Bioactive potential of Lagotis cashmeriana: a study on morphology, phytochemicals, and antioxidant activity.

The present study aimed to investigate the morphological features, phytochemicals, phenolic content, and antioxidant activity in different parts of Lagotis cashmeriana. The morphological features depicted that the plant is 7.9 ± 1.699 cm tall with flowers arranged into an inflorescence. The length of inflorescence was 2.597 ± 0.796 cm. Basal leaves were measuring 2.99 ± 0.58 cm. Besides, the number of basal leaves and inflorescence ranged from 4-9 and 0-4 respectively. Methanolic extract of leaves displayed the highest phenolic content (169.5 µg/mL of GAE), followed by inflorescences (157 µg/mL of GAE). Among aqueous extracts, leaves displayed the highest phenolic content (88.38 µg/mL of GAE), followed by inflorescences (76.95 µg/mL of GAE). The results of antioxidant study revealed that the methanolic extracts of leaves possessed the highest antioxidant potential (180.76 µg/mL of AAE). Interestingly, for each extract, there was a positive correlation between the phenolic content and the antioxidant activity.

Synthesis and Biological Evaluation of Novel Uracil Derivatives as Thymidylate Synthase Inhibitors.

Cell division is driven by nucleic acid metabolism, and thymidylate synthase (TYMS) catalyzes a rate-limiting step in nucleotide synthesis. As a result, thymidylate synthase has emerged as a critical target in chemotherapy. 5-Fluorouracil (5-FU) is currently being used to treat a wide range of cancers, including breast, pancreatic, head and neck, colorectal, ovarian, and gastric cancers The objective of this study was to establish a new methodology for the low-cost, one-pot synthesis of uracil derivatives (UD-1 to UD-5) and to evaluate their therapeutic potential in BC cells. One-pot organic synthesis processes using a single solvent were used for the synthesis of drug analogues of Uracil. Integrated bioinformatics using GEPIA2, UALCAN, and KM plotter were utilized to study the expression pattern and prognostic significance of TYMS, the key target gene of 5-fluorouracil in breast cancer patients. Cell viability, cell proliferation, and colony formation assays were used as in vitro methods to validate the in silico lead obtained. BC patients showed high levels of thymidylate synthase, and high expression of thymidylate synthase was found associated with poor prognosis. In silico studies indicated that synthesized uracil derivatives have a high affinity for thymidylate synthase. Notably, the uracil derivatives dramatically inhibited the proliferation and colonization potential of BC cells in vitro. In conclusion, our study identified novel uracil derivatives as promising therapeutic options for breast cancer patients expressing the augmented levels of thymidylate synthase.

Adapalene inhibits the growth of triple-negative breast cancer cells by S-phase arrest and potentiates the antitumor efficacy of GDC-0941.

Although advances in diagnostics and therapeutics have prolonged the survival of triple-negative breast cancer (TNBC) patients, metastasis, therapeutic resistance, and lack of targeted therapies remain the foremost hurdle in the effective management of TNBC. Thus, evaluation of new therapeutic agents and their efficacy in combination therapy is urgently needed. The third-generation retinoid adapalene (ADA) has potent antitumor activity, and using ADA in combination with existing therapeutic regimens may improve the effectiveness and minimize the toxicities and drug resistance. The current study aimed to assess the anticancer efficacy of adapalene as a combination regimen with the PI3K inhibitor (GDC-0941) in TNBC in vitro models. The Chou-Talalay's method evaluated the pharmacodynamic interactions (synergism, antagonism, or additivity) of binary drug combinations. Flow cytometry, Western blotting, and in silico studies were used to analyze the mechanism of GDC-ADA synergistic interactions in TNBC cells. The combination of GDC and ADA demonstrated a synergistic effect in inhibiting proliferation, migration, and colony formation of tumor cells. Accumulation of reactive oxygen species upon co-treatment with GDC and ADA promoted apoptosis and enhanced sensitivity to GDC in TNBC cells. The findings indicate that ADA is a promising therapeutic agent in treating advanced BC tumors and enhance sensitivity to GDC in inhibiting tumor growth in TNBC models while reducing therapeutic resistance.

Adapalene and Doxorubicin Synergistically Promote Apoptosis of TNBC Cells by Hyperactivation of the ERK1/2 Pathway Through ROS Induction.

Doxorubicin is a commonly used chemotherapeutic agent to treat several malignancies, including aggressive tumors like triple-negative breast cancer. It has a limited therapeutic index owing to its extreme toxicity and the emergence of drug resistance. As a result, there is a pressing need to find innovative drugs that enhance the effectiveness of doxorubicin while minimizing its toxicity. The rationale of the present study is that combining emerging treatment agents or repurposed pharmaceuticals with doxorubicin might increase susceptibility to therapeutics and the subsequent establishment of improved pharmacological combinations for treating triple-negative breast cancer. Additionally, combined treatment will facilitate dosage reduction, reducing the toxicity associated with doxorubicin. Recently, the third-generation retinoid adapalene was reported as an effective anticancer agent in several malignancies. This study aimed to determine the anticancer activity of adapalene in TNBC cells and its effectiveness in combination with doxorubicin, and the mechanistic pathways in inhibiting tumorigenicity. Adapalene inhibits tumor cell growth and proliferation and acts synergistically with doxorubicin in inhibiting growth, colony formation, and migration of TNBC cells. Also, the combination of adapalene and doxorubicin enhanced the accumulation of reactive oxygen species triggering hyperphosphorylation of Erk1/2 and caspase-dependent apoptosis. Our results demonstrate that adapalene is a promising antitumor agent that may be used as a single agent or combined with present therapeutic regimens for TNBC treatment.

Expression Pattern and Prognostic Significance of Chemokines in Breast cancer: An Integrated Bioinformatics Analysis.

BACKGROUND: Breast cancer (BC), one of the most prevalent malignancies, is the second major cause of mortality from cancer among women worldwide. Even though substantial progress has been made in breast cancer treatment, metastasis still accounts for the majority of the deaths. The tumor microenvironment (TME) comprising stromal and non-stromal components is central to tumor growth and development and is partly regulated by chemokines. Chemokines regulate immune cell trafficking, the development of stroma and play a key role in inflammation, a cancer hallmark. METHODS: In the present study, we used a bioinformatics approach to identify highly deregulated chemokines in BC patients. We performed expression analysis, survival analysis, gene ontology analysis, KEGG analysis, and protein-protein interaction network analysis of the deregulated chemokines using Gepia2, UALCAN, Kaplan-Meier Plotter, DAVID, and STRING tools. RESULTS: We identified >2-fold change (FC) increase in CXCL9/10/11/13 and >-2 FC decrease in CCL14/21/28, CXCL2/12 CX3CL1. Also, increased expression of CCL14, CCL21, CXCL13, CXCL9, CXCL12 correlated with better overall survival (OS) of BC patients. CONCLUSIONS: Our results strongly indicate that chemokines may have potential biomarker characteristics, and the constructed PPI network contributed to an in-depth understanding of the chemokine networks. The deregulated chemokines may prove to be therapeutic targets for the effective management of BC.

Chemokines in triple-negative breast cancer heterogeneity: New challenges for clinical implications.

Tumor heterogeneity is a hallmark of cancer and one of the primary causes of resistance to therapies. Triple-negative breast cancer (TNBC), which accounts for 15-20% of all breast cancers and is the most aggressive subtype, is very diverse, connected to metastatic potential and response to therapy. It is a very diverse disease at the molecular, pathologic, and clinical levels. TNBC is substantially more likely to recur and has a worse overall survival rate following diagnosis than other breast cancer subtypes. Chemokines, low molecular weight proteins that stimulate chemotaxis, have been shown to control the cues responsible for TNBC heterogeneity. In this review, we have focused on tumor heterogeneity and the role of chemokines in modulating tumor heterogeneity, since this is the most critical issue in treating TNBC. Additionally, we examined numerous cues mediated by chemokine networks that contribute to the heterogeneity of TNBC. Recent developments in our knowledge of the chemokine networks that regulate TNBC heterogeneity may pave the way for developing effective therapeutic modalities for effective treatment of TNBC.

The tumor microenvironment as driver of stemness and therapeutic resistance in breast cancer: New challenges and therapeutic opportunities.

BACKGROUND: Breast cancer (BC), the second most common cause of cancer-related deaths, remains a significant threat to the health and wellness of women worldwide. The tumor microenvironment (TME), comprising cellular components, such as cancer-associated fibroblasts (CAFs), immune cells, endothelial cells and adipocytes, and noncellular components such as extracellular matrix (ECM), has been recognized as a critical contributor to the development and progression of BC. The interplay between TME components and cancer cells promotes phenotypic heterogeneity, cell plasticity and cancer cell stemness that impart tumor dormancy, enhanced invasion and metastasis, and the development of therapeutic resistance. While most previous studies have focused on targeting cancer cells with a dismal prognosis, novel therapies targeting stromal components are currently being evaluated in preclinical and clinical studies, and are already showing improved efficacies. As such, they may offer better means to eliminate the disease effectively. CONCLUSIONS: In this review, we focus on the evolving concept of the TME as a key player regulating tumor growth, metastasis, stemness, and the development of therapeutic resistance. Despite significant advances over the last decade, several clinical trials focusing on the TME have failed to demonstrate promising effectiveness in cancer patients. To expedite clinical efficacy of TME-directed therapies, a deeper understanding of the TME is of utmost importance. Secondly, the efficacy of TME-directed therapies when used alone or in combination with chemo- or radiotherapy, and the tumor stage needs to be studied. Likewise, identifying molecular signatures and biomarkers indicating the type of TME will help in determining precise TME-directed therapies.

Both BRCA1-wild type and -mutant triple-negative breast cancers show sensitivity to the NAE inhibitor MLN4924 which is enhanced upon MLN4924 and cisplatin combination treatment.

Triple-negative breast cancer (TNBC) shows limited therapeutic efficacy. PARP inhibitor has been approved to treat advanced BRCA-mutant breast cancer but shows high resistance. Therefore, the development of new therapeutics that sensitize TNBC irrespective of BRCA status is urgently needed. The neddylation pathway plays a critical role in many physiological processes by regulating the degradation of proteins. MLN4924, a selective inhibitor of the key neddylation enzyme NEDD8 Activation Enzyme (NAE1), shows higher sensitivity to both BRCA1-wild type and -mutant TNBCs compared to other breast cancer subtypes. MLN4924 induced re-replication with >4N DNA content leading to robust DNA damage. Accumulation of unrepaired DNA damage resulted in S and G2/M arrest causing apoptosis and senescence, due to the stabilization of the replication initiation protein CDT1 and the accumulation of cell cycle proteins upon MLN4924 treatment. Moreover, adding MLN4924 to the standard TNBC chemotherapeutic agent cisplatin increased the DNA damage level, further enhancing the sensitivity. In vivo, MLN4924 reduced tumor growth in a NOD-SCID mouse xenograft model by inducing DNA damage which was further augmented with the MLN4924 and cisplatin cotreatment. NAE1 is overexpressed in TNBC cell lines and in patients compared to other breast cancer subtypes suggesting that NAE1 status is prognostic of MLN4924 treatment response and outcome. Taken together, we demonstrated the mechanism of TNBC sensitization by the MLN4924 and MLN4924/cisplatin treatments irrespective of BRCA1 status, provided a strong justification for using MLN4924 alone or in combination with cisplatin, and identified a genetic background in which this combination will be particularly effective.

Modulation of dietary folate with age confers selective hepatocellular epigenetic imprints through DNA methylation.

The present study has been designed to determine the effect of folate modulation (deficiency/supplementation) with aging on the promoter methylation of tumor suppressor and proto-oncogenes to understand the underlying mechanism of epigenetic alterations. Folate deficiency was induced for 3 and 5 months in weanling, young and adult groups, and after 3 months of folate deficiency, they were repleted with physiological folate (2 mg/kg diet) and folate oversupplementation (8 mg/kg diet) for another 2 months. The methylation facet in the present study revealed that the combined effect of folate deficiency and aging decreased the methylation index. Folate deficiency with age resulted in the up-regulation of proto-oncogenes (C-MYC and C-JUN) and cell cycle regulator gene Cyclin E as a result of promoter hypomethylation. However, in case of tumor suppressor genes (p53, p15ink4b and p16ink4a), the expression levels were found to be decreased at transcriptional level due to promoter hypermethylation. Upon repletion with physiological folate and folate oversupplementation, we found down-regulation of proto-oncogenes and up-regulation of tumor suppressor genes as a result of promoter hypermethylation and hypomethylation, respectively. Deregulation of these important genes due to folate deficiency may contribute toward the pathogenesis at cellular level.

Reprograming of Glucose Metabolism by Zerumbone Suppresses Hepatocarcinogenesis.

Hepatocellular carcinoma (HCC) is the most prevalent and highly aggressive liver malignancy with limited therapeutic options. Here, the therapeutic potential of zerumbone, a sesquiterpene derived from the ginger plant Zingiber zerumbet, against HCC was explored. Zerumbone inhibited proliferation and clonogenic survival of HCC cells in a dose-dependent manner by arresting cells at the G2-M phase and inducing apoptosis. To elucidate the underlying molecular mechanisms, a phosphokinase array was performed that showed significant inhibition of the PI3K/AKT/mTOR and STAT3 signaling pathways in zerumbone-treated HCC cells. Gene expression profiling using microarray and analysis of microarray data by Gene Set Enrichment Analysis (GSEA) and Ingenuity Pathway Analysis (IPA) revealed that zerumbone treatment resulted in significant deregulation of genes regulating apoptosis, cell cycle, and metabolism. Indeed, tracing glucose metabolic pathways by growing HCC cells with 13C6-glucose and measuring extracellular and intracellular metabolites by 2D nuclear magnetic resonance (NMR) spectroscopy showed a reduction in glucose consumption and reduced lactate production, suggesting glycolytic inhibition. In addition, zerumbone impeded shunting of glucose-6-phosphate through the pentose phosphate pathway, thereby forcing tumor cells to undergo cell-cycle arrest and apoptosis. Importantly, zerumbone treatment suppressed subcutaneous and orthotopic growth and lung metastasis of HCC xenografts in immunocompromised mice. In conclusion, these findings reveal a novel and potentially effective therapeutic strategy for HCC using a natural product that targets cancer cell metabolism.Implications: Dietary compounds, like zerumbone, that impact cell cycle, apoptosis, and metabolic processes may have therapeutic benefits for HCC patients. Mol Cancer Res; 16(2); 256-68. ©2017 AACR.

RAGE mediates S100A7-induced breast cancer growth and metastasis by modulating the tumor microenvironment.

RAGE is a multifunctional receptor implicated in diverse processes including inflammation and cancer. In this study, we report that RAGE expression is upregulated widely in aggressive triple-negative breast cancer (TNBC) cells, both in primary tumors and in lymph node metastases. In evaluating the functional contributions of RAGE in breast cancer, we found that RAGE-deficient mice displayed a reduced propensity for breast tumor growth. In an established model of lung metastasis, systemic blockade by injection of a RAGE neutralizing antibody inhibited metastasis development. Mechanistic investigations revealed that RAGE bound to the proinflammatory ligand S100A7 and mediated its ability to activate ERK, NF-κB, and cell migration. In an S100A7 transgenic mouse model of breast cancer (mS100a7a15 mice), administration of either RAGE neutralizing antibody or soluble RAGE was sufficient to inhibit tumor progression and metastasis. In this model, we found that RAGE/S100A7 conditioned the tumor microenvironment by driving the recruitment of MMP9-positive tumor-associated macrophages. Overall, our results highlight RAGE as a candidate biomarker for TNBCs, and they reveal a functional role for RAGE/S100A7 signaling in linking inflammation to aggressive breast cancer development.

Reduced expression of folate transporters in kidney of a rat model of folate oversupplementation.

Folic acid is the key one-carbon donor required for de novo nucleotide and methionine synthesis. Its deficiency is associated with megaloblastic anemia, cancer and various complications of pregnancy. However, its supplementation results in reduction of neural tube defects and prevention of several types of cancer. The intake of folic acid from fortified food together with the use of nutritional supplements creates a state of folate oversupplementation. Fortification of foods is occurring worldwide with little knowledge of the potential safety and physiologic consequences of intake of such high doses of folic acid. So, we planned to examine the effects of acute and chronic folate oversupplementation on the physiology of renal folate transport in rats. Male Wistar rats were procured and divided into two groups. Rats in group I were given semisynthetic diets containing 2 mg folic acid/kg diet (control) and those in group II were given folate-oversupplemented rat diet, i.e., 20 mg folic acid/kg diet (oversupplemented). Six animals from group I and group II received the treatment for 10 days (acute treatment) and remaining six for 60 days (chronic treatment). In acute folate-oversupplemented rats, 5-[(14)C]-methyltetrahydrofolate uptake was found to be significantly reduced, as compared to chronic folate-oversupplemented and control rats. This reduction in uptake was associated with a significant decrease in the mRNA and protein levels of the folate transporters. Results of the present investigation showed that acute oversupplementation led to a specific and significant down-regulation of renal folate uptake process mediated via transcriptional and translational regulatory mechanism(s).

Mechanistic insights of intestinal absorption and renal conservation of folate in chronic alcoholism.

Folate mediated one-carbon metabolism is of fundamental importance for various cellular processes, including DNA synthesis and methylation of biological molecules. Due to the exogenous requirement of folate in mammals, there exists a well developed epithelial folate transport system for regulation of normal folate homeostasis. The intestinal and renal folate uptake is tightly and diversely regulated and disturbances in folate homeostasis like in alcoholism have pathological consequences. The study was sought to delineate the regulatory mechanism of folate uptake in intestine and reabsorption in renal tubular cells that could evaluate insights of malabsorption during alcoholism. The folate transporters PCFT and RFC were found to be associated with lipid rafts of membrane surfaces in intestine and kidney. Importantly, the observed lower intestinal and renal folate uptake was associated with decreased levels of folate transporter viz. PCFT and RFC in lipid rafts of intestinal and renal membrane surfaces. The decreased association of folate transporters in lipid rafts was associated with decreased protein and mRNA levels. In addition, immunohistochemical studies showed that alcoholic conditions deranged that localization of PCFT and RFC. These findings could explain the possible mechanistic insights that may result in folate malabsorption during alcoholism.

Decreased activity of folate transporters in lipid rafts resulted in reduced hepatic folate uptake in chronic alcoholism in rats.

Folic acid is an essential nutrient that is required for one-carbon biosynthetic processes and for methylation of biomolecules. Deficiency of this micronutrient leads to disturbances in normal physiology of cell. Chronic alcoholism is well known to be associated with folate deficiency, which is due in part to folate malabsorption. The present study deals with the regulatory mechanisms of folate uptake in liver during chronic alcoholism. Male Wistar rats were fed 1 g/kg body weight/day ethanol (20 % solution) orally for 3 months, and the molecular mechanisms of folate uptake were studied in liver. The characterization of the folate transport system in liver basolateral membrane (BLM) suggested it to be a carrier mediated and acidic pH dependent, with the major involvement of proton coupled folate transporter and folate binding protein in the uptake. The folate transporters were found to be associated with lipid raft microdomain of liver BLM. Moreover, ethanol ingestion decreased the folate transport by altering the Vmax of folate transport process and downregulated the expression of folate transporters in lipid rafts. The decreased transporter levels were associated with reduced protein and mRNA levels of these transporters in liver. The deranged folate uptake together with reduced folate transporter levels in lipid rafts resulted in reduced folate levels in liver and thereby to its reduced levels in serum of ethanol-fed rats. The chronic ethanol ingestion led to decreased folate uptake in liver, which was associated with the decreased number of transporter molecules in the lipid rafts that can be ascribed to the reduced synthesis of these transporters.

Adaptive transport of folic acid across renal epithelia in folate-deficient rats.

Folate (vitamin B(9)) is an essential vitamin for a wide spectrum of biochemical reactions; however, unlike bacteria and plants, mammals are devoid of folate biosynthesis and thus must obtain this cofactor from exogenous sources. The activities of folate transporters on the kidneys play an important role in conserving folate excretion and reabsorption across the apical membrane of the renal proximal tubules. The different transport system activities may become identifiable in response to external stimuli, such as folate availability and exposure to chemotherapeutic agents. We have explored the effect of folate deficiency on the activity and expression of folate transporters in rat kidneys. Wistar rats were fed a folate-containing diet (2 mg folic acid kg(-1) diet) or a folic acid-free diet over a 3-month period, and mechanisms of folate transport were studied in renal brush border membrane vesicles and basolateral membrane vesicles. The renal folate uptake process is saturable and pH dependent, and it involves the folate receptor and reduced folate carrier (RFC) systems and possibly the proton coupled folate transporter (PCFT) system. We found that folate deficiency increased the renal brush border membrane and basolateral folate uptake by increasing the number of transporter molecules. The observed up-regulation of mRNA expression was also associated with a significant increase in RFC and PCFT expression at the protein level.

Mechanism of intestinal folate transport during folate deficiency in rodent model.

BACKGROUND & OBJECTIVES: Folate deficiency is a public health problem and is the most notable for its association with neural tube defect in developing embryo, megaloblastic anaemia, cancers and cardiovascular diseases. The mechanisms of the intestinal folate uptake process have been earlier characterized. However, much less is known about regulation. In this study we evaluated the mechanistic insights of folate absorption in an in vivo model of folate deficiency. METHODS: Male Wistar rats were fed folate-containing diet (2 mg/kg folic acid) or a folic acid-free diet over 3 months and folate transport was studied in intestinal brush border membrane vesicles (BBMV). RESULTS: The characterization of the folate transport system in intestinal brush border membrane (BBM) suggested it to be a carrier mediated, acidic pH stimulated, and Na⁺ independent. Folate deficiency increased the folate transport by altering the Vmax without changing the Km of folate transport process. The increased transport efficiency of the BBM was associated with upregulation of folate transporters at both mRNA and protein level. INTERPRETATION & CONCLUSIONS: Folate deficiency resulted in significant upregulation of intestinal folate uptake, by increasing number of transporters without any change in specificity of transporters towards its substrate. The observed upregulation was associated with significant increase in reduced folate carrier (RFC) and proton coupled folate transporter (PCFT) expressions, suggesting the transcriptional and translational regulation of folate uptake during folate deficiency.

Folate malabsorption is associated with down-regulation of folate transporter expression and function at colon basolateral membrane in rats.

Folates, an essential component (important B vitamin) in the human diet, are involved in many metabolic pathways, mainly in carbon transfer reactions such as purine and pyrimidine biosynthesis and amino acid interconversions. Deficiency of this micronutrient leads to the disruption of folate-dependent metabolic pathways that lead to the development of clinical abnormalities ranging from anaemia to growth retardation. Folate deficiency due to alcohol ingestion is quite common, primarily due to malabsorption. The present study dealt with the mechanistic insights of folate malabsorption in colonic basolateral membrane (BLM). Wistar rats (n 12) were fed 1 g/kg body weight per d ethanol (20 %) solution orally for 3 months and folate transport was studied in the isolated colonic BLM. The folate exit across colon BLM shows characteristics of carrier-mediated process with the major involvement of reduced folate carrier (RFC). The chronic ethanol ingestion decreased the uptake by decreasing the affinity by 46 % (P < 0·01) and the number of transport molecules by 43 % (P < 0·001) at the colon BLM. The decreased uptake was associated with down-regulation of proton-coupled folate transporter (PCFT) and RFC expression at mRNA and protein levels. The extent of decrease was 44 % (P < 0·01) and 24 % (P < 0·05) for PCFT and 23 % (P < 0·01) and 57 % (P < 0·01) for RFC at mRNA and protein levels, respectively. Moreover, folate transporters were associated with lipid rafts (LR) of colon BLM, and chronic alcoholism decreased the association of these transporters with LR.

Alcohol-associated folate disturbances result in altered methylation of folate-regulating genes.

Folate plays a critical role in maintaining normal metabolic, energy, differentiation and growth status of all mammalian cells. The steady-state accumulation of folate seems to depend on the activity of two enzymes: folylpolyglutamate synthetase (FPGS), which adds glutamate residues, and gamma-glutamyl hydrolase (GGH), which removes them, enabling it to be transported across the biological membranes. Overexpression of GGH and downregulation of FPGS would be expected to decrease intracellular folate in its polyglutamylated form, thereby increasing efflux of folate and its related molecules, which might lead to resistance to drugs or folate deficiency. The study was sought to delineate the activity of GGH and expression FPGS in tissues involved in folate homeostasis during alcoholism and the epigenetic regulation of these enzymes and transporters regulating intracellular folate levels. We determined the activity of GGH and expression of FPGS in tissues after 3 months of ethanol feeding to rats at 1 g/kg body weight/day. The results showed that there was not any significant change in the activity of folate hydrolyzing enzyme GGH in ethanol-fed rats while there was significant down regulation in the expression of FPGS. Ethanol feeding decreased the total as well as polyglutamated folate levels. There was tissue-specific hyper/hypo methylation of folate transporter genes viz. PCFT and RFC by chronic ethanol feeding. Moreover, hypermethylation of FPGS gene was observed in intestine and kidney without any change in methylation levels of GGH in the ethanol-fed rats. In conclusion, the initial deconjugation of polyglutamylated folate by GGH was not impaired in ethanol-fed rats while the conversion of monoglutamylated folate to polyglutamylated form might be impaired. There was tissue-specific altered methylation of folate transporter genes by chronic ethanol feeding.

Biochemical and molecular mechanisms of folate transport in rat pancreas; interference with ethanol ingestion.

Folic acid is an essential nutrient that is required for one-carbon biosynthetic processes and for methylation of biomolecules. Deficiency of this micronutrient leads to disturbances in normal physiology of cell. Chronic alcoholism is well known to be associated with folate deficiency which is due, in part to folate malabsorption. The present study deals with the mechanistic insights of reduced folate absorption in pancreas during chronic alcoholism. Male Wistar rats were fed 1 g/kg body weight/day ethanol (20% solution) orally for 3 months and the mechanisms of alcohol associated reduced folate uptake was studied in pancreas. The folate transport system in the pancreatic plasma membrane (PPM) was found to be acidic pH dependent one. The transporters proton coupled folate transporter (PCFT) and reduced folate carrier (RFC) are involved in folate uptake across PPM. The folate transporters were found to be associated with lipid raft microdomain of the PPM. Ethanol ingestion decreased the folate transport by reducing the levels of folate transporter molecules in lipid rafts at the PPM. The decreased transport efficiency of the PPM was reflected as reduced folate levels in pancreas. The chronic ethanol ingestion led to decreased pancreatic folate uptake. The decreased levels of PCFT and RFC expression in rat PPM were due to decreased association of these proteins with lipid rafts (LR) at the PPM.

Reduced levels of folate transporters (PCFT and RFC) in membrane lipid rafts result in colonic folate malabsorption in chronic alcoholism.

We studied the effect of chronic ethanol ingestion on folate transport across the colonic apical membranes (CAM) in rats. Male Wistar rats were fed 1 g/kg body weight/day ethanol (20%) solution orally for 3 months and folate transport was studied in the isolated colon apical membrane vesicles. The folate transport was found to be carrier mediated, saturable, with pH optima at 5.0. Chronic ethanol ingestion reduced the folate transport across the CAM by decreasing the affinity of transporters (high Km) for the substrate and by decreasing the number of transporter molecules (low Vmax) on the colon luminal surface. The decreased transport activity at the CAM was associated with down-regulation of the proton-coupled folate transporter (PCFT) and the reduced folate carrier (RFC) which resulted in decreased PCFT and RFC protein levels in the colon of rats fed alcohol chronically. Moreover, the PCFT and the RFC were found to be distributed in detergent insoluble fraction of the CAM in rats. Floatation experiments on Optiprep density gradients demonstrated the association of the PCFT and the RFC protein with lipid rafts (LR). Chronic alcoholism decreased the PCFT and the RFC protein levels in the CAM LR in accordance with the decreased synthesis. Hence, we propose that downregulation in the expression of the PCFT and the RFC in colon results in reduced levels of these transporters in colon apical membrane LR as a mechanism of folate malabsorption during chronic alcoholism.