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Tumor Discovery Sorafenib induces MVPs in NSCLC

significantly blocked CPAF and sorafenib-induced, but cell viability by imipramine alone at the 48-h time point
not PMA-induced MVP release in both the cell lines (Figure 4B and D), indicating a chemopreventive ability
(Figure 3A and B), indicating the involvement of the PAFR of this repurposed drug, providing a rationale for it to be
signaling in MVP release. On the other hand, imipramine explored in combination with other therapeutic agents.
significantly blocked CPAF-, PMA-, and sorafenib-induced Taken together, these results suggest that imipramine
MVP release, indicating that involvement of an aSMase in enhances the antiproliferative effects of sorafenib, through
MVP release (Figure 3A and B). These data also indicate its ability to inhibit aSMase-mediated pathways, thereby
that regardless of the nature of the stimuli used, inhibiting reducing ceramide production and MVP release, as
aSMase blocks MVP release. These data are consistent with shown in Figure 5. These findings highlight the potential
our previous findings, 14,18,19 demonstrating that other ROS- implication of imipramine to enhance the efficacy of
generating stimuli induce MVP release in a PAFR and sorafenib in NSCLC.
aSMase-dependent manner.
4. Discussion
3.3. Imipramine enhances the antiproliferative
1-3
effect of sorafenib As NSCLC continues to pose challenges, sorafenib,
Given that aSMase inhibitors block MVP release and have a multikinase inhibitor, has demonstrated variable
been evaluated in cancer patients, 34,48 the next studies antitumor effects in NSCLC models by targeting multiple
tested if blocking aSMase could increase the efficacy of signaling pathways, including those associated with
3,4
sorafenib. To evaluate the synergy of an aSMase inhibitor angiogenesis and ROS generation. Although ROS can
on sorafenib-mediated growth inhibition in NSCLC cells, mediate cytotoxicity in tumors, elevated levels of ROS may
A549 and H1299 cells were pre-treated with imipramine paradoxically enhance survival and promote resistance
5,6
(20 µM for 1 h), followed by treatment with or without through compensatory pathways. Consequently,
14
sorafenib at a lower concentration (4 µM), consistent with combination approaches that both exploit sorafenib’s
prior studies utilizing lower micromolar concentrations of cytotoxic potential and suppress parallel pro-survival
sorafenib in combination strategies. 41,49 The cell survival was pathway have garnered significant attention in efforts to
26
assessed using the SRB assay at 24- and 48-h time points. As improve NSCLC outcomes.
shown in Figure 4A-D, imipramine enhanced the cytotoxic A growing body of evidence implicates MVP as a
effect of sorafenib resulting in a significant reduction mediator of therapy resistance, tumor progression, and
in cell viability compared to sorafenib monotherapy. immune evasion in multiple cancer models, including
We also noticed a modest but significant inhibition of NSCLC. 23,25 By encapsulating pro-survival factors,

A B

Figure 3. Effects of PAFR antagonist and aSMase inhibitor on sorafenib-induced MVP release. A549 (A) and H1299 (B) cells were pre-treated with
WEB2086 (a PAFR antagonist, 10 µM, 1 h) or imipramine (an aSMase inhibitor, 20 µM, 1 h) followed by the treatments with or without CPAF (100 nM),
PMA (100 nM), or sorafenib (8 µM). These cell lines were also treated with vehicle (0.1% DMSO), WEB2086 (10 µM) and imipramine (20 µM) alone.
After 4 h of incubation, MVP were isolated and analyzed. Data are presented as mean ± scanning electron microscope of three independent biological
replicates, normalized per 1 × 10 cells. The statistically significant differences were observed between control and CPAF, PMA, and sorafenib alone
6
groups; CPAF and WEB+CPAF; SF and WEB+SF; CPAF and IMI+CPAF; PMA and IMI + PMA; and SF and IMI + SF.
Notes: **p<0.01, ***p<0.001 compared with control; p<0.001 compared with CPAF; p<0.001 compared with SF; p<0.001 compared with CPAF; p<0.05
§
‡
†
&
compared with PMA; p<0.001 compared with PMA; p<0.001 compared with SF.
#
¶
Abbreviations: aSMase: Acid sphingomyelinase; CPAF: Carbamoyl-platelet-activating factor; IMI: Imipramine; MVP: Microvesicle particles;
PAFR: Platelet-activating factor-receptor; PMA: Phorbol myristate acetate; SF: Sorafenib; WEB: WEB2086.
Volume 4 Issue 3 (2025) 85 doi: 10.36922/TD025110019