Phytol and α-Bisabolol Synergy Induces Autophagy and Apoptosis in A549 Cells and Additional Molecular Insights through Comprehensive Proteome Analysis via Nano LC-MS/MS.

BACKGROUND: Non-Small Cell Lung Cancer (NSCLC) is a malignancy with a significant prevalence and aggressive nature, posing a considerable challenge in terms of therapeutic interventions. Autophagy and apoptosis, two intricate cellular processes, are integral to NSCLC pathophysiology, each affecting the other through shared signaling pathways. Phytol (Phy) and α -bisabolol (Bis) have shown promise as potential anticancer agents individually, but their combined effects in NSCLC have not been extensively investigated. OBJECTIVE: The present study was to examine the synergistic impact of Phy and Bis on NSCLC cells, particularly in the context of autophagy modulation, and to elucidate the resulting differential protein expression using LCMS/ MS analysis. METHODS: The A549 cell lines were subjected to the patented effective concentration of Phy and Bis, and subsequently, the viability of the cells was evaluated utilizing the MTT assay. The present study utilized real-time PCR analysis to assess the expression levels of crucial apoptotic genes, specifically Bcl-2, Bax, and Caspase-9, as well as autophagy-related genes, including Beclin-1, SQSTM1, Ulk1, and LC3B. The confirmation of autophagy marker expression (Beclin-1, LC3B) and the autophagy-regulating protein SQSTM1 was achieved through the utilization of Western blot analysis. Differentially expressed proteins were found using LC-MS/MS analysis. RESULTS: The combination of Phy and Bis demonstrated significant inhibition of NSCLC cell growth, indicating their synergistic effect. Real-time PCR analysis revealed a shift towards apoptosis, with downregulation of Bcl-2 and upregulation of Bax and Caspase-9, suggesting a shift towards apoptosis. Genes associated with autophagy regulation, including Beclin-1, SQSTM1 (p62), Ulk1, and LC3B, showed significant upregulation, indicating potential induction of autophagy. Western blot analysis confirmed increased expression of autophagy markers, such as Beclin-1 and LC3B, while the autophagy-regulating protein SQSTM1 exhibited a significant decrease. LC-MS/MS analysis revealed differential expression of 861 proteins, reflecting the modulation of cellular processes. Protein-protein interaction network analysis highlighted key proteins involved in apoptotic and autophagic pathways, including STOML2, YWHAB, POX2, B2M, CDA, CAPN2, TXN, ECHS1, PEBP1, PFN1, CDC42, TUBB1, HSPB1, PXN, FGF2, and BAG3, emphasizing their crucial roles. Additionally, PANTHER pathway analysis uncovered enriched pathways associated with the differentially expressed proteins, revealing their involvement in a diverse range of biological processes, encompassing cell signaling, metabolism, and cellular stress responses. CONCLUSION: The combined treatment of Phy and Bis exerts a synergistic inhibitory effect on NSCLC cell growth, mediated through the interplay of apoptosis and autophagy. The differential protein expression observed, along with the identified proteins and enriched pathways, provides valuable insights into the underlying molecular mechanisms. These findings offer a foundation for further exploration of the therapeutic potential of Phy and Bis in the management of NSCLC.

Synergistic induction of apoptosis in lung cancer cells through co-delivery of PLGA phytol/α-bisabolol nanoparticles.

This study explored the potential of poly-(lactic-co-glycolic) acid (PLGA) nanoparticles to enhance the effectiveness of anticancer treatments through combination therapy with phytol and α-bisabolol. The encapsulation efficiency of the nanoparticles was investigated, highlighting the role of ionic interactions between the drugs and the polymer. Characterization of PLGA-Phy+Bis nanoparticles was carried out using DLS with zeta potential and HR-TEM for size determination. Spectrophotometric measurements evaluated the encapsulation efficiency, loading efficiency, and in vitro drug release. FTIR analysis assessed the chemical interactions between PLGA and the drug actives, ensuring nanoparticle stability. GC-MS was employed to analyze the chemical composition of drug-loaded PLGA nanocarriers. Cytotoxicity was evaluated via the MTT assay, while Annexin V-FITC/PI staining and western blot analysis confirmed apoptotic cell death. Additionally, toxicity tests were performed on L-132 cells and in vivo zebrafish embryos. The study demonstrates high encapsulation efficiency of PLGA-Phy+Bis nanoparticles, which exhibit monodispersity and sizes of 189.3±5nm (DLS) and 268±54 nm (HR-TEM). Spectrophotometric analysis confirmed efficient drug encapsulation and release control. FTIR analysis revealed nanoparticle structural stability without chemical interactions. MTT assay results demonstrated the promising anticancer potential of all the three nanoparticle types (PLGA-Phy, PLGA-Bis, and PLGA-Phy+Bis) against lung cancer cells. Apoptosis was confirmed through Annexin V-FITC/PI staining and western blot analysis, which also revealed changes in Bax and Bcl-2 protein expression. Furthermore, the nanoparticles exhibited non-toxicity in L-132 cells and zebrafish embryo toxicity tests. PLGA-Phy+Bis nanoparticles exhibited efficient encapsulation, controlled release, and low toxicity. Apoptosis induction in A549 cells and non-toxicity in healthy cells highlight their clinical potential.

α-bisabolol ß-d-fucopyranoside (ABFP) ameliorates scopolamine-induced memory deficits through cholinesterase inhibition and attenuation of oxidative stress in zebrafish (Danio rerio).

Alzheimer's disease (AD) is one of the major devastating neurodegenerative disorders associated with the gradual decline of an individual's memory, cognition, and ability to carry out day-to-day activities. In the present study, the neuroprotective ability of α-bisabolol ß-d-fucopyranoside (ABFP) was assessed via measurement of antioxidant parameters like lipid peroxidation, glutathione peroxidation, glutathione, protein carbonyl content assays, and caspase-3 activity estimation. Moreover, the acute toxicity of ABFP was estimated in the zebrafish larval model. The results showed that ABFP exhibits little to no toxicity at lower concentrations in the acute toxicity test. ABFP-pretreated and scopolamine-exposed fish exhibited more exploratory behavior in the behavior assay than scopolamine-only induced groups. Additionally, the results of antioxidant enzyme assays revealed reduced oxidative stress and damage in ABFP-treated fish, while enzyme activity experiments carried out with brain homogenate from ABFP-treated fish showed decreased acetylcholinesterase enzyme activity. Overall, it can be concluded that ABFP has the potential to be a promising agent for the treatment of AD in the future.

Hesperidin Methyl Chalcone reduces extracellular Aß(25-35) peptide aggregation and fibrillation and also protects Neuro 2a cells from Aß(25-35) induced neuronal dysfunction.

In the present study, we evaluated the neuroprotective potential of Hesperidin Methyl Chalcone (HMC) against the neurotoxicity induced by Aß(25-35) peptide. HMC demonstrated higher free-radical scavenging activity than Hesperidin in initial cell-free studies. Investigations using the fluorescent dye thioflavin T with Aß(25-35) peptide showed that HMC has the ability to combat extracellular amyloid aggregation by possessing anti-aggregation property against oligomers and by disaggregating mature fibrils. Also, the results of the molecular simulation studies show that HMC ameliorated oligomer formation. Further, the anti-Alzheimer's property of HMC was investigated in in vitro cell conditions by pre-treating the neuro 2a (N2a) cells with HMC before inducing Aß(25-35) toxicity. The findings demonstrate that HMC increased cell viability, reduced oxidative stress, prevented macromolecular damage, allayed mitochondrial dysfunction, and exhibited anticholinesterase activity. HMC also reduced Aß induced neuronal cell death by modulating caspase-3 activity, Bax expression and Bcl2 overexpression, demonstrating that HMC pre-treatment reduced mitochondrial damage and intrinsic apoptosis induced by Aß(25-35).In silico evaluation against potential AD targets reveal that HMC could be a potent inhibitor of BACE-1, inhibiting the formation of toxic Aß peptides. Overall, the findings imply that the neuroprotective efficacy of HMC has high prospects for addressing a variety of pathogenic consequences caused by amyloid beta in AD situations and alleviating cognitive impairments.

Chitosan based encapsulation increased the apoptotic efficacy of thymol on A549 cells and exhibited non toxic response in swiss albino mice.

Thymol is a plant-derived natural phenolic compound abundantly present in Thymus vulgaris species. In the present study, we developed a chitosan-based drug delivery system to deliver thymol to A549 cells. The physicochemical properties of thymol-loaded chitosan nanoparticles (thymol-NP) were characterized using polyphasic techniques viz., FTIR, XRD, DLS, and SEM. Thymol-NP exhibited a size of 282.5 nm and encapsulation efficiency of 74.08 ± 0.73%. The IC50 of thymol-NP against A549 cells was 99.57 µg/ml at 24 h, which was lower than that of the pure form. Clear apoptotic features such as cellular morphology, cell shrinkage, and augmentation of dead cells were observed in both the thymol and thymol-NP treated A549 cells. The percentage of apoptotic cells in the thymol-NP IC50 treated cells was >90% which was considerably higher than the group treated with thymol alone. In vivo toxicity study showed that the swiss albino mice treated up to a concentration of 1000 mg/kg of thymol-NP neither showed signs of toxicity nor death up to 14 days. Also, no significant influence was observed on behavior, body weight, organ weight, and organ histology. Overall, the data concluded that thymol-NP can be considered a safe and potent drug candidate against A549 cells.

Autophagy: A Potential Therapeutic Target of Polyphenols in Hepatocellular Carcinoma.

Autophagy is a conserved biological phenomenon that maintains cellular homeostasis through the clearing of damaged cellular components under cellular stress and offers the cell building blocks for cellular survival. Aberrations in autophagy subsidize to various human pathologies, such as dementia, cardiovascular diseases, leishmaniosis, influenza, hepatic diseases, and cancer, including hepatocellular carcinoma (HCC). HCC is the fifth common mortal type of liver cancer globally, with an inhomogeneous topographical distribution and highest incidence tripled in men than women. Existing treatment procedures with liver cancer patients result in variable success rates and poor prognosis due to their drug resistance and toxicity. One of the pathophysiological mechanisms that are targeted during the development of anti-liver cancer drugs is autophagy. Generally, overactivated autophagy may lead to a non-apoptotic form of programmed cell death (PCD) or autophagic cell death or type II PCD. Emerging evidence suggests that manipulation of autophagy could induce type II PCD in cancer cells, acting as a potential tumor suppressor. Hence, altering autophagic signaling offers new hope for the development of novel drugs for the therapy of resistant cancer cells. Natural polyphenolic compounds, including flavonoids and non-flavonoids, execute their anticarcinogenic mechanism through upregulating tumor suppressors and autophagy by modulating canonical (Beclin-1-dependent) and non-canonical (Beclin-1-independent) signaling pathways. Additionally, there is evidence signifying that plant polyphenols target angiogenesis and metastasis in HCC via interference with multiple intracellular signals and decrease the risk against HCC. The current review offers a comprehensive understanding of how natural polyphenolic compounds exhibit their anti-HCC effects through regulation of autophagy, the non-apoptotic mode of cell death.