Functional characterisation of OPCML clinical mutations in ovarian cancer and correlate to OPCML crystal structure

Ovarian cancer is a heterogeneous disease, considered the most lethal of all gynaecological malignancies. With an initially high response rate to treatment, ovarian cancer can suddenly relapse, with advanced stages showing loco-regional dissemination. Despite enormous efforts to tackle ovarian cancer, current treatments fail to bring complete remission or significantly extend patient life. OPCML is a promising candidate in ovarian cancer treatment, identified as a tumour suppressor frequently inactivated in ovarian and other cancers. OPCML is a negative regulator of RTKs and regulates different cellular activities. The aims of this PhD were to identify and functionally characterise clinical OPCML mutations in ovarian cancer guided by the newly resolved crystal structure of OPCML. The project describes how TCGA and COSMIC databases have been used here to identify somatic missense mutations in several tumours. A panel of OPCML mutant constructs was then created and these selected constructs were utilised to transduce three ovarian cancer cell lines (SKOV3, PEA1, and PEO1) for domain I, and SKOV3 cells for domain II and III. Selected transduced mutants were used to characterise the mutants’ localisation, interaction with receptor tyrosine kinases, proliferation, cellular transformation, invasion, migration, and their potential role in the adhesion to the extracellular matrix in vitro. Furthermore, two in vivo models were used to characterise OPCML mutants: the athymic mice model and the chick embryo chorioallantoic membrane assay. The results showed that the OPCML mutants in domain I behave differently when they interact with receptor tyrosine kinase AXL and FGFR1 compared to wild-type OPCML. These OPCML mutants preserve the ability to inhibit growth in vitro, but have a complete loss of function in vivo. They also block the OPCML inhibitory activity on anchorage-independent growth, invasion, migration, and adhesion to extracellular matrix components laminin I, fibronectin, and fibrinogen. The OPCML mutants in domain II showed partial loss of function in adhesion to collagen I and IV, and complete loss of tumour suppressor function in cell proliferation in vitro and in vivo, anchorage-independent growth, invasion, migration, and adhesion to laminin I, fibronectin, and fibrinogen. The OPCML mutants in domain III preserve adhesion to collagen I and IV, but show complete loss of function in the inhibition of tumour growth in vitro and in vivo, anchorage-independent growth, invasion, migration, and adhesion to laminin I, fibronectin, and fibrinogen. Therefore, this study identifies a new mechanism of OPCML inactivation by mutations and shows that single amino acid change can abolish OPCML function. These findings suggest that OPCML is an excellent candidate for a new and novel therapeutic strategy in ovarian cancer and other malignancies, as well as other non-cancer diseases.Open Acces

Facile, polyherbal drug-mediated green synthesis of CuO nanoparticles and their potent biological applications

Copper oxide nanoparticles (CuO NPs) were synthesized using ayurvedic medicine septilin. The septilin-mediated CuO NPs were characterized using UV–Vis, fourier-transform infrared spectroscopy, X-ray diffraction (XRD), scanning electron microscope (SEM), and transmission electron microscope (TEM). The average particle size of CuO NPs was 8 nm as evident from TEM. Minimum inhibitory concentration of CuO NPs against Escherichia coli, Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus (MRSA), and Candida albicans was found in the range of 1–2.5 mg·mL−1. CuO NPs dose-dependently decreased the biofilm formation from 0.0315 to 2 mg·mL−1, at the highest dose of 2 mg·mL−1 of CuO NPs; 92.91%, 79.84%, and 71.57% decrease in biofilm was observed for P. aeruginosa, MRSA, and C. albicans, respectively. Down-regulation of biofilm upon treatment with nanoparticles (NPs) was also observed by SEM analysis. SEM analysis also showed the change in morphological structure, and deformities in bacterial and fungal cells upon treatment of NPs. Furthermore, the anticancer efficacy of NPs was assessed using colon cancer (HCT-116). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay clearly showed the anticancer potential of NPs, as the concentration of CuO NPs increased, the number of viable cells decreased. The produced CuO NPs have promise for future investigations in many biological and therapeutic domains, including the treatment of microbial biofilm infections, as well as the inhibition of cancer cell growth