A study on the anticancer activity of imidazolyl benzamide derivative-IMUEB on a 549 lung cancer cell line.

Background: Cancer is a deadly disease, which is due to the uncontrolled division of cells with abnormal or unusual characteristics. It is a consequence of lethal mutations occurring due to various chemical and physical carcinogens, affecting many cellular signalling pathways and leading to uncontrolled proliferation. In this study, we analyzed the effect of 4-(1H-imidazol-1-yl)-N-(2-(3-(4-methylbenzyl) ureido) ethyl)benzamide (IMUEB), an imidazole derivative, on A549 cells (lung cancer cells). Methods: The MTT and LDH assays were performed to measure the cytotoxicity of IMUEB against A549 cells. Apoptotic mode of cell death of A549 cells was determined by fluorescence imaging by using different stains. Flow cytometry was performed to detect the cell cycle arrest. Western blotting was performed to determine the levels of apoptotic protein. Wound healing assay was performed to find the effect of IMUEB on cell migration. In silico molecular docking of IMUEB was performed to predict its affinity towards apoptotic proteins and metastasis related enzymes. Result and Discussion: The MTT assay showed an increase in cytotoxicity with increasing concentrations of IMUEB. In addition, it was found that IMUEB arrests cell cycle at G1 phase as detected by flow cytometry analysis and induces apoptosis. The treatment with IMUEB drastically decreased the migratory potential of A549 cells as evaluated by migration and invasion assay. By Western blotting analysis, it was found that the concentration of caspase-3 was increased after the treatment with IMUEB. Conclusion: Altogether, our results indicate that IMUEB shows antitumor activity by inhibiting proliferation and inducing apoptosis in A549 cells.

Role of indole curcumin in the epigenetic activation of apoptosis and cell cycle regulating genes.

Background: Head-and-neck squamous cell carcinoma is associated with the epigenetic silencing of various genes such as DAPK, ataxia telangiectasia mutated (ATM), BRCA1, p16INK4a, pVHL, p16, and RASSF1A. The most common epigenetic change observed in these genes is DNA methylation that directs the studies toward finding inhibitors for DNA methyltransferases (DNMTs), the protagonist in the action. The present study focuses on analyzing the possibility whether indole curcumin can reverse epigenetic changes of the various tumor suppressor genes, characteristically silenced by methylation, by inhibiting the major methylation enzyme DNA methyltransferase 1 or DNMT1. Materials and Methods: The cytotoxic effects of indole curcumin were studied through the MTT and lactate dehydrogenase assays. To determine the apoptosis-mediated death of HEp-2 cells, fluorescence imaging using different stains was done. Gene or mRNA expression analysis was done for p53, ATM, and DAPK genes. Results: The results obtained from this study clearly indicate that the indole analog of curcumin plays a remarkable role in activating genes involved in cell cycle regulation and apoptosis induction through epigenetic regulation. The influence that the drug has on the methylation status of gene promoter sequence of the ATM gene is also very significant. Conclusion: Indole curcumin, being an analog of curcumin, promises to be a very useful drug molecule having various potential targets. The target selected for this study was DNMT1 enzyme and the drug seems to actually show the effects; it was predicted to be having on the target molecule.

Indole curcumin combats metastatic HBV-positive hepatocellular carcinoma by inhibiting cell proliferation, migration, and matrix metalloproteinase-9 activity.

Background: Chemical modification of the natural products and molecules can lead us toward drugs with lesser off-target effects for chemotherapeutic use against cancers. In this study, we explored the effect of an indole analog of the molecule curcumin, for the first time against HBV-positive hepatocellular carcinoma (HCC) cells in vitro. Materials and Methods: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase assays were used to measure the cytotoxic effects of indole curcumin against Hep3B cells. The mode of cell death was established through acridine orange/ethidium bromide fluorescence staining, propidium iodide fluorescence staining, and the comet assay. The effect of the compound on cell migration behavior was studied through wound healing assay, whereas the effect on matrix metalloproteinase (MMP) activity was evaluated using gelatin zymography. In silico molecular docking was performed to predict the affinity of indole curcumin toward probable intracellular interacting partners. Results and Discussion: Indole curcumin had an antiproliferative effect on Hep3B cells, induced apoptotic mode of cell death, inhibited cell migration in time- and dose-dependent assays, and decreased MMP-9 activity levels. Molecular docking results suggest that the interaction of PI3K with indole curcumin may have led to downregulation of MMP-9 expression, thereby contributing to the overall reduction in MMP-9 activity. Conclusion: Our study establishes that indole curcumin is an effective cytotoxic and antimetastatic agent against hepatitis virus-B positive HCC cells. Hence, it can be a possible candidate for the treatment of hepatocarcinoma induced/promoted by the presence of chronic hepatitis B infection.

Implications of Three-Dimensional Cell Culture in Cancer Therapeutic Research.

Replicating the naturalistic biomechanical milieu of cells is a primary requisite to uncover the fundamental life processes. The native milieu is significantly not replicated in the two-dimensional (2D) cell cultures. Alternatively, the current three-dimensional (3D) culture techniques can replicate the properties of extracellular matrix (ECM), though the recreation of the original microenvironment is challenging. The organization of cells in a 3D manner contributes to better insight about the tumorigenesis mechanism of the in vitro cancer models. Gene expression studies are susceptible to alterations in their microenvironment. Physiological interactions among neighboring cells also contribute to gene expression, which is highly replicable with minor modifications in 3D cultures. 3D cell culture provides a useful platform for identifying the biological characteristics of tumor cells, particularly in the drug sensitivity area of translational medicine. It promises to be a bridge between traditional 2D culture and animal experiments and is of great importance for further research in tumor biology. The new imaging technology and the implementation of standard protocols can address the barriers interfering with the live cell observation in a natural 3D physiological environment.

Epigenetic modulation and apoptotic induction by a novel imidazo-benzamide derivative in human lung adenocarcinoma cells.

PURPOSE: Lung cancer is the most commonly diagnosed and leading cause of cancer death worldwide. Imidazo-benzamides are considered to be good anti-cancer agents. The present study was aimed to investigate the cytotoxicity of a novel imidazo-benzamide derivative N-(2-(3-(tert-butyl)ureido)ethyl)-4-(1H-imidazol-1-yl)benzamide (TBUEIB) in lung cancer cell line A549. METHODS: The antiproliferative activity of TBUEIB was investigated using MTT, LDH and trypan blue assay. The apoptotic potential was investigated using various staining techniques and further confirmed by DNA fragmentation assay and western blotting. RESULTS: TBUEIB inhibited fifty precent A549 cells at a dose of 106 µM. The novel compound was found to exert a modulatory effect on apoptotic marker caspase-3 as well as epigenetic regulatory proteins like DNA Methyltransferase 1 (DNMT1). In silico studies with the compound and other epigenetic proteins such as Histone deacetylase (HDAC) and ubiquitin-like with PHD (plant homeodomain) and RING (Really Interesting New Gene) finger domains 1(UHRF1) showed good modulatory effects. CONCLUSION: The overall results obtained in the study conclude that the novel compound TBUEIB has potential anti-cancer activities, mainly by targeting the expression of DNMT1 enzyme, which may have re-activated the major tumor suppressor genes involved in the cell cycle, leading to the apoptosis of the cancer cells. The results also indicate that the compound has more than one target in the epigenetic pathway implying that the compound may be a potential multi-target compound.

A novel anticancer chromeno-pyrimidine analogue inhibits epithelial-mesenchymal transition in lung adenocarcinoma cells.

Cancer is the second most dreaded disease worldwide. It is either acquired or inherited leading to the accompanying undesirable changes in the affected cells. Most existing chemotherapeutic drugs show enormous side effects. To minimize such effects, constant progress has been observed in the field of cancer by screening the anti-cancer effects of different chemical analogues. In the current study, we investigated the mechanism of action of a novel anticancer chromeno-pyrimidine analogue. We employed MTT, LDH assay to study cytotoxicity. DNA fragmentation, fluorescence imaging, and flow cytometric techniques have been carried out to study apoptosis, ROS generation, and cell cycle respectively. Wound healing assay and western blotting were used to evaluate the markers of epithelial-mesenchymal transition associated with metastasis. Molecular docking was used to predict possible protein targets that bind to this compound. The novel analogue induced apoptosis in lung adenocarcinoma cells and exhibited anti-metastatic activity. Increased expression of E-cadherin and inhibition of epithelial-mesenchymal transition was also observed. Docking studies with metastasis-related proteins such as Frizzled-7 (CRD), and Snail1 predict a high binding affinity of CP4b to both proteins. The novel analogue is therefore an anti-metastatic compound with EMT-inhibiting property and is hypothesized to act via binding to multiple targets in cancer cells.

Long Non-Coding RNAs Regulating Immunity in Insects.

Recent advances in modern technology have led to the understanding that not all genetic information is coded into protein and that the genomes of each and every organism including insects produce non-coding RNAs that can control different biological processes. Among RNAs identified in the last decade, long non-coding RNAs (lncRNAs) represent a repertoire of a hidden layer of internal signals that can regulate gene expression in physiological, pathological, and immunological processes. Evidence shows the importance of lncRNAs in the regulation of host-pathogen interactions. In this review, an attempt has been made to view the role of lncRNAs regulating immune responses in insects.

Crowding interactions perturb structure and stability by destabilizing the stable core of the α-subunit of tryptophan synthase.

The consequences of crowding derived from relatively small and intrinsically disordered proteins are not clear yet. We report the effect of ficoll-70 on the structure and stability of native and partially folded states of the 29 kDa alpha subunit of tryptophan synthase (αTS). Overall, combining the changes in the circular dichroism and fluorescence spectra, in conjunction with the gradual loss of cooperativity under urea denaturation in the presence of increasing amounts of ficoll, it may be concluded that the crowding agent perturbs not only the native state but also the partially folded state of αTS. Importantly, NMR data indicate that ficoll interacts with the residues that constitute the stable core of the protein thus shedding light on the origin of the observed perturbation.

Antiproliferative and apoptotic effects of Sesbania grandiflora leaves in human cancer cells.

Natural phytochemicals and their derivatives are good drug candidates for anticancer therapeutic approaches against multiple targets. We report here the initial findings from our studies on the anticancer properties of the leaves of the medicinal plant Sesbania grandiflora. In the current study, five different solvent fractions from the leaves of S. grandiflora were tested on cancer cell lines such as MCF-7, HepG2, Hep-2, HCT-15, and A549. The methanolic fraction of S. grandiflora was found to exert potent antiproliferative effects especially in the human lung cancer cell line, A549. Caspase 3 was activated in the methanolic fraction treated A549 cells thereby leading to cell death by apoptosis. DAPI staining, DNA laddering, and decrease in mitochondrial membrane potential further confirmed the apoptotic mode of cell death. The high levels of ROS intermediates as evidenced by DCF-DA staining could have played a role in the apoptotic induction. Decrease in levels of cyclin D1 and decrease in the activation of NFkB were observed in A549 cells on treatment with methanolic fraction, giving a hint on the possible mechanism of action. These results prove that the medicinal plant S. grandiflora can be explored further for promising candidate molecules to combat cancer, especially lung cancer.

Innate lymphoid cells responding to IL-33 mediate airway hyperreactivity independently of adaptive immunity.

BACKGROUND: Asthma has been considered an immunologic disease mediated by T(H)2 cells and adaptive immunity. However, clinical and experimental observations suggest that additional pathways might regulate asthma, particularly in its nonallergic forms, such as asthma associated with air pollution, stress, obesity, and infection. OBJECTIVES: Our goal was to understand T(H)2 cell-independent conditions that might lead to airway hyperreactivity (AHR), a cardinal feature of asthma. METHODS: We examined a murine model of experimental asthma in which AHR was induced with glycolipid antigens, which activate natural killer T (NKT) cells. RESULTS: In this model AHR developed rapidly when mice were treated with NKT cell-activating glycolipid antigens, even in the absence of conventional CD4(+) T cells. The activated NKT cells directly induced alveolar macrophages to produce IL-33, which in turn activated NKT cells, as well as natural helper cells, a newly described non-T, non-B, innate lymphoid cell type, to increase production of IL-13. Surprisingly, this glycolipid-induced AHR pathway required not only IL-13 but also IL-33 and its receptor, ST2, because it was blocked by an anti-ST2 mAb and was greatly reduced in ST2(-/-) mice. When adoptively transferred into IL-13(-/-) mice, both wild-type natural helper cells and NKT cells were sufficient for the development of glycolipid-induced AHR. CONCLUSION: Because plant pollens, house dust, and some bacteria contain glycolipids that can directly activate NKT cells, these studies suggest that AHR and asthma can fully develop or be greatly enhanced through innate immune mechanisms involving IL-33, natural helper cells, and NKT cells.

Clinical Toxicities of Histone Deacetylase Inhibitors.

The HDAC inhibitors are a new family of antineoplastic agents. Since the entry of these agents into our therapeutic armamentarium, there has been increasing interest in their use. Although this family comprises chemical compounds from unrelated chemical classes that have different HDAC isoform specificities, they surprisingly have very similar toxicity profiles. In contrast, the observed toxicity profile is somewhat different from that of traditional cytotoxic chemotherapeutic agents and from other epigenetic agents. While some of the side effects may be familiar to the oncologist, others are less commonly seen. As some patients remain on therapy for a prolonged period of time, the long-term sequelae need to be characterized. In addition, since preclinical models suggest promising activity when used in combination with other antineoplastic agents, combination trials are being pursued. It will thus be important to distinguish the relative toxicity attributed to these agents and be alert to the exacerbation of toxicities observed in single agent studies. Notably, few of the agents in this class have completed phase 2 testing. Consequently, more clinical experience is needed to determine the relative frequency of the observed side effects, and to identify and develop approaches to mitigate potential clinical sequelae.

CXCR4/CXCL12 hyperexpression plays a pivotal role in the pathogenesis of lupus.

Among various surface molecules screened, CXCR4 was significantly up-regulated on monocytes, neutrophils, B cell subsets, and plasma cells in multiple murine models of lupus with active nephritis, including B6.Sle1Yaa, BXSB, and MRL.lpr. TLR-mediated signaling and inflammatory cytokines accounted in part for this increase. Increased CXCR4 expression was associated with functional consequences, including increased migration and enhanced B cell survival. Simultaneously, the ligand for CXCR4, CXCL12, was significantly up-regulated in the nephritic kidneys. Treatment with a peptide antagonist of CXCR4 prolonged survival and reduced serum autoantibodies, splenomegaly, intrarenal leukocyte trafficking, and end organ disease in a murine model of lupus. These findings underscore the pathogenic role of CXCR4/CXCL12 in lymphoproliferative lupus and lupus nephritis and highlight this axis as a promising therapeutic target in this disease.

A Tlr7 translocation accelerates systemic autoimmunity in murine lupus.

The y-linked autoimmune accelerating (yaa) locus is a potent autoimmune disease allele. Transcription profiling of yaa-bearing B cells revealed the overexpression of a cluster of X-linked genes that included Tlr7. FISH analysis demonstrated the translocation of this segment onto the yaa chromosome. The resulting overexpression of Tlr7 increased in vitro responses to Toll-like receptor (TLR) 7 signaling in all yaa-bearing males. B6.yaa mice are not overtly autoimmune, but the addition of Sle1, which contains the autoimmune-predisposing Slam/Cd2 haplotype, causes the development of fatal lupus with numerous immunological aberrations. B6.Sle1yaa CD4 T cells develop the molecular signature for T(FH) cells and also show expression changes in numerous cytokines and chemokines. Disease development and all component autoimmune phenotypes were inhibited by Sles1, a potent suppressor locus. Sles1 had no effect on yaa-enhanced TLR7 signaling in vitro, and these data place Sles1 downstream from the lesion in innate immune responses mediated by TLR7, suggesting that Sles1 modulates the activation of adaptive immunity in response to innate immune signaling.

The importance of epistatic interactions in the development of autoimmunity.

Genetic predisposition is the main element in susceptibility to a variety of autoimmune diseases, including systemic lupus erythematosus (SLE). Epistatic interactions between susceptibility loci play a major role in the progression of autoimmunity, as our recent work, associating a common haplotype of the SLAM/CD2 receptor family with autoimmune susceptibility in specific genomic contexts, has illustrated. Furthermore, these interactions can be abrogated in the presence of appropriate modifier loci. We postulate that susceptibility to autoimmunity may be a consequence of imbalances in immune regulation that are elicited by specific combinations of common alleles at multiple, immunoregulatory loci.

Epistatic suppression of systemic lupus erythematosus: fine mapping of Sles1 to less than 1 mb.

Sle is a susceptibility locus for systemic autoimmunity derived from the lupus-prone NZM2410 mouse. The New Zealand White-derived suppressive modifier Sles1 was identified as a specific modifier of Sle1 and prevents the development of IgG anti-chromatin autoantibodies mediated by Sle1 on the C57BL/6 (B6) background. Fine mapping of Sles1 with truncated congenic intervals localizes it to a approximately 956-kb segment of mouse chromosome 17. Sles1 completely abrogates the development of activated T and B cell populations in B6.Sle1. Despite this suppression of the Sle1-mediated cell surface activation phenotypes, B6.Sle1 Sles1 splenic B cells still exhibit intrinsic ERK phosphorylation. Classic genetic complementation tests using the nonautoimmmune 129/SvJ mouse suggests that this strain possesses a Sles1 allele complementary to that of New Zealand White, as evidenced by the lack of glomerulonephritis, splenomegaly, and antinuclear autoantibody production seen in (129 x B6.Sle1 Sles1)F(1)s. These findings localize and characterize the suppressive properties of Sles1 and implicate 129 as a useful strain for aiding in the identification of this elusive epistatic modifier gene.

Selective expression of the 21-kilodalton tyrosine-phosphorylated form of TCR zeta promotes the emergence of T cells with autoreactive potential.

T cells undergo negative selection in the thymus to eliminate potentially autoreactive cells. The signals generated through the alphabeta TCR following receptor interactions with peptide/MHC complexes in the thymus control these selection processes. Following receptor ligation, a fraction of the TCR zeta subunit appears as two distinct tyrosine-phosphorylated forms of 21 and 23 kDa (p21 and p23). Previous data have reported elevated levels of p21 in some murine models of autoimmunity. We have examined the contributions of both the p21 and p23 to T cell negative selection in the HY TCR-transgenic system using ITAM-substituted TCR zeta and CD3 epsilon transgenic mice. Expression of just p21, in the absence of p23, partially impairs negative selection of self-reactive HY-specific T cells. This results in the emergence of potentially autoreactive peripheral T cells and an elevated population of CD11b(+)B220(+) B cells in the spleen. These data clearly identify a specific and unique role for p21 during negative selection.

Association of extensive polymorphisms in the SLAM/CD2 gene cluster with murine lupus.

Susceptibility to autoimmunity in B6.Sle1b mice is associated with extensive polymorphisms between two divergent haplotypes of the SLAM/CD2 family of genes. The B6.Sle1b-derived SLAM/CD2 family haplotype is found in many other laboratory mouse strains but only causes autoimmunity in the context of the C57Bl/6 (B6) genome. Phenotypic analyses have revealed variations in the structure and expression of several members of the SLAM/CD2 family in T and B lymphocytes from B6.Sle1b mice. T lymphocytes from B6.Sle1b mice have modified signaling responses to stimulation at 4-6 weeks of age. While autoimmunity may be mediated by a combination of genes in the SLAM/CD2 family cluster, the strongest candidate is Ly108, a specific isoform of which is constitutively upregulated in B6.Sle1b lymphocytes.

Fenfluramine-induced hypothermia is associated with cutaneous dilation in conscious rats.

The antiobesity agent, fenfluramine, produces hypothermia in rodents by an, as yet, uncharacterized mechanism. The present study was conducted in conscious rats to determine if fenfluramine-induced hypothermia was associated with cutaneous dilation. In animals maintained at 16 degrees C, core body temperature (T(CORE)) was measured telemetrically, and tail surface temperature was monitored with thermocouples fixed to the tail (T(TAIL)). D-Fenfluramine (10 mg/kg ip) produced a rapid increase in T(TAIL) of 7.7+/-0.4 degrees C (P<.001) and a decline in T(CORE) of 4+/-0.3 degrees C (P<.001). Two findings indicate that the increase in T(TAIL) was due to the withdrawal of a sympathetic vasoconstrictor tone. First, pretreatment with the ganglionic blocker, pentolinium, prevented fenfluramine-induced changes in T(TAIL). Second, when sympathetic tone to the tail was physiologically withdrawn by increasing the environmental temperature to 28 degrees C, fenfluramine treatment produced no increase in T(TAIL). Moreover, the effects of fenfluramine on T(TAIL) and T(CORE) depended on the uptake of fenfluramine into serotonergic neurons because these effects were markedly attenuated by pretreatment with the selective serotonin re-uptake inhibitor, fluoxetine. The hypothermic effect of fenfluramine occurred despite the fact that total body oxygen consumption increased by 20%. The results suggest that heat loss due to the dilation of the cutaneous circulation contributes to fenfluramine-induced hypothermia.

Sympathetic activation by fenfluramine depletes brown adipose tissue norepinephrine content in rats.

The antiobesity agent fenfluramine (FEN) has been reported to produce an activation of sympathetic neurons to brown adipose tissue (BAT) resulting in thermogenesis. The present study was conducted to determine if FEN-induced activation of BAT is affected by the ambient temperature at which animals are maintained. Body temperature was determined in conscious male Sprague-Dawley rats using implanted temperature transmitters, and sympathetic activation of BAT was determined by measuring norepinephrine (NE) content. Animals maintained at 22 and 4 degrees C incurred a significant decline in core body temperature following FEN administration. FEN produced a significant depletion of BAT NE content, and the magnitude of BAT NE depletion was related to the dose of FEN (-57.4% at 3 mg/kg dose and -75.9% at 10 mg/kg dose). However, the extent of BAT NE depletion was equal at 22 and 4 degrees C. BAT NE depletion by FEN appears to depend on activation of sympathetic neurons because it was prevented by pretreatment with the ganglionic blocker pentolinium (10 mg/kg ip). Furthermore, NE depletion by FEN could be blocked with fluoxetine (10 mg/kg ip) pretreatment, indicating that the action of FEN is mediated through the release of serotonin.