Papain induces Mitochondria-mediated apoptosis and modulates inflammatory signaling in leukemic cells

Leukemia remains a major therapeutic challenge due to frequent treatment resistance and relapse. Novel agents that preferentially target leukemic cells are urgently needed. Papain, a cysteine protease derived from Carica papaya, has been reported to exhibit antioxidant, anti-inflammatory, and anticancer properties; however, its molecular effects in leukemia cells remain poorly understood.

This study aimed to evaluate the antileukemic effects of papain on HL-60 and K562 cells, focusing on apoptosis induction, cell-cycle regulation, inflammatory modulation, and gene/protein expression changes.

HL-60 and K562 cells were treated with papain at 123 µM and 386-483 µM, respectively, for 24-48 h. Concentrations were selected based on IC₂₅ values for HL-60 and IC₅₀ values for K562 obtained from viability assays. Cell viability was assessed using the MTS assay. Apoptosis, cell-cycle distribution, and surface expression of **Sema3A-a semaphorin family member involved in immune regulation and leukemic cell signaling-**were analyzed using flow cytometry. RT-qPCR quantified expression of apoptosis-related genes (P53, PTEN, BAX, CASP3, BCL-2, AKT-1, NF-κB), and ELISA measured Cytochrome c, Cleaved Caspase-3, TNF-α, IL-6, IL-10, GPX4, and ACSL4.

Papain significantly reduced cell viability in both cell lines in a dose- and time-dependent manner. Increased Cytochrome c and Cleaved Caspase-3 levels suggested apoptosis induction. Papain exposure led to predominant accumulation of cells in the G₀/G₁ phase accompanied by a reduction in the G₂/M population in HL-60 cells, whereas in K562 cells it induced a significant and dose-dependent accumulation in the S phase rather than G₂/M arrest. Papain induced significant alterations in apoptosis-related gene expression in both leukemia cell lines. These changes reflected activation of apoptotic signaling pathways rather than a uniform dose-dependent or directionally consistent transcriptional pattern. Furthermore, papain significantly downregulated Sema3A surface expression and altered cytokine profiles, increasing TNF-α and IL-10 in both cell lines and IL-6 in K562 cells.

Papain exhibits antileukemic effects in vitro, associated with mitochondrial-mediated apoptosis, altering cell-cycle progression, suppressing Sema3A expression, and modulating inflammatory responses. These findings suggest that papain may represent a candidate for further preclinical investigation in leukemia models.