<p>Novel Molecular Mechanism of Aspirin and Celecoxib Targeting Mammalian Neuraminidase-1 Impedes Epidermal Growth Factor Receptor Signaling Axis and Induces Apoptosis in Pancreatic Cancer Cells</p>

Abstract

Background: Aspirin (acetylsalicylic acid) and celecoxib have been used as potential anticancer therapies. Aspirin exerts its therapeutic effect in both cyclooxygenase (COX)-dependent and-independent pathways to reduce tumor growth and disable tumorigenesis. Celecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor, reduces factors that cause inflammation and pain. The question is whether aspirin and celecoxib have other molecular targets of equal or more therapeutic efficacy with significant anti-cancer preventive benefits. Aim: Here, we propose that aspirin and celecoxib exert their anti-cancer effects by targeting and inhibiting mammalian neuraminidase-1 (Neu-1). Neu-1 has been reported to regulate the activation of several receptor tyrosine kinases (RTKs) and TOLL-like receptors and their downstream signaling pathways. Neu-1 in complex with matrix metalloproteinase-9 (MMP-9) and G proteincoupled receptors (GPCRs) has been reported to be tethered to RTKs at the ectodomain. Materials and Methods: The WST-1 cell viability assay, Caspase 3/7 assay, and Annexin V assay were used to evaluate the cell viability and detect apoptotic and necrotic cells following treatment in MiaPaCa-2, PANC-1 and the gemcitabine-resistant PANC-1 variant (PANC-1 GemR) cells. Microscopic imaging, lectin cytochemistry, and flow cytometry were used to detect levels of α-2,3 sialic acid. Epidermal growth factor (EGF)-stimulated live cell sialidase assays and neuraminidase assays were used to detect Neu-1 activity. Immunocytochemistry was used to detect levels of EGFR and phosphorylated EGFR (pEGFR) following treatment. Results: For the first time, aspirin and celecoxib were shown to significantly inhibit Neu-1 sialidase activity in a dose-and time-dependent manner following stimulation with EGF. Aspirin blocked Neu-1 desialylation of α-2,3-sialic acid expression following 30 min stimulation with EGF. Aspirin and celecoxib significantly and dose-dependently inhibited isolated neuraminidase (Clostridium perfringens) activity on fluorogenic substrate 2ʹ-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid (4-MUNANA). Aspirin inhibited phosphorylation of the EGFR in EGF-stimulated cells. Aspirin dose-and time-dependently induced CellEvent caspase-3/7 + cells as well as apoptosis and necrosis on PANC-1 cells. Conclusion: These findings signify a novel multimodality mechanism(s) of action for aspirin and celecoxib, specifically targeting and inhibiting Neu-1 activity, regulating EGF-induced growth receptor activation and inducing apoptosis and necrosis in a dose-and time-dependent manner. Repurposing aspirin and celecoxib as anti-cancer agents may also upend other critical targets involved in multistage tumorigenesis regulated by mammalian neuraminidase-1. Significance: These findings may be the missing link connecting the anti-cancer efficacy of NSAIDs to the role of glycosylation in inflammation and tumorigenesis.

Next Generation of Cancer Drug Repurposing: Therapeutic Combination of Aspirin and Oseltamivir Phosphate Potentiates Gemcitabine to Disable Key Survival Pathways Critical for Pancreatic Cancer Progression

“…This may be due to the reduced clonogenic potential of cells following treatment, suggesting that ASA+OP+GEM treatment preferentially targets malignant cells with stem cell-like properties, such as regenerative capacity (Figures 4 and 5). Given that we have reported on the role of OP [23,28] and ASA [22] in targeting and shutting down Neu-1 activity in complex with growth factor receptors, these findings are not surprising. A recent study reported on Neu-1 suppressing bladder cancer progression by inhibiting the fibronectin-integrin α5β1 interaction and the Akt signaling pathway [47].…”

“…We believe that the combination of ASA+OP can interfere with EMT that is triggered by chemotherapy, and this is likely the reason for its effectiveness. In support of this premise, we have previously reported that GEM-resistant PANC-1 (PANC-1-GemR) cells treated with increasing concentrations of ASA (0.1 to 10 mM) revealed a significant concentration-and time-dependent decrease in cell viability [22]. It is noteworthy that resistance to GEM can involve multiple mechanisms, such as altered apoptotic regulating genes and altered expressions or sensitivities of enzyme targets.…”

“…We have previously reported that repurposing ASA as an anti-cancer agent can upend critical targets involved in multistage tumorigenesis regulated by mammalian Neu-1 [22]. Our group has also described a novel signaling paradigm implicated in multistage tumorigenesis [25].…”

“…This trimeric complex plays critical roles in ligandinduced activation of several RTKs, including the EGFR [23], insulin receptor (IR) [50], the nerve growth factor (NGF) TrkA receptor [51], and TOLL-like receptors (TLRs) [24,26,52], all of which are upregulated in cancer. We have reported that therapeutic targeting of Neu-1 with OP [28] and ASA [22] disables this intrinsic receptor signaling platform for cancer cell survival in human pancreatic cancer with acquired chemoresistance. Here, we reported on the therapeutic efficacy of ASA and OP in sensitizing and potentiating the efficacy of standard of care GEM for the treatment of pancreatic cancer.…”

“…We recently reported on the missing link connecting the anti-cancer efficacy of ASA to the role of glycosylation in inflammation and tumorigenesis [22]. We showed that ASA exerts anti-cancer effects by targeting and inhibiting mammalian neuraminidase-1 (Neu-1).…”