OAR@UM Community:

Investigating the toxic effects of mTOR inhibition

2026-01-01T00:00:00Z

Title: Investigating the toxic effects of mTOR inhibition Abstract: Background: Although survival rates have increased due to significant breakthroughs in targeted cancer therapy, treatment-associated cardiotoxicity is still a significant clinical burden (Lyon et al., 2022). Pharmacological disruption of the PI3K/Akt/mTOR signalling pathways is a dual-purpose therapeutic approach because it is essential for both tumour growth and cardiomyocyte homeostasis. Compared to first-generation rapalogs, AZD2014, a second generation, dual mTORC1/2 inhibitor, offers a more extensive pathway suppression, but its cardiovascular safety profile requires careful evaluation prior to clinical incorporation (Middleton et al., 2025). Aim: The purpose of this study is to use complimentary in vitro and in vivo models to examine the cardiotoxic potential of AZD2014, with an emphasis on finding a concentration that can produce anti-cancer action while minimising harmful cardiac toxicities. Method: H9C2 rat cardiomyoblasts were exposed to AZD2014 across a concentration range of 0.2–1.2 μM at 24-, 48- and 72-hour timepoints, with cell viability assessed via the PrestoBlue® assay. For in vivo evaluation, the Fish Embryo Toxicity (FET) test was conducted in accordance with OECD Test Guideline 236 using wild-type Tübingen Long-fin zebrafish embryos exposed to concentrations of 0.75–10 μM over 96 hours post-fertilisation, with monitoring of lethal and sublethal developmental endpoints. Results: In H9C2 cardiomyoblasts, AZD2014 caused concentration and time dependent cytotoxicity, with significant decreases in cell viability at 48 and 72 hours. Higher concentrations in zebrafish embryos resulted in statistically significant increases in coagulation, absence of heartbeat, defective tail detachment, and lack of somite development and secondary defects such as haemorrhage, cardiac oedema, and delayed hatching. Conclusions: These result show that AZD2014 has a potential distinct cardiotoxic profile and suggest a possible narrow therapeutic window. In order to maximise anti-cancer efficacy while reducing cardiovascular risk, the results offer crucial preliminary data to support the future development of a triple combinatory treatment strategy. Description: B.Sc. (Hons)(Melit.)

Investigating treatment difference in oncogene-addicted 2D and 3D lung cancer models

2026-01-01T00:00:00Z

Title: Investigating treatment difference in oncogene-addicted 2D and 3D lung cancer models Abstract: Background: Lung cancer remains a leading global cause of cancer mortality (Bray et al., 2024), particularly oncogene-addicted subtypes which are highly associated with treatment resistance. Preliminary stages in in vitro research primarily rely on 2D cell culture work, which unfortunately poorly replicates in vivo characteristics. In recent years, this gap has been significantly bridged with the introduction of 3D cell cultures (Ohguro et al., 2024). Aim: The principal aim of this study was to investigate the effect of a dual mTOR inhibitor, AZD2014, on an oncogene-addicted non-small cell lung cancer (NSCLC) cell line. To this end, three objectives were pursued: (1) To optimise the generation and maintenance of 3D cell culture spheroids of H1975, an oncogene-addicted NSCLC cell line (2) To investigate the antiproliferative and antimetastatic effect of AZD2014 on 2D and 3D cell cultures (3) To analyse the difference in such anticancer properties of AZD2014 in 2D and 3D cell cultures Methods: A 3D oncogene-addicted lung cancer model was optimised using agarose micromould-based spheroid culture of H1975 cells. Cell viability was assessed using the PrestoBlue resazurin-based assay (2D) and the CellTiter-Glo luminescent ATP assay (3D) at 24, 48 and 72 hour time points across a concentration range of 0.2-1.2 μM (2D) and 5 and 10 μM (3D). Spheroid morphological parameters including diameter, circularity, and compactness were quantified using AnaSP. An in vitro wound healing (scratch) assay was also performed to evaluate the anti-migratory effects of AZD2014 at 0.2 μM, 0.6 μM and 1.2 μM, with wound closure measured at 24 hour intervals over 4 days. Results: AZD2014 produced a time- and concentration-dependent reduction in H1975 cell viability in both 2D and 3D models. In 2D, the repeated-measures Two-Way ANOVA confirmed highly significant effects of treatment concentration and incubation time on cell viability (both p<0.001). In the 3D model, significant viability reductions were observed at 5 μM and 10 μM across all time points, with a one-Way ANOVA confirming overall group differences at 24hrs (p=0.007), 48hrs (p=0.001) and 72hrs (p<0.001). In the wound healing assay, AZD2014 produced a concentration-dependent reduction in H1975 migration, with statistically significant effects of both time and treatment concentrations (both p<0.001). Conclusion: AZD2014 demonstrated a time- and concentration-dependent reduction in H1975 cell viability in both 2D and 3D models along with a decrease in migration in the wound healing assay. The 3D model required substantially higher concentrations to achieve equivalent inhibitory effects relative to the 2D, which is consistent with multicellular resistance mechanisms inherent to spheroid architecture. Spheroid diameter was reduced without a disruption of the overall structural integrity, suggesting a primarily cytostatic rather than cytotoxic mechanism of action. AZD2014 also demonstrated significant anti-migratory effects at sub-IC50 concentrations. Collectively, these findings support the importance of 3D models as physiologically relevant pre-clinical plaƞorms and reinforce the rationale for combining antisense oligonucleotides targeting HSP27 and TCTP within the LCRM trifactor therapeutic strategy. Description: B.Sc. (Hons)(Melit.)

A study of parental use of paracetamol in young children based on the theory of planned behaviour

2026-01-01T00:00:00Z

Title: A study of parental use of paracetamol in young children based on the theory of planned behaviour Abstract: Paracetamol is one of the most commonly used over the counter medications worldwide for the treatment on pain in adults and children over the age of 2 months. Although paracetamol is known to have a favourable safety profile, it has also been documented that parents may use it unconventionally as a means of social control. This includes calming an irritable child or helping the child sleep. The aim of this study was to assess the parental attitudes and practices regarding paracetamol use in children under five years of age in Malta by applying the Theory of Planned Behaviour (TPB) to identify the psychological predictors of use. A cross-sectional study was conducted using an anonymous questionnaire distributed via social media. Data from 379 parents/guardians were collected over a 3-month period. The questionnaire measured the constructs of the TPB – Attitude, Subjective Norms (SN), Perceived Behavioural Control (PBC) and Intention – using 10-point Likert scales. Responses were analysed statistically using Guttman’s Lambda 2 for reliability statistics, Pearson correlations and hierarchical multiple regression. Results found that the majority of respondents were female (85.5%) and completed tertiary education (63.3%). The primary sources of information regarding the use of paracetamol were reported to be the doctors (93.3%) and the pharmacists (56.7%), which may be supported by the fact that locally, paracetamol is only dispensed over the counter. Hierarchical regression revealed that Attitude (β = 0.44, p < 0.001) and SN (β = 0.28, p < 0.001) were the strongest predictors of the Intention to administer paracetamol. While the correlation between PBC and Intention was significant, the effect of PBC was mediated by Attitude and SN. Intention significantly predicted Behaviour (β = 0.34, p < 0.001). The findings indicate that the administration of paracetamol in young children in Malta is a behaviour mainly driven by Attitudes and perceived social pressures rather than parental confidence. The findings suggest that psychological predictors of Behaviour require assessment to develop effective strategies focused on reshaping these constructs. Promoting the appropriate use of paracetamol requires the reshaping of parental attitudes and reaffirming that not every challenge requires a pharmacological solution. Description: B.Sc. (Hons)(Melit.)

Bioinformatic and functional analysis of a variant in the promoter region of the neuropharmacology-associated CNR1 gene

2026-01-01T00:00:00Z

Title: Bioinformatic and functional analysis of a variant in the promoter region of the neuropharmacology-associated CNR1 gene Abstract: Introduction: The cannabinoid receptor 1 (CB1), encoded by CNR1, is one of the most abundant G-protein coupled receptors in the brain and a key component of the endocannabinoid system, which regulates mood, reward, appetite, pain and stress responses. Although CNR1 variation has been associated with neuropsychiatric, behavioural and metabolic phenotypes, the functional impact of common non-coding variants remains poorly understood. This is important because most disease- and trait-associated variants identified by genome-wide association studies map to the non-coding genome, where regulatory consequences are difficult to establish. Given the role of promoter architecture and transcription factor binding in controlling CNR1 expression, this study aimed to prioritise and functionally assess a common CNR1 promoter variant in a human neuronal cell model. Methods: A 500 bp region upstream of the CNR1 transcription start site was screened in silico using genomic and regulatory resources to identify an uncharacterised candidate variant. Wild‐type and variant CNR1 promoter fragments were cloned upstream of Firefly luciferase in constructs co‐expressing Renilla luciferase, then transfected into SH‐SY5Y neuroblastoma cells under optimised conditions. Promoter activity was quantified under basal and acetylcholine‐stimulated conditions by normalising Firefly to Renilla luminescence, with relative luciferase activity compared between constructs using the Mann-Whitney U test. Results: Thirty‐six variants were identified within the selected CNR1 promoter sequence, none of which had established clinical or pharmacogenetic annotations. Rs9344756 (chr6:88166822 G>A) was prioritised as a common SNP located within an epigenomically active promoter segment and predicted to alter multiple transcription factor binding motifs. Under basal conditions, the CNR1 wild‐type and rs9344756 variant constructs showed mean relative luciferase activities of 0.76 ± 0.34 and 0.58 ± 0.19, respectively. Following acetylcholine stimulation, activity increased to 1.04 ± 0.55 for the wild‐type and 0.80 ± 0.36 for the variant, corresponding to an approximate 1.3-fold wild-type/variant difference in both conditions. Non‐normal data distributions led to use of the Mann-Whitney U test, which showed no significant difference between wild‐type and variant constructs (basal: p = 0.753; acetylcholine-stimulated: p = 0.916). Conclusion: rs9344756 did not produce a statistically significant change in CNR1 promoter activity in this SH-SY5Y reporter model. Although the variant construct showed descriptively lower mean luciferase activity than the wild-type under both basal and acetylcholine-stimulated conditions, interpretation is limited by the use of a single experimental plate with technical replicates only. These findings suggest that rs9344756 is unlikely to have a large independent effect on promoter activity under the conditions tested, although a subtle or context-dependent regulatory role cannot be excluded. This study provides a preliminary experimental assessment of a common, uncharacterised CNR1 promoter variant and demonstrates a framework for integrating bioinformatic prioritisation with functional validation of non-coding variants. Description: B.Sc. (Hons)(Melit.)