Analysis of the Effect of Gentian Violet on Apoptosis and Proliferation in Cutaneous T-Cell Lymphoma in an In Vitro Study.

IMPORTANCE

Triggering the extrinsic apoptotic pathway is an effective way to kill cutaneous T-cell lymphoma (CTCL) cells in vitro and ex vivo.

OBJECTIVE

To compare small molecules that induce extrinsic apoptosis in CTCL to identify and analyze compounds that induce high levels of tumor cell death and block tumor cell growth.

DESIGN, SETTING, AND PARTICIPANTS: From November 5, 2014, to January 30, 2018, this study performed high-throughput small molecule screening of 1710 compounds followed by detailed analysis of the ability of gentian violet (GV) to promote apoptosis and inhibit proliferation of CTCL cells.

EXPOSURES

In vitro and ex vivo analyses using enzyme-linked immunosorbent assays, flow cytometry, and immunoblotting.

MAIN OUTCOMES AND MEASURES

Apoptosis, cleaved caspases, extrinsic apoptotic death receptors and ligands, cell proliferation, nuclear factor-κB expression, and other factors.

RESULTS

This study used high-throughput screening to detect cleaved caspase 8 induced in CTCL cells by 1710 unique compounds. The nonprescription, topical antimicrobial remedy GV induced more total apoptosis than did nitrogen mustard (mechlorethamine). Furthermore, GV induced 4 to 6 times greater apoptosis in CTCL lines than in normal keratinocytes, suggesting a favorable topical toxicity profile. In addition to increasing caspase 8, GV also upregulated death receptors 4 and 5, tumor necrosis factor (TNF)-related apoptosis-inducing ligand, and Fas ligand but not the Fas receptor, TNF receptor, or TNF-α ligand. These results are consistent with induction of extrinsic apoptosis via the Fas and TNF-related apoptosis-inducing ligand pathways. Increased phosphorylation of phospholipase C-γ1, Ca2+ influx, and reactive oxygen species were also detected, indicating that the mechanism of Fas ligand upregulation involves key elements of the activation-induced cell death pathway. In ex vivo studies, 1-μmol/L GV induced up to 90% CTCL apoptosis in Sézary blood cells. In addition, GV reduced expression of antiapoptotic myeloid cell leukemia 1 and proproliferative nuclear factor-κB components and increased inhibitory κB levels. This finding was associated with cell cycle arrest and reduced CTCL tumor cell proliferation. Furthermore, the CTCL killing associated with GV was augmented when used in combination with methotrexate.

CONCLUSIONS AND RELEVANCE

This study found that GV attacked tumor viability and growth in CTCL. Although purple at neutral pH, GV can be rendered colorless by altering its pH. These preclinical findings may help to broaden knowledge of the antineoplastic features of GV and provide a rationale for clinical studies of its use as a novel, inexpensive, topical therapy for CTCL that is available worldwide.