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Tumor Discovery Sorafenib induces MVPs in NSCLC

that have enhanced survival outcomes, particularly in NSCLC. 23-26 By sequestering and exporting oncogenic
patients with early-stage or resectable disease. Notably, proteins, nucleic acids, and even chemotherapeutic agents,
3
advances in immune checkpoint inhibitors and targeted MVP can diminish drug accumulations in tumor cells and
therapies have provided tailored options based on tumor modulate the surrounding microenvironment to favor
characteristics such as programmed cell death ligand 1 cancer progression. 23,24 It has been demonstrated that
(PD-L1) expression and specific genetic mutations (i.e., PAF and related lipid mediators can be packaged within
epidermal growth factor receptor [EGFR] and anaplastic MVP, enabling inflammatory and immune-modulating
lymphoma kinase) leading to better disease control and responses that further compromise treatment efficacy. 18,20-22
prolonged survival. Despite these advances, emergence These findings underscore that in addition to targeting
3
of resistance mechanisms remains a significant challenge, primary oncogenic pathways, strategies that disrupt MVP
which includes on-target mutations, bypass pathways, and release may aid in overcoming drug resistance.
histological transformation. 3 Imipramine, a tricyclic antidepressant, has garnered
Among targeted therapies, tyrosine kinase inhibitors attention as an effective aSMase inhibitor that disrupts
(TKIs), including sorafenib, have been used to treat ceramide biosynthesis, a key lipid mediator in MVP
NSCLC. Sorafenib, a multikinase inhibitor, has formation and NSCLC pathophysiology. 27-32 By reducing
3,4
emerged as a promising agent targeting multiple ceramide production, imipramine decreases the budding
pathways involved in tumor progression, angiogenesis, of vesicles, thus curtailing MVP release. 27,33 Therefore,
and resistance mechanisms. Notably, reactive oxygen imipramine and other sphingolipid-targeted drugs have
4
species (ROS) generation is one of the key mechanisms been of interest as adjunct therapies. 27,34-37 For example,
32
through which TKIs induce cytotoxic effects; however, studies by Irep et al., have demonstrated enhanced
elevated ROS levels activate resistance mechanisms inhibitory effects on cisplatin/etoposide by targeting
enabling the tumor to evade therapy and continue to small extracellular vesicles (also referred to as exosomes)
grow. For example, oxidative modifications of EGFR and synthesis and trafficking in a small cell lung cancer model.
5
associated downstream signaling pathways enhance tumor In NSCLC, sorafenib’s efficacy has also been shown to
progression and resistance to EGFR TKIs. These findings significantly improve with dual-therapy approaches. 4,38-40
6
highlight the paradoxical nature of ROS in NSCLC therapy. However, no approach to aSMase inhibition, such as with
While ROS generation is critical for the effectiveness of imipramine, has been investigated.
TKIs, the adaptive responses of NSCLC cells to oxidative
stress underscore the need for combination strategies 2. Materials and methods
that both amplify ROS cytotoxicity and inhibit resistance 2.1. Reagents
pathways to improve therapeutic outcomes. Among these Culture media was obtained from GE Healthcare
signaling pathways, ROS non-enzymatically cleaves lipid Biosciences (Marlborough, MA, USA), with fetal bovine
membranes to produce oxidized glycerophosphocholines serum (FBS) from Corning (Corning, NY, USA). Penicillin-
(Ox-GPCs) that exhibit platelet-activating factor (PAF) streptomycin was acquired from Hyclone (Logan, UT,
agonistic properties, which mediate angiogenesis, tumor USA) and antibiotic-antimycotic solution was purchased
growth, metastasis, and immune modulation. 7-10 In from Gibco (Gaithersburg, MD, USA). The PAFR agonist
addition, PAF-like molecules are often upregulated in carbamoyl-PAF (CPAF), the antagonist WEB2086, and
response to radiation and chemotherapy, exacerbating the aSMase inhibitor imipramine were all obtained from
immune suppression and therapy resistance. 10-13 Our Cayman Chemicals Co. (Ann Arbor, MI, USA). Sorafenib
group has shown that in NSCLC models, both tumor tosylate was procured from Millipore Sigma (St. Louis,
and its environment are modulated by PAF and platelet- MO, USA). All other reagents were purchased from Sigma-
activating factor-receptor (PAFR) signaling. 9,14,15 Moreover, Aldrich (St. Louis, MO, USA).
PAFR plays a significant role in vesicular formation and is
dependent on pathways such as mitogen-activated protein 2.2. Cell lines
kinase (MAPK), nuclear factor kappa B (NF-κB), and acid Human NSCLC lines, A549 and H1299, were used for
sphingomyelinase (aSMase). 16-19 Notably, formed vesicles all experiments as both express PAFR at similar levels.
14
have been shown to contain PAF-like agonists and serve as These cell lines were a kind gift from Dr. Weiwen Long
bioactive molecules. 18-22 (Department of Biochemistry and Molecular Biology
Mounting evidence points to these large extracellular at Wright State University). A549 cells were maintained
vesicles, also referred to as microvesicle particles (MVP), in F-12K medium supplemented with 10% FBS, 2.5 mL
as critical mediators of treatment resistance, including penicillin-streptomycin, 2.5 mL antibiotic-antimycotic,

Volume 4 Issue 3 (2025) 82 doi: 10.36922/TD025110019