2,3,7,8-Tetrachlorodibenzo-p-dioxin induces multigenerational alterations in the expression of microRNA in the thymus through epigenetic modifications.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a potent AhR ligand, is an environmental contaminant that is known for mediating toxicity across generations. However, whether TCDD can induce multigenerational changes in the expression of microRNAs (miRs) has not been previously studied. In the current study, we investigated the effect of administration of TCDD in pregnant mice (F0) on gestational day 14, on the expression of miRs in the thymus of F0 and subsequent generations (F1 and F2). Of the 3200 miRs screened, 160 miRs were dysregulated similarly in F0, F1, and F2 generations, while 46 miRs were differentially altered in F0 to F2 generations. Pathway analysis revealed that the changes in miR signature profile mediated by TCDD affected the genes that regulate cell signaling, apoptosis, thymic atrophy, cancer, immunosuppression, and other physiological pathways. A significant number of miRs that showed altered expression exhibited dioxin response elements (DRE) on their promoters. Focusing on one such miR, namely miR-203 that expressed DREs and was induced across F0 to F2 by TCDD, promoter analysis showed that one of the DREs expressed by miR-203 was functional to TCDD-mediated upregulation. Also, the histone methylation status of H3K4me3 in the miR-203 promoter was significantly increased near the transcriptional start site in TCDD-treated thymocytes across F0 to F2 generations. Genome-wide chromatin immunoprecipitation sequencing study suggested that TCDD may cause alterations in histone methylation in certain genes across the three generations. Together, the current study demonstrates that gestational exposure to TCDD can alter the expression of miRs in F0 through direct activation of DREs as well as across F0, F1, and F2 generations through epigenetic pathways.

AhR Activation Leads to Massive Mobilization of Myeloid-Derived Suppressor Cells with Immunosuppressive Activity through Regulation of CXCR2 and MicroRNA miR-150-5p and miR-543-3p That Target Anti-Inflammatory Genes.

The compound 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), an environmental contaminant, is a potent ligand for aryl hydrocarbon receptor (AhR). In the current study, we made an exciting observation that naive C57BL/6 mice that were exposed i.p. to TCDD showed massive mobilization of myeloid-derived suppressor cells (MDSCs) in the peritoneal cavity. These MDSCs were highly immunosuppressive and attenuated Con A-induced hepatitis upon adoptive transfer. TCDD administration in naive mice also led to induction of several chemokines and cytokines in the peritoneal cavity and serum (CCL2, CCL3, CCL4, CCL11, CXCL1, CXCL2, CXCL5, CXCL9, G-CSF, GM-CSF, VEGF, and M-CSF) and chemokine receptors on MDSCs (CCR1, CCR5, and CXCR2). Treatment with CXCR2 or AhR antagonist in mice led to marked reduction in TCDD-induced MDSCs. TCDD-induced MDSCs had high mitochondrial respiration and glycolytic rate and exhibited differential microRNA (miRNA) expression profile. Specifically, there was significant downregulation of miR-150-5p and miR-543-3p. These two miRNAs targeted and enhanced anti-inflammatory and MDSC-regulatory genes, including IL-10, PIM1, ARG2, STAT3, CCL11 and its receptors CCR3 and CCR5 as well as CXCR2. The role of miRs in MDSC activation was confirmed by transfection studies. Together, the current study demonstrates that activation of AhR in naive mice triggers robust mobilization of MDSCs through induction of chemokines and their receptors and MDSC activation through regulation of miRNA expression. AhR ligands include diverse compounds from environmental toxicants, such as TCDD, that are carcinogenic to dietary indoles that are anti-inflammatory. Our studies provide new insights on how such ligands may regulate health and disease through induction of MDSCs.

Computational prediction and in vitro validation of VEGFR1 as a novel protein target for 2,3,7,8-tetrachlorodibenzo-p-dioxin.

The toxic manifestations of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), an environmental contaminant, primarily depend on its ability to activate aryl hydrocarbon receptor (AhR), which is a ligand-dependent transcription factor belonging to the superfamily of basic-helix-loop-helix DNA-binding proteins. In the present study, we aimed to identify novel protein receptor targets for TCDD using computational and in vitro validation experiments. Interestingly, results from computational methods predicted that Vascular Endothelial Growth Factor Receptor 1 (VEGFR1) could be one of the potential targets for TCDD in both mouse and humans. Results from molecular docking studies showed that human VEGFR1 (hVEGFR1) has less affinity towards TCDD compared to the mouse VEGFR1 (mVEGFR1). In vitro validation results showed that TCDD can bind and phosphorylate hVEGFR1. Further, results from molecular dynamic simulation studies showed that hVEGFR1 interaction with TCDD is stable throughout the simulation time. Overall, the present study has identified VEGFR1 as a novel target for TCDD, which provides the basis for further elucidating the role of TCDD in angiogenesis.

MicroRNA-92 Expression in CD133+ Melanoma Stem Cells Regulates Immunosuppression in the Tumor Microenvironment via Integrin-Dependent Activation of TGFß.

In addition to being refractory to treatment, melanoma cancer stem cells (CSC) are known to suppress host antitumor immunity, the underlying mechanisms of which need further elucidation. In this study, we established a novel role for miR-92 and its associated gene networks in immunosuppression. CSCs were isolated from the B16-F10 murine melanoma cell line based on expression of the putative CSC marker CD133 (Prominin-1). CD133+ cells were functionally distinct from CD133- cells and showed increased proliferation in vitro and enhanced tumorigenesis in vivo. CD133+ CSCs also exhibited a greater capacity to recruit immunosuppressive cell types during tumor formation, including FoxP3+ Tregs, myeloid-derived suppressor cells (MDSC), and M2 macrophages. Using microarray technology, we identified several miRs that were significantly downregulated in CD133+ cells compared with CD133- cells, including miR-92. Decreased expression of miR-92 in CSCs led to higher expression of target molecules integrin αV and α5 subunits, which, in turn, enhanced TGFß activation, as evidenced by increased phosphorylation of SMAD2. CD133+ cells transfected with miR-92a mimic and injected in vivo showed significantly decreased tumor burden, which was associated with reduced immunosuppressive phenotype intratumorally. Using The Cancer Genome Atlas database of patients with melanoma, we also noted a positive correlation between integrin α5 and TGFß1 expression levels and an inverse association between miR-92 expression and integrin alpha subunit expression. Collectively, this study suggests that a miR-92-driven signaling axis involving integrin activation of TGFß in CSCs promotes enhanced tumorigenesis through induction of intratumoral immunosuppression. SIGNIFICANCE: CD133+ cells play an active role in suppressing melanoma antitumor immunity by modulating miR-92, which increases influx of immunosuppressive cells and TGFß1 expression.

Role of microRNA in CB1 antagonist-mediated regulation of adipose tissue macrophage polarization and chemotaxis during diet-induced obesity.

Although cannabinoid receptor 1 (CB1) antagonists have been shown to attenuate diet-induced obesity (DIO) and associated inflammation, the precise molecular mechanisms involved are not clear. In the current study, we investigated the role of microRNA (miR) in the regulation of adipose tissue macrophage (ATM) phenotype following treatment of DIO mice with the CB1 antagonist SR141716A. DIO mice were fed high-fat diet (HFD) for 12 weeks and then treated daily with SR141716A (10 mg/kg) for 4 weeks while continuing HFD. Treated mice experienced weight loss, persistent reduction in fat mass, improvements in metabolic profile, and decreased adipose inflammation. CB1 blockade resulted in down-regulation of several miRs in ATMs, including the miR-466 family and miR-762. Reduced expression of the miR-466 family led to induction of anti-inflammatory M2 transcription factors KLF4 and STAT6, whereas down-regulation of miR-762 promoted induction of AGAP-2, a negative regulator of the neuroimmune retention cues, Netrin-1 and its coreceptor UNC5B. Furthermore, treatment of primary macrophages with SR141716A up-regulated KLF4 and STAT6, reduced secretion of Netrin-1, and increased migration toward the lymph node chemoattractant CCL19. These studies demonstrate for the first time that CB1 receptor blockade attenuates DIO-associated inflammation through alterations in ATM miR expression that promote M2 ATM polarization and macrophage egress from adipose tissue. The current study also identifies additional novel therapeutic targets for diet-induced obesity and metabolic disorder.

Role of miRNA in the regulation of cannabidiol-mediated apoptosis in neuroblastoma cells.

Neuroblastoma (NBL) is one of the most common childhood cancers that originate from the immature nerve cells of the sympathetic system. Studies with NBL cancers have also shown that miRNAs are dysregulated and may play a critical role in pathogenesis. Cannabidiol (CBD) is a non-psychoactive compound found in marijuana which has been previously shown by our laboratory and others to induce apoptosis in cancer cells. However, there are no studies reported to test if CBD mediates these effects through regulation of miRNA. In the current study, therefore, we investigated if CBD induces apoptosis in human NBL cell lines, SH SY5Y and IMR-32, and if it is regulated by miRNA. Our data demonstrated that CBD induces apoptosis in NBL cells through activation of serotonin and vanilloid receptors. We also found that caspase-2 and -3 played an important role in the induction of apoptosis. CBD also significantly reduced NBL cell migration and invasion in vitro. Furthermore, CBD blocked mitochondrial respiration and caused a shift in metabolism towards glycolysis. CBD altered the expression of miRNA specifically, down-regulating hsa-let-7a and upregulating hsa-mir-1972. Downregulation of let-7a increased expression of target caspase-3, and growth arrest specific-7 (GAS-7) genes. Upregulation of hsa-mir-1972 caused decreased expression of BCL2L1 and SIRT2 genes. Together, our studies suggest that CBD-mediated apoptosis in NBL cells is regulated by miRNA.

Diethylstilbestrol (DES) induces autophagy in thymocytes by regulating Beclin-1 expression through epigenetic modulation.

Diethylstilbestrol (DES) is an endocrine disruptor that was used to prevent adverse effects of pregnancy in women in late 1940s until early 1970s. Its use was banned following significant toxicity and negative effects not only in the mothers but also transgenerationally. Previous studies from our laboratory showed that DES induces thymic atrophy and immunosuppression in mice. In this study, we investigated the molecular mechanisms through which DES triggers thymic atrophy, specifically autophagy. To that end, we treated C57BL/6 mice with DES, and determined expression of two autophagy-related proteins, microtubule-associated protein-1 light chain 3 (LC3) and Beclin-1 (Becn1). We observed that DES-induced thymic atrophy was associated with increased autophagy in thymocytes and significant upregulation in the expression of both Becn1 and LC3. DES also caused downregulation in the expression of miR-30a in thymocytes, and transfection studies revealed that miR-30a targeted Becn1. Upon examination of methylation status of Becn1, we noted hypomethylation of Becn1 in thymocytes of mice exposed to DES. Together, these data demonstrate for the first time that DES induces autophagy in thymocytes potentially through epigenetic changes involving hypomethylation of Becn1 and down-regulation of miR-30a expression.

Genistein induces macrophage polarization and systemic cytokine to ameliorate experimental colitis.

Mucosal changes in Crohn's disease (CD) and ulcerative colitis (UC), two major forms of inflammatory bowel disease (IBD), are characterized by a prominent infiltration of inflammatory cells including lymphocytes, macrophages, T cells and neutrophils. The precise etiology of IBD is unknown but it involves a complex interplay of factors associated with the immune system, environment, host genotype and enteric commensal bacteria. As there is no known safe cure for IBD, natural alternative therapeutic options without side effects are urgently needed. To this end, Soy-based foods, which have been eaten for centuries in Asian countries, have potential benefits, including lowering the incidence of coronary heart disease, atherosclerosis, type-2 diabetes, allergic response, and autoimmune diseases. This study describes the effect of Soy isoflavons 4', 5, 7 Trihydroxyisoflavone (genistein) on dextran sodium sulphate (DSS) induced experimental colitis. The extent and severity of disease was analyzed through body weight, histopathological analysis, cellular immune response, systemic cytokine levels, and inflammation score using a disease activity index. Genistein treatment significantly attenuated DSS-induced colitis severity and resulted in increase in body weight, colon length and reduction in inflammation score. Genistein also skews M1 macrophages towards the M2 phenotype. Further, gen also reduced the systemic cytokine levels as compared to vehicle control. This serves as the first detailed study towards natural soya based product that shows the polarization of M1 towards M2 macrophages, and reduction of systemic cytokine in part to attenuate the colitis symptoms. Thus, our work demonstrates that genistein, a soya compound, may be useful for the treatment of IBD.

Differential role of CXCR3 in inflammation and colorectal cancer.

Chemokines (CXCR3) and their ligands (CXCL9, CXCL10, and CXCL11) exert exquisite control over T-cell trafficking and are critical for activation, differentiation and effector T cell function. CXCR3 is important for CD4 Th1 cells, CD8 effectors, memory cells, and for the function of natural killer and natural killer T cells. The presence of high cytotoxic CXCR3 ligand expression on CD8 T cells in colorectal cancerous tissue has been well documented in the past. CXCR3 and its ligands are differentially expressed at sites of inflammation and within the tumors. Further, the expression of CXCR3 and its ligands has been correlated with both the presence of effector T cells within tumor tissue and disease-free survival of patients. However, effector T cell infiltration into primary and metastatic tumors is highly variable and, in fact, often absent. Thus, understanding why T cells fail to infiltrate into tumors and determining the way to improve effector T cell entry into tumors would be important advances in efforts to harness the power of the immune system to fight cancer. To this end, the recent exciting discovery that CXCR3 is functionally expressed on regulatory T cells and also induces the differentiation of peripheral CD4 T cells into regulatory T cells, might address the novel clinically relevant question of the therapeutic potential of the CXCR3 system. This is also coupled with the fact that increases in CXCR3 expression also improves effector T cell function. This review describes the differential role of CXCR3 induction on peripheral and tumor microenvironment inflammation. Further, this review, tied with important findings from our laboratory, demonstrates that polyphenols induce CXCR3 expression on regulatory T cells and increases CXCR3 ligands in the tumor microenvironment, which act together to suppress colorectal cancer through a differential mechanism discussed herewith.

Resveratrol-Mediated Attenuation of Staphylococcus aureus Enterotoxin B-Induced Acute Liver Injury Is Associated With Regulation of microRNA and Induction of Myeloid-Derived Suppressor Cells.

Resveratrol (RES) is a polyphenolic compound found abundantly in plant products including red grapes, peanuts, and mulberries. Because of potent anti-inflammatory properties of RES, we investigated whether RES can protect from Staphylococcal enterotoxin B (SEB)-induced acute liver injury in mice. SEB is a potent super antigen that induces robust inflammation and releases inflammatory cytokines that can be fatal. We observed that SEB caused acute liver injury in mice with increases in enzyme aspartate transaminase (AST) levels, and massive infiltration of immune cells into the liver. Treatment with RES (100 mg/kg body weight) attenuated SEB-induced acute liver injury, as indicated by decreased AST levels and cellular infiltration in the liver. Interestingly, RES treatment increased the number of myeloid derived suppressor cells (MDSCs) in the liver. RES treatment led to alterations in the microRNA (miR) profile in liver mononuclear cells (MNCs) of mice exposed to SEB, and pathway analysis indicated these miRs targeted many inflammatory pathways. Of these, we identified miR-185, which was down-regulated by RES, to specifically target Colony Stimulating Factor (CSF1) using transfection studies. Moreover, the levels of CSF1 were significantly increased in RES-treated SEB mice. Because CSF1 is critical in MDSC induction, our studies suggest that RES may induce MDSCs by down-regulating miR-185 leading to increase the expression of CSF1. The data presented demonstrate for the first time that RES can effectively attenuates SEB-induced acute liver injury and that this may result from its action on miRs and induction of MDSCs.

Antibody blockade of CLEC12A delays EAE onset and attenuates disease severity by impairing myeloid cell CNS infiltration and restoring positive immunity.

The mechanism of dendritic cells (DCs) recruitment across the blood brain barrier (BBB) during neuroinflammation has been the least explored amongst all leukocytes. For cells of myeloid origin, while integrins function at the level of adhesion, the importance of lectins remains unknown. Here, we identified functions of one C-type lectin receptor, CLEC12A, in facilitating DC binding and transmigration across the BBB in response to CCL2 chemotaxis. To test function of CLEC12A in an animal model of multiple sclerosis (MS), we administered blocking antibody to CLEC12A that significantly ameliorated disease scores in MOG35-55-induced progressive, as well as PLP138-151-induced relapsing-remitting experimental autoimmune encephalomyelitis (EAE) mice. The decline in both progression and relapse of EAE occurred as a result of reduced demyelination and myeloid cell infiltration into the CNS tissue. DC numbers were restored in the spleen of C57BL/6 and peripheral blood of SJL/J mice along with a decreased TH17 phenotype within CD4+ T-cells. The effects of CLEC12A blocking were further validated using CLEC12A knockout (KO) animals wherein EAE disease induction was delayed and reduced disease severity was observed. These studies reveal the utility of a DC-specific mechanism in designing new therapeutics for MS.

Fatty acid amide hydrolase (FAAH) blockade ameliorates experimental colitis by altering microRNA expression and suppressing inflammation.

Inflammatory bowel disease (IBD), including Crohn's disease (CD) and ulcerative colitis (UC), which is thought to result from immune-mediated inflammatory disorders, leads to high morbidity and health care cost. Fatty acid amide hydrolase (FAAH) is an enzyme crucially involved in the modulation of intestinal physiology through anandamide (AEA) and other endocannabinoids. Here we examined the effects of an FAAH inhibitor (FAAH-II), on dextran sodium sulphate (DSS)-induced experimental colitis in mice. Treatments with FAAH-II improved overall clinical scores by reversing weight loss and colitis-associated pathogenesis. The frequencies of activated CD4+ T cells in spleens, mesenteric lymph nodes (MLNs), Peyer's patches (PPs), and colon lamina propiria (LP) were reduced by FAAH inhibition. Similarly, the frequencies of macrophages, neutrophils, natural killer (NK), and NKT cells in the PPs and LP of mice with colitis declined after FAAH blockade, as did concentrations of systemic and colon inflammatory cytokines. Microarray analysis showed that 26 miRNAs from MLNs and 217 from PPs had a 1.5-fold greater difference in expression after FAAH inhibition. Among them, 8 miRNAs were determined by reverse-transcription polymerase chain reaction (RT-PCR) analysis to have anti-inflammatory properties. Pathway analysis demonstrated that differentially regulated miRNAs target mRNA associated with inflammation. Thus, FAAH-II ameliorates experimental colitis by reducing not only the number of activated T cells but also the frequency of macrophages, neutrophils, and NK/NKT cell, as well as inflammatory miRNAs and cytokine at effector sites in the colon. These studies demonstrate for the first time that FAAH-II inhibitor may suppress colitis through regulation of pro-inflammatory miRNAs expression.

The comet assay: Reflections on its development, evolution and applications.

The study of DNA damage and its repair is critical to our understanding of human aging and cancer. This review reflects on the development of a simple technique, now known as the comet assay, to study the accumulation of DNA damage and its repair. It describes my journey into aging research and the need for a method that sensitively quantifies DNA damage on a cell-by-cell basis and on a day-by-day basis. My inspirations, obstacles and successes on the path to developing this assay and improving its reliability and sensitivity are discussed. Recent modifications, applications, and the process of standardizing the technique are also described. What was once untried and unknown has become a technique used around the world for understanding and monitoring DNA damage. The comet assay's use has grown exponentially in the new millennium, as emphasis on studying biological phenomena at the single-cell level has increased. I and others have applied the technique across cell types (including germ cells) and species (including bacteria). As it enters new realms and gains clinical relevance, the comet assay may very well illuminate human aging and its prevention.

Effects of radiation from a radiofrequency identification (RFID) microchip on human cancer cells.

PURPOSE: Radiofrequency identification (RFID) microchips are used to remotely identify objects, e.g. an animal in which a chip is implanted. A passive RFID microchip absorbs energy from an external source and emits a radiofrequency identification signal which is then decoded by a detector. In the present study, we investigated the effect of the radiofrequency energy emitted by a RFID microchip on human cancer cells. MATERIALS AND METHODS: Molt-4 leukemia, BT474 breast cancer, and HepG2 hepatic cancer cells were exposed in vitro to RFID microchip-emitted radiofrequency field for 1 h. Cells were counted before and after exposure. Effects of pretreatment with the spin-trap compound N-tert-butyl-alpha-phenylnitrone or the iron-chelator deferoxamine were also investigated. Results We found that the energy effectively killed/retarded the growth of the three different types of cancer cells, and the effect was blocked by the spin-trap compound or the iron-chelator, whereas an inactive microchip and energy from the external source had no significant effect on the cells. Conclusions Data of the present study suggest that radiofrequency field from the microchip affects cancer cells via the Fenton Reaction. Implantation of RFID microchips in tumors may provide a new method for cancer treatment.

Chemokine and cytokine levels in inflammatory bowel disease patients.

Crohn's disease (CD) and ulcerative colitis (UC), two forms of inflammatory bowel disease (IBD), are chronic, relapsing, and tissue destructive lesions that are accompanied by the uncontrolled activation of effector immune cells in the mucosa. Recent estimates indicate that there are 1.3 million annual cases of IBD in the United States, 50% of which consists of CD and 50% of UC. Chemokines and cytokines play a pivotal role in the regulation of mucosal inflammation by promoting leukocyte migration to sites of inflammation ultimately leading to tissue damage and destruction. In recent years, experimental studies in rodents have led to a better understanding of the role played by these inflammatory mediators in the development and progression of colitis. However, the clinical literature on IBD remains limited. Therefore, the aim of this study was to evaluate systemic concentrations of key chemokines and cytokines in forty-two IBD patients with a range of disease activity compared to levels found in ten healthy donors. We found a significant increase in an array of chemokines including macrophage migration factor (MIF), CCL25, CCL23, CXCL5, CXCL13, CXCL10, CXCL11, MCP1, and CCL21 in IBD patients as compared to normal healthy donors (P<0.05). Further, we also report increases in the inflammatory cytokines IL-16, IFN-γ, IL-1ß and TNF-α in IBD patients when compared to healthy donors (P<0.05). These data clearly indicate an increase in circulating levels of specific chemokines and cytokines that are known to modulate systemic level through immune cells results in affecting local intestinal inflammation and tissue damage in IBD patients. Blockade of these inflammatory mediators should be explored as a mechanism to alleviate or even reverse symptoms of IBD.

DNA damage in dihydroartemisinin-resistant Molt-4 cells.

Artemisinin generates carbon-based free radicals when it reacts with iron, and induces molecular damage and apoptosis. Its toxicity is more selective toward cancer cells because cancer cells contain a higher level of intracellular free iron. Dihydroartemisinin (DHA), an analog of artemisinin, has selective cytotoxicity toward Molt-4 human lymphoblastoid cells. A major concern is whether cancer cells could develop resistance to DHA, thus limiting its therapeutic efficacy. We have developed a DHA-resistant Molt-4 cell line (RTN) and found out that these cells exhibited resistance to DHA but no significant cross- resistance to artemisinin-tagged holotransferrin (ART-TF), a synthetic artemisinin compound. In the present study, we investigated DNA damage induced by DHA and ART-TF in both Molt-4 and RTN cells using the comet assay. RTN cells exhibited a significantly lower level of basal and X-ray-induced DNA damage compared to Molt-4 cells. Both DHA and ART-TF induced DNA damage in Molt-4 cells, whereas DNA damage was induced in RTN cells by ART-TF, and not DHA. The result of this study shows that by the cell selection method, it is possible to generate a Molt-4 cell line which is not sensitive to DHA, but sensitive to ART-TF, as measured by DNA damage.

Exposure to diethylstilbestrol during pregnancy modulates microRNA expression profile in mothers and fetuses reflecting oncogenic and immunological changes.

Prenatal exposure to diethylstilbestrol (DES) is known to cause an increased susceptibility to a wide array of clinical disorders in humans. Previous studies from our laboratory demonstrated that prenatal exposure to DES induces thymic atrophy and apoptosis in the thymus. In the current study, we investigated if such effects on the thymus result from alterations in the expression of microRNA (miR). To that end, pregnant C57BL/6 mice who were exposed to DES and miR profiles in thymocytes of both the mother and fetuses on postnatal day 3 (gestation day 17) were studied. Of the 609 mouse miRs examined, we noted 59 altered miRs that were common for both mothers and fetuses, whereas 107 altered miRs were specific to mothers only and 101 altered miRs were specific to fetuses only. Upon further analyses in the fetuses, we observed that DES-mediated changes in miR expression may regulate genes involved in important functions, such as apoptosis, autophagy, toxicity, and cancer. Of the miRs that showed decreased expression following DES treatment, miR-18b and miR-23a were found to possess complementary sequences and binding affinity for 3' untranslated regions of the Fas ligand (FasL) and Fas, respectively. Transfection studies confirmed that DES-mediated downregulation of miR-18b and miR-23a led to increased FasL and Fas expression. These data demonstrated that prenatal DES exposure can cause alterations in miRs, leading to changes in the gene expression, specifically, miR-mediated increased expression in FasL and Fas causing apoptosis and thymic atrophy.

Additive cytotoxic effects of dihydroartemisinin and sodium salicylate on cancer cells.

BACKGROUND/AIM: The standard methods of chemotherapy in cancer treatment are expensive and pose serious health effects. The present study investigates an alternative chemotherapy by testing the combined treatment of two drugs on leukemia cells: dihydroartemisinin (DHA) and sodium salicylate (SS). MATERIALS AND METHODS: Cells were divided into 4 treatment groups: a control, treatment with DHA-only, treatment with SS-only, and treatment with both DHA and SS. Cells were counted immediately before the addition of any reagents (0-h count), and at 24, 48, and 72 h after treatment. RESULTS AND CONCLUSION: At low concentrations, the combination of DHA and SS significantly reduced cancer cell proliferation, although no synergistic interaction between the two drugs was found. Even without a clear synergistic interaction, the combination of DHA and SS provides a safe and affordable form of cancer treatment.

Development of a dihydroartemisinin-resistant Molt-4 leukemia cell line.

Artemisinin generates cytotoxic free radicals when it reacts with iron. Its toxicity is more selective toward cancer cells because cancer cells contain a higher level of intracellular-free iron. We previously reported that dihydroartemisinin (DHA), an active metabolite of artemisinin, has selective cytotoxicity toward Molt-4 human lymphoblastoid cells. A concern is whether cancer cells could develop resistance to DHA after repeated administration, thus limiting its therapeutic efficacy. In the present study, we developed a DHA-resistant Molt-4 cell line (RTN) by exposing Molt-4 cells to gradually increasing concentrations of DHA in vitro. The half-maximal inhibitory concentration (IC50) of DHA for RTN cells is 7.1-times higher than that of Molt-4 cells. RTN cells have a higher growth rate than Molt-4 cells. In addition, we investigated the toxicities of two more potent synthetic artemisinin compounds, artemisinin dimer-alcohol and artemisinin-tagged holotransferrin toward RTN cells; RTN cells showed no significant cross-resistance to these compounds.