Cigarette smoke-induced galectin-3 as a diagnostic biomarker and therapeutic target in lung tissue remodeling.

Galectin-3 (Gal-3), a multifunctional carbohydrate-binding lectin, has emerged as a key player in various biological processes including inflammation, cancer, cardiovascular diseases and fibrotic disorders, however it remains unclear if Gal-3 is a bystander or drives lung tissue remodeling (LTR). Persistent exposure to cigarette smoke (CS) is the leading cause of oxidative and inflammatory damage to the lung tissues. CS-induced pathological increase in Gal-3 expression has been implicated in the pathogenesis of various respiratory conditions, such as chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), and lung cancer. We and others have reported that CS induces Gal-3 synthesis and secretion, which modulates the pathological signaling pathways in lung epithelial cells implicating Gal-3 as a novel diagnostic marker and a factor driving LTR in CS-exposed lungs. Therefore, pharmacological interventions targeting Gal-3 and its upstream and downstream signaling pathways can help combat CS-induced LTR. Excitingly, preclinical models have demonstrated the efficacy of interventions such as Gal-3 expression inhibition, Gal-3 receptor blockade, and signaling pathways modulation open up promising avenues for future therapeutic interventions. Furthermore, targeting extracellular vesicles-mediated Gal-3 release and the potential of microRNA-based therapy are emerging as novel therapeutic approaches in CS-induced LTR and have been discussed in this article.

Cigarette smoke induces epithelial-to-mesenchymal transition, stemness, and metastasis in lung adenocarcinoma cells via upregulated RUNX-2/galectin-3 pathway.

AIMS: An elevated level of galectin-3, a carbohydrate-binding lectin implicated in tumorigenesis, metastasis, and epithelial-mesenchymal transition (EMT), has been found in cigarette smokers. However, the regulation of its expression and role in the pathogenesis of CS-induced EMT and lung cancer metastasis is unclear. Here, we have investigated the mechanism of CS-induced and galectin-3-mediated EMT in airway epithelial cells (AECs). MAIN METHODS: A549 adenocarcinoma cells and primary small airway epithelial cells cultured on an air-liquid interface (ALI) were exposed to cigarette smoke extract (CSE), and MTT, trypan blue, migration, invasion, tumor spheroid and colony formation assays were performed to assess EMT phenotype. Immunoblotting was performed to assess EMT and stemness markers and other regulatory proteins. KEY FINDINGS: CSE exposure affected cell survival and morphology, migration, invasion, and clonogenicity of AECs, which were concomitant with an increase in the expression of EMT markers, galectin-3, and runt-related transcription factor-2 (RUNX-2), an osteogenic transcription factor and upstream regulator of galectin-3. Chemical inhibition or silencing of RUNX-2 downregulated galectin-3 and modulated EMT marker expression, migration, invasion, and clonogenicity in CSE-exposed AECs. Recombinant human galectin-3 also induced EMT and stemness-associated changes in the AECs, and GB1107, a galectin-3 inhibitor, ameliorated these changes. Further, CSE-induced intracellular ROS enabled an increase in RUNX-2 and galectin-3 expression, which were reversed by n-acetyl-cysteine. SIGNIFICANCE: These results provide a novel mechanistic insight into CSE-induced EMT via RUNX-2/galectin-3 axis mediated through ROS, which promoted EMT-associated changes, including invasion, migration, and stemness in AECs, which could be implicated in CS-induced lung cancer progression.

First Record of Clonostachys rosea (Ascomycota: Hypocreales) Entomopathogenic Fungus in the Mango Hopper Amritodus atkinsoni (Hemiptera: Cicadellidae).

Mango hopper (Amritodus atkinsoni Lethierry) causes devastations in the early vegetative stage of the mango crop. The classical management of mango hopper is with systemic insecticides but their overuse has caused environmental pollution. Here, we have evaluated the entomopathogenic role of Clonostachys rosea through bioassay and optimized media for its large-scale culturing. The current study reveals the potentiality of C. rosea as entomopathogenic on A. atkinsoni. Initially, morphological and molecular characterization was used to validate local isolates' identity as C. rosea. Further, we have evaluated the entomopathogenic role of C. rosea through a bioassay, where the highest mean mortality in A. atkinsoni was observed at a treatment concentration of 3 × 108 conidia/mL, with 96.67% mortality after 168 h of infection. This work also provides insight into the laboratory-based media standardization for C. rosea, resulting in oatmeal agar media and broth as the most suitable artificial media, and 20 °C temperature for its mass culture. Thus, C. rosea is a novo-entomopathogenic fungus on A. atkinsoni and has a high potency to be included in the management of mango hopper pests.

Cellular and molecular insights into the roles of visfatin in breast cancer cells plasticity programs.

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.

Method of Preparation of Cigarette Smoke Extract to Assess Lung Cancer-Associated Changes in Airway Epithelial Cells.

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.

Air-Liquid Interface Culture Model to Study Lung Cancer-Associated Cellular and Molecular Changes.

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.

Fisetin suppresses cigarette smoke extract-induced epithelial to mesenchymal transition of airway epithelial cells through regulating COX-2/MMPs/ß-catenin pathway.

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 ß-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/ß-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.

COVID-19 severity in obese patients: Potential mechanisms and molecular targets for clinical intervention.

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.

Induction of growth cessation by acacetin via ß-catenin pathway and apoptosis by apoptosis inducing factor activation in colorectal carcinoma cells.

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 ß-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.