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Airway epithelial cells arrayed in the inner lining of the airways of the lung are believed to be the major source for the development of malignancy of the lung. The advent of in vitro cell culture model made it easy to understand the molecular mechanism of carcinogenesis at a cellular level, where the airway epithelial cells are cultured on a 2D surface submerged in the culture media. However, this method of culturing airway epithelial cells does not reflect their true nature, and thus results obtained have their limitations. Further, they exhibit dissimilar morphology, transcriptome, and secretome when compared to the cells in vivo. Thus, the experimental data obtained from 2D culture models are inconclusive and, in most cases, could not be validated further in in vivo settings. These limitations can be addressed by culturing the airway epithelial cells on air-liquid interface (ALI), where they develop ciliated morphology similar to that of the lung. Experiments performed with this 3D model provide reliable data that are more realistic, and, in many cases, could replace the requirement of further in vivo validation. Here, we provide the detailed protocol of a 3D culture system called ALI culture for growing human-derived primary small airway epithelial cells to study the cellular and molecular changes associated with lung cancer.

Smoking tobacco is a major risk factor for the development of lung cancer, COPD, and other lung pathologies in smokers. Cigarette smoke (CS), which is comprised of several toxic components, is known to cause oxidative stress and inflammation-induced lung damage. Since airway epithelial cells act as the primary barrier, they protect the lung tissues from environmental insults, including CS. Upon exposure to these insults, airway epithelial cells act as the initial site of injury and orchestrate the pathophysiology of lung cancer. Scientists have been using cigarette smoke extract (CSE) in the preclinical model of in vitro cell culture to understand the effect of CS on the cellular, biochemical, and molecular changes in the lung epithelial cells. However, the standard procedure to prepare the CSE in the laboratory with a low-cost assembly and obtaining a reproducible quality of CSE in different batches is a challenge. Here, in this chapter, we delineate the method for the preparation of CSE using a discontinuous puff-based system which is an economical and reproducible method to prepare CSE in the laboratory. This method is suitable for studying CSE-induced molecular changes in lung diseases, including lung cancer, using in vitro models of lung adenocarcinoma cells.

Obesity has reached a pandemic proportion and is responsible for the augmentation of multimorbidity including certain cancers. With the rise in obesity amongst the female population globally, a concomitant increase in breast cancer (BC) incidence and related mortality has been observed. In the present review, we have elucidated the cellular and molecular insight into the visfatin-mediated cellular plasticity programs such as Epithelial to mesenchymal transition (EMT) and Endothelial to mesenchymal transition (EndoMT), and stemness-associated changes in BC cells. EMT and EndoMT are responsible for inducing metastasis in cancer cells and conferring chemotherapy resistance, immune escape, and infinite growth potential. Visfatin, an obesity-associated adipokine implicated in metabolic syndrome, has emerged as a central player in BC pathogenesis. Several studies have indicated the presence of visfatin in the tumor microenvironment (TME) where it augments EMT and EndoMT of BC cells. Further, Visfatin also modulates the TME by acting on the tumor stroma cells such as adipocytes, infiltrated immune cells, and adipose-associated stem cells that secrete factors such as cytokines, and extracellular vesicles responsible for augmenting cellular plasticity program. Visfatin induced altered metabolism of the cancer cells and molecular determinants such as non-coding RNAs involved in EMT and EndoMT have been discussed. We have also highlighted specific therapeutic targets that can be exploited for the development of effective BC treatment. Taken together, these advanced understandings of cellular and molecular insight into the visfatin-mediated cellular plasticity programs may stimulate the development of better approaches for the prevention and therapy of BC, especially in obese patients.

With the global spread of SARS-CoV-2, millions of people have been affected leading to the declaration of coronavirus disease 2019 (COVID-19) as a pandemic by the WHO. Several studies have linked the severity of COVID-19 cases and increased fatality in patients with obesity and other comorbid conditions such as diabetes, cardiovascular diseases, hypertension, and kidney disease. Obesity, a metabolically deranged condition, establishes a low-grade chronic inflammation in the body, which affects different organs and promotes the development of several other diseases. The ways in which SARS-CoV-2 infection aggravates the already overloaded body organs with inflammation or vice versa has perplexed the researchers. As a result, there is an intensified search for the clear-cut mechanism to understand the link of obesity with the increased severity of COVID-19 in obese patients. In this article we have discussed various mechanisms linking obesity, inflammation, and COVID-19 to enhance the understanding of the disease process and help the clinicians and scientists develop potential cellular, molecular and metabolic targets for clinical intervention and management of COVID-19 severity in obese patients.

Cigarette smoke exposure leads to upregulation of cyclooxygenase-2 (COX-2), an inducible enzyme that synthesizes prostaglandin E2 (PGE2) and promotes airway inflammation. COX-2 overexpression is frequently implicated in inflammation, invasion, metastasis, and epithelial-mesenchymal transition (EMT). However, its detailed molecular mechanism in cigarette smoke induced EMT is not clear. Further, fisetin, a bioflavonoid, exhibits antioxidant and anti-inflammatory properties, but its effect in modulating COX-2-mediated inflammation and downstream sequelae remains unexplored. Therefore, we have investigated the mechanism of cigarette smoke-induced COX-2-mediated EMT in airway epithelial cells and examined the role of fisetin in controlling this aberration. MTT, trypan blue staining, gelatin zymography, Western blotting, invasion, wound healing, and tumor sphere formation assays in cigarette smoke extract (CSE) and/or fisetin treated airway epithelial cells, and in-silico molecular docking studies were performed. Results revealed that CSE exposure increased the expression and activity of COX-2, MMP-2/9, and β-catenin and also enhanced expression of EMT markers leading to higher migration and invasion potential of airway epithelial cells. A specific COX-2 inhibitor NS-398 as well as fisetin treatment reversed the expression of EMT biomarkers, reduced the activity of MMP-2/9, and blocked the migration and invasion potential induced by CSE. Further, PGE2 also increased MMPs activity, invasion, and migration potential similar to CSE, which were significantly reversed by fisetin. In-silico studies showed a high binding affinity of fisetin to key EMT associated proteins, validating its anti-EMT potential. Thus, our study firstly unearths the mechanism of CSE-induced EMT in airway epithelial cells via COX-2/MMP/β-catenin pathway, and secondly, it reveals that fisetin could significantly reverse CSE-induced EMT by inhibiting COX-2, indicating that fisetin could be an effective drug candidate for cigarette smoke-induced lung dysfunction.

Acacetin, a bioflavanoid, contains anti-inflammatory and anti-cancer activities as shown in different experimental models. However, its anticancer potential and mechanism of action against colorectal cancer cells is largely unknown. Here, we have investigated the efficacy of acacetin using two colorectal adenocarcinoma SW480 and HCT-116 cell lines. Cell survival was examined by Trypan-blue exclusion and MTT assays, cell cycle analysis by FACS, apoptosis was assessed using Annexin V FITC assay and nuclear condensation by Hoechst staining, ROS level by DCFDA and Mitosox, and protein expression level by Western blotting. Acacetin reduced the cell survival and proliferation of both types of cells, and induced S- and G2-M phase arrest and also reduced the levels of β-catenin and its downstream target c-myc. Further, acacetin induced apoptosis as examined by Annexin-V FITC and nuclear condensation. It increased intracellular ROS production, especially mitochondrial ROS. Acacetin increased mitochondrial membrane potential depolarization and Bax:Bcl-2 ratio. Although significant changes in caspases -8 and -9 and PARP level was not observed, acacetin could induce the truncation and subsequent translocation of activated AIF from mitochondria to cytosol, which could further induce chromosomal breakage leading to apoptosis. In conclusion, Acacetin induces mitochondrial ROS-mediated cell death in a caspase-independent manner in SW480 and HCT-116 colon carcinoma cells by inducing apoptosis inducing factor (AIF), which may potentiate its anticancer and chemotherapeutic prospects against colorectal carcinoma.