Asrar H. Wafai

Tectorigenin ablates the inflammation-induced epithelial–mesenchymal transition in a co-culture model of human lung carcinoma

OBJECTIVES Tumors not only manage to escape from the host immune system, but they effectively contrive to benefit from infiltrating immune cells by modifying their functions so as to create a pro-inflammatory microenvironment favorable for tumor progression and metastasis. In this study we investigated if tectorigenin could suppress lung cancer-induced pro-inflammatory response generated from monocytes. MATERIALS AND METHODS A549:THP1 co-culture model was set-up favoring release of pro-inflammatory cytokines interleukin (IL)-6 and tumor necrosis factor alpha (TNF-α). Effect of tectorigenin on A549 imparted invasive phenotype of A549:THP-1 co-culture was monitored by cytokine release from monocytes, and metastasis/epithelial-mesenchymal transitiom (EMT) in A549 cells. RESULTS In a contact A549:THP1 co-culture model, THP-1 cells were activated by A549 cells favoring secretion of pro-inflammatory cytokines, TNF-α and IL-6. However, priming of A549 cells with tectorigenin for 24h repressed A549 cell-induced secretion of TNF-α and IL-6 by THP-1 cells. Tectorigenin induced change in functional phenotype of A549 cells rendered THP-1 cells non-responsive for the secretion of IL-6 and TNF-α in a contact co-culture setup. Additionally, conditioned media from this non-responsive A549:THP-1 co-culture suppressed metastatic potential of A549 cells as confirmed by the wound healing and transwell migration assays. These finding were further corroborated by decrease in expression of Snail with a concomitant increase in E-cadherin, the two signature markers of EMT. CONCLUSION These results clearly demonstrate the therapeutic potential of tectorigenin to prevent lung cancer elicited inflammatory and pro-metastatic response in monocytes and warrants further investigations to elucidate its mechanism of action.

Relative expression of CsZCD gene and apocarotenoid biosynthesis during stigma development in Crocus sativus L.

AbstractCrocus sativus is a triploid sterile plant characterized by its red stigmas, which produce significant quantities of carotenoid derivatives formed from the oxidative cleavage of β-carotene and zeaxanthin. The accumulation of three major carotenoid derivatives- crocin, picrocrocin, and safranal- is responsible for the color, bitter taste, and aroma of saffron, which is obtained from the dried stigma of Crocus. Maximum apocarotenoid accumulation occurs during fully developed scarlet stage of stigma development. Zeaxanthin is the precursor for biosynthesis of apocarotenoids. Crocus zeaxanthin 7, 8 (7, 8)-cleavage dioxygenase gene (CsZCD) encodes a chromoplast enzyme that initiates the biogenesis of these apocarotenoids by cleaving zeaxanthin. The Reverse Transcription-PCR analysis revealed that CsZCD gene expression followed different patterns during stigma development. Highest levels of CsZCD gene expression was observed in fully developed scarlet stage of stigma. Real Time PCR analysis showed that there is a sharp increase in gene expression from yellow to orange and orange to scarlet stages of stigma development. Increase in CsZCD gene expression parallels with the apocarotenoid content during the development of stigma, suggesting its regulatory role for apocarotenoid biosynthesis and stigma development in saffron.

Irigenin , a novel lead from Western Himalayan chemiome inhibits Fibronectin-Extra Domain A induced metastasis in Lung cancer cells

Several lines of evidence indicate that Fibronectin Extra Domain A (EDA) promotes metastatic capacity of tumor cells by engaging cell surface α9β1 integrins. This interaction mediated by the C-C loop of EDA activates pro-oncogenic signaling pathways leading to epithelial to mesenchymal transition (EMT) of tumor cells, thus signifying its importance in control of metastatic progression. In this context the present study was designed to explore the active compounds from selected ethno-medicinal plants of western Himalayan region for targeting EDA of Fibronectin in lung carcinoma cells. Structure based informatics for drug designing and screening was employed to generate a lead compound(s) feed that were conformationally and energetically viable. Out of 120 compounds selected, Irigenin showed best binding-affinity with C-C loop of EDA. Irigenin specifically targeted α9β1 and α4β1 integrin binding sites on EDA comprising LEU46, PHE47, PRO48, GLU58, LEU59 and GLN60 in its C-C loop as evaluated by energy decomposition per residue of Irigenin–EDA complex. In-vitro cell motility assays complemented with EDA knock-in and knockdown assays distinctively demonstrated that Irigenin prevents metastatic capacity of lung cancer cells by selectively blocking EDA. The results presented thus project Irigenin as a lead compound to overcome Fibronectin EDA induced metastatic progression in lung carcinoma cells.

Relative expression of apocarotenoid biosynthetic genes in developing stigmas of Crocus sativus L.

Saffron, the desiccated stigmas of Crocus sativus, is recognized for its attractive color, flavor, and aroma which are due to the accumulation of crocin, picrocrocin, and safranal, respectively. HPLC analysis demonstrated maximum apocarotenoid accumulation during the fully developed scarlet stage of stigma development followed by the orange and yellow stages of stigma development. Reverse Transcription-PCR analysis revealed a concurrent expression pattern of CsZCD and CsLYC genes in a developmental stagespecific manner. However, CsBCH and CsGT2 genes were specifically expressed during the mature, scarlet stage of stigma development. Real-Time PCR analysis showed a sharp increase in gene expression of CsLYC gene during stigma development indicative of its possible regulatory role in apocarotenoid biosynthesis or stigma development. Results suggest that genetic manipulation of this gene can help to improve the quality of stigma in saffron; besides highlighting its potential to monitor stigma development during in vitro experimentation.

Comparative expression analysis of senescence gene CsNAP and B-class floral development gene CsAP3 during different stages of flower development in Saffron ( Crocus sativus L .)

Crocus sativus, a monocot triploid species belonging to the Iridaceae family, is cultivated for its red stigmatic lobes of the carpel that constitute saffron. Flower development has been extensively studied in different plants. Different floral developmental pathways have been deciphered in many plants. In Crocus sativus, flower is the most important part and understanding the pathway underlying the flower development can pave the way for new avenues to improve its productivity and quality. The combination of class A genes (including APETALA1; CsAP1 and APETALA2; CsAP2), class B genes (including APETALA3; CsAP3 and PISTILLATA; CsPI) and class C genes (including AGAMOUS; CsAG) that are active in each whorl, determines the identity of the organs that will later develop in that whorl. CsAP3 is a class B homeotic gene which promotes petal and stamen formation and has a very important role in flower development. It also activates other genes playing pivotal role in flower development. It has been earlier reported that CsAP3 gene has direct role in activation of CsNAP gene which promotes senescence in plants. Present work was focused on study of relative gene expression changes of CsAP3 and CsNAP gene during different stages of flower development. CsAP3 gene expression was found maximum during late-preanthesis stages of stigma development. Expression increases from stage 5 to stage 6 of flower development and then reduces again from stage 6 to stage 7. CsNAP gene had moderate expression during stage 3 to stage 4 transition and its expression increased abruptly from stage 6 to stage 7 of flower development. There is no direct concordance in the expression of CsAP3 and CsNAP gene expression in saffron. We may conclude that some other factor(s) may be responsible for initiation of CsNAP expression and CsAP3 gene may directly/indirectly be involved in regulating the factors responsible for CsNAP activation.

A novel kinase mutation in VEGFR-1 predisposes its αC-helix/activation loop towards allosteric activation: Atomic insights from protein simulation.

Vascular endothelial growth factor receptor 1 (VEGFR-1) has been implicated in diverse pathologies, including cancers. Although VEGFR-1 is considered as functionally impaired kinase, its decoy characteristics make it an important regulator of VEGFR-mediated signaling, particularly in tumor angiogenesis. VEGFR-1 conveys signaling via its tyrosine kinase (TK) domain whose activation is regulated by phosphorylation of specific tyrosine residues. Thus dysregulation of VEGFR-1 signaling, as reported in most of the cancers, might be a consequence of altered phosphorylation that could be attributed to genotypic variations in its TK domain. Considering the importance of TK domain of VEGFR-1, we carried out its mutational screening in 84 clinically validated and histopathologically confirmed colorectal cancer patients. By means of direct DNA sequencing and SNP analyses, eight novel variations, including one synonymous, two deletion, one missense and four intronic variations, were reported in the TK domain of VEGFR-1. rs730882263:C>G variation specifically reported in colon cancer, representing a single-atomic change (Sulfur to Oxygen) in the predicted (p.Cys1110Ser) protein, was observed as potentially deleterious variation as assessed by multiple single-nucleotide polymorphism prediction servers. Molecular dynamics simulations of VEGFR-1 Wt and (p.Cys1110Ser) variant models revealed major conformational changes in variant protein presumptuously generating an open conformation thereby exposing the activation domain and consequently increasing the probability of phosphorylation events: a condition frequently reported in cancers.