Background:
Pseudomyxoma peritonei (PMP) is a rare malignancy with limited treatment options. Mitomycin C (MMC) is a frontline chemotherapeutic used in hyperthermic intraperitoneal chemotherapy (HIPEC) due to its potent cytotoxicity via DNA cross-linking. However, its severe systemic toxicity restricts use to localized administration. TRAABA24, a lipid nanoparticle (LNP) formulation, was developed as a safer alternative with a distinct mechanism of action, offering sustained efficacy and improved tolerability. This study evaluates TRAABA24’s effects on PMP cell viability, its mechanism of action, and therapeutic potential.

Methods:
PMP cells were treated with TRAABA24 LNP, MMC, and doxorubicin. Cell viability was assessed at 24 and 48 hours using the Promega CellTiter-Glo 3D assay, and ED50 values were calculated using a three-parameter log-logistic model. To evaluate safety, cytotoxicity assays were also conducted in HIEC-6 normal intestinal epithelial cells (1×10⁴ cells/well) under identical conditions. A cAMP quantification assay was performed to validate TRAABA24’s interaction with Gs alpha signaling.

Results:
At 24 hours MMC exhibited the highest cytotoxicity in PMP cells (ED50: 17.4 µM), reducing viability more rapidly than TRAABA24 (ED50: 22.7 µM). However, by 48 hours, TRAABA24 achieved comparable cytotoxicity, suggesting a delayed but sustained effect. MMC induced rapid apoptosis via DNA cross-linking, whereas TRAABA24 triggered an autophagic response, enabling a controlled and tolerable therapeutic effect suitable for systemic administration and sustained oral maintenance therapy. To assess TRAABA24’s selectivity and safety cytotoxicity was evaluated in HIEC-6 normal intestinal epithelial cells. The ED50 values were 221 µM for MMC, 162 µM for Doxorubicin, and 509 µM for TRAABA24, indicating that TRAABA24 exhibited significantly lower toxicity in normal cells compared to MMC and Doxorubicin. 

TRAABA24 was identified through in silico screening for its high binding affinity to the Gs alpha subunit, particularly in the GNAS 201C mutant crystal structure (PDB: 6AU6). Additionally, TRAABA24 binds to tubulin monomers, tubulin dimers, and taxol binding sites A and B, suggesting a role in microtubule dynamics. GNAS mutations cause constitutive Gs alpha activation, leading to elevated cAMP levels, disrupted cellular signaling, and uncontrolled mucin secretion in PMP. A cAMP quantification assay confirmed TRAABA24’s ability to modulate cAMP signaling, reinforcing its distinct mechanism compared to MMC and Doxorubicin.