Revisiting the NPcis mouse model: A new tool to model plexiform neurofibroma.

Neurofibromatosis Type I (NF1) is a rare genetic disorder. NF1 patients frequently develop a benign tumor in peripheral nerve plexuses called plexiform neurofibroma. In the past two decades, tissue-specific Nf1 knockout mouse models were developed using commercially available tissue-specific Cre recombinase and the Nf1 flox mice to mimic neurofibroma development. However, these models develop para-spinal neurofibroma, recapitulating a rare type of neurofibroma found in NF1 patients. The NPcis mouse model developed a malignant version of neurofibroma called malignant peripheral nerve sheath tumor (MPNST) within 3 to 6 months but intriguingly without apparent benign precursor lesion. Here, we revisited the NPcis model and discovered that about 20% display clinical signs similar to Nf1 tissue-specific knockout mice models. However, a systematic histological analysis could not explain the clinical signs we observed although we noticed lesions reminiscent of a neurofibroma in a peripheral nerve, a cutaneous neurofibroma, and para-spinal neurofibroma on rare occasions in NPcis mice. We also observed that 10% of the mice developed a malignant peripheral nerve sheath tumor (MPNST) spontaneously, coinciding with their earring tag identification. Strikingly, half of the sciatic nerves from NPcis mice developed plexiform neurofibroma within 1-6 months when intentionally injured. Thus, we provided a procedure to turn the widely used NPcis sarcoma model into a model recapitulating plexiform neurofibroma.

Unraveling the molecular mechanism of l-menthol against cervical cancer based on network pharmacology, molecular docking and in vitro analysis.

Cervical cancer is a major cause of gynecological related mortalities in developing countries. Cisplatin, a potent chemotherapeutic agent used for treating advanced cervical cancer exhibits side effects and resistance development. The current study was aimed to investigate the repurposing of l-menthol as a potential therapeutic drug against cervical cancer. L-menthol was predicted to be non-toxic with good pharmacokinetic properties based on SwissADME and pkCSM analysis. Subsequently, 543 and 1664 targets of l-menthol and cervical cancer were identified using STITCH, BATMAN-TCM, PharmMapper and CTD databases. STRING and Cytoscape analysis of the merged protein-protein interaction network revealed 107 core targets of l- menthol against cervical cancer. M-CODE identified highly connected clusters between the core targets which through KEGG analysis were found to be enriched in pathways related to apoptosis and adherence junctions. Molecular docking showed that l- menthol targeted E6, E6AP and E7 onco-proteins of HPV that interact and inactivate TP53 and Rb1 in cervical cancer, respectively. Molecular docking also showed good binding affinity of l-menthol toward proteins associated with apoptosis and migration. Molecular dynamics simulation confirmed stability of the docked complexes. In vitro analysis confirmed that l-menthol was cytotoxic towards cervical cancer CaSki cells and altered expression of TP53, Rb1, CDKN1A, E2F1, NFKB1, Akt-1, caspase-3, CDH1 and MMP-2 genes identified through network pharmacology approach. Schematic representation of the work flow depicting the potential of l-menthol to target cervical cancer.

Plumbagin downregulates UHRF1, p-Akt, MMP-2 and suppresses survival, growth and migration of cervical cancer CaSki cells.

Plumbagin is a natural compound known to impede growth of cancerous cells. However, anti-cervical cancer effects of plumbagin and its underlying molecular mechanism still remains elusive. In this study, plumbagin reduced the viability of CaSki cells in a concentration dependent manner and suppressed their colony formation potential. It led to G2/M phase arrest with downregulation of E2F1 and upregulation of p21. Plumbagin reduced mitochondrial membrane potential and concomitantly increased the percentage of apoptotic cells as revealed by annexin V-propidium iodide staining. Real Time PCR and western blotting confirmed that plumbagin induced apoptosis by reducing the expression of pAkt, procaspase 9 and full-length PARP. Furthermore, scratch assay showed that plumbagin suppressed migratory potential of CaSki cells which could be due to the reduced expression and activity of MMP-2 and upregulation of TIMP2. Interestingly, plumbagin also downregulated UHRF1 expression. Transient silencing of UHRF1 like plumbagin, induced G2/M phase arrest, enhanced apoptosis and suppressed metastasis of CaSki cells suggesting the role of UHRF1 in mediating anti-cancer activities of plumbagin. Plumbagin at IC20 (1 µM) interacted synergistically with cisplatin and reduced its IC50 value by 13.23 fold with improved effectivity as revealed by augmented apoptosis in CaSki cells.

3-indoleacetonitrile attenuates biofilm formation and enhances sensitivity to imipenem in Acinetobacter baumannii.

Acinetobacter baumannii poses a global danger due to its ability to resist most of the currently available antimicrobial agents. Furthermore, the rise of carbapenem-resistant A. baumannii isolates has limited the treatment options available. In the present study, plant auxin 3-indoleacetonitrile (3IAN) was found to inhibit biofilm formation and motility of A. baumannii at sublethal concentration. Mechanistically, 3IAN inhibited the synthesis of the quorum sensing signal 3-OH-C12-HSL by downregulating the expression of the abaI autoinducer synthase gene. 3IAN was found to reduce the minimum inhibitory concentration of A. baumannii ATCC 17978 against imipenem, ofloxacin, ciprofloxacin, tobramycin, and levofloxacin, and significantly decreased persistence against imipenem. Inhibition of efflux pumps by downregulating genes expression may be responsible for enhanced sensitivity and low persistence. 3IAN reduced the resistance to imipenem in carbapenem-resistant A. baumannii isolates by downregulating the expression of OXA ß-lactamases (blaoxa-51 and blaoxa-23), outer membrane protein carO, and transporter protein adeB. These findings demonstrate the therapeutic potential of 3IAN, which could be explored as an adjuvant with antibiotics for controlling A. baumannii infections.

Synergistic anti-cancer action of salicylic acid and cisplatin on HeLa cells elucidated by network pharmacology and in vitro analysis.

AIM: To investigate the anti-cancer potential of salicylic acid and cisplatin combination in HeLa cells and the underlying mechanism. MAIN METHODS: Drugs and disease targets were extracted from DrugBank, BATMAN-TCM, STITCH, PharmMapper and Comparative Toxigenomics Database. Cytoscape 3.8.2 was used to merge the protein-protein interaction networks and select core targets. GO and KEGG analysis was done using Metascape and WebGestalt. Effect of salicylic acid and cisplatin alone and in combination on cells viability was studied by MTT assay. The type of interaction between salicylic acid and cisplatin was determined by CompuSyn. Apoptosis was evaluated by molecular docking, Rhodamine-123, DAPI, AO/EtBr staining, flow cytometry, qRT-PCR and western blotting. Metastasis was studied using scratch assay and western blotting. UHRF1 transient silencing was performed by siRNA. KEY FINDINGS: Out of 420, 1863 and 1362 respective targets of salicylic acid, cisplatin and cervical cancer, 18 core proteins were enriched in apoptosis and cell migration related pathways. IC50 value of cisplatin was reduced by 14 fold in combination with salicylic acid at IC20 (4 µM). There was loss of mitochondrial membrane potential and downregulation of UHRF1, pAkt, full length PARP and pro-caspase 3 expression. Transient silencing of UHRF1 also induced mitochondrial depolarization and apoptosis. The combination also exhibited anti-metastasis effect as it suppressed migration, upregulated PAX1 and downregulated MMP-2. SIGNIFICANCE: Reduction in cisplatin concentration, enhanced anti-cancer effects and UHRF1 downregulation due to synergistic interaction between salicylic acid and cisplatin underscores the therapeutic importance of the combination to overcome chemo-resistance and side effects of cisplatin.

Multicopper oxidase of Acinetobacter baumannii: Assessing its role in metal homeostasis, stress management and virulence.

A putative multicopper oxidase, encoded as CopA in the proteome of Acinetobacter baumannii 19606, and designated as AbMCO, was expressed heterologously in E. coli (pET-28a) and purified by Ni-NTA affinity chromatography. The purified AbMCO exhibited in vitro oxidase activities upon exogenous addition of ≥1 µM copper ions. Kinetic studies revealed its phenol oxidase activity as it could catalyze the oxidation of substrates viz. 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), guaiacol, pyrogallol and catechol. Additionally, AbMCO displayed siderophore oxidase activity which depicted its role in metal homeostasis and protection from the toxic redox states of copper and iron. Importantly, expression of abMCO increased manifold upon challenge with high concentrations of copper sulphate (CuSO4, 1.5 mM) and sodium chloride (NaCl, 700 mM) which suggested its protective role in stress adaptation and management. Intra-macrophage assay of abMCO-expressing and abMCO-non expressing cells depicted no significant change in the survival rate of A. baumannii inside the macrophages. These findings indicate that A. baumannii encodes a multicopper oxidase, conferring copper tolerance and survival under stress conditions but had no role in virulence of this pathogen.

UHRF1: The key regulator of epigenetics and molecular target for cancer therapeutics.

UHRF1 is a master regulator of epigenome as it coordinates DNA methylation and histone modifications. Compelling evidence suggests a strong link between UHRF1 overexpression and tumorigenesis, substantiating its ability to act as a potential biomarker for cancer diagnosis and prognosis. UHRF1 also mediates repair of damaged DNA that makes cancer cells resistant toward cytocidal drugs. Hence, understanding the molecular mechanism of UHRF1 regulation would help in developing cancer therapeutics. Natural compounds have shown applicability to downregulate UHRF1 leading to growth arrest and apoptosis in cancer cells.