1. Involvement of proline oxidase (PutA) in programmed cell death of Xanthomonas
Arun Sharma, Satyendra Gautam, Surbhi Wadhawan PLoS One . 2014 May 1;9(5):e96423. doi: 10.1371/journal.pone.0096423.
Xanthomonas campestris strains have been reported to undergo programmed cell death (PCD) in a protein rich medium. Protein hydrolysates used in media such as nutrient broth comprise of casein digest with abundance of proline and glutamate. In the current study, X. campestris pv. campestris (Xcc) cells displayed PCD when grown in PCD inducing medium (PIM) containing casein tryptic digest. This PCD was also observed in PCD non-inducing carbohydrate rich medium (PNIM) fortified with either proline or proline along with glutamate. Surprisingly, no PCD was noticed in PNIM fortified with glutamate alone. Differential role of proline or glutamate in inducing PCD in Xcc cells growing in PNIM was studied. It was found that an intermediate product of this oxidation was involved in initiation of PCD. Proline oxidase also called as proline utilization A (PutA), catalyzes the two step oxidation of proline to glutamate. Interestingly, higher PutA activity was noticed in cells growing in PIM, and PCD was found to be inhibited by tetrahydro-2-furoic acid, a competitive inhibitor of this enzyme. Further, PCD was abolished in Xcc ΔputA strain generated using a pKNOCK suicide plasmid, and restored in Xcc ΔputA strain carrying functional PutA in a plasmid vector. Xanthomonas cells growing in PIM also displayed increased generation of ROS, as well as cell filamentation (a probable indication of SOS response). These filamented cells also displayed enhanced caspase-3-like activity during in situ labeling using a fluorescent tagged caspase-3 inhibitor (FITC-DEVD-FMK). The extent of PCD associated markers such as DNA damage, phosphatidylserine externalization and membrane depolarization were found to be significantly enhanced in wild type cells, but drastically reduced in Xcc ΔputA cells. These findings thus establish the role of PutA mediated proline oxidation in regulating death in stressed Xanthomonas cells.
2. E series prostaglandins alter the proliferative, apoptotic and migratory properties of T98G human glioma cells in vitro
Renata N Gomes, Alison Colquhoun Lipids Health Dis . 2012 Dec 11;11:171. doi: 10.1186/1476-511X-11-171.
Background:In many types of cancer, prostaglandin E2 (PGE2) is associated with tumour related processes including proliferation, migration, angiogenesis and apoptosis. However in gliomas the role of this prostanoid is poorly understood. Here, we report on the proliferative, migratory, and apoptotic effects of PGE(1), PGE(2) and Ibuprofen (IBP) observed in the T98G human glioma cell line in vitro.Methods:T98G human glioma cells were treated with IBP, PGE(1) or PGE(2) at varying concentrations for 24-72 hours. Cell proliferation, mitotic index and apoptotic index were determined for each treatment. Caspase-9 and caspase-3 activity was measured using fluorescent probes in live cells (FITC-LEHD-FMK and FITC-DEVD-FMK respectively). The migratory capacity of the cells was quantified using a scratch migration assay and a transwell migration assay.Results:A significant decrease was seen in cell number (54%) in the presence of 50 μM IBP. Mitotic index and bromodeoxyuridine (BrdU) incorporation were also decreased 57% and 65%, respectively, by IBP. The apoptotic index was increased (167%) and the in situ activity of caspase-9 and caspase-3 was evident in IBP treated cells. The inhibition of COX activity by IBP also caused a significant inhibition of cell migration in the monolayer scratch assay (74%) and the transwell migration assay (36%). In contrast, the presence of exogenous PGE(1) or PGE(2) caused significant increases in cell number (37% PGE(1) and 45% PGE(2)). When mitotic index was measured no change was found for either PG treatment. However, the BrdU incorporation rate was significantly increased by PGE(1) (62%) and to a greater extent by PGE(2) (100%). The apoptotic index was unchanged by exogenous PGs. The addition of exogenous PGs caused an increase in cell migration in the monolayer scratch assay (43% PGE(1) and 44% PGE(2)) and the transwell migration assay (28% PGE(1) and 68% PGE(2)).Conclusions:The present study demonstrated that treatments which alter PGE(1) and PGE(2) metabolism influence the proliferative and apoptotic indices of T98G glioma cells. The migratory capacity of the cells was also significantly affected by the change in prostaglandin metabolism. Modifying PG metabolism remains an interesting target for future studies in gliomas.
3. Rutamarin, an Active Constituent from Ruta angustifolia Pers., Induced Apoptotic Cell Death in the HT29 Colon Adenocarcinoma Cell Line
Sok Lai Hong, Sri Nurestri Abdul Malek, Shafinah Ahmad Suhaimi Pharmacogn Mag . 2017 Jul;13(Suppl 2):S179-S188. doi: 10.4103/pm.pm_432_16.
Background:Ruta angustifoliaPers. is a perennial herb that is cultivated worldwide, including Southeast Asia, for the treatment of various diseases as traditional medicine.Objective:The purpose of the study was to identify an active principle ofR. angustifoliaand to investigate its effect on the HT29 cell death.Materials and methods:The methanol and fractionated extracts (hexane, chloroform, ethyl acetate, and water) ofR. angustifoliaPers. were initially investigated for their cytotoxic activity against two human carcinoma cell lines (MCF7 and HT29) and a normal human colon fibroblast cell line (CCD-18Co) using sulforhodamine B cytotoxicity assay. Eight compounds including rutamarin were isolated from the active chloroform extract and evaluated for their cytotoxic activity against HT29 human colon carcinoma cell line and CCD-18Co noncancer cells. Further studies on the induction of apoptosis such as morphological examinations, biochemical analyses, cell cycle analysis, and caspase activation assay were conducted in rutamarin-treated HT29 cells.Results:Rutamarin exhibited remarkable cytotoxic activity against HT29 cells (IC50value of 5.6 μM) but was not toxic to CCD-18Co cells. The morphological and biochemical hallmarks of apoptosis including activation of caspases 3, 8, and 9 were observed in rutamarin-treated HT29 cells. These may be associated with cell cycle arrest at the G0/G1 and G2/M checkpoints, which was also observed in HT29 cells.Conclusions:The present study describes rutamarin-induced apoptosis in the HT29 cell line for the first time and suggests that rutamarin has the potential to be developed as an anticancer agent.Summary:Rutamarin was cytotoxic to HT29 colon cancer cells but exerted no damage to normal colon cellsRutamarin induced morphological and biochemical hallmarks of apoptosis in HT29 cellsRutamarin induced cell cycle arrest at the G0/G1 and G2/M checkpoints in a dose-dependent manner in HT29 cellsRutamarin activated caspases 3, 8, and 9 in a dose-dependent manner in HT29 cells.Abbreviations used:ACN: Acetonitrile, ANOVA: One-way analysis of variance, BrdU: Bromodeoxyuridine, 13C-NMR: Carbon-13 Nuclear magnetic resonance, CAD: Caspase-activated endonuclease, CCD-18Co: Human colon normal, DLD1: Human Duke's type C colorectal adenocarcinoma, DMRT: Duncan's multiple range test, DMSO: Dimethyl sulfoxide, DNA: Deoxyribonucleic acid, DR4/5: Death receptor 4/5 protein, EMEM: Eagle's minimum essential media, FBS: Fetal bovine serum, FITC Annexin V: Annexin V conjugated with fluorescein isothiocyanate, FITC-DEVD-FMK: Fluorescein isothiocyanate conjugate of caspase inhibitor Asp-Glu-Val-Asp-fluoromethyl ketone, FITC-IETD-FMK: Fluorescein isothiocyanate conjugate of caspase inhibitor Ile-Glu-Thr-Asp-fluoromethyl ketone, FITC-LEHD-FMK: Fluorescein isothiocyanate conjugate of caspase inhibitor Leu-Glu-His-Asp-fluoromethyl ketone, G0: Quiescent phase of cell cycle, G1: Gap 1 phase of cell cycle, G2: Gap 2 phase of cell cycle, GC-MS: Gas chromatography-mass spectrometry, HeLa: Human cervical adenocarcinoma, HPLC: High performance liquid chromatography, HT29: Human colon adenocarcinoma, Huh7.5: Human hepatocellular carcinoma, IC50: Half maximal inhibitory concentration, KSHV: Kaposi's sarcoma-associated herpesvirus, M phase: Mitotic phase of cell cycle, MCF7: Human breast adenocarcinoma, NMR: Nuclear magnetic resonance, PBS: Phosphate-buffered saline, PI: Propidium iodide, RNase: Ribonuclease, rt: Retention time, S phase: Synthesis phase of cell cycle, SD: Standard deviation, SRB: Sulforhodamine B, TCA: Trichloroacetic acid, TLC: Thin layer chromatography, TNF-R1: Tumor necrosis factor receptor 1 protein, TUNEL: Terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling, UV: Ultraviolet.
