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

Abstract

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.

Figure 1.. Gentian Violet (GV)–Induced Apoptosis in Cutaneous T-Cell Lymphoma Cells

Cells were treated with GV, nitrogen mustard (mechlorethamine), or dimethyl sulfoxide (DMSO) at 1 μmol/L for 24 hours; apoptosis was detected by flow cytometry. The Jurkat T-cell acute lymphoblastic luekemia cell line line was included for comparison. A, P < .01 for GV compared with DMSO for all. B, P < .01 between each GV-treated column with the DMSO-treated column. SS1 indicates Sézary syndrome 1; SS2, Sézary syndrome 2. ^aP < .01 compared with nitrogen mustard.

Figure 2.. Gentian Violet (GV)–Induced Apoptosis Involving the Extrinsic and Intrinsic Pathways

Cells were treated with GV, nitrogen mustard (mechlorethamine), or dimethyl sulfoxide (DMSO) at 1 μmol/L for 24 hours, and cleaved caspases 8, 9, 3, and 2 were measured by flow cytometry. MFI indicates mean fluorescence intensity; SS1, Sézary syndrome 1; SS2, Sézary syndrome 2. ^aP < .01 compared with DMSO. ^bP < .01 compared with nitrogen mustard.

Figure 3.. Gentian Violet (GV)–Induced Upregulation of Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand (TRAIL) Pathway Factors and Fas Ligand (FasL)

Cells were treated with GV, nitrogen mustard (mechlorethamine), or dimethyl sulfoxide (DMSO) at 1 μmol/L for 24 hours. These different proteins or ions were detected by flow cytometry. DR indicates death receptor; MFI, mean fluorescence intensity; SS1, Sézary syndrome 1; SS2, Sézary syndrome 2. ^aP < .01 compared with DMSO. ^bP < .01 compared with nitrogen mustard.

Figure 4.. Gentian Violet (CV) Inhibition of Cell Proliferation and Induction of Cell Cycle Arrest at G2/S Phases in Cutaneous T-Cell Lymphoma Lines

Cells were treated with GV (1 μmol/L) or dimethyl sulfoxide (DMSO) (1 μmol/L). Cell cycle and proliferation were measured by propidium iodide staining and flow cytometry and MTT assay, respectively. A, Flow cytometry plots show cell cycle arrest after GV treatment (48 hours). Arrowheads indicate the the median fluorescence intensity of the G0/G1 and G2 populations. B, A570 absorbance comparison of GV- and DMSO-treated cells at days 1 to 5 shows decreased cell proliferation.

Figure 5.. Methotrexate-Induced Increase in Gentian Violet (GV)–Induced Apoptosis

Cells were treated with GV (1 μmol/L), methotrexate (10 μmol/L), methotrexate and GV, or dimethyl sulfoxide (DMSO) (1 μmol/L) for 24 hours; apoptosis was detected by flow cytometry. There was no significant difference (P > .05) between methotrexate and GV vs GV treatment in SeAx cells. The Jurkat T-cell acute lymphoblastic luekemia line was included for comparison. ^aP < .001 for methotrexate and GV treatment vs DMSO, methotrexate, and GV. ^bP < .001 for methotrexate and GV treatment vs DMSO and methotrexate.