Small molecules that affect the p53 pathway and their potential use in the treatment of cancer

The tumor suppressor p53 was identified 35 years ago and has since then been studied extensively, but despite all efforts, no drug or therapy directly involving it has been clinically approved - yet! A lot of potential new drugs are on their way that can reactivate p53 function by various mechanisms. Even a whole new approach called cyclotherapy has been established, during which p53 is activated in normal cells to protect patients from the adverse effects of chemotherapy while tumor cells are still being killed efficiently. In this thesis, 16 drug combinations are being described in this context (paper I). Four individual p53-activating compounds, i.e. tenovin-6, leptomycin B (LMB), nutlin-3 and actinomycin D at low doses (LDactD), were used prior to the addition of each one clinically approved chemotherapeutic agent, i.e vinblastine, vinorelbine, cytosine arabinoside or gemcitabine. LDactD, which is clinically approved, showed the most promising results. Unexpectedly, we identified two compounds that can inhibit p53’s ability to induce p21, i.e. the novel SirT2 inhibitor tenovin-D3 (paper II) and the widely used histone deacetylase inhibitor (HDACi) trichostatin A (TSA) (paper III). Inhibition of p21 in tumor cells might be desirable during cancer treatment to prevent tumor cells from undergoing cell cycle arrest, which would make them more vulnerable to classic chemotherapy. On the other hand, an inhibition of cell cycle arrest in normal cells might occur, which may worsen the side effects caused by chemotherapy. However, SirT2 plays a role in neurodegenerative diseases, and hence compounds like tenovin-D3 may be of use in the treatment thereof. Furthermore, the decrease in p21 levels may be a contributing factor in the previously observed increase in efficacy during the generation of induced pluripotent stem cells upon treatment with TSA; also tenovin-D3 could be useful in this context. With the aid of a cell-based screen we identified two small molecules that can activate p53: 1) MJ05 was one of the most active hit compounds and was very selective (paper IV); it was highly cytotoxic in ARN8, especially when combined with nutlin-3, whereas it was cytostatic or had a very mild effect in other tumor cell lines and normal cells. It inhibited tumor growth in vivo, an effect that was enhanced upon co-treatment with nutlin-3. Furthermore, MJ05 selectively killed chronic myelogenous leukemia stem cells ex vivo while having milder effects in leukocyte stem cells derived from cord blood. Preliminary data strongly suggest that MJ05 acts by inhibition of pyrimidine (deoxy-) nucleotide synthesis. 2) Despite being a hit compound in our screen, MJ25 was not very potent at activating p53 (paper V). Nevertheless, its ability to inhibit thiredoxin reductase 1 (TrxR1) and its selectivity towards melanoma cell lines compared with normal cells were interesting features. We compared it with the TrxR1 inihibitor auranofin, which was very potent and selective at killing melanoma cells in cell viability assays. The insolubility of MJ25 at concentrations required for in vivo studies prevented us from testing it on xenografts in mice. Furthermore, MJ25 might not be specific for TrxR1, so the identification of additional targets could be investigated in the future. Auranofin, the other hand, has a more defined mechanism of action and is clinically approved for the treatment of rheumatoid arthritis. These traits combined with its potentially selective cytotoxic effect at low micromolar concentrations in melanoma cells may turn this compound into a potential drug candidate to be tested in patients suffering from malignant melanoma. In the final study presented in this thesis (paper VI) we tested the small molecule tenovin-6 in zebrafish embryos The compound had been described previously by our group. The original aim of this study was to investigate if the activation of p53 in an organism could affect the ability of tumor cells to disseminate. Even though tenovin-6 did not activate wild-type p53 under the conditions tested, in vivo activity of the compound was still detectable, since embryos expressing mutant p53 (M214K) displayed an increase in p53 protein levels; furthermore, the compound was lethal in a dose- and time-dependent manner, and the embryos lost most of their brown/black pigmentation. The exact mechanism behind the latter observation could not be elucidated in the course of the project. However, tyrosinase, a key enzyme in melanogenesis, was not inhibited by tenovin-6, and the combination of data obtained by others on mutated or pharmacologically inhibited vacuolar H+-ATPase (V- ATPase) and yeast mutant strains suggested that the compound may target V-ATPase

EXOSOME NANOCARRIERS: BASIC BIOLOGY, DIAGNOSIS, NOVEL AND PERSPECTIVE APPROACH IN DRUG DELIVERY SYSTEMS: A REVIEW

Exosomes are the extracellular vesicles surrounded by a phospholipid bilayer shed from all cell varieties and plays a significant role in the communication and Transportation of materials between the cells due to their ability to transfer the proteins and nucleic acids from One cell to the another cell. Analogous in size and performance to synthetic nanoparticles, exosomes provide several Advantages, rendering them the foremost promising candidates for targeted drug or gene delivery vehicles. This review highlights the isolation techniques and delivery potential of exosomes, and equally presents research or analysis gaps for enhancing the use of natural vesicles for delivery functions. Exosome-based drug formulations can be applied to an extensive variety of diseases such as various infectious, cardiovascular, cancer and neurodegenerative disorders. Mostly, exosomes combine the benefits of both synthetic nanocarriers and cell-mediated drug delivery systems however avoiding their limitations

DEVELOPMENT AND VALIDATION OF STABILITY INDICATING HPLC METHOD FOR THE DETERMINATION OF ULIPRISTAL ACETATE IN PHARMACEUTICAL DOSAGE FORM

A simple, novel, precise and accurate stability indicating RP-HPLC method was developed and validated for the estimation of Ulipristal Acetate in pharmaceutical dosage form. A Phenoxneome C18 (150 mm x 4.6 mm, 5 µm) column was used as stationary phase with mobile phase consisting of 0.1% ortho phosphoric acid and acetonitrile in the ratio of 50:50 v/v (pH was adjusted to 4.0 with triethyl amine). The flow rate was maintained at 1.0 mL/min and effluents were monitored at 223 nm. The retention time was 1.895 min. The linearity of the method was observed in the concentration range of 20-100 µg/mL with correlation coefficient of 0.999. The method developed was validated for linearity, precision, accuracy, system suitability and forced degradation studies like acidic, alkaline, oxidative and neutral stress conditions were performed as per ICH guidelines. The results obtained in the study were within the acceptable limits and hence this method can be used for the estimation of Ulipristal Acetate in pharmaceutical dosage form

Evaluation of an Actinomycin D/VX-680 aurora kinase inhibitor combination in p53-based cyclotherapy

p53-based cyclotherapy is proving to be a promising approach to palliate undesired effects of chemotherapy in patients with tumours carrying p53 mutations. For example, pre-treatment of cell cultures with Nutlin-3, a highly-selective inhibitor of the p53-mdm2 interaction, has been successfully used as a cytostatic agent to protect normal cells, but not p53-defective cells, from subsequent treatment with mitotic poisons or S-phase specific drugs. Here we sought to evaluate whether low doses of Actinomycin D (LDActD), a clinically-approved drug and potent p53 activator, could substitute Nutlin-3 in p53-based cyclotherapy. We found that pre-treatment with LDActD before adding the aurora kinase inhibitor VX-680 protects normal fibroblasts from polyploidy and nuclear morphology abnormalities induced by VX-680. However, and although to a lower extent than normal fibroblasts, tumour cell lines bearing p53 mutations were also protected by LDActD (but not Nutlin-3) from VX-680-induced polyploidy. We also report that a difference between the response of p53 wild-type cells and p53-defective cells to the LDActD/VX-680 sequential combination is that only the former fail to enter S-phase and therefore accumulate in G1/G0. We propose that drugs that incorporate into DNA during S-phase may perform better as second drugs than mitotic poisons in cyclotherapy approaches using LDActD as a cytostatic agent

Small satellites for space science : A COSPAR scientific roadmap

This is a COSPAR roadmap to advance the frontiers of science through innovation and international collaboration using small satellites. The world of small satellites is evolving quickly and an opportunity exists to leverage these developments to make scientific progress. In particular, the increasing availability of low-cost launch and commercially available hardware provides an opportunity to reduce the overall cost of science missions. This in turn should increase flight rates and encourage scientists to propose more innovative concepts, leading to scientific breakthroughs. Moreover, new computer technologies and methods are changing the way data are acquired, managed, and processed. The large data sets enabled by small satellites will require a new paradigm for scientific data analysis. In this roadmap we provide several examples of long-term scientific visions that could be enabled by the small satellite revolution. For the purpose of this report, the term “small satellite” is somewhat arbitrarily defined as a spacecraft with an upper mass limit in the range of a few hundred kilograms. The mass limit is less important than the processes used to build and launch these satellites. The goal of this roadmap is to encourage the space science community to leverage developments in the small satellite industry in order to increase flight rates, and change the way small science satellites are built and managed. Five recommendations are made; one each to the science community, to space industry, to space agencies, to policy makers, and finally, to COSPAR

The collagen prolyl hydroxylases are bifunctional growth regulators in melanoma

Appropriate post-translational processing of collagen requires prolyl hydroxylation, catalyzed by the prolyl 3- (C-P3H) and prolyl 4- (C-P4H) hydroxylases is essential for normal cell function. Here we have investigated the expression, transcriptional regulation and function of the C-P3H and C-P4H families in melanoma. We show that the CP3H family exemplified by Leprel1 and Leprel2 are subject to methylation-dependent transcriptional silencing in primary and metastatic melanoma consistent with a tumour suppressor function. In contrast, although there is transcriptional silencing of P4HA3 in a sub-set of melanomas, the CP4H family members P4HA1, P4HA2 and P4HA3 are often over-expressed in melanoma, expression being prognostic of worse clinical outcomes. Consistent with tumour suppressor function, ectopic expression of Leprel1 and Leprel2 inhibits melanoma proliferation, whereas P4HA2 and P4HA3 increase proliferation and particularly invasiveness of melanoma cells. Pharmacological inhibition with multiple selective C-P4H inhibitors reduces proliferation and inhibits invasiveness of melanoma cells. Together, our data identify the C-P3H and C-P4H families as potentially important regulators of melanoma growth and invasiveness and suggest that selective inhibition of C-P4H is an attractive strategy to reduce the invasive properties of melanoma cells

The Renin Angiotensin System (RAS) mediates bifunctional growth regulation in melanoma and is a novel target for therapeutic intervention

Despite emergence of new systemic therapies, metastatic melanoma remains a challenging and often fatal form of skin cancer. The renin–angiotensin system (RAS) is a major physiological regulatory pathway controlling salt–water equilibrium, intravascular volume and blood pressure. Biological effects of the RAS are mediated by the vasoactive hormone angiotensin II (AngII) via two receptor subtypes, AT1R (encoded by AGTR1) and AT2R (encoded by AGTR2). We report decreasing expression and increasing CpG island methylation of AGTR1 in metastatic versus primary melanoma and detection in serum of methylated genomic DNA from the AGTR1 CpG island in metastatic melanoma implying that AGTR1 encodes a tumour suppressor function in melanoma. Consistent with this hypothesis, antagonism of AT1R using losartan or shRNA-mediated knockdown in melanoma cell lines expressing AGTR1 resulted in acquisition of the ability to proliferate in serum-free conditions. Conversely, ectopic expression of AGTR1 in cell lines lacking endogenous expression inhibits proliferation irrespective of the presence of AngII implying a ligand-independent suppressor function for AT1R. Treatment of melanoma cell lines expressing endogenous AT2R with either AngII or the AT2R-selective agonist Y6AII induces proliferation in serum-free conditions whereas the AT2R-specific antagonists PD123319 and EMA401 inhibit melanoma growth and angiogenesis and potentiate inhibitors of BRAF and MEK in cells with BRAF V600 mutations. Our results demonstrate that the RAS has both oncogenic and tumour suppressor functions in melanoma. Pharmacological inhibition of AT2R may provide therapeutic opportunities in melanomas expressing this receptor and AGTR1 CpG island methylation in serum may serve as a novel biomarker of metastatic melanoma

Uncommon presentation of common clinical case

Lipoma is the most common benign tumor of the mesenchyme. Common location being trunk and limbs. Lipoma can occur rarely in the head and neck region with most common location being the posterior neck. We report a case of a 62 year old male who presented with a huge swelling (26x16cms), in the chin region with ulceration and bleeding. On further evaluation the swelling was found to be a benign tumor. The swelling was excised along with the overlying skin and on histopathologic examination it was found to be a simple lipoma. On postoperative follow up for 1year there was no recurrence and good aesthetic appearance was achieved. Awareness regarding such uncommon presentations of common clinical conditions like lipoma should always be considered

Enhancing Cloud Communication Security: A Blockchain-Powered Framework with Attribute-Aware Encryption

The global production of information continuously increases in quantity and variety. However, the tools and technologies developed to handle such large volumes of data have not adequately met the security and privacy requirements. Existing cloud security systems, often managed by a trusted third party, are susceptible to various security risks. To address these challenges and ensure the protection of personal information, blockchain technology emerges as a crucial solution with substantial potential. This research uses the blockchain-powered attribute-aware encryption method to establish a real-time secure communication approach over the cloud. By employing attribute-based encryption technology, data owners can implement fine-grained search permissions for data users. The proposed solution incorporates accessible encryption technology to enable secure access to encrypted data and facilitate keyword searches on the blockchain. This study provides a functional comparison of recently developed attribute-based encryption algorithms. The access control strategy comprises two access tree types and a linear secret-sharing system, serving as the main components. The elliptic curve’s base field was set to 512b, and the bilinear pairing parameter type used was Type-A. This approach involves storing keywords on a remote server and encrypting them using attribute-based encryption. Furthermore, the encrypted data blockchain and the corresponding ciphertext are stored in the blockchain. Numerical experiments were conducted to evaluate the system’s key generation, trapdoor building, and keyword retrieval capabilities