Heavy-chain antibody targeting of CD38 NAD+ hydrolase ectoenzyme to prevent fibrosis in multiple organs.

The functionally pleiotropic ectoenzyme CD38 is a glycohydrolase widely expressed on immune and non-hematopoietic cells. By converting NAD+ to ADP-ribose and nicotinamide, CD38 governs organismal NAD+ homeostasis and the activity of NAD+-dependent cellular enzymes. CD38 has emerged as a major driver of age-related NAD+ decline underlying adverse metabolic states, frailty and reduced health span. CD38 is upregulated in systemic sclerosis (SSc), a chronic disease characterized by fibrosis in multiple organs. We sought to test the hypothesis that inhibition of the CD38 ecto-enzymatic activity using a heavy-chain monoclonal antibody Ab68 will, via augmenting organismal NAD+, prevent fibrosis in a mouse model of SSc characterized by NAD+ depletion. Here we show that treatment of mice with a non-cytotoxic heavy-chain antibody that selectively inhibits CD38 ectoenzyme resulted in NAD+ boosting that was associated with significant protection from fibrosis in multiple organs. These findings suggest that targeted inhibition of CD38 ecto-enzymatic activity could be a potential pharmacological approach for SSc fibrosis treatment.

Dissection of paracrine/autocrine interplay in lung tumor microenvironment mimicking cancer cell-monocyte co-culture models reveals proteins that promote inflammation and metastasis.

BACKGROUND: Tumor cell-monocyte interactions play crucial roles in shaping up the pro-tumorigenic phenotype and functional output of tumor-associated macrophages. Within the tumor microenvironment, such heterotypic cell-cell interactions are known to occur via secretory proteins. Secretory proteins establish a diabolic liaison between tumor cells and monocytes, leading to their recruitment, subsequent polarization and consequent tumor progression. METHODS: We co-cultured model lung adenocarcinoma cell line A549 with model monocytes, THP-1 to delineate the interactions between them. The levels of prototypical pro-inflammatory cytokines like TNF-𝛼, IL-6 and anti-inflammatory cytokines like IL-10 were measured by ELISA. Migration, invasion and attachment independence of lung cancer cells was assessed by wound healing, transwell invasion and colony formation assays respectively. The status of EMT was evaluated by immunofluorescence. Identification of secretory proteins differentially expressed in monocultures and co-culture was carried out using SILAC LC-MS/MS. Various insilico tools like Cytoscape, Reacfoam, CHAT and Kaplan-Meier plotter were utilized for association studies, pathway analysis, functional classification, cancer hallmark relevance and predicting the prognostic potential of the candidate secretory proteins respectively. RESULTS: Co-culture of A549 and THP-1 cells in 1:10 ratio showed early release of prototypical pro-inflammatory cytokines TNF-𝛼 and IL-6, however anti-inflammatory cytokine, IL-10 was observed to be released at the highest time point. The conditioned medium obtained from this co-culture ratio promoted the migration, invasion and colony formation as well as the EMT of A549 cells. Co-culturing of A549 with THP-1 cells modulated the secretion of proteins involved in cell proliferation, migration, invasion, EMT, inflammation, angiogenesis and inhibition of apoptosis. Among these proteins Versican, Tetranectin, IGFBP2, TUBB4B, C2 and IFI30 were found to correlate with the inflammatory and pro-metastatic milieu observed in our experimental setup. Furthermore, dysregulated expression of these proteins was found to be associated with poor prognosis and negative disease outcomes in lung adenocarcinoma compared to other cancer types. Pharmacological interventions targeting these proteins may serve as useful therapeutic approaches in lung adenocarcinoma. CONCLUSION: In this study, we have demonstrated that the lung cancer cell-monocyte cross-talk modulates the secretion of IFI30, RNH1, CLEC3B, VCAN, IGFBP2, C2 and TUBB4B favoring tumor growth and metastasis.

Ala307Thr variation modulates FSHR structure and impairs its binding affinity for FSH: Implications in polycystic ovarian syndrome.

Follicle-stimulating hormone receptor (FSHR) belongs to the family of G-protein coupled receptors and acts as a cognate receptor for follicle-stimulating hormone (FSH). Among the various polymorphic changes reported in FSHR, rs6165 polymorphism leading to Ala307Thr variation in the extracellular domain of the FSHR (FSHRED ) is widely reported. Therefore we attempted to evaluate the functional implications of this variation by studying its effects on FSHRED structure as well as FSH binding. Our atomic-scale investigations reveal that the hinge region, a key hormone interaction site in the extracellular domain of Wt FSHR, exhibits significantly more flexibility compared with the variant structure. Moreover, the Wt receptor in complex with FSH was observed to form a pocket-like structure in its hinge region whereas such a structure was not detected in the variant. The study further reveals that the key residue, sTyr335, required for FSH recognition and FSHR activation, exhibits lower binding free energy in the variant structure as compared to the Wt. In conclusion, our results point out that Ala307Thr variation leads to structural and conformational anomalies in FSHRED which may alter its FSH binding and affect its activation.

Shielding and nurturing: Fibronectin as a modulator of cancer drug resistance.

Resistance to chemotherapy and targeted therapies constitute a common hallmark of most cancers and represent a dominant factor fostering tumor relapse and metastasis. Fibronectin, an abundant extracellular matrix glycoprotein, has long been proposed to play an important role in the pathobiology of cancer. Recent research has unraveled the role of Fibronectin in the onset of chemoresistance against a variety of antineoplastic drugs including DNA-damaging agents, hormone receptor antagonists, tyrosine kinase inhibitors, microtubule destabilizing agents, etc. The current review summarizes the role played by Fibronectin in mediating drug resistance against diverse anticancer drugs. We have also discussed how the aberrant expression of Fibronectin drives the oncogenic signaling pathways ultimately leading to drug resistance through the inhibition of apoptosis, promotion of cancer cell growth and proliferation.

Identification of novel inhibitors of tetranectin-plasminogen interaction to suppress breast cancer invasion: an integrated computational and cell-based investigation.

Tetranectin-plasminogen interaction plays a defining role in extracellular matrix degradation, enabling tumor cell invasion and metastasis. This interaction occurs via the carbohydrate recognition domain (CRD) and Kringle 4 domain of tetranectin and plasminogen, respectively, leading to activation of the plasminogen-cascade that triggers the proteolytic processes. Thus targeting this interaction represents an important strategy to suppress tumor cell migration and invasion. In this direction, we attempted to target the CRD of tetranectin to inhibit its interaction with the Kringle-4 domain of plasminogen using natural bioactive compounds. A cheminformatics pipeline for drug designing and screening was utilized to obtain lead compound(s) that exhibit conformationally and energetically viable CRD binding. Out of 206 compounds screened, diosgenin and scytonemin displayed the most favorable interactions with CRD. Short-term molecular dynamics simulations of 20 ns were employed to further study the conformational stability of both compounds with tetranectin CRD which reflected at the increased stability of diosgenin in the CRD binding pocket compared to scytonemin. Finally, an extended molecular dynamic simulation of 100 ns affirmed the robust and stable interaction of diosgenin with CRD. Furthermore, diosgenin was observed to exert a pronounced anti-proliferative effect on high tetranectin-expressing MDA-MB-231 breast cancer cells. The inhibitory effect of diosgenin on the tetranectin-plasminogen interaction was corroborated by the reduced migration and invasiveness of MDA-MB-231 cells under diosgenin treatment. Overall the study presents an alternate and safer approach to impede breast cancer metastasis and delineates the novel anti-metastatic activity of diosgenin.Communicated by Ramaswamy H. Sarma.

Exploring novel and potent molecules for disrupting DEPTOR-mTOR interaction through structure-steered screening, extra-exactitude molecular docking, prime binding free energy estimation and voguish molecular dynamics.

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.

In vitro and In silico Evaluation of Structurally Diverse Benzyl-pyrrolidine-3-ol Analogues as Apoptotic Agents via Caspase Activation.

The activation of caspases is central to apoptotic process in living systems. Defects in apoptosis have been implicated with carcinogenesis. Need to develop smart agents capable of inducing apoptosis in tumor cells is obvious. With this motive, diversity oriented synthesis of 1-benzylpyrrolidin-3-ol analogues was envisaged. The multi component Ugi reaction synthesized library of electronically diverse analogues was explored for cytotoxic propensity towards a panel of human cancer cell lines at 10 µM. The lead compounds exhibit a selective cytotoxicity towards HL-60 cells as compared to cell lines derived from solid tumors. Besides, their milder cytotoxic effect on non-cancerous cell lines reaffirm their selective action towards cancer cells only. The lead molecules were tested for their ability to target caspase-3, as a vital protease triggering apoptosis. The lead compounds were observed to induce apoptosis in HL-60 cells around 10 µM concentration. The lead compounds exhibited various non-covalent supra type interactions with caspase-3 key residues around the active site. The binding ability of lead compounds with caspase-3 was studied via molecular docking and molecular dynamic (MD) simulations. MD simulations indicated the stability of compound-caspase-3 complex throughout the 50 ns simulation run. The stability and bio-availability of the lead compounds under physiological conditions was assessed by their interaction with Bovine Serum Albumin (BSA) as model protein. BSA interactions of lead compounds were studied by various bio-physical methods and further substantiated with in silico MD simulations.

Genetic analysis of colorectal carcinoma using high throughput single nucleotide polymorphism genotyping technique within the population of Jammu and Kashmir.

BACKGROUND: SNP genotyping has become increasingly more common place to understand the genetic basis of complex diseases like cancer. SNP-genotyping through MassARRAY™ is a cost-effective method to quantitatively analyse the variation of gene expression in multiple samples, making it a potential tool to identify the underlying causes of colorectal carcinogenesis. METHODS: In the present study, SNP genotyping was carried out using Agena MassARRAY™, which is a cost-effective, robust, and sensitive method to analyse multiple SNPs simultaneously. We analysed 7 genes in 492 samples (100 cases and 392 controls) associated with CRC within the population of Jammu and Kashmir. These SNPs were selected based on their association with multiple cancers in literature. RESULTS: This is the first study to explore these SNPs with colorectal cancer within the J&K population.7 SNPs with a call rate of 90% were selected for the study. Out of these, five SNPs rs2234593, rs1799966, rs2229080, rs8034191, rs1042522 were found to be significantly associated with the current study under the allelic model with an Odds Ratio OR = 2.981(1.731-5.136 at 95% CI); p value = 4.81E-05 for rs2234593,OR = 1.685(1.073-2.647 at 95% CI);; p value = 0.02292 for rs1799966, OR = 1.5 (1.1-2.3 at 95% CI), p value = 0.02 for rs2229080, OR = 1.699(1.035-2.791 at 95% CI); p value = 0.03521 for rs8034191, OR = 20.07 (11.26-35.75); p value = 1.84E-34 for rs1042522 respectively. CONCLUSION: This is the first study to find the relation of Genetic variants with the colorectal cancer within the studied population using high throughput MassARRAY™ technology. It is further anticipated that the variants should be evaluated in other population groups that may aid in understanding the genetic complexity and bridge the missing heritability.

Lung cancer cell-derived EDA-containing fibronectin induces an inflammatory response from monocytes and promotes metastatic tumor microenvironment.

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.

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 α9ß1 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 α9ß1 and α4ß1 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.

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.

Selective Killing of Breast Cancer Cells by Doxorubicin-Loaded Fluorescent Gold Nanoclusters: Confocal Microscopy and FRET.

Fluorescent gold nanoclusters (AuNCs) capped with lysozymes are used to deliver the anticancer drug doxorubicin to cancer and noncancer cells. Doxorubicin-loaded AuNCs cause the highly selective and efficient killing (90 %) of breast cancer cells (MCF7) (IC50 =155 nm). In contrast, the killing of the noncancer breast cells (MCF10A) by doxorubicin-loaded AuNCs is only 40 % (IC50 =4500 nm). By using a confocal microscope, the fluorescence spectrum and decay of the AuNCs were recorded inside the cell. The fluorescence maxima (at ≈490-515 nm) and lifetime (≈2 ns), of the AuNCs inside the cells correspond to Au10-13 . The intracellular release of doxorubicin from AuNCs is monitored by Förster resonance energy transfer (FRET) imaging.

Comparative proteomics and global genome-wide expression data implicate role of ARMC8 in lung cancer.

BACKGROUND: Cancer loci comprise heterogeneous cell populations with diverse cellular secretions. Therefore, disseminating cancer-specific or cancer-associated protein antigens from tissue lysates could only be marginally correct, if otherwise not validated against precise standards. MATERIALS AND METHODS: In this study, 2DE proteomic profiles were examined from lysates of 13 lung-adenocarcinoma tissue samples and matched against the A549 cell line proteome. A549 matched-cancer-specific hits were analyzed and characterized by MALDI-TOF/MS. RESULTS: Comparative analysis identified a total of 13 protein spots with differential expression. These proteins were found to be involved in critical cellular functions regulating pyrimidine metabolism, pentose phosphate pathway and integrin signaling. Gene ontology based analysis classified majority of protein hits responsible for metabolic processes. Among these, only a single non-predictive protein spot was found to be a cancer cell specific hit, identified as Armadillo repeat-containing protein 8 (ARMC8). Pathway reconstruction studies showed that ARMC8 lies at the centre of cancer metabolic pathways. CONCLUSIONS: The findings in this report are suggestive of a regulatory role of ARMC8 in control of proliferation and differentiation in lung adenocarcinomas.

Atomic Insight into the Altered O6-Methylguanine-DNA Methyltransferase Protein Architecture in Gastric Cancer.

O6-methylguanine-DNA methyltransferase (MGMT) is one of the major DNA repair protein that counteracts the alkalyting agent-induced DNA damage by replacing O6-methylguanine (mutagenic lesion) back to guanine, eventually suppressing the mismatch errors and double strand crosslinks. Exonic alterations in the form of nucleotide polymorphism may result in altered protein structure that in turn can lead to the loss of function. In the present study, we focused on the population feared for high exposure to alkylating agents owing to their typical and specialized dietary habits. To this end, gastric cancer patients pooled out from the population were selected for the mutational screening of a specific error prone region of MGMT gene. We found that nearly 40% of the studied neoplastic samples harbored missense mutation at codon151 resulting into Serine to Isoleucine variation. This variation resulted in bringing about the structural disorder, subsequently ensuing into a major stoichiometric variance in recognition domain, substrate binding and selectivity loop of the active site of the MGMT protein, as observed under virtual microscope of molecular dynamics simulation (MDS). The atomic insight into MGMT protein by computational approach showed a significant change in the intra molecular hydrogen bond pattern, thus leading to the observed structural anomalies. To further examine the mutational implications on regulatory plugs of MGMT that holds the protein in a DNA-Binding position, a MDS based analysis was carried out on, all known physically interacting amino acids essentially clustered into groups based on their position and function. The results generated by physical-functional clustering of protein indicated that the identified mutation in the vicinity of the active site of MGMT protein causes the local and global destabilization of a protein by either eliminating the stabilizing salt bridges in cluster C3, C4, and C5 or by locally destabilizing the "protein stabilizing hing" mapped on C3-C4 cluster, preceding the active site.

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.

Affinity proteomics led identification of vimentin as a potential biomarker in colon cancers: insights from serological screening and computational modelling.

Proteomic analysis using multiplex affinity reagents is perhaps the most reliable strategy to capture differentially expressed proteins that are slightly or immensely modified. In addition to expressional variation, it is comprehensively evident that the immunogenicity of a protein can be a deciding factor for instigating an inflammation afflicted-carcinogenesis. Considering both these factors, a simple and systematic strategy was designed to capture the immunogenic cancer biomarkers from sera of colorectal cancer patients. The affinity reagent, in the form of an antibody repertoire against the secretome of the HT29 cell line was used to grade the sera samples on the basis of the degree of immuno-reactivity and to capture differentially expressed antigens from the patient sera. Following affinity based 2DE-MALDI-TOF; the proteins were identified as (1) soluble vimentin; and (2) TGF-beta-inhibited membrane-associated protein (PP16B), in colon cancer sera and (3) keratin, type II cytoskeletal protein in rectal cancer sera. Pathway reconstruction and protein-protein networking of identified proteins predicted only Vimentin to be physically and genetically engaged in close proximity with the most established colorectal cancer associated tumorigenic pathways. Furthermore, our findings suggest that a possible surface stoichiometric shift in the structure of protein could be due to mutations in the coding sequence of Vimentin that may elicit its enhanced secretion possibly due to protein-hyperphosphorylation. Of the three proteins identified, only Vimentin showed higher expression in sera of colon cancer patients alone. Thus, it could be argued that vimentin might help in predicting individuals at higher risk of developing colon cancers. Our data are therefore suggestive of using vimentin as an antigen for tumor vaccination in an autologous set-up for colon cancers.