RESUMO
Tumor-associated macrophages (TAMs) play a pivotal role in facilitating tumor growth and metastasis. This tumor-promoting propensity of TAMs sets in as a result of their complex cross-talk with tumor cells mediated primarily by tumor cell-secreted proteins in the tumor microenvironment. To explore such interactions, we employed an immunoscreening approach involving the immunization of Balb-c mice with model human lung carcinoma cell line, A549. From serological examination combined with mass spectrometric analysis, EDA-containing fibronectin (EDAFN ) was identified as a conspicuous immunogenic protein in A549 cell secretome. We showed that A549 secreted EDAFN engages TLR-4 on THP-1 monocytes to drive the proinflammatory response via NF-κB signaling cascade. Conversely, A549 derived EDAFN potentiates their metastatic capacity by inducing epithelial-mesenchymal transition through its autocrine activity. In conclusion, the study proposes a possible mechanism of cellular cross-talk between lung cancer cells and associated monocytes mediated by lung cancer-derived EDAFN and resulting in the establishment of proinflammatory and metastatic tumor microenvironment.
Assuntos
Fibronectinas/metabolismo , Neoplasias Pulmonares/metabolismo , Monócitos/metabolismo , Células A549 , Animais , Western Blotting , Transição Epitelial-Mesenquimal/fisiologia , Imunofluorescência , Células HT29 , Células HeLa , Humanos , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos BALB C , Microambiente Tumoral/fisiologiaRESUMO
Dep domain containing mTOR interacting protein (DEPTOR) has critical implications in the development and progression of human malignancies. Increased expression of DEPTOR promotes the growth of tumor cells by inhibiting the mTORC1, which alleviates the negative feedback inhibition by mTORC1 downstream target S6Ks on PI3K/AKT pathway thereby promotes cell survival and prevents apoptosis. This clearly suggests that targetting DEPTOR-mTOR interactions through small molecules may prove as an effective strategy for circumventing distinct cancers. In this study, we employed a top-down approach for finding three novel molecules which may prove effective in disrupting Deptor-mTOR interaction. Following DEPTOR modelling and validation we performed grid-directed structure-based screening by specifying the residues of DEPTOR known to interact with mTOR. A library of 10,000 protein-protein disrupting molecules was screened against the defined region of DEPTOR. From the screened molecules, 30 molecules with highest binding affinity were chosen for molecular docking. Thirty (30) extra-precision molecular docking experiments and 30 molecular mechanics generalized born surface area (MMGBSA) assays were performed. Following this top 10 molecules in terms of binding affinity were selected and the interaction profile of their corresponding docked files was generated. The top three molecules were finally selected after taking all the three parameters including docking score, binding energy value and interaction profile into consideration. For atomistic insights regarding DEPTOR-topmost hit interactions, molecular dynamics was performed for 100 ns. This molecule after further evaluation may prove as promising candidate for anticancer therapy.Communicated by Ramaswamy H. Sarma.
Assuntos
Simulação de Dinâmica Molecular , Fosfatidilinositol 3-Quinases , Humanos , Simulação de Acoplamento Molecular , Fosfatidilinositol 3-Quinases/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Serina-Treonina Quinases TOR/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismoRESUMO
The overexploitation of medicinal plants is depleting gene pool at an alarming rate. In this scenario inducing the genetic variability through targeted mutations could be beneficial in generating varieties with increased content of active compounds. The present study aimed to develop a reproducible protocol for in vitro multiplication and mutagenesis of Hyoscyamus niger targeting putrescine N-methyltransferase (PMT) and 6ß-hydroxy hyoscyamine (H6H) genes of alkaloid biosynthetic pathway. In vitro raised callus were treated with different concentrations (0.01% - 0.1%) of Ethyl Methane Sulfonate (EMS). Emerging multiple shoots and roots were obtained on the MS media supplemented with cytokinins and auxins. Significant effects on morphological characteristics were observed following exposure to different concentrations of EMS. EMS at a concentration of 0.03% was seen to be effective in enhancing the average shoot and root number from 14.5±0.30 to 22.2 ±0.77 and 7.2±0.12 to 8.8±0.72, respectively. The lethal dose (LD50) dose was calculated at 0.08% EMS. The results depicted that EMS has an intense effect on PMT and H6H gene expression and metabolite accumulation. The transcripts of PMT and H6H were significantly upregulated at 0.03-0.05% EMS compared to control. EMS treated explants showed increased accumulation of scopolamine (0.639 µg/g) and hyoscyamine (0.0344µg/g) compared to untreated.