Rescue of Methionine Dependence by Cobalamin in a Human Colorectal Cancer Cell Line.

Methionine dependence is a characteristic of most cancer cells where they are unable to proliferate when the essential amino acid methionine is replaced with its precursor homocysteine in the growing media. Normal cells, on the other hand, thrive under these conditions and are referred to as methionine-independent. The reaction that adds a methyl group from 5-methyltetrahydrofolate to homocysteine to regenerate methionine is catalyzed by the enzyme methionine synthase with the cofactor cobalamin (vitamin B12). However, decades of research have shown that methionine dependence in cancer is not due to a defect in the activity of methionine synthase. Cobalamin metabolism has been tied to the dependent phenotype in rare cell lines. We have identified a human colorectal cancer cell line in which the cells regain the ability to proliferation in methionine-free, L-homocystine-supplemented media when cyanocobalamin is supplemented at a level of 1 µg/mL. In human SW48 cells, methionine replacement with L-homocystine does not induce any measurable increase in apoptosis or reactive oxygen species production in this cell line. Rather, proliferation is halted, then restored in the presence of cyanocobalamin. Our data show that supplementation with cyanocobalamin prevents the activation of the integrated stress response (ISR) in methionine-deprived media in this cell line. The ISR-associated cell cycle arrest, characteristic of methionine-dependence in cancer, is also prevented, leading to the continuation of proliferation in methionine-deprived SW48 cells with cobalamin. Our results highlight differences between cancer cell lines in the response to cobalamin supplementation in the context of methionine dependence.

Evaluation of Ulcer Protective Activity of Morus alba L. Extract-Loaded Chitosan Microspheres in Ethanol-Induced Ulcer in Rat Model.

Due to an unhealthy lifestyle, gastric ulcers have become a very common disease these days. Moreover, the side effects linked with the prolonged use of conventional treatments have shifted the paradigm towards herbal therapies. The leaves of Morus alba L. (Family-Moraceae) have been traditionally used for a large number of metabolic diseases. In the present research, we focused on the development of chitosan microspheres using extracts of leaves of Morus alba L. and their evaluation for gastroprotective efficacy against ethanol-induced ulcers in experimental rats. The process of development of M. alba extract microsphere (MEM) is also optimized using the Box-Behnken design. The formulation was prepared at optimized conditions (chitosan concentration (1.66% w/w), volume of glutaraldehyde (4.69 mL), and stirrer rotation per minute, RPM, 854.8), and the percentage yield (Y 1) of the resulted microspheres is ∼95% with an encapsulation efficiency (EE) of (Y 2(rutin)) ∼86%, Y 2(quercetin)) ∼85%, and particle size (Y 3) of ∼40 µm. The MEM prepared at optimized conditions can also be characterized for various parameters to ensure the uniformity of parameters. Also, the drug release studies indicated that the percentage release of rutin and quercetin from MEM was enhanced as compared to M. alba extract (ME) alone. Furthermore, in vivo analysis of the antiulcer potential of pretreatment with ME and MEM (500 mg/kg p.o.) in rats indicated that mucosal lesions, gastric juice volume, and total acidity were significantly altered as compared to ethanol-treated animals. Histopathology of tissue sections also confirmed the protection of gastric mucosa on pretreatment with MEM at 500 mg/kg p.o. On the basis of these findings, we can conclude that prepared microspheres can be used to develop a sustained release formulation of extract for the management of gastric ulcers. However, additional research is needed to establish the specific mechanisms of M. alba's antiulcer efficacy.

Methionine dietary supplementation potentiates ionizing radiation-induced gastrointestinal syndrome.

Methionine is an essential amino acid needed for a variety of processes in living organisms. Ionizing radiation depletes tissue methionine concentrations and leads to the loss of DNA methylation and decreased synthesis of glutathione. In this study, we aimed to investigate the effects of methionine dietary supplementation in CBA/CaJ mice after exposure to doses ranging from 3 to 8.5 Gy of 137Cs of total body irradiation. We report that mice fed a methionine-supplemented diet (MSD; 19.5 vs. 6.5 mg/kg in a methionine-adequate diet, MAD) developed acute radiation toxicity at doses as low as 3 Gy. Partial body irradiation performed with hindlimb shielding resulted in a 50% mortality rate in MSD-fed mice exposed to 8.5 Gy, suggesting prevalence of radiation-induced gastrointestinal syndrome in the development of acute radiation toxicity. Analysis of the intestinal microbiome demonstrated shifts in the gut ecology, observed along with the development of leaky gut syndrome and bacterial translocation into the liver. Normal gut physiology impairment was facilitated by alterations in the one-carbon metabolism pathway and was exhibited as decreases in circulating citrulline levels mirrored by decreased intestinal mucosal surface area and the number of surviving crypts. In conclusion, we demonstrate that a relevant excess of methionine dietary intake exacerbates the detrimental effects of exposure to ionizing radiation in the small intestine.NEW & NOTEWORTHY Methionine supplementation, instead of an anticipated health-promoting effect, sensitizes mice to gastrointestinal radiation syndrome. Mechanistically, excess of methionine negatively affects intestinal ecology, leading to a cascade of physiological, biochemical, and molecular alterations that impair normal gut response to a clinically relevant genotoxic stressor. These findings speak toward increasing the role of registered dietitians during cancer therapy and the necessity of a solid scientific background behind the sales of dietary supplements and claims regarding their benefits.

Characterization of transgenic Gfrp knock-in mice: implications for tetrahydrobiopterin in modulation of normal tissue radiation responses.

AIMS: The free radical scavenger and nitric oxide synthase cofactor, 5,6,7,8-tetrahydrobiopterin (BH4), plays a well-documented role in many disorders associated with oxidative stress, including normal tissue radiation responses. Radiation exposure is associated with decreased BH4 levels, while BH4 supplementation attenuates aspects of radiation toxicity. The endogenous synthesis of BH4 is catalyzed by the enzyme guanosine triphosphate cyclohydrolase I (GTPCH1), which is regulated by the inhibitory GTP cyclohydrolase I feedback regulatory protein (GFRP). We here report and characterize a novel, Cre-Lox-driven, transgenic mouse model that overexpresses Gfrp. RESULTS: Compared to control littermates, transgenic mice exhibited high transgene copy numbers, increased Gfrp mRNA and GFRP expression, enhanced GFRP-GTPCH1 interaction, reduced BH4 levels, and low glutathione (GSH) levels and differential mitochondrial bioenergetic profiles. After exposure to total body irradiation, transgenic mice showed decreased BH4/7,8-dihydrobiopterin ratios, increased vascular oxidative stress, and reduced white blood cell counts compared with controls. INNOVATION AND CONCLUSION: This novel Gfrp knock-in transgenic mouse model allows elucidation of the role of GFRP in the regulation of BH4 biosynthesis. This model is a valuable tool to study the involvement of BH4 in whole body and tissue-specific radiation responses and other conditions associated with oxidative stress.