Dihydrofolate Reductase Expression Research Articles

Taurine and enzymatically modified isoquercitrin protected against methotrexate-induced deteriorations in the conductivity and rhythmicity of the heart in rats: Antioxidant, anti-inflammatory, and histological architecture approach.

Cardiotoxicity is one of the most devastating complications of cancer treatment by methotrexate (MTX). The present study aimed to investigate the potential anti-cardiotoxic efficacy of taurine (Tau) and enzymatically modified isoquercitrin (EMIQ) alone or combined against MTX-induced cardiotoxicity in adult male rats. A total of 36 rats were randomly divided into six groups (six animals each): control, MTX (a single i.p. dose of 20 mg/kg), EMIQ + MTX (26 mg/kg of EMIQ, p.o. for 16 days), Tau + MTX (500 mg/kg of Tau, p.o. for 16 days), EMIQ + Tau + MTX at the same previous doses, and (EMIQ + Tau)½+ MTX. MTX reduced the percentage of body weight change, the expression of dihydrofolate reductase (DHFR) and folypolyglutamyl synthetase (FPGS), the cleaved tumor necrosis factor alpha (TNF-α) level in the cardiac tissue, and the elevated serum TNF-α level. MTX extensively deteriorated the electrocardiography (ECG), inducing tachycardia with shortening of the time intervals between successive heartbeats (R-R interval), associated with elongation of ventricular depolarization (QRS interval), and the corrected total time for ventricular de- and repolarization (QTc) duration. Treatment with MTX resulted in a significant reduction in atrial depolarization (P amplitude) and rapid repolarization (T amplitude) and a significant elevation in plateau phase (ST height). MTX treatment resulted in swelling of cardiomyocytes with extensive vacuolization of sarcoplasm with numerous variably sized vacuoles in addition to apoptotic cells. Tau and EMIQ protected against MTX-induced deteriorations in the conductivity and rhythmicity of the heart through antioxidative, anti-inflammatory, and antiapoptotic activities. Treatment with tau and EMIQ combined at high or low doses offered superior protection to the heart than using each agent alone.

Dysregulation of hepatic one-carbon metabolism in classical homocystinuria: Implications of redox-sensitive DHFR repression and tetrahydrofolate depletion for pathogenesis and treatment.

Cystathionine beta-synthase-deficient homocystinuria (HCU) is a life-threatening disorder of sulfur metabolism. HCU can be treated by using betaine to lower tissue and plasma levels of homocysteine (Hcy). Here, we show that mice with severely elevated Hcy and potentially deficient in the folate species tetrahydrofolate (THF) exhibit a very limited response to betaine indicating that THF plays a critical role in treatment efficacy. Analysis of a mouse model of HCU revealed a 10-fold increase in hepatic levels of 5-methyl -THF and a 30-fold accumulation of formiminoglutamic acid, consistent with a paucity of THF. Neither of these metabolite accumulations were reversed or ameliorated by betaine treatment. Hepatic expression of the THF-generating enzyme dihydrofolate reductase (DHFR) was significantly repressed in HCU mice and expression was not increased by betaine treatment but appears to be sensitive to cellular redox status. Expression of the DHFR reaction partner thymidylate synthase was also repressed and metabolomic analysis detected widespread alteration of hepatic histidine and glutamine metabolism. Many individuals with HCU exhibit endothelial dysfunction. DHFR plays a key role in nitric oxide (NO) generation due to its role in regenerating oxidized tetrahydrobiopterin, and we observed a significant decrease in plasma NOx (NO2 + NO3) levels in HCU mice. Additional impairment of NO generation may also come from the HCU-mediated induction of the 20-hydroxyeicosatetraenoic acid generating cytochrome CYP4A. Collectively, our data shows that HCU induces dysfunctional one-carbon metabolism with the potential to both impair betaine treatment and contribute to multiple aspects of pathogenesis in this disease.

Reduced Malignancy of Super Methotrexate-resistant Osteosarcoma Cells With Dihydrofolate Reductase Amplification Despite Paradoxical Gain of Oncogenic PI3K/AKT/mTOR and c-MYC expression.

Methotrexate (MTX) resistance in osteosarcoma leads to a very poor prognosis. In the present study, in order to further understand the basis and ramifications of MTX resistance in osteosarcoma, we selected an osteosarcoma cell line that has a 5,500-fold-increased MTX IC50 Materials and Methods: The super MTX-resistant 143B osteosarcoma cells (143B-MTXSR) were selected from MTX-sensitive parental human 143B osteosarcoma cells (143B-P) by continuous culture with step-wise increased amounts of MTX. To compare the malignancy of 143B-MTXSR and 143B-P, colony-formation capacity was compared with clonogenic assays on plastic and in soft agar. In addition, tumor growth was compared with orthotopic xenograft mouse models of osteosarcoma. Expression of dihydrofolate reductase (DHFR), phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR), and myelocytomatosis oncogene (MYC) was examined with western immunoblotting and compared in 143B-MTXSR and 143B-P cells. 143B-MTXSR had a 5,500-fold increase in the MTX IC50 compared to the parental 143B-P cells. Expression of DHFR was increased 10-fold in 143B-MTXSR compared to 143B-P (p<0.01). 143B-MTXSR cells had reduced colony-formation capacity on plastic (p=0.032) and in soft agar (p<0.01) compared to 143B-P and reduced tumor growth in orthotopic xenograft mouse models (p<0.001). These results demonstrate that 143B-MTXSR had reduced malignancy. 143B-MTXSR also showed an increased expression of PI3K (p<0.01), phosphorylated (activated) AKT (p=0.031), phosphorylated mTOR (p=0.043), and c-MYC (p=0.024) compared to 143B-P. The present study demonstrates that the increased expression of DHFR, PI3K/AKT/mTOR and c-MYC appears to be linked to super MTX resistance and, paradoxically, to reduced malignancy. The present results suggest that DHFR may be a powerful tumor suppressor when highly amplified.

Abstract LB404: ATR inhibitors synergize with DHFR inhibitors in rhabdomyosarcoma cells by disrupting DNA damage checkpoint

Abstract Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children and adolescents. RMS tumors are classified into four subtypes: embryonal, alveolar, spindle cell/sclerosing, and pleomorphic. Treatment options for RMS include a combination of surgery, chemotherapy, and radiation therapy and regimens for patients with relapsed and metastatic RMS remain limited. However, a recent study has reported that pharmacological inhibitors targeting ataxia telangiectasia and Rad3-related protein (ATR) are currently undergoing phase I/II trials and may be utilized for the treatment of alveolar RMS. Interestingly, we identified that the gene signature of replication stress positively correlated with dihydrofolate reductase (DHFR) expression in the cancer cell line database (CellMinerCDB, discover.nci.nih.gov/cellminercdb; Pearson correlation (r)=0.66). DHFR is an important therapeutic target for cancer and autoimmune diseases, and its inhibitors are routinely used and clinically approved. DHFR inhibitors are known to exhibit their anticancer effects by inhibiting nucleotide synthesis and DNA replication. In this study, we aimed to explore a potential synergistic interaction using a combination treatment of ATR inhibitors (elimusertib and ceralasertib) and DHFR inhibitors (pralatrexate, methotrexate, and raltitrexated) in multiple RMS cell lines. We used a drug matrix reduction format and found that combined treatment of ATR inhibitors and DHFR inhibitors induced a synergistically decreased RMS cell viability, showing higher synergy in fusion-positive RMS cells than in fusion-negative RMS cells. Mechanistically, ATR inhibitors abrogated DNA damage response checkpoint CHK1 activation caused by DHFR inhibitor, leading to increased γH2AX and decreased DNA replication activity. Accordingly, the accumulation of DNA damage in combination with ATR inhibitor and DHFR inhibitor triggered higher cell cycle arrest and apoptotic cell death than single agent treatment of DHFR inhibitor. Our findings warrant further research into identifying genomic regions where the DNA damage is enriched in combination treatment by using END-sequencing and synthesis-associated with repair sequencing (SAR-seq). In conclusion, this study provides promising rationale for the combination treatment of ATR and DHFR inhibitors to treat RMS patients, including those with advanced, relapsed, and metastatic states. Citation Format: Ukhyun Jo, George K Annor, Jack F. Shern. ATR inhibitors synergize with DHFR inhibitors in rhabdomyosarcoma cells by disrupting DNA damage checkpoint [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB404.

Astaxanthin Co-treatment with Low Dose Methotrexate Increases the Cell Cycle Arrest and Ameliorates the Methotrexate-induced Inflammatory Response in NALM-6.

Methotrexate (MTX), an antimetabolite agent, is widely used for acute lymphoblastic leukemia treatment, despite its association with significant organ dysfunction. Astaxanthin (AST) is a natural carotenoid which has recently been emerged as a promising anti-tumor and anti-inflammatory agent. In this study, we aimed to evaluate the effectiveness of astaxanthin and low-dose methotrexate co-treatment in acute lymphoblastic leukemia cell line. The expression of Dihydrofolate reductase (DHFR), Thymidylate synthase (TYMS), apoptotic, anti-apoptotic as well as inflammatory genes was investigated using qRT-PCR. Flow cytometry was performed for cell cycle quantitative evaluation. Clonogenic assay was used to assess NALM6 cells proliferation capacity following treatment with AST, MTX, and co-treatment. To compare the antioxidant property of each group, the ferric ion reducing anti-oxidant power assay was performed. A reduction in viability was observed in the presence of MTX, AST, and their combined treatment. Both AST alone and in combination with MTX caused cell cycle arrest and a reduction in the expression of DHFR and TYMS. While MTX, AST, and their combination could reduce STAT3 and BCL-XL gene expression, they could act as positive regulators for the expression of BAX and CASP3, TNFα, and IL6. AST and MTX co-treatment inhibited the colony formation ability. FRAP assay also revealed that AST and AST+MTX increased the antioxidant capacity. Our data suggests that AST can improve MTX treatment efficacy and their combination therapy can be considered as a promising strategy for the management of acute lymphoblastic leukemia.

Novel insights into the ecDNA formation mechanism involving MSH3 in methotrexate‑resistant human colorectal cancer cells.

Extrachromosomal DNAs (ecDNAs), also known as double minutes (DMs), can induce a fast increase in gene copy numbers and promote the development of cancer, including drug resistance. MutS homolog 3 (MSH3), a key protein in mismatch repair, has been indicated to participate in the regulation of DNA double‑strand break (DSB) repair, which has been reported to be associated with the formation of ecDNAs. However, it remains unclear whether MSH3 can influence drug resistance via ecDNAs in cancer. In the present study, high MSH3 expression was observed in methotrexate (MTX)‑resistant HT29 cells [DM‑ and homogeneously staining region (HSR)‑containing cells] compared with parental HT29 cells. Additionally, decreased amounts of ecDNAs, HSRs and amplified genes locating on ecDNAs and HSRs were detected following depletion of MSH3 and this could be reversed by overexpressing MSH3 in DM‑containing cells. No corresponding changes were found in HSR‑containing cells. The present study further verified the involvement of MSH3‑regulated DNA DSB repair pathways in the formation of ecDNAs by detecting the expression of core proteins and pathway activity. Furthermore, expulsion of ecDNAs/HSRs was detected and increased frequencies of micronuclei/nuclear buds with dihydrofolate reductase (DHFR) signals were observed in MSH3‑depleted DM‑containing cells. Finally, changes in MSH3 expression could affect DHFR amplification‑derived DHFR expression and cell sensitivity to MTX, suggesting that MSH3 may influence cancer drug resistance by altering the amount of ecDNAs. In conclusion, the present study revealed a novel mechanism involving MSH3 in the regulation of ecDNAs by DSB repair, which will have clinical value in the treatment of ecDNA‑based drug resistance in cancer.

Bioinformatics and Experimental Identification of circ_0001535 Associated with Diagnosis and Development of Alzheimer's Disease.

Alzheimer's disease (AD) is a type of disease frequently occurs in the elderly population. Diagnosis and treatment methods for this disease are still lacking, and more research is required. In addition, little is known about the function of the circular RNAs (circRNAs) in AD. In this research, RNA expression data of AD from the Gene Expression Omnibus (GEO) database were downloaded. The expression levels of circRNAs in cerebrospinal fluid samples of healthy participants and AD patients were measured by reverse transcription‑quantitative PCR (RT-qPCR). The diagnosed value of differential expressed circRNAs was analyzed with the Receiver operating characteristic curve (ROC). Pathways related to circ_0001535 were found using gene set enrichment analysis (GSEA) and Metascape. The direct interactions between circ_0001535 and E2F transcription factor 1 (E2F1) or E2F1 and dihydrofolate reductase (DHFR) were verified using Chromatin immunoprecipitation (ChIP) and RNA Binding Protein Immunoprecipitation (RIP) assays. Cell Counting Kit-8 (CCK8) and flow cytometry were used to identify the function of circ_0001535/E2F1/DHFR axis on the proliferation and apoptosis of AD cells. In total, 12 circRNAs have been linked to AD diagnosis. The expression levels of 7 circRNAs differed between AD patients and control groups. Circ_0001535 had the most diagnose value among these circRNAs. Hence, circ_0001535 was regarded as a key circRNA in the present study. E2F1/DHFR axis was predicted to be regulated by circ_0001535. In addition, IP assays experiment results showed that E2F1 could bind to the promoter region of DHFR and be regulated by circ_0001535. In vitro results showed that circ_0001535 overexpression could promote DHFR expression, while E2F1 knock down could inhibit DHFR expression in SH-SY5Y cells. Finally, rescue experiments suggested that circ_0001535 could reduce Aβ25-35-induced SH-SY5Y cell proliferation and facilitate apoptosis through E2F1/DHFR axis. Our research in AD circRNA can offer important information regarding the role of specific circRNAs in the AD environment and point to specific future areas of therapeutic intervention in AD.

Gallic Acid Restores the Sulfonamide Sensitivity of Multidrug-Resistant Streptococcus suis via Polypharmaceology Mechanism

Due to the large amount of antibiotics used for human therapy, agriculture, and even aquaculture, the emergence of multidrug-resistant Streptococcus suis (S. suis) led to serious public health threats. Antibiotic-assisted strategies have emerged as a promising approach to alleviate this crisis. Here, the polyphenolic compound gallic acid was found to enhance sulfonamides against multidrug-resistant S. suis. Mechanistic analysis revealed that gallic acid effectively disrupts the integrity and function of the cytoplasmic membrane by dissipating the proton motive force of bacteria. Moreover, we found that gallic acid regulates the expression of dihydrofolate reductase, which in turn inhibits tetrahydrofolate synthesis. As a result of polypharmacology, gallic acid can fully restore sulfadiazine sodium activity in the animal infection model without any drug resistances. Our findings provide an insightful view into the threats of antibiotic resistance. It could become a promising strategy to resolve this crisis.

Phosphorylation of Thymidylate Synthase and Dihydrofolate Reductase in Cancer Cells and the Effect of CK2α Silencing.

Our previous research suggests an important regulatory role of CK2-mediated phosphorylation of enzymes involved in the thymidylate biosynthesis cycle, i.e., thymidylate synthase (TS), dihydrofolate reductase (DHFR), and serine hydroxymethyltransferase (SHMT). The aim of this study was to show whether silencing of the CK2α gene affects TS and DHFR expression in A-549 cells. Additionally, we attempted to identify the endogenous kinases that phosphorylate TS and DHFR in CCRF-CEM and A-549 cells. We used immunodetection, immunofluorescence/confocal analyses, reverse transcription-quantitative polymerase chain reaction (RT-qPCR), in-gel kinase assay, and mass spectrometry analysis. Our results demonstrate that silencing of the CK2α gene in lung adenocarcinoma cells significantly increases both TS and DHFR expression and affects their cellular distribution. Additionally, we show for the first time that both TS and DHFR are very likely phosphorylated by endogenous CK2 in two types of cancer cells, i.e., acute lymphoblastic leukaemia and lung adenocarcinoma. Moreover, our studies indicate that DHFR is phosphorylated intracellularly by CK2 to a greater extent in leukaemia cells than in lung adenocarcinoma cells. Interestingly, in-gel kinase assay results indicate that the CK2α' isoform was more active than the CK2α subunit. Our results confirm the previous studies concerning the physiological relevance of CK2-mediated phosphorylation of TS and DHFR.

Procyanidin B2 ameliorates endothelial dysfunction induced by nicotine via the induction of tetrahydrobiopterin synthesis

• Nicotine decreases the expression of DHFR and GCH1 to inhibit BH 4 and NO production. • PCB2 prevents nicotine-induced DHFR and GCH1 loss. • PCB2 improves endothelium-dependent vasorelaxation impaired by nicotine. • PCB2 induces vasorelaxation through a PPARδ-BH 4 dependent manner. Smoking is an independent risk factor for the cardiovascular diseases. Nicotine, a major component of tobacco, is responsible for the impaired endothelial-dependent vasorelaxation in smokers. Procyanidin B2 (PCB2), a natural flavonoid, has been reported to possess several potential beneficial effects on the cardiovascular system. However, whether PCB2 prevents nicotine-induced endothelial dysfunction remains unknown. In this study, we demonstrated that PCB2 improved nicotine-impaired endothelium-dependent vasorelaxation in mouse thoracic aortas. Mechanistically, PCB2 increased the expression levels of dihydrofolate reductase (DHFR) and GTP cyclohydrolase 1 (GCH1), two important enzymes in the synthesis of tetrahydrobiopterin (BH 4 ), in mouse aortas and cultured ECs. GSK0660, a selective antagonist of peroxisome proliferator-activated receptor δ (PPARδ), abolished the PCB2-induced effects on BH 4 synthesis, nitric oxide (NO) production and vasodilation. Collectively, we demonstrated that PCB2 mitigates nicotine-induced endothelial dysfunction through a PPARδ-BH 4 dependent manner, thereby suggesting a novel role of PCB2 in preventing vascular dysfunction caused by nicotine exposure.

Palbociclib regulates the expression of dihydrofolate reductase and the cell cycle to inhibit t (11;14) multiple myeloma.

BackgroundMultiple myeloma (MM) with t (11;14) has a unique biology. New combinations of novel agents are needed to improve the prognosis of patients with t (11;14) MM. As a selective inhibitor of cyclin-dependent kinases 4 and 6 (CDK4/6), palbociclib (PAL) has been used to treat various malignancies, including breast cancer, ovarian cancer, and so on. However, the effects of PAL on MM remain unclear, and thus, further investigations are warranted.MethodsTwo t (11;14) MM cell lines, U266 and KMS27, were used in this study. The cell viability and cell cycle were detected to evaluate the anti-myeloma effect of the combination of pralatrexate (PTX) and methotrexate (MTX) with PAL. Three-dimensional (3D) spheroid and mouse models were built to verify the effect of the combination treatment. Western blot was used to detect the expressions of Minichromosome Maintenance Complex Component 2 (MCM2), Minichromosome Maintenance Complex Component 3 (MCM3), and dihydrofolate reductase (DHFR).ResultsIt was observed that PAL had obvious anticancer effects on U266 and KMS27 cells, which had synergistic effects together with PTX and MTX. Importantly, it was found that PAL could promote cell cycle genes including MCM2 and MCM3, and increase the number of MM cells in the G1 phase. Moreover, PAL reduced the expression level of DHFR and exerted its anticancer effects on MM cells by targeting DHFR. It was also determined that PAL exerted anticancer effects on MM in the spheroid and mouse models.ConclusionsPAL exerted obvious anticancer effects on t (11;14) MM cells, which had synergistic effects together with PTX and MTX. PAL could promote cell cycle genes and the G1 phase of MM cells. Moreover, PAL reduced the expression level of DHFR and exerted its anticancer effects on t (11;14) MM cells by targeting DHFR.

An Approach to Derive Functional Peptide Inhibitors of Transcription Factor Activity.

We report the development of a high-throughput, intracellular “transcription block survival” (TBS) screening platform to derive functional transcription factor antagonists. TBS is demonstrated using the oncogenic transcriptional regulator cJun, with the development of antagonists that bind cJun and prevent both dimerization and, more importantly, DNA binding remaining a primary challenge. In TBS, cognate TRE sites are introduced into the coding region of the essential gene, dihydrofolate reductase (DHFR). Introduction of cJun leads to TRE binding, preventing DHFR expression by directly blocking RNA polymerase gene transcription to abrogate cell proliferation. Peptide library screening identified a sequence that both binds cJun and antagonizes function by preventing DNA binding, as demonstrated by restored cell viability and subsequent in vitro hit validation. TBS is an entirely tag-free genotype-to-phenotype approach, selecting desirable attributes such as high solubility, target specificity, and low toxicity within a complex cellular environment. TBS facilitates rapid library screening to accelerate the identification of therapeutically valuable sequences.

CRIF1 Deficiency Increased Homocysteine Production by Disrupting Dihydrofolate Reductase Expression in Vascular Endothelial Cells.

Elevated plasma homocysteine levels can induce vascular endothelial dysfunction; however, the mechanisms regulating homocysteine metabolism in impaired endothelial cells are currently unclear. In this study, we deleted the essential mitoribosomal gene CR6 interacting factor 1 (CRIF1) in human umbilical vein endothelial cells (HUVECs) and mice to induce endothelial cell dysfunction; then, we monitored homocysteine accumulation. We found that CRIF1 downregulation caused significant increases in intracellular and plasma concentrations of homocysteine, which were associated with decreased levels of folate cycle intermediates such as 5-methyltetrahydrofolate (MTHF) and tetrahydrofolate (THF). Moreover, dihydrofolate reductase (DHFR), a key enzyme in folate-mediated metabolism, exhibited impaired activity and decreased protein expression in CRIF1 knockdown endothelial cells. Supplementation with folic acid did not restore DHFR expression levels or MTHF and homocysteine concentrations in endothelial cells with a CRIF1 deletion or DHFR knockdown. However, the overexpression of DHFR in CRIF1 knockdown endothelial cells resulted in decreased accumulation of homocysteine. Taken together, our findings suggest that CRIF1-deleted endothelial cells accumulated more homocysteine, compared with control cells; this was primarily mediated by the disruption of DHFR expression.

Characterization of newly established Pralatrexate-resistant cell lines and the mechanisms of resistance

BackgroundPralatrexate (PDX) is a novel antifolate approved for the treatment of patients with relapsed/refractory peripheral T-cell lymphoma, but some patients exhibit intrinsic resistance or develop acquired resistance. Here, we evaluated the mechanisms underlying acquired resistance to PDX and explored potential therapeutic strategies to overcome PDX resistance.MethodsTo investigate PDX resistance, we established two PDX-resistant T-lymphoblastic leukemia cell lines (CEM and MOLT4) through continuous exposure to increasing doses of PDX. The resistance mechanisms were evaluated by measuring PDX uptake, apoptosis induction and folate metabolism-related protein expression. We also applied gene expression analysis and methylation profiling to identify the mechanisms of resistance. We then explored rational drug combinations using a spheroid (3D)-culture assay.ResultsCompared with their parental cells, PDX-resistant cells exhibited a 30-fold increase in half-maximal inhibitory concentration values. Induction of apoptosis by PDX was significantly decreased in both PDX-resistant cell lines. Intracellular uptake of [14C]-PDX decreased in PDX-resistant CEM cells but not in PDX-resistant MOLT4 cells. There was no significant change in expression of dihydrofolate reductase (DHFR) or folylpolyglutamate synthetase (FPGS). Gene expression array analysis revealed that DNA-methyltransferase 3β (DNMT3B) expression was significantly elevated in both cell lines. Gene set enrichment analysis revealed that adipogenesis and mTORC1 signaling pathways were commonly upregulated in both resistant cell lines. Moreover, CpG island hypermethylation was observed in both PDX resistant cells lines. In the 3D-culture assay, decitabine (DAC) plus PDX showed synergistic effects in PDX-resistant cell lines compared with parental lines.ConclusionsThe resistance mechanisms of PDX were associated with reduced cellular uptake of PDX and/or overexpression of DNMT3B. Epigenetic alterations were also considered to play a role in the resistance mechanism. The combination of DAC and PDX exhibited synergistic activity, and thus, this approach might improve the clinical efficacy of PDX.

Intratumoral gene expression of dihydrofolate reductase and folylpoly-c-glutamate synthetase affects the sensitivity to 5-fluorouracil in non-small cell lung cancer

BackgroundVarious factors related to the sensitivity of non-small cell lung carcinoma (NSCLC) to 5-fluorouracil (5-FU) have been reported, and some of them have been clinically applied. In this single-institutional prospective analysis, the mRNA expression level of five folic acid-associated enzymes was evaluated in surgical specimens of NSCLC. We investigated the correlation between the antitumor effect of 5-FU in NSCLC using an anticancer drug sensitivity test and the gene expression levels of five enzymes.Materials and methodsForty patients who underwent surgery for NSCLC were enrolled, and the antitumor effect was measured using an in vitro anticancer drug sensitivity test (histoculture drug response assay) using freshly resected specimens. In the same sample, the mRNA expression levels of five enzymes involved in the sensitivity to 5-FU were measured in the tumor using real-time PCR. The expression levels and the result of the sensitivity test were compared.ResultsNo correlation was found between dihydropyrimidine dehydrogenase (DPD), orotate phosphoribosyltransferase (OPRT), or DPD/OPRT expression and the antitumor effects of 5-FU. On the other hand, a correlation was found between thymidylate synthase (TS), folylpoly-c-glutamate synthetase (FPGS), and dihydrofolate reductase (DHFR) expression and 5-FU sensitivity.ConclusionExpression of FPGS and DHFR may be useful for predicting the efficacy of 5-FU-based chemotherapy for NSCLC.

Plasma thymidylate synthase and dihydrofolate reductase mRNA levels as potential predictive biomarkers of pemetrexed sensitivity in patients with advanced non-small cell lung cancer.

BackgroundHigh levels of thymidylate synthase (TS) and dihydrofolate reductase (DHFR) expression in tumour tissues are an indicator of ineffective responses to pemetrexed-based chemotherapy in various tumours, including non-small cell lung cancer (NSCLC). However, tumour tissues are highly heterogeneous, so a single biopsy may not reflect genetic alterations during disease progression. This study investigated the potential use of plasma TS and DHFR mRNA levels as biomarkers for predicting sensitivity to pemetrexed-based chemotherapy.MethodsPlasma samples were obtained from 245 patients with advanced NSCLC and 30 healthy donors. Total RNA was extracted from the plasma samples, and TS and DHFR mRNA levels were determined via real-time PCR. TS and DHFR mRNA levels between cancer patients and healthy controls were compared. The association between plasma TS and DHFR mRNA levels and tumour response to pemetrexed/cisplatin chemotherapy was analysed.ResultsThe plasma TS and DHFR mRNA levels decreased in patients with advanced NSCLC compared with healthy controls. Moreover, plasma TS and DHFR mRNA levels negatively correlated with tumour response to pemetrexed/cisplatin chemotherapy in patients with advanced NSCLC. Overall survival time was prolonged in patients with low TS mRNA expression compared with those with high TS mRNA expression, although the difference was not statistically significant.ConclusionsLow expression levels of plasma TS and DHFR mRNA confer increased tumour sensitivity to pemetrexed/cisplatin chemotherapy in patients with advanced NSCLC. The results suggested that plasma TS and DHFR mRNA levels are promising biomarkers for choosing patients who are likely to respond and benefit the most from pemetrexed-based chemotherapy.

Tetrahydrobiopterin prevents chronic ischemia-related lower urinary tract dysfunction through the maintenance of nitric oxide bioavailability

This study aimed to investigate the influence of chronic ischemia on nitric oxide biosynthesis in the bladder and the effect of administering tetrahydrobiopterin (BH4), a cofactor for endothelial nitric oxide synthase (eNOS), on chronic ischemia-related lower urinary tract dysfunction (LUTD). This study divided male Sprague–Dawley rats into Control, chronic bladder ischemia (CBI) and CBI with oral BH4 supplementation (CBI/BH4) groups. In the CBI group, bladder capacity and bladder muscle strip contractility were significantly lower, and arterial wall was significantly thicker than in Controls. Significant improvements were seen in bladder capacity, muscle strip contractility and arterial wall thickening in the CBI/BH4 group as compared with the CBI group. Western blot analysis of bladder showed expressions of eNOS (p = 0.043), HIF-1α (p < 0.01) and dihydrofolate reductase (DHFR) (p < 0.01), which could regenerate BH4, were significantly higher in the CBI group than in Controls. In the CBI/BH4 group, HIF-1α (p = 0.012) and DHFR expressions (p = 0.018) were significantly decreased compared with the CBI group. Our results suggest that chronic ischemia increases eNOS and DHFR in the bladder to prevent atherosclerosis progression. However, DHFR could not synthesize sufficient BH4 relative to the increased eNOS, resulting in LUTD. BH4 supplementation protects lower urinary tract function by promoting eNOS activity.

Targeting feed-forward signaling of TGFβ/NOX4/DHFR/eNOS uncoupling/TGFβ axis with anti-TGFβ and folic acid attenuates formation of aortic aneurysms: Novel mechanisms and therapeutics

In the present study we aimed to identify novel mechanisms and therapeutics for thoracic aortic aneurysm (TAA) in Fbn1C1039G/+ Marfan Syndrome (MFS) mice. The expression of mature/active TGFβ and its downstream effector NOX4 were upregulated while tetrahydrobiopterin (H4B) salvage enzyme dihydrofolate reductase (DHFR) was downregulated in Fbn1C1039G/+ mice. In vivo treatment with anti-TGFβ completely attenuated NOX4 expression, restored DHFR protein abundance, reduced ROS production, recoupled eNOS and attenuated aneurysm formation. Intriguingly, oral administration with folic acid (FA) to recouple eNOS markedly alleviated expansion of aortic roots and abdominal aortas in Fbn1C1039G/+ mice, which was attributed to substantially upregulated DHFR expression and activity in the endothelium to restore tissue and circulating levels of H4B. Notably, circulating H4B levels were accurately predictive of tissue H4B bioavailability, and negatively associated with expansion of aortic roots, indicating a novel biomarker role of circulating H4B for TAA. Furthermore, FA diet abrogated TGFβ and NOX4 expression, disrupting the feed-forward loop to inactivate TGFβ/NOX4/DHFR/eNOS uncoupling axis in vivo and in vitro, while PTIO, a NO scavenger, reversed this effect in cultured human aortic endothelial cells (HAECs). Besides, expression of the rate limiting H4B synthetic enzyme GTP cyclohydrolase 1 (GTPCHI), was downregulated in Fbn1C1039G/+ mice at baseline. In cultured HAECs, RNAi inhibition of fibrillin resulted in reduced GTPCHI expression, while this response was abrogated by anti-TGFβ, indicating TGFβ-dependent downregulation of GTPCHI in response to fibrillin deficiency. Taken together, our data for the first time reveal that uncoupled eNOS plays a central role in TAA formation, while anti-TGFβ and FA diet robustly abolish aneurysm formation via inactivation of a novel TGFβ/NOX4/DHFR/eNOS uncoupling/TGFβ feed-forward pathway. Correction of fibrillin deficiency is additionally beneficial via preservation of GTPCHI function.

MiR-192 overexpression effect on DHFR and TYMS in acute lymphoblastic leukemia

PurposeNon-coding RNAs play a critical role in gene regulation in cancer cells. Reduced expression of microRNA-192 has been detected in many cancers. Here, we investigated the role of miR-192 in Dihydrofolate reductase (DHFR) and Thymidylate Synthase (TYMS) expression level and in an acute lymphoblastic leukemia cell line. Basic procedures20 patients diagnosed with acute lymphoblastic leukemia (ALL) were studied for the level of (micro RNA-192) miR-192, DHFR and TYMS expression level by qRT-PCR. NALM-6 cells were transduced using recombinant lentiviruses for overexpression of miR-192 and its backbone, then the relative changes in DHFR and TYMS genes expression were studied by qRT-PCR. DHFR Protein level changes were analyzed by Western blotting. Main findingsALL patients with relapse, experience lower levels of miR-192 and higher levels of DHFR and TYMS in comparison with treated patients. Overexpression of miR-192 in NALM-6 cells resulted in decreased expression of DHFR and TYMS. Moreover, the protein level of DHFR was decreased after overexpression of miR-192. Principal conclusionsThese results suggest the tumor suppression effects of miR-192 and its correlation with decreased DHFR and TYMS level in acute lymphoblastic leukemia cells for the first time.

Generation of pralatrexate resistant T-cell lymphoma lines reveals two patterns of acquired drug resistance that is overcome with epigenetic modifiers.

While pralatrexate (PDX) has been successfully developed for the treatment of T‐cell lymphoma, the mechanistic basis for its T‐cell selectivity and acquired resistance remains elusive. In an effort to potentially identify synergistic combinations that might circumnavigate or delay acquired PDX resistance, we generated resistant cells lines over a broad concentration range. PDX‐resistant cell lines H9‐12 and H9‐200 were developed, each exhibiting an IC50 of 35 and over 1000 nM, respectively. These lines were established in vitro from parental H9 cells. Expression analysis of the proteins known to be important determinants of antifolate pharmacology revealed increase expression of dihydrofolate reductase (DHFR) due to gene amplification, and reduced folate carrier1 downregulation, as the putative mechanisms of resistance in H9‐12 and H9‐200 cells. Cross resistance was only seen with methotrexate but not with romidepsin, azacitidine (AZA), decitabine, gemcitabine, doxorubicin, or bortezomib. Resistance to PDX was reversed by pretreatment with hypomethylating agents in a concentration‐dependent fashion. Comparison of gene expression profiles of parental and resistant cell lines confirmed markedly different patterns of gene expression, and identified the dual specificity phosphatase four (DUSP4) as one of the molecular target of PDX activity. Reduced STAT5 phosphorylation following exposure to PDX was observed in the H9 but not in the H9‐12 and H9‐200 cells. These data suggest that combination with hypomethylating agents could be potent, and that DUSP4 and STAT5 could represent putative biomarkers of PDX activity.