Mohammad Rasheduzzaman

Exosomes are small extracellular vesicles secreted by cells and are known to play a key role in intercellular communication. Several studies have associated exosomes with various roles in tumorigenesis and explored their potential as a source of biomarkers for diagnosis and prognosis in cancer research. Exosomes can be isolated from several body fluids, including those that are noninvasively accessible, such as human saliva. This book chapter provides a step-by-step detailed description of techniques that are used for the isolation, quantification, and characterization of exosomes from saliva. These include ultracentrifugation for the isolation, nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and western blot (WB) for characterization of exosomes. The NTA approach explores the Brownian motion and light scattering of particles to predict size and concentration. TEM enables visualization of the exosomes which often present a cup-shaped morphology. Western blot is used to detect commonly expressed exosome-associated proteins. Finally, salivary exosomes isolated using these protocols can further be characterized for downstream analysis according to their cargo (proteins, DNA, RNA, miRNA) and utilized for cancer biomarker discovery.

Alzheimer’s disease (AD) is characterized by the formation of intracellular aggregate composed of heavily phosphorylated tau protein and extracellular deposit of amyloid-β (Aβ) plaques derived from proteolysis cleavage of amyloid precursor protein (APP). Autophagy refers to the lysosomal-mediated degradation of cytoplasmic constituents, which plays a critical role in maintaining cellular homeostasis. Importantly, recent studies reported that dysregulation of autophagy is associated in the pathogenesis of AD, and therefore, autophagy modulation has gained attention as a promising approach to treat AD pathogenesis. In AD, both the maturation of autolysosomes and its retrograde transports have been obstructed, which causes the accumulation of autophagic vacuoles and eventually leads to degenerating and dystrophic neurites function. However, the mechanism of autophagy modulation in APP processing and its pathogenesis have not yet been fully elucidated in AD. In the early stage of AD, APP processing and Aβ accumulation-mediated autophagy facilitate the removal of toxic protein aggregates via mTOR-dependent and -independent pathways. In addition, a number of autophagy-related genes (Atg) and APP are thought to influence the development of AD, providing a bidirectional link between autophagy and AD pathology. In this review, we summarized the current observations related to autophagy regulation and APP processing in AD, focusing on their modulation associated with the AD progression. Moreover, we emphasizes the application of small molecules and natural compounds to modulate autophagy for the removal and clearance of APP and Aβ deposits in the pathological condition of AD.

Autophagy refers to the degradation of cytoplasmic constituents by a lysosomal-mediated pathway, which plays a critical role in maintaining cellular homeostasis. Importantly, dysregulation of autophagy has been implicated in multiple neurodegenerative disorders. Previous studies reported that autophagy affects the processing of amyloid precursor protein (APP), thus stimulating β‐amyloid (Aβ) production in Alzheimer’s disease (AD) eventually. Although the mechanism of autophagy modulation on APP processing and its pathogenesis has not yet been fully elucidated at the molecular level, but modulation of autophagy has received considerable attention as a promising approach for the treatment of AD. In the early stage of AD, Aβ may prompt autophagy to facilitate its removal via mTOR‐independent as well as-dependent pathways. However, a recent study proposed that autophagy processes are not properly regulated as AD continues to progress, and consequently, the production of Aβ tends to accumulate rapidly. Meanwhile, a number of autophagy-related genes (Atg) as well as APP genes are also thought to influence the development of AD, which may serve as a bi‐directional link to autophagy and AD pathology. In this review, we summarized current observations related to autophagy regulation and APP processing, focusing on their dynamic modifications associated with the progression of AD. Recent findings together highlight the essential role of autophagy in the removal and clearance of APP and Aβ deposition in the pathological condition of AD.

A major concern in the clinical application of tumor necrosis factor related apoptosis‐inducing ligand (TRAIL) in tumors is the development of resistance. Therefore, agents that can potentially restore TRAIL sensitivity are important therapeutic targets for cancer treatment. Herein, we evaluated lanatoside c and digoxin, both of which are widely used cardiac glycosides (CGs), for their ability to sensitize human hepatocellular carcinoma cells (Huh‐7 and HepG2) through TRAIL‐induced apoptosis. CGs functionalize TRAIL as shown by its effect on intracellular reactive oxygen species (ROS) generation, which damages mitochondrial integrity and thereby confers intrinsic apoptotic caspase cascade during combined treatment. Caspase activation is dependent on ROS as shown by the ability of CGs to generate ROS and the ROS‐N‐acetylcysteine (NAC) relationship, which inhibits apoptosis during cotreatment by preventing the formation of caspase‐8 and ‐3. Furthermore, CGs triggered p38MAPK phosphorylation and NAC pre‐exposure blocked p38MAPK phosphorylation, which demonstrated that p38MAPK was dependent upon ROS generation. Additionally, CGs were found to be potent inducers of AMPK‐mediated protective autophagy as pharmacological and genetic autophagy inhibition reached the higher threshold of TRAIL‐mediated apoptosis. Finally, CGs downregulated the expression of the antiapoptotic protein Bcl‐2 and increased the translocation of proapoptotic protein cytochrome c, thereby inducing apoptosis. Collectively, these results indicate that CGs potentiate the enhanced cytotoxic capacity to TRAIL through ROS generation, p38MAPK phosphorylation, cell survival protein downregulation, and protective autophagy inhibition.

Enhancement of TRAIL-induced apoptosis by 5-fluorouracil requires activating Bax and p53 pathways in TRAIL-resistant lung cancers