(T1330-03-20) Synthesis, Structural Characterization, and Inhibitory Effects of Caffeic Acid Phenethyl ester (CAPE) Derivatives on Human Multiple Myeloma Cell Growth

Purpose: Multiple Myeloma (MM) is a hematologic cancer that develops due to the proliferation of malignant plasma cells in the bone marrow. The accumulation of MM cells leads to a variety of complications such as anemia, bone disease, and renal damage. MM remains challenging to treat due to its drug resistance and the associated side effects, leading researchers to explore naturally derived compounds as adjuvants with current treatments. Caffeic acid phenethyl ester (CAPE) has demonstrated various pharmacological properties, including anti-cancer activities. Previous studies have reported that CAPE inhibits the growth of MM cells through the induction of apoptosis and oxidative stress. The objective of this project is to determine if newly synthesized CAPE derivatives are more potent inhibitors of MM cell growth than CAPE.

Methods: Synthesis of CAPE derivatives: CAPE derivatives were synthesized through coupling reactions involving caffeic acid with derivatives of phenylalanine and tyrosine (Figure 1). NMR and MS confirmed the presence of pure CAPE derivatives. MM cell line, RPMI 8226, was cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. Cytotoxicity: The MM cell line, RPMI 8226, was cultured according to the manufacturer’s protocol and treated with CAPE and synthesized derivatives (0 – 40 μM) for 24, 48, or 72 hours prior to cell viability assay tests using Presto Blue™. Cell death mechanism: The induction of apoptosis and ferroptosis in RPMI8226 MM cells was determined through western blotting for proapoptotic proteins (cleaved caspase-3 and cleaved PARP-1) and ferroptosis-related proteins (GPX4 and CHAC1). House-keeping proteins, β-actin and α-tubulin, are used as the loading control. Statistical analyses used included ANOVA and Dunnett’s multiple comparisons tests with GraphPad Prism software 9.3.1 to determine statistical significance.

Results: CAPE derivatives were evaluated for their effects on RPMI 8226 cell viability, which decreased in a time- and dose-dependent manner. Western blot analysis showed that, while CAPE induced caspase-3-mediated apoptosis, it is not quite mechanism of cell death for CAPE derivatives, prompting further investigation into their potential link to ferroptosis. When combined with the ferroptosis inducer, Erastin, a significant reduction in cell viability was observed. In contrast, no significant effect occurred with the ferroptosis inhibitor, Ferrostatin-1. Additionally, Western blot analysis showed a notable decrease in GPX4 expression and increased CHAC1 expression when combined with Erastin. The opposite results were seen when the derivatives were combined with Ferrostatin-1, with an increase in GPX4 expression and a decrease in CHAC1 expression.

Conclusion: The results from the cytotoxicity study differentiate the inhibitory effects of those CAPE derivatives on the MM cell growth and identify more potent CAPE derivatives than the parental compound. Ferroptosis induction may serve as the main mechanism behind the inhibitory effects of CAPE derivatives compared to apoptosis induction. The structure-activity relationship of CAPE and its derivatives reveals that replacing the ester group with an amide results in a stronger cell death response, as seen in two more potent derivatives compared to CAPE. The data highlights how structural modifications can either preserve or diminish CAPE's effects through different cell death modes, emphasizing the critical role of structural changes in modulating its activity and mechanism of action.

Schematic illustration of the synthesis of CAPE derivatives.