miRNAs as novel immunoregulators in cancer.

The immune system is a well-known vital regulator of tumor growth, and one of the main hallmarks of cancer is evading the immune system. Immune system deregulation can lead to immune surveillance evasion, sustained cancer growth, proliferation, and metastasis. Tumor-mediated disruption of the immune system is accomplished by different mechanisms that involve extensive crosstalk with the immediate microenvironment, which includes endothelial cells, immune cells, and stromal cells, to create a favorable tumor niche that facilitates the development of cancer. The essential role of non-coding RNAs such as microRNAs (miRNAs) in the mechanism of cancer cell immune evasion has been highlighted in recent studies. miRNAs are small non-coding RNAs that regulate a wide range of post-transcriptional gene expression in a cell. Recent studies have focused on the function that miRNAs play in controlling the expression of target proteins linked to immune modulation. Studies show that miRNAs modulate the immune response in cancers by regulating the expression of different immune-modulatory molecules associated with immune effector cells, such as macrophages, dendritic cells, B-cells, and natural killer cells, as well as those present in tumor cells and the tumor microenvironment. This review explores the relationship between miRNAs, their altered patterns of expression in tumors, immune modulation, and the functional control of a wide range of immune cells, thereby offering detailed insights on the crosstalk of tumor-immune cells and their use as prognostic markers or therapeutic agents.

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.

Cytokine-chemokine network driven metastasis in esophageal cancer; promising avenue for targeted therapy.

Esophageal cancer (EC) is a disease often marked by aggressive growth and poor prognosis. Lack of targeted therapies, resistance to chemoradiation therapy, and distant metastases among patients with advanced disease account for the high mortality rate. The tumor microenvironment (TME) contains several cell types, including fibroblasts, immune cells, adipocytes, stromal proteins, and growth factors, which play a significant role in supporting the growth and aggressive behavior of cancer cells. The complex and dynamic interactions of the secreted cytokines, chemokines, growth factors, and their receptors mediate chronic inflammation and immunosuppressive TME favoring tumor progression, metastasis, and decreased response to therapy. The molecular changes in the TME are used as biological markers for diagnosis, prognosis, and response to treatment in patients. This review highlighted the novel insights into the understanding and functional impact of deregulated cytokines and chemokines in imparting aggressive EC, stressing the nature and therapeutic consequences of the cytokine-chemokine network. We also discuss cytokine-chemokine oncogenic potential by contributing to the Epithelial-Mesenchymal Transition (EMT), angiogenesis, immunosuppression, metastatic niche, and therapeutic resistance development. In addition, it discusses the wide range of changes and intracellular signaling pathways that occur in the TME. Overall, this is a relatively unexplored field that could provide crucial insights into tumor immunology and encourage the effective application of modulatory cytokine-chemokine therapy to EC.

An insight into the cancer stem cell survival pathways involved in chemoresistance in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is the most complex, aggressive and fatal subtype of breast cancer. Owing to the lack of targeted therapy and heterogenic nature of TNBC, chemotherapy remains the sole treatment option for TNBC, with taxanes and anthracyclines representing the general chemotherapeutic regimen in TNBC therapy. But unfortunately, patients develop resistance to the existing chemotherapeutic regimen, resulting in approximately 90% treatment failure. Breast cancer stem cells (BCSCs) are one of the major causes for the development of chemoresistance in TNBC patients. After surviving the chemotherapy damage, the presence of BCSCs results in relapse and recurrence of TNBC. Several pathways are known to regulate BCSCs' survival, such as the Wnt/ß-catenin, Hedgehog, JAK/STAT and HIPPO pathways. Therefore it is imperative to target these pathways in the context of eliminating chemoresistance. In this review we will discuss the novel strategies and various preclinical and clinical studies to give an insight into overcoming TNBC chemoresistance. We present a detailed account of recent studies carried out that open an exciting perspective in relation to the mechanisms of chemoresistance.

Chemokine-Cytokine Networks in the Head and Neck Tumor Microenvironment.

Head and neck squamous cell carcinomas (HNSCCs) are aggressive diseases with a dismal patient prognosis. Despite significant advances in treatment modalities, the five-year survival rate in patients with HNSCC has improved marginally and therefore warrants a comprehensive understanding of the HNSCC biology. Alterations in the cellular and non-cellular components of the HNSCC tumor micro-environment (TME) play a critical role in regulating many hallmarks of cancer development including evasion of apoptosis, activation of invasion, metastasis, angiogenesis, response to therapy, immune escape mechanisms, deregulation of energetics, and therefore the development of an overall aggressive HNSCC phenotype. Cytokines and chemokines are small secretory proteins produced by neoplastic or stromal cells, controlling complex and dynamic cell-cell interactions in the TME to regulate many cancer hallmarks. This review summarizes the current understanding of the complex cytokine/chemokine networks in the HNSCC TME, their role in activating diverse signaling pathways and promoting tumor progression, metastasis, and therapeutic resistance development.

Recent Advances in Head and Neck Tumor Microenvironment-Based Therapy.

Head and neck squamous cell carcinomas (HNSCCs) are a group of heterogeneous aggressive tumors affecting more than half a million patients worldwide annually. While the tobacco- and alcohol-associated HNSCC tumors are declining, human papillomavirus (HPV)-induced tumors are on rise. Despite recent advances in multimodality therapeutic interventions including surgery in combination with chemoradiation therapy (CRT), the overall 5-year survival has not improved more than 50%. The underlying reasons for this dismal prognosis is the intrinsic or acquired resistance to CRT. While previous studies were focused to target tumor cells, recent findings have implicated the involvement of tumor microenvironment (TME) on tumor progression and response to therapy. HNSCC TME includes cancer-associated fibroblasts (CAFs), endothelial cells, immune cells, endocrine cells, and the extracellular matrix (ECM) proteins including collagen and fibronectin. Understanding the crosstalk between TME and cancer cells is important to formulate more effective novel therapies and to overcome resistance mechanisms. Here, we summarized the current literature on recent advances on HNSCC TME with special emphasis on novel cell-cell interactions and therapies currently under development.

miRNA-122 Protects Mice and Human Hepatocytes from Acetaminophen Toxicity by Regulating Cytochrome P450 Family 1 Subfamily A Member 2 and Family 2 Subfamily E Member 1 Expression.

Acetaminophen toxicity is a leading cause of acute liver failure (ALF). We found that miRNA-122 (miR-122) is down-regulated in liver biopsy specimens of patients with ALF and in acetaminophen-treated mice. A marked decrease in the primary miR-122 expression occurs in mice on acetaminophen overdose because of suppression of its key transactivators, hepatocyte nuclear factor (HNF)-4α and HNF6. More importantly, the mortality rates of male and female liver-specific miR-122 knockout (LKO) mice were significantly higher than control mice when injected i.p. with an acetaminophen dose not lethal to the control. LKO livers exhibited higher basal expression of cytochrome P450 family 2 subfamily E member 1 (CYP2E1) and cytochrome P450 family 1 subfamily A member 2 (CYP1A2) that convert acetaminophen to highly reactive N-acetyl-p-benzoquinone imine. Upregulation of Cyp1a2 primary transcript and mRNA in LKO mice correlated with the elevation of aryl hydrocarbon receptor (AHR) and mediator 1 (MED1), two transactivators of Cyp1a2. Analysis of ChIP-seq data in the ENCODE (Encyclopedia of DNA Element) database identified association of CCCTC-binding factor (CTCF) with Ahr promoter in mouse livers. Both MED1 and CTCF are validated conserved miR-122 targets. Furthermore, depletion of Ahr, Med1, or Ctcf in Mir122-/- hepatocytes reduced Cyp1a2 expression. Pulse-chase studies found that CYP2E1 protein level is upregulated in LKO hepatocytes. Notably, miR-122 depletion sensitized differentiated human HepaRG cells to acetaminophen toxicity that correlated with upregulation of AHR, MED1, and CYP1A2 expression. Collectively, these results reveal a critical role of miR-122 in acetaminophen detoxification and implicate its therapeutic potential in patients with ALF.

Sorafenib and 2-Deoxyglucose Synergistically Inhibit Proliferation of Both Sorafenib-Sensitive and -Resistant HCC Cells by Inhibiting ATP Production.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related deaths globally. Sorafenib is the only first-line systemic drug for advanced HCC, but it has very limited survival benefits because patients treated with sorafenib either suffer from side effects or show disease progression after initial response. Thus, there is an urgent need to develop novel strategies for first-line and second-line therapies. The association between sorafenib resistance and glycolysis prompted us to screen several drugs with known antiglycolytic activity to identify those that will sensitize cells to sorafenib. We demonstrate that the combination of glycolytic inhibitor 2-deoxyglucose (2DG) and sorafenib drastically inhibits viability of sorafenib-sensitive and -resistant cells. However, the combination of other antiglycolytic drugs like lonidamine, gossypol, 3-bromopyruvate, and imatinib with sorafenib does not show synergistic effect. Cell cycle analysis revealed that the combination of 2DG and sorafenib induced cell cycle arrest at G0/G1. Mechanistic investigation suggests that the cell cycle arrest is due to depletion of cellular ATP that activates AMP-activated protein kinase (AMPK), which, in turn, inhibits mammalian target of rapamycin (mTOR) to induce cell cycle arrest. This study provides strong evidence for the therapeutic potential of the combination of sorafenib and 2DG for HCC.

Cannabinoid receptor-2 agonist inhibits macrophage induced EMT in non-small cell lung cancer by downregulation of EGFR pathway.

JWH-015, a cannabinoid receptor 2 (CB2) agonist has tumor regressive property in various cancer types. However, the underlying mechanism by which it acts in lung cancer is still unknown. Tumor associated macrophage (TAM) intensity has positive correlation with tumor progression. Also, macrophages recruited at the tumor site promote tumor growth by enhancing epithelial to mesenchymal (EMT) progression. In this study, we analyzed the role of JWH-015 on EMT and macrophage infiltration by regulation of EGFR signaling. JWH-015 inhibited EMT in NSCLC cells A549 and also reversed the mesenchymal nature of CALU-1 cells by downregulation of EGFR signaling targets like ERK and STAT3. Also, in vitro co-culture experiments of A549 with M2 polarized macrophages provided evidence that JWH-015 decreased migratory and invasive abilities which was proved by reduced expression of FAK, VCAM1, and MMP2. Furthermore, it decreased macrophage induced EMT in A549 by attenuating the mesenchymal character by downregulating EGFR and its targets. These results were confirmed in an in vivo subcutaneous syngenic mouse model where JWH-015 blocks tumor growth and also inhibits macrophage recruitment and EMT at the tumor site which was regulated by EGFR pathway. Finally, JWH-015 reduced lung tumor lesions in an in vivo tumorigenicity mouse model. These data confer the impact of this cannabinoid on anti-proliferative and anti-tumorigenic effects, thus enhancing our understanding of its therapeutic efficacy in NSCLC. Our findings open new avenues for cannabinoid receptor CB2 agonist-JWH-015 as a novel and potential therapeutic target based on EGFR downregulation mechanisms in NSCLC. © 2016 Wiley Periodicals, Inc.

C-X-C motif chemokine 12/C-X-C chemokine receptor type 7 signaling regulates breast cancer growth and metastasis by modulating the tumor microenvironment.

INTRODUCTION: Although C-X-C motif chemokine 12 (CXCL12) has been shown to bind to C-X-C chemokine receptor type 7 (CXCR7), the exact molecular mechanism regulations by CXCL12/CXCR7 axis in breast tumor growth and metastasis are not well understood. CXCR7 expression has been shown to be upregulated during pathological processes such as inflammation and cancer. METHODS: Breast cancer cell lines were genetically silenced or pharmacologically inhibited for CXCR7 and/or its downstream target signal transducer and activator of transcription 3 (STAT3). 4T1 or 4T1 downregulated for CXCR7 and 4T1.2 breast cancer cell lines were injected in mammary gland of BALB/c mice to form tumors, and the molecular pathways regulating tumor growth and metastasis were assessed. RESULTS: In this study, we observed that CXCL12 enhances CXCR7-mediated breast cancer migration. Furthermore, genetic silencing or pharmacologic inhibition of CXCR7 reduced breast tumor growth and metastasis. Further elucidation of mechanisms revealed that CXCR7 mediates tumor growth and metastasis by activating proinflammatory STAT3 signaling and angiogenic markers. Furthermore, enhanced breast tumorigenicity and invasiveness were associated with macrophage infiltration. CXCR7 recruits tumor-promoting macrophages (M2) to the tumor site through regulation of the macrophage colony-stimulating factor (M-CSF)/macrophage colony-stimulating factor receptor (MCSF-R) signaling pathway. In addition, CXCR7 regulated breast cancer metastasis by enhancing expression of metalloproteinases (MMP-9, MMP-2) and vascular cell-adhesion molecule-1 (VCAM-1). We also observed that CXCR7 is highly expressed in invasive ductal carcinoma (IDC) and metastatic breast tissue in human patient samples. In addition, high CXCR7 expression in tumors correlates with worse prognosis for both overall survival and lung metastasis-free survival in IDC patients. CONCLUSION: These observations reveal that CXCR7 enhances breast cancer growth and metastasis via a novel pathway by modulating the tumor microenvironment. These findings identify CXCR7-mediated STAT3 activation and modulation of the tumor microenvironment as novel regulation of breast cancer growth and metastasis. These studies indicate that new strategies using CXCR7 inhibitors could be developed for antimetastatic therapy.

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.

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.

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.

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.

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.

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.

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.

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.

Regulatory mechanisms of intestinal folate uptake in a rat model of folate oversupplementation.

Folic acid is essential for numerous biological functions, ranging from nucleotide biosynthesis to the remethylation of homocysteine. Folic acid is unable to cross the biological membranes by simple diffusion, so there exists a well-developed epithelial folate transport system for the regulation of normal folate homeostasis in the intestine. Any perturbances in the folate uptake system might lead to a state of folate deficiency, which in turn is strongly associated with the risk of various cancers, birth defects and CVD. Countries with obligatory folate fortification of food (USA and Canada) have documented a significant decrease in neural tube defects in newborns. However, the effect of folate oversupplementation on the intestinal absorption of folic acid has not been studied. We studied the process of folate transport and the expression of folate transporters in the rat intestine after folate oversupplementation. Rats were oversupplemented with tenfold the normal requirement of folic acid for periods of 10 and 60 d. Folate uptake in intestinal brush-border membrane vesicles followed saturable kinetics with pH optimum at 5·5. Acute, but not chronic, folate oversupplementation led to a significant down-regulation in intestinal folate uptake at acidic pH optima and was associated with a decrease in Vmax without any significant change in the Km of the folate uptake process. The decrease in folate uptake was also associated with the down-regulation in the protein levels of major folate transporters, proton-coupled folate transporter (PCFT) and reduced folate carrier (RFC), without altering their mRNA levels. Hence, it was concluded that acute folate oversupplementation results in a significant decrease in intestinal folate uptake by down-regulating the expressions of RFC and PCFT, via some post-transcriptional or translational mechanisms.