation of bcl2 gene. Betaine treatment could attenuate the apoptotic-related changes induced by high glucose concentration in granulosa cells. According to the results of the present study, betaine could ameliorate the adverse effects of hyperglycemia on the physiological function of ovarian granulosa cells. The results highlight the potential role of betaine for the intervention of ovarian dysfunction in diabetic patients. Abbreviations: AABA: Betaine-α-aminobutyric acid; AGEs: Advanced glycation end products; bax: bcl2 Associated X; bcl2: B-cell lymphoma 2; AMPK: AMP-activated protein kinase; BHMT: Betaine homocysteine methyltransferase; C/EBP: CCAAT-enhancer-binding proteins; Cyp11a1: Cholesterol side-chain cleavage cytochrome P450; Cyp19a1: Cytochrome P450 aromatase; DM: Diabetes mellitus; E2: Estradiol; ERS: Endoplasmic reticulum stress; GCs: Granulosa cells; GLUT: Glucose transporter; FSH: Follicle-stimulating hormone; 3ßHSD: 3ß-hydroxysteroid dehydrogenase; IL-1ß: interleukin-1ß; LH: Luteinizing hormone; MDCK: Madin-Darby Canine Kidney cell; MT: Methionine synthase, MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NLRP3: NLR Family Pyrin Domain Containing 3; NF-κB: Nuclear factor κB; P4: Progesterone; ROS: Reactive oxygen species; SGLT: Sodium dependent glucose transporter; SLC7A6: Solute Carrier Family 7 Member 6; StAR: Steroidogenic acute regulatory protein; STZ: Streptozotocin; Tumor necrosis factor α: TNF-α; TXNIP: Thioredoxin interacting protein.