Altered expression of methylenetetrahydrofolate reductase modifies response to methotrexate and 5-fluorouracil in mice

Folates are essential cofactors that are required for the synthesis of nucleotides, the precursors of DNA and RNA. Two widely used anti-metabolite chemotherapeutic drugs, anti-folate methotrexate (MTX) and the pyrimidine antagonist 5-fluorouracil (5-FU), inhibit DNA and RNA synthesis and induce apoptosis, through their effects on the folate pathway. A common polymorphism (677C→T) in methylenetetrahydrofolate reductase (MTHFR), a critical folate-metabolizing enzyme in nucleotide synthesis, may modify the chemosensitivity of MTX and 5-FU.In this thesis, two mouse models (a well-characterized mouse model deficient in MTHFR (Mthfr-/- and Mthfr +/-) and a new mouse model that overexpresses MTHFR (MTHFR-Tg )) were used to investigate the effect of MTHFR expression on response to MTX and 5-FU. The latter mouse model, MTHFR-Tg, was generated and characterized in this thesis. MTHFR-Tg mice had an increase in methionine in brain, a decrease in cysteine in duodenum, an increase in glutathione in liver and decreases in 10-formyltetrahydrofolate levels in liver and duodenum.Mthfr-deficient and MTHFR-Tg mice and their wild-type littermates were injected with MTX or 5-FU (with saline as a control) and assessed for hematological parameters (hematocrit, hemoglobin, red and white blood cell numbers), plasma homocysteine levels, serum nephrotoxicity and hepatotoxicity markers, splenic dUTP/dTTP ratios and apoptosis.MTHFR overexpression increased the effect of MTX on hematopoietic cells, through reduced DNA synthesis and deoxyribonucleotide imbalance (higher dUTP/dTTP ratios)-induced splenic apoptosis, with a protection against MTX-induced hyperhomocysteinemia. MTHFR deficiency had similar effects on MTX-treated hematopoietic cells, through hyperhomocysteinemia-induced splenic apoptosis, with enhanced MTX-induced hyperhomocysteinemia and nephrotoxicity.Furthermore, in transformed mouse embryonic fibroblasts, MTHFR overexpression was protective against MTX-induced apoptosis, possibly through its protective effect against hyperhomocysteinemia.Lastly, MTHFR overexpression enhanced 5-FU-induced leucopoenia and splenic apoptosis while reducing plasma Hcy levels.The major problem with MTX and 5-FU therapies is the variability in therapeutic outcome and toxicity which is the primary reason for discontinuation of therapy. Our studies illustrate the critical effect of MTHFR expression on the chemosensitivity to MTX and 5-FU, and the importance of pharmacogenetic testing for the MTHFR polymorphism and possibly polymorphisms in other folate-metabolizing enzymes to maximize efficacy and minimize toxicity

The effect of ACE2 receptor, IFN-?, and TNF-? polymorphisms on the severity and prognosis of the disease in SARS-CoV-2 infection

To investigate the effect of genetic variations in the angiotensin converting enzyme (ACE), interferon (IFNG) and tumor necrosis factor (TNF-alpha) genes on the severity of coronavirus disease (COVID-19). Between September and December 2021, 33 patients with COVID-19 were included in this prospective study. The patients were classified and compared according to disease severity: mild&moderate (n = 26) vs severe&critical (n = 7). These groups were evaluated to assess possible relationships with ACE, TNF-alpha and IFNG gene variations using univariate and multivariable analyses. The median age of the mild&moderate group was 45.5 (22-73), and that of the severe&critical group was 58 (49-80) years (p = 0.014). Seventeen (65.4%) of the mild&moderate patients and 3 (42.9%) of severe&critical patients were female (p = 0.393). According to results of univariate analysis, the percentage of patients with the c.418-70C>G variant of the ACE gene was significantly higher in the mild&moderate group (p = 0.027). The ACE gene polymorphisms, c.2312C>T, c.3490G>A, c.3801C>T, and c.731A>G, were each only seen in separate patients with critical disease. The following variants were observed more frequently in the mild&moderate group: c.582C>T, c.3836G>A, c.511+66A>G, c.1488-58T>C, c.3281+25C>T, c.1710-90G>C, c.2193A> G, c.3387T>C for ACE; c.115-3delT for IFNG; and c.27C>T for TNF. It can be expected that patients carrying the ACE gene c.418-70C>G variant may present with a mild clinical manifestation of COVID-19. Several genetic polymorphisms may be associated with pathophysiology, as they appear to help predict COVID-19 severity and enable early identification of the patients requiring aggressive treatment.Turkiye Modern Cerrahi Egitim ve Arasxtirma Dernegi; Turkiye Solunum Arasxtirmalari Dernegi [ADF/17]The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This research received support from two organizations(Turkiye Modern Cerrahi Egitim ve Arasxtirma Dernegi, Turkiye Solunum Arasxtirmalari Dernegi (ADF/17)

HDAC Inhibition Overcomes Acute Resistance to MEK Inhibition in BRAF-Mutant Colorectal Cancer by Downregulation of c-FLIPL

PURPOSE: Activating mutations in the BRAF oncogene are found in 8-15% of colorectal cancer (CRC) patients and have been associated with poor survival. In contrast to BRAF mutant (MT) melanoma, inhibition of the MAPK pathway is ineffective in the majority of BRAFMT CRC patients. Therefore, identification of novel therapies for BRAFMT CRC is urgently needed. EXPERIMENTAL DESIGN: BRAFMT and WT CRC models were assessed in vitro and in vivo. Small molecule inhibitors of MEK1/2, MET and HDAC were employed, over-expression and siRNA approaches were applied, and cell death was assessed by flow cytometry, Western blotting, cell viability and caspase activity assays. RESULTS: Increased c-MET-STAT3 signalling was identified as a novel adaptive resistance mechanism to MEK inhibitors (MEKi) in BRAFMT CRC models in vitro and in vivo. Moreover, MEKi treatment resulted in acute increases in transcription of the endogenous caspase-8 inhibitor c-FLIP(L) in BRAFMT cells, but not in BRAFWT cells, and inhibition of STAT3 activity abrogated MEKi-induced c-FLIP(L) expression. In addition, treatment with c-FLIP-specific siRNA or HDAC inhibitors abrogated MEKi-induced upregulation of c-FLIP(L) expression and resulted in significant increases in MEKi-induced cell death in BRAFMT CRC cells. Notably, combined HDAC inhibitor/MEKi treatment resulted in dramatically attenuated tumor growth in BRAFMT xenografts. CONCLUSIONS: Our findings indicate that c-MET/STAT3-dependent upregulation of c-FLIP(L) expression is an important escape mechanism following MEKi treatment in BRAFMT CRC. Thus, combinations of MEKi with inhibitors of c-MET or c-FLIP (eg. HDAC inhibitors) could be potential novel treatment strategies for BRAFMT CRC