Platelet-Activating Factor-Receptor Signaling Mediates Targeted Therapies-Induced Microvesicle Particles Release in Lung Cancer Cells

Abstract

Microvesicle particles (MVP) secreted by a variety of cell types in response to reactive oxygen species (ROS)-generating pro-oxidative stressors have been implicated in modifying the cellular responses including the sensitivity to therapeutic agents. Our previous studies have shown that expression of a G-protein coupled, platelet-activating factor-receptor (PAFR) pathway plays critical roles in pro-oxidative stressors-mediated cancer growth and MVP release. As most therapeutic agents act as pro-oxidative stressors, the current studies were designed to determine the role of the PAFR signaling in targeted therapies (i.e., gefitinib and erlotinib)-mediated MVP release and underlying mechanisms using PAFR-expressing human A549 and H1299 non-small cell lung cancer (NSCLC) cell lines. Our studies demonstrate that both gefitinib and erlotinib generate ROS in a dose-dependent manner in a process blocked by antioxidant and PAFR antagonist, verifying their pro-oxidative stressor’s ability, and the role of the PAFR in this effect. We observed that these targeted therapies induce MVP release in a dose- and time-dependent manner, similar to a PAFR-agonist, carbamoyl-PAF (CPAF), and PAFR-independent agonist, phorbol myristate acetate (PMA), used as positive controls. To confirm the PAFR dependency, we demonstrate that siRNA-mediated PAFR knockdown or PAFR antagonist significantly blocked only targeted therapies- and CPAF-mediated but not PMA-induced MVP release. The use of pharmacologic inhibitor strategy suggested the involvement of the lipid ceramide-generating enzyme, acid sphingomyelinase (aSMase) in MVP biogenesis, and observed that regardless of the stimuli used, aSMase inhibition significantly blocked MVP release. As mitogen-activated protein kinase (MAPK; ERK1/2 and p38) pathways crosstalk with PAFR, their inhibition also significantly attenuated targeted therapies-mediated MVP release. These findings indicate that PAFR signaling could be targeted to modify cellular responses of targeted therapies in lung cancer cells.

Effects of miRNA-149-5p and Platelet-Activating Factor-Receptor Signaling on the Growth and Targeted Therapy Response on Lung Cancer Cells

“…As previously reported, we found that both A549 and H1299 cell lines express endogenous PAFR and miR-149 [ 11 , 29 ]. The functional studies confirmed that PAFR activation induced an increased proliferation of both the cell lines in a dose-dependent manner, similarly to what has been shown in various tumor models [ 19 , 29 , 31 ]. Consistent with the previous findings, in our study we demonstrated that transfection with miR-149-specific mimics reduced the proliferation of A549 cells in a dose-dependent manner compared to the normal control or scrambled-miRNA transfection control, verifying its tumor suppressor activity.…”

“…Based upon this rationale, in the current study we sought to determine the effects of miR-149-5p and PAFR on the growth and cytotoxic responses of targeted therapies using NSCLC cellular models [ 31 ]. We hypothesized that the tumor suppressive activity of miR-149-5p would circumvent the PAFR-mediated growth enhancing effect or reduce the therapeutic response in our lung cancer model.…”

“…KBP cells were created from the PAFR-negative human epidermal KB cell line via transduction with the MSCV2.1 retroviral vector, containing human PAFR cDNA, whereas KBM cells were transduced with MSCV2.1 retroviral vector [ 38 ]. The human lung epithelial carcinoma A549 and H1299 NSCLC cell lines were grown as previously reported [ 31 ].…”

“…Total RNA samples were extracted as described above. A High-Capacity cDNA Reverse Transcription kit containing random hexamers was used to transcribe RNA samples to cDNA as per the manufacturer’s instructions, and a SYBR green-based quantitative qPCR method was performed as per the manufacturer’s instructions and as previously reported [ 31 , 39 ]. The fluorescence was detected using a StepOne Real-Time PCR machine (Applied Biosystems, Foster City, CA, USA).…”

Evaluation of SARS-CoV-2 Spike S1 Protein Response on PI3K-Mediated IL-8 Release

“…The fetal bovine serum (FBS) was from Corning (Corning, NY, USA), antibiotic-antimycotic was from Gibco (Gaithersburg, MD, USA), and penicillin-streptomycin was purchased from Hyclone (Logan, UT, USA). Human nasopharyngeal carcinoma KBP cells were grown in DMEM high glucose media, and non-small cell lung cancer A549 cells were grown in F-12K media supplemented with 10% FBS and antibiotic/antimycotic, as previously described [30,31].…”