Targeting p53/p21 Signaling: Linarin as a Novel Modulator of 5-FU Sensitization in Colorectal Cancer
TL;DR
Linarin demonstrates the capacity to potentiate 5-FU cytotoxicity by reactivating the p53/p21 pathway in CRC models harboring functional p53 by reactivating the p53/p21 pathway in linarin's sensitizing action in p53-competent cells.
Abstract
Purpose: We assessed linarin, a plant-derived flavone glycoside, for its ability to overcome intrinsic 5-fluorouracil (5-FU) resistance by reinstating p53/p21-mediated tumor suppression in colorectal cancer (CRC) cell models, and we explicitly present these findings as a mechanistic proof of concept within a defined in vitro scope. Methods: Cell viability was measured using the MTT assay; synergy was assessed using the combination index (CI) with isobologram validation; and programmed cell death was evaluated by internucleosomal DNA fragmentation ELISA and Western blot analysis of caspase-dependent markers. Underlying pathways were probed by immunoblotting, quantitative RT-PCR, and targeted siRNA-mediated knockdown of p53 and p21, complemented by pharmacological inhibition with pifithrin-α and UC2288. Results: Linarin induced concentration-dependent cell killing in CRC lines, yielding IC₅₀ values of 122.8–210.2 μM at 48 hours, alongside selective sparing of normal colon cells (NCM460; selectivity index approximately 2.0–3.3-fold). The p53 status-dependent sensitivity gradient (HCT116 wild-type < SW480 mutant < HCT116 p53⁻/⁻) provided initial evidence of p53-dependent cytotoxicity. Dual therapy with 5-FU yielded robust synergy (CI 0.52–0.88) in p53-proficient lines, with near-additive effects in p53-null cells (CI 0.77), and notably reduced the 5-FU IC₅₀ in intrinsically resistant LoVo cells from 19.0 μM to 5.7 μM. Linarin promoted p53 stabilization and Ser15/Ser20 phosphorylation, elevated p21 mRNA levels to 5.4-fold the control level, and suppressed MDM2 mRNA levels to 0.42-fold the control level. This was accompanied by caspase-dependent cell death and amplified DNA damage signaling (elevated γ-H2AX). Functional validation via siRNA knockdown and pharmacological inhibition confirmed the centrality of the p53/p21 pathway in linarin's sensitizing action in p53-competent cells. Conclusion: As a mechanistic proof-of-concept, linarin demonstrates the capacity to potentiate 5-FU cytotoxicity by reactivating the p53/p21 pathway in CRC models harboring functional p53. Future investigations should prioritize models of acquired resistance and formulation optimization to advance these findings toward preclinical translation.