Evaluating the Role of Inflammation In Glioblastoma Multiform Induced Depression Disorder for Alternate Therapeutics

Abstract

Major depressive disorder (MDD) is the leading cause of disability worldwide. The incidence of depression and anxiety in Glioblastoma Multiforme (GBM) patients is as high as 90% and 97% respectively which not only affect the quality of life but also worsen the progression and survival. Chemotherapeutic drugs such as Temozolomide further deteriorate the depressive condition. MDD and GBM share intricate immune pathways, dysregulation of which lead to failure of treatment strategies and increased morbidity and mortality. Owing to the unsatisfactory efficacy of conventional antidepressants and anti-cancers, particularly in comorbid scenario, there is a need to explore novel therapies. This is only possible through development of better screening methods for drug candidates and identification of genetic factors potentially associated with disease risk and pathogenesis. The current study aimed to explore the antidepressant and anti-cancer potential of thymoquinone, a natural compound with anti-inflammatory activity and proposed its underlying mechanism of action in vitro and in vivo. Besides, a three-Dimensional Quantitative Structure-Activity Relationship Model for Blood-Brain Barrier (BBB) permeability by utilizing GRid INdependent molecular Descriptors derived from Molecular Interaction Fields has been developed. To identify genetic factors associated with disease pathogenesis, the regulatory role of single nucleotide polymorphisms (SNPs) reported in Genome-Wide Association Studies and their proxy SNPs was elucidated via Regulome DB. In vitro anticancer activity of thymoquinone was checked by MTT assay, single-cell tracking through live cell time- lapse fluorescent microscopy and Fluorescent Activated Cell Sorting. Thymoquinone showed cytotoxic potential against the U87-mg cell line in the MTT assay. Thymoquinone decreased the migration speed, track length, displacement, and track straightness of cancer cells. Thymoquinone showed a modest yet significant arrest of the cell cycle at the G2/M and S phase in the U87-MG cell line 24 hours post-treatment. In the next phase, we developed GBM induced depression mice model and an unpredictable chronic mild stress (UCMS) induced depression model to evaluate anti-depressant effect of thymoquinone. Our results indicate that GBM induced behavioural deficit, decreased capacity of social interaction, and increased latency to feed as quantified by social interaction test, and novelty suppressed feeding test respectively. Thymoquinone improved latency to feed and social interaction while standard chemotherapeutic drug, Temozolomide aggravated the anxiety-like behaviour and latency to feed. The FDA approved anti-depressant Fluoxetine reinstated only latency to feed in orthotopic xenograft GBM mice model. Interestingly, thymoquinone improved latency to feed and resignation behaviour affected by UCMS. In xiv conclusion, thymoquinone pose better behavioural effects as compared to standard antidepressant and anticancer drug (which has worsened the condition) against GBM induced depression as well as chronic stress-induced depression. To identify the underlying mechanism of action responsible for behavioural changes, mRNA expression of neurogenesis (NeuN), neural plasticity (BDNF) proliferation (Ki67), and inflammatory (IL1β, IL6, and TNF-α) markers were analysed in the tumour, hippocampus, and amygdala in both models. The results indicate that GBM and UCMS induced changes in the pro-inflammatory cytokines were effectively reversed by the thymoquinone treatment. Moreover, thymoquinone reversed the opposing effect of stress on BDNF in hippocampus and amygdala. However, thymoquinone didn’t not increase proliferation and neuronal marker expression which points towards the proposition that the behavioural effects of thymoquinone are due to its anti-inflammatory potential and independent to neurogenesis. To develop a better screening method for drug candidates for brain instigated disorders, a three-Dimensional Quantitative Structure-Activity Relationship model was developed for blood-brain permeability prediction. The results demonstrate that an increase in the distance between the hydrophobic group and hydrogen bond donor correlated with an increase in the Blood-Brain Barrier Permeability (BBBP). Moreover, the shape of the molecules also plays a critical role in the determination of permeability across BBB. The approach presented in the current study thus provides a relatively quick yet inexpensive method to derive a potentially reliable 3D-QSAR model for BBBP. To identify genetic components and proteins playing a significant role in GBM and MDD pathogenesis, non-coding single SNPs acting as eQTL, and their respective regulatory proteins were elucidated using SNAP and Regulome DB. For GBM, 6 SNPs and for MDD 230 SNPs were predicted to pose significant regulatory function. Two SNPs (rs8051216C>T and rs7206735T>C) showed significant association when tested on GBM patients. Taken together, our study reports the anti-cancer effect of thymoquinone in in vitro as well as the anti depressant effect in in vivo models of glioblastoma and chronic stress. Along with the proposed model of BBB permeability and identification of novel SNPs responsible for disease susceptibility, our results add significant knowledge to the understanding of the MDD and GBM induced depression and highlight the shared mechanisms of their pathogenesis along with the proposition of a potential compound with better safety profile than currently available drug regimens.