Cytosolic phospholipase A2 (cPLA2) IVA as a potential signature molecule in cigarette smoke condensate induced pathologies in alveolar epithelial lineages.

BACKGROUND: Smoking is one of the leading causes of millions of deaths worldwide. During cigarette smoking, most affected and highly exposed cells are the alveolar epithelium and generated oxidative stress in these cells leads to death and damage. Several studies suggested that oxidative stress causes membrane remodeling via Phospholipase A2s but in the case of cigarette smokers, mechanistically study is not yet fully defined. In view of present perspective, we evaluated the involvement of cytosolic phospholipase A2 (cPLA2) IVA as therapeutic target in cigarette smoke induced pathologies in transformed type I and type II alveolar epithelial cells. METHODS: Transformed type I (WI26) and type II (A549) alveolar epithelial cells were used for the present study. Cigarette smoke condensate (CSC) was prepared from most commonly used cigarette (Gold Flake with filter) by the Indian population. CSC-induced molecular changes were evaluated through cell viability using MTT assay, reactive oxygen species (ROS) measurement using 2,7 dichlorodihydrofluorescin diacetate (DCFH-DA), cell membrane integrity using fluorescein diacetate (FDA) and ethidium bromide (EtBr) staining, super oxide dismutase (SOD) levels, cPLA2 activity and molecular involvement of specific cPLA2s at selected 24 h time period. RESULTS: CSC-induced response on both type of epithelial cells shown significantly reduction in cell viability, declined membrane integrity, with differential escalation of ROS levels in the range of 1.5-15 folds and pointedly increased cPLA2 activity (p < 0.05). Likewise, we observed distinction antioxidant potential in these two types of lineages as type I cells had considerably higher SOD levels when compared to type II cells (p < 0.05). Further molecular expression of all cPLA2s increased significantly in a dose dependent manner, specifically cytosolic phospholipase A2 IVA with maximum manifestation of 3.8 folds. Interestingly, CSC-induced ROS levels and cPLA2s expression were relatively higher in A549 cells as compared to WI26 cells. CONCLUSIONS: The present study indicates that among all cPLA2s, specific cPLA2 IVA are the main enzymes involved in cigarette smoke induced anomalies in type I and type II lung epithelial cells and targeting them holds tremendous possibilities in cigarette smoke induced lung pathologies.

Secretory Phospholipase A2 (sPLA2) Isozymes as Potential Targets in Tobacco Condensate- induced Colon Damage.

AIMS: To find out the role of secretory phospholipase A2 (sPLA2) isozymes as potential targets in tobacco condensate-induced colon damage. BACKGROUND: The effects of cigarette smoke condensate (CSC) and the molecular mechanisms involved in the regulation of phospholipase A2 (PLA2) and its isozymes in colon cells, which are still unclear and emerging, are studied. OBJECTIVES: The study aimed to check the effect of CSC on cell viability and reactive oxygen species (ROS) and superoxide. Also, the effect of CSC on gene expression of different secretory phospholipase A2 (sPLA2) was evaluated. Moreover, the impact of inhibition of sPLA2 on various cell properties i.e. cell viability, cell proliferation, membrane damage and free radicals' generation is also studied. METHODS: CSC-induced changes were evaluated in cell viability by MTT assay, followed by the evaluation of membrane modulation by flow cytometry, free radical generation by fluorescent dyes, PLA2 isoforms gene expression patterns and their suppression by small interfering RNA (siRNA) studied in HCT-15 male and HT-29 female colon cells. RESULTS: Our results demonstrate that HCT-15 and HT-29 cells treated with CSC significantly reduced the cell viability by 50% within 48 h and significantly enhanced the total reactive oxygen species (ROS) by 2 to 10-fold, and mitochondrial ROS (mtROS) and superoxide radicals (SOR) by 2-fold each. Treatment with CSC significantly unregulated secretory phospholipase A2 (sPLA2) IID group and down-regulated IB and cytosolic phospholipase (cPLA2) IVA groups in HCT-15 cells without affecting them in HT-29 cells. Silencing the sPLA2 IID group results in an increase in cell viability and a decrease in ROS. Silencing the PLA2 IVA gene in the HCT-15 cells showed a reduced expression which had no impact on the CSC-induced cell proliferation, membrane damage and free radicals (ROS, mtROS, and SOR) generation. CONCLUSION: Therefore, identifying cell-specific sPLA2 isozymes seems to play a key role in controlling the ROSinduced damage by CSC and helps develop specific therapeutic strategies.