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. 2024 Mar 12;25(6):3226.
doi: 10.3390/ijms25063226.

Repurposed Drugs Celecoxib and Fmoc-L-Leucine Alone and in Combination as Temozolomide-Resistant Antiglioma Agents-Comparative Studies on Normal and Immortalized Cell Lines, and on C. elegans

Łukasz Uram  1 , Natalia Pieńkowska  2 , Maria Misiorek  1 , Żaneta Szymaszek  1 , Magdalena Twardowska  1 , Michał Siorek  1 , Stanisław Wołowiec  3
Affiliations

Repurposed Drugs Celecoxib and Fmoc-L-Leucine Alone and in Combination as Temozolomide-Resistant Antiglioma Agents-Comparative Studies on Normal and Immortalized Cell Lines, and on C. elegans

Łukasz Uram et al. Int J Mol Sci. .

Abstract

Glioblastoma multiforme therapy remains a significant challenge since there is a lack of effective treatment for this cancer. As most of the examined gliomas express or overexpress cyclooxygenase-2 (COX-2) and peroxisome proliferator-activated receptors γ (PPARγ), we decided to use these proteins as therapeutic targets. Toxicity, antiproliferative, proapoptotic, and antimigratory activity of COX-2 inhibitor (celecoxib-CXB) and/or PPARγ agonist (Fmoc-L-Leucine-FL) was examined in vitro on temozolomide resistant U-118 MG glioma cell line and comparatively on BJ normal fibroblasts and immortalized HaCaT keratinocytes. The in vivo activity of both agents was studied on C. elegans nematode. Both drugs effectively destroyed U-118 MG glioma cells via antiproliferative, pro-apoptotic, and anti-migratory effects in a concentration range 50-100 µM. The mechanism of action of CXB and FL against glioma was COX-2 and PPARγ dependent and resulted in up-regulation of these factors. Unlike reports by other authors, we did not observe the expected synergistic or additive effect of both drugs. Comparative studies on normal BJ fibroblast cells and immortalized HaCaT keratinocytes showed that the tested drugs did not have a selective effect on glioma cells and their mechanism of action differs significantly from that observed in the case of glioma. HaCaTs did not react with concomitant changes in the expression of COX-2 and PPARγ and were resistant to FL. Safety tests of repurposing drugs used in cancer therapy tested on C. elegans nematode indicated that CXB, FL, or their mixture at a concentration of up to 100 µM had no significant effect on the entire nematode organism up to 4th day of incubation. After a 7-day treatment, CXB significantly shortened the lifespan of C. elegans at 25-400 µM concentration and body length at 50-400 µM concentration.

Keywords: ATP level; C. elegans; Fmoc-L-Leucine; HaCaT immortalized cells; apoptosis; celecoxib; glioma U-118 MG; migration; normal human fibroblasts BJ; toxicity.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
The viability of U-118 MG, BJ, and HaCaT cells after 24 h exposure for CXB, FL, or CXB+FL mixture, estimated with neutral red assay. Results are presented as medians of triplicate assays from three independent experiments, expressed as a % of non-treated controls. The whiskers are lower (25%) and upper (75%) quartile ranges. * p < 0.05; Kruskal–Wallis test (against non-treated control), arrows indicate statistical significantly differences between drugs at appropriate concentrations (U Mann–Whitney test).
Figure 2
Figure 2
An antiproliferative effect of CXB, FL, or mixture of CXB and FL against U-118 MG, BJ, and HaCaT cells after 72 h incubation. Results are presented as medians and expressed as a % of non-treated controls. The whiskers are lower (25%) and upper (75%) quartile ranges. * p < 0.05; Kruskal–Wallis test (against non-treated control), arrows indicate statistical significantly differences between drugs at equal concentrations (U Mann–Whitney test).
Figure 3
Figure 3
Intracellular ATP level per cell (column A) and caspase 3/7 activity per cell (column B) in U-118 MG, BJ, and HaCaT cells after 24 h treatment with CXB, FL, or equimolar mixture of both drugs. Results are presented as medians, expressed as a % of non-treated controls. The whiskers are lower (25%) and upper (75%) quartile ranges. * p < 0.05; Kruskal–Wallis test (against non-treated control), arrows indicate statistical significantly differences between drugs at equal concentrations (U Mann–Whitney test).
Figure 4
Figure 4
The influence of CXB, FL, or their equimolar mixture on U-118 MG, BJ, and HaCaT cells migration. Results are presented as medians of triplicate assay from three independent experiments, expressed as a % of non-treated controls. * p < 0.05; Kruskal–Wallis test (against non-treated control).
Figure 5
Figure 5
(A) The COX-2 level in U-118 MG, BJ, and HaCaT cells after 24 h treatment with CXB, FL, or CXB+FL mixture at non-toxic concentrations and IC50. Results are presented as medians expressed as percent of non-treated control. The whiskers are lower (25%) and upper (75%) quartile ranges. Symbol * shows statistically significant difference against control, p < 0.05, Kruskal–Wallis test. (B) Image of immunoblots.
Figure 6
Figure 6
(A) Changes of PPARγ level in U-118 MG, HaCaT, BJ, and SCC-15 cells after 24 h incubation with CXB, FL, and CXB+FL mixture at non-toxic concentrations and IC50. Results are presented as medians expressed as percent of non-treated control. The whiskers are lower (25%) and upper (75%) quartile ranges. Symbol * shows statistically significant difference against DMSO-treated control, p < 0.05, Kruskal–Wallis test. (B) Image of immunoblots.
Figure 7
Figure 7
(A) The Kaplan–Meier survival curves of C. elegans during 7 days of incubation with CXB, FL, or CXB+FL. Results are presented as cumulative proportion surviving. Statistically significant differences against DMSO-treated control obtained in Gehan’s Wilcoxon test are marked with asterisks * (p ≤ 0.05) in the colors corresponding to the tested concentrations. (B) Changes of body length after 7 days treatment. Statistically significant differences against DMSO-treated control are marked with asterisks * (p ≤ 0.05, Kruskal–Wallis test).
Figure 8
Figure 8
Morphology of C. elegans after 7 days incubation with CXB, FL, or CXB+FL. Red arrows show individuals with significantly shorter body length.
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