The delivery of N-myc downstream-regulated gene 2 (NDRG2) self-amplifying mRNA via modified lipid nanoparticles as a potential treatment for drug-resistant and metastatic cancers

THE DELIVERY OF N-MYC DOWNSTREAM-REGULATED GENE 2 (NDRG2) SELF-AMPLIFYING MRNA VIA MODIFIED LIPID NANOPARTICLES AS A POTENTIAL TREATMENT FOR DRUG-RESISTANT AND METASTATIC CANCERS Medical Review (Rights reserved) (-) The delivery of N-myc downstream-regulated gene 2 (NDRG2) self-amplifying mRNA via modified lipid nanoparticles as a potential treatment for drug-resistant and metastatic cancers / Reznik, Sandra E. (Rights reserved) (-

MicroRNA and Cancer

MicroRNAs (miRs) are small noncoding RNAs that function as post-transcriptional regulators of gene expression and have important roles in almost all biological pathways. Deregulated miR expression has been detected in numerous cancers, where miRs act as both oncogene and tumor suppressors. Due to their important roles in tumorigenesis, miRs have been investigated as prognostic and diagnostic biomarkers and as useful targets for therapeutic intervention. From a therapeutic point of view, two modalities can serve to rectify gene networks in cancer cells. For oncomiRs, a rational means is downregulation through antagomirs. Moreover, observations of the pathological reductions in tumor-suppressive miRs have inspired the concept of “miR replacement therapy” to enhance the amount of these miRs, thereby restoring them to normal levels. However, the clinical applicability of miR-based therapies is severely limited by the lack of effective delivery systems. Therefore, to understand the role of this new class of regulators, we need to identify the mRNA targets regulated by individual miRs as well as to develop specific, efficient, and safe delivery systems for therapeutic miRs

Targeting STAT3 and STAT5 in Cancer

Every minute, 34 new patients are diagnosed with cancer globally. Although over the past 50 years treatments have improved and survival rates have increased dramatically for several types of cancers, many remain incurable. Several aggressive types of blood and solid cancers form when mutations occur in a critical cellular signaling pathway, the JAK-STAT pathway; (Janus Kinase-Signal Transducer and Activator of Transcription). Currently, there are no clinically available drugs that target the oncogenic STAT3/5 proteins in particular or their Gain of Function hyperactive mutant products. Here, we summarize targeting approaches on STAT3/5, as the field moves towards clinical applications as well as we illuminate on upstream or downstream JAK-STAT pathway interference with kinase inhibitors, heat shock protein blockers or changing nuclear import/export processes. We cover the design paradigms and medicinal chemistry approaches to illuminate progress and challenges in understanding the pleiotropic role of STAT3 and STAT5 in oncogenesis, the microenvironment, the immune system in particular, all culminating in a complex interplay towards cancer progression

Evolution of the Molecular Biology of Brain Tumors and the Therapeutic Implications

A dramatic increase in knowledge regarding the molecular biology of brain tumors has been established over the past few years. In particular recent new avenues regarding the role of stem cells and microRNAs along with further understanding of the importance of angiogenesis, immunotherapy and explanations for the resistance of the tumors to chemotherapeutic agents and radiation therapy has been developed. It is hopeful that this new information will lead to efficacious treatment strategies for these tumors which remain a challenge. In this book a review of the latest information on these topics along with a variety of new therapeutic treatment strategies with an emphasis on molecular targeted therapies is provided

Development of Novel Therapy Targeting Autophagy Against Advanced and Chemo-resistant Pancreatic Cancer.

Autophagy is a highly conserved recycling process carried out ubiquitously in humans that primarily involves both selective and non-selective degradation of cellular materials ranging from small proteins to organelles as a means to provide an alternate nutrient source. This process is generally regarded as a survival pathway by resisting cell-mediated death (i.e. apoptosis) and sustaining cellular resources under stressful conditions (i.e. hypoxia, and glucose and nutrient starvation). Unfortunately, PDAC is a devastating cancer subtype that is closely linked with a highly aggressive and, metastatic and chemoresistant phenotype. As an intricately regulated pathway with a highly complex upstream canonical network, mutations in the PDAC genome often dysregulate homeostatic autophagy. As a result of its markedly rapid growth and progression, PDAC tumours are frequently associated with intense metabolic and mechanical stressors that significantly contribute to the augmented autophagy in PDAC. Hence, autophagy has been comprehensively linked to PDAC progression and could be a key mechanism responsible for poor therapeutic efficacy in this disease. While therapeutic, diagnostic and prognostic advancements in the treatment and management of PDAC have been at the forefront of recent research, patient survival rate has only marginally improved over the last 20 years. Addressing the treatment of PDAC, the inhibition of autophagy is a therapeutic strategy that has been trialled in the past with only the late-stage inhibitors, Chloroquine and Hydroxychloroquine, reaching clinical trials. While these agents are shown to synergise with other currently used PDAC chemotherapeutics, they have limited potency and are known to induce various adverse effects. As a fundamental pathway involved in the survival and progression of PDAC, the development and validation of novel therapies targeting autophagy could be pivotal in overcoming chemoresistant PDAC. In the first results chapter of this thesis (i.e. Chapter 3), we demonstrate a novel chemotherapeutic strategy involving the potent suppression of the two central autophagy initiation complexes, primarily coordinated by ULK1 and Beclin-1, and the synergistic combination with Gemcitabine in both parent and chemoresistant PDAC cell lines. Initially, the targeted gene silencing of either ULK1 or BECN1 induced a counteractive cellular response to upregulate the expression of the other. After the development of two Gemcitabine-resistant PDAC cellular models from parent PANC-1 and MIA PaCa-2 cells, we proceeded to assess the effectiveness of two ULK1 complex inhibitors (i.e. MRT68921 and SBI-0206965) and two Beclin-1/VPS34 complex inhibitors (i.e. SAR405 and Spautin-1) with Gemcitabine treatment on proliferation, apoptosis and migration. Our results demonstrated that the targeted inhibition of either autophagic initiation complex increased PDAC chemosensitivity to Gemcitabine in the parent and resistant cell lines. The simultaneous inhibition of both the ULK1 complex and Beclin-1/VPS34 complex also resulted in a synergistic anti-proliferative effect on both PDAC phenotypes and were typically more effective in the resistant cells. We determined that SAR405 and MRT68921 were the most potent autophagy inhibitors individually, in combination with each other and in combination with Gemcitabine. Combining these concepts together, we employed these drugs as a triple combination treatment and observed a strong synergism in the resistant PDAC cells. Considering the importance of stress-induced autophagy in PDAC, serum and glucose starvation conditions were commissioned to more accurately mimic the stressful tumour microenvironment associated with PDAC. The same treatment strategy was performed in the PDAC cells after autophagy was confirmed to be upregulated from nutrient or glucose starvation. The combination composed of Gemcitabine, SAR405 and MRT68921 demonstrated a potent and synergistic anti-proliferative effect in the metabolically stressed parent and resistant PDAC cells. Next, the metabolic stress conditions (i.e. serum and glucose starvation) were applied together to further stress the PDAC cells and allow for a detailed assessment of the drugs on apoptosis. This study demonstrated that SAR405 and MRT68921 can effectively induce apoptosis individually and promote Gemcitabine-induced apoptosis in the parent and resistant PDAC cells. Moreover, SAR405, SBI-0206965 and MRT68921 were highly effective at limiting PDAC migration individually and in combination with Gemcitabine, which was unable to affect migration independently. Considering the synergy between Gemcitabine, SAR405 and MRT68921, an in vivo assessment was performed using a subcutaneous xenograft model with both parent and resistant PDAC. Although, the triple combination treatment was only significantly effective at reducing tumour weight and size in the tumours from parent PDAC, while a statistically non-significant decrease was observed in the chemoresistant tumours. Finally, we observed that high levels of p62, which is a marker of dysfunctional autophagy, was associated with improved patient overall survival and was an independent prognostic factor. Collectively, these results demonstrate the importance of autophagy activity in PDAC cells and indicate that its inhibition is an effective treatment strategy at overcoming chemoresistant PDAC. In Chapter 4, we examined the mechanisms of acquired chemoresistance using Gemcitabine-resistant PDAC cell models. The underlying mutational composition is unique to each PDAC patient. As a result, the modern pitfalls of chemotherapy in PDAC are often associated with chemoresistant mechanisms responsible for reduced therapeutic efficacy. Understanding the intricate cellular mechanisms in acquired chemoresistance is vital for optimising PDAC treatment. By investigating the transcriptome, proteome, and metabolome of Gemcitabine-resistant PDAC cells, we highlighted novel insights into this network and identified AMPK as a crucial upstream regulator of acquired chemoresistance. We observed that Gemcitabine-resistant PANC-1 and MIA PaCa-2 cells elicit significantly upregulated autophagic activity, major upstream dysregulation to the MAPK and PI3K/AKT pathways and a hyperactivated metabolism when compared to the parent cells. These networks are all linked through AMPK, which was shown to be activated overall and specifically from the catalytic subunit AMPKα2. We also identified RASSF1 in both PANC-1 and MIA PaCa-2 resistant cells as an activated upstream regulator of autophagy. As RASSF1 is a known autophagy regulator in the literature, we propose it as a vital protein in PDAC-acquired chemoresistance. Emphasising this, we demonstrated that reduced AMPKα2 levels increased the sensitivity of resistant PDAC cells to Gemcitabine. Finally, AMPKα2 was shown to be pharmacologically inhibited by SBI-0206965 as a potential autophagy targeting therapy in chemoresistant PDAC. In the final results chapter (i.e. Chapter 5), we assessed the potential of a novel biomarker panel at predicting chemoresponse in PDAC patients. The currently available biomarkers in PDAC are limited and ineffective at predicting and managing patient response to neoadjuvant chemotherapy (NAC). Improving these biomarkers could help personalise the clinical therapeutic strategy for patients based on their response to NAC and improve survival outcomes. This chapter demonstrates that a promising biomarker panel in serum composed of the existing CA19-9 and the novel PDAC marker CA-125 could be used as a predictive tool in NAC-treated PDAC patients. The panel was confirmed to significantly predict PDAC patient’s response to NAC. In all aspects of its assessment, it was superior to the currently used CA19-9. Thus, implicating further validation of this biomarker panel as a valuable clinical aide. In conclusion, this thesis has demonstrated the value of autophagic inhibition in PDAC and its use as a viable therapy against chemoresistant PDAC through the nomination of an effective combination regime. Moreover, we identified that AMPK and its major downstream effector pathway (i.e. autophagy) are activated in Gemcitabine resistant PDAC. Finally, we propose a novel biomarker panel that can predict patient response to NAC. Collectively, this thesis proposes novel and promising strategies across various stages of PDAC treatment that can be used to improve patient survival outcomes

Antioxidant and DPPH-Scavenging Activities of Compounds and Ethanolic Extract of the Leaf and Twigs of Caesalpinia bonduc L. Roxb.

Antioxidant effects of ethanolic extract of Caesalpinia bonduc and its isolated bioactive compounds were evaluated in vitro. The compounds included two new cassanediterpenes, 1α,7α-diacetoxy-5α,6β-dihydroxyl-cass-14(15)-epoxy-16,12-olide (1)and 12α-ethoxyl-1α,14β-diacetoxy-2α,5α-dihydroxyl cass-13(15)-en-16,12-olide(2); and others, bonducellin (3), 7,4’-dihydroxy-3,11-dehydrohomoisoflavanone (4), daucosterol (5), luteolin (6), quercetin-3-methyl ether (7) and kaempferol-3-O-α-L-rhamnopyranosyl-(1Ç2)-β-D-xylopyranoside (8). The antioxidant properties of the extract and compounds were assessed by the measurement of the total phenolic content, ascorbic acid content, total antioxidant capacity and 1-1-diphenyl-2-picryl hydrazyl (DPPH) and hydrogen peroxide radicals scavenging activities.Compounds 3, 6, 7 and ethanolic extract had DPPH scavenging activities with IC50 values of 186, 75, 17 and 102 μg/ml respectively when compared to vitamin C with 15 μg/ml. On the other hand, no significant results were obtained for hydrogen peroxide radical. In addition, compound 7 has the highest phenolic content of 0.81±0.01 mg/ml of gallic acid equivalent while compound 8 showed the highest total antioxidant capacity with 254.31±3.54 and 199.82±2.78 μg/ml gallic and ascorbic acid equivalent respectively. Compound 4 and ethanolic extract showed a high ascorbic acid content of 2.26±0.01 and 6.78±0.03 mg/ml respectively.The results obtained showed the antioxidant activity of the ethanolic extract of C. bonduc and deduced that this activity was mediated by its isolated bioactive compounds