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. 2022 Apr 14:12:852940.
doi: 10.3389/fonc.2022.852940. eCollection 2022.

Lucanthone Targets Lysosomes to Perturb Glioma Proliferation, Chemoresistance and Stemness, and Slows Tumor Growth In Vivo

Daniel P Radin  1   2 , Gregory Smith  1 , Victoria Moushiaveshi  1 , Alexandra Wolf  1 , Robert Bases  3   4 , Stella E Tsirka  1
Affiliations

Lucanthone Targets Lysosomes to Perturb Glioma Proliferation, Chemoresistance and Stemness, and Slows Tumor Growth In Vivo

Daniel P Radin et al. Front Oncol. .

Abstract

Glioblastoma is the most common and aggressive primary brain tumor in adults. Median survival time remains at 16-20 months despite multimodal treatment with surgical resection, radiation, temozolomide and tumor-treating fields therapy. After genotoxic stress glioma cells initiate cytoprotective autophagy, which contributes to treatment resistance, limiting the efficacy of these therapies and providing an avenue for glioma recurrence. Antagonism of autophagy steps has recently gained attention as it may enhance the efficacy of classical chemotherapies and newer immune-stimulating therapies. The modulation of autophagy in the clinic is limited by the low potency of common autophagy inhibitors and the inability of newer ones to cross the blood-brain barrier. Herein, we leverage lucanthone, an anti-schistosomal agent which crosses the blood-brain barrier and was recently reported to act as an autophagy inhibitor in breast cancer cells. Our studies show that lucanthone was toxic to glioma cells by inhibiting autophagy. It enhanced anti-glioma temozolomide (TMZ) efficacy at sub-cytotoxic concentrations, and suppressed the growth of stem-like glioma cells and temozolomide-resistant glioma stem cells. In vivo lucanthone slowed tumor growth: reduced numbers of Olig2+ glioma cells, normalized tumor vasculature, and reduced tumor hypoxia. We propose that lucanthone may serve to perturb a mechanism of temozolomide resistance and allow for successful treatment of TMZ-resistant glioblastoma.

Keywords: angiogenesis; autophagy; cancer stem cell; glioma; hypoxia; lucanthone.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Lucanthone compromises glioma cell growth. (A) Chemical structures of lucanthone and chloroquine. (B) Effects of long-term treatment of KR158 and GLUC2 cultures with 10 μM lucanthone on glioma cell proliferation. (C) Acridine orange (AO) marks lysosomes as punctae staining after 48 hour of lucanthone treatment. (D) LC3 marks autophagosome punctae levels after 48 hour treatment with lucanthone. (E, F) Effect of lucanthone on P62 and Cathepsin D levels in GLUC2 and KR158 cells, respectively. Scale bar = 30 μm. Bars are mean +/- SEM. N= 3-4 independent experiments. *p < 0.05, ****p < 0.0001, student’s t-test.
Figure 2
Figure 2
Interaction between lucanthone and temozolomide. (A) KR158 and GLUC2 cells were treated with Lucanthone for 72 hours, after which an MTT assay was performed. Bars are mean +/- SEM, N=3-7 independent experiment. ANOVA p<0.0001. *p < 0.05, **p < 0.01, ****p < 0.0001, Dunnett’s multiple comparison test to control-treated cells. (B, C) KR158 and GLUC2 cells were treated with lucanthone, TMZ, or the combination for 4 days and then allowed to recover in drug-free medium for 3 days. The cells were PFA-fixed and stained with crystal violet. Crystal violet-stained cells were then lysed and relative absorbance was measured to approximate culture viability. Representative wells are shown in (B). (C) Quantification of crystal-violet stained cultures. Bars are mean +/- SEM, N=3-4 independent experiments. *p < 0.05, **p < 0.01, Dunnett’s multiple comparison test to control-treated cells. +p < 0.05, King’s synergy test, demonstrating significant interactions between lucanthone and TMZ in both cell lines. (D) Representative micrographs of γH2AX stained GLUC2 cells and quantification of γH2AX intensity per number of cells in the field of view in experiments where the GLUC2 cells were incubated with TMZ, or the combination of lucanthone and TMZ. (E) Representative micrographs of γH2AX stained KR158 cells. Quantification of γH2AX intensity per number of cells in the field of view in experiments where the KR158 cells were incubated with TMZ, or the combination of lucanthone and TMZ. Bars are mean +/- SEM, N=4 independent experiments. *p < 0.05, Mann-Whitney test.
Figure 3
Figure 3
Lucanthone targeted GSC and overcame acquired resistance to temozolomide. GLUC2 and KR158 spheroids were mechanically dissociated, plated overnight and treated with increasing concentrations of lucanthone for 5 days. After treatment, they were stained with Calcein-AM to visualize viable cells. (A) Representative images of KR158 and GLUC2 GSC treated with increasing concentrations of Lucanthone for 5 days; (B) Spheroid area distribution. ****p < 0.0001, Kolmogorov-Smirnov test comparing distributions to control-treated cultures; ND, Not Detected. (C) Spheroid number per field of view; (D) Viability of cultures as determined by MTT assay. Bars are mean +/- SEM, N=3-4 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, Dunnett’s multiple comparison test to control-treated cells. (E) LC3 staining in GLUC2 and KR158 spheroid cultures treated with media or 10 μM lucanthone for 48 hours; (F) LC3 intensity measured in the same cultures. *p < 0.05, Mann-Whitney test; (G) Olig2 staining in GLUC2 spheroid cultures treated with media (Control) and 10 μM lucanthone-treated for 48 hours; (H) Olig2 intensity and mRNA expression in the same cultures. **p < 0.01, t-test. N=3-4 independent experiments; (I) Immunoblot analysis of p62 and LC3 in protein extracts from GLUC2 and KR158 spheroids with media or 10 μM lucanthone for 48 hours; (J) GLUC2 cells treated with 5 cycles of TMZ stained for the stemness marker CD133 and for the proliferation marker Ki67; (K) TMZ-resistant GLUC2 cells treated with media or 10 μM lucanthone for 5 days.
Figure 4
Figure 4
Patient-derived glioma cells are susceptible to lucanthone. (A) GBM43 cells were treated with lucanthone and assessed for changes in acridine orange staining, (B) LC3 and (C) p62 levels. (D) GBM43 CSCs were treated with lucanthone for 5 days and then an MTT assay was performed. (E) GBM43 GSC were treated with media or lucanthone for 5 days, after which spheroids were visualized by Calcein-AM and Ethidium homodimer staining. Data are representative of 4 independent experiments. ****p < 0.0001, t-test. Dotted line represents culture viability prior to any treatment.
Figure 5
Figure 5
Lucanthone mitigated the growth of dissociated GLUC2 spheroids in vivo. (A) Treatment scheme used for the study. (B) Representative images of in vivo luminescent imaging on Days 7, 14 and 21. (C) Fold increase in luminescence from day 7 to day 21. ***p < 0.001, Mann-Whitney test. (D) Tumor volume of control- and lucanthone-treated animals with representative images shown in (E) **p < 0.01, Mann-Whitney test. (F) Body mass depicted as a percentage of the start of treatment on day 7. ***p < 0.001, Mann-Whitney test, compared to relative body mass on day 7. Bars are mean +/- SEM, N=7-8 animals.
Figure 6
Figure 6
Lucanthone reduced Olig2+ positivity in tumors in vivo. (A) Representative immunohistochemical images of Olig2 and Ki67 in tumors and surrounding stroma in saline- and lucanthone-treated mice. (B) Expression of Olig2 in different areas in human glioblastomas adapted from the Ivy Glioblastoma Atlas. ****p < 0.0001 Kruskal-Wallis test, demonstrating significant differences in Olig2 expression among various tumor areas. *p < 0.05, ****p < 0.0001 Dunn’s test, compared to infiltrating tumor. +p < 0.05, ++++p < 0.0001, Dunn’s test, compared to cellular tumor. (C, D) Olig2 expression in tumor periphery and tumor core in both treatment conditions with intensity quantifications in (E) Two-way ANOVA p < 0.05. **p < 0.01, Bonferroni multiple comparison test. Bars are mean +/- SEM, N=4 animals per group.
Figure 7
Figure 7
Tumor microenvironmental changes induced by Lucanthone. (A) Representative images of blood vessels marked by CD31 of control- and lucanthone-treated tumors. (B) Blood vessel area. (C) Luminal area/blood vessel area. (D) Blood vessel circularity. ****p < 0.0001, Kolmogorov-Smirnov test. Bars are mean +/- SEM, N=4-5 animals per group. (E, F) Representative images of Glut1 levels in control- and lucanthone-treated tumors, respectively. (G) Quantification of Glut1 intensity in the tumor microenvironment. Bars are mean +/- SEM. N=5 mice **p < 0.01, t-test. (H) Glut1 expression in necrotic areas in clinical specimens. Data adapted from the Ivy Glioblastoma Atlas. ****p < 0.0001, Kruskal Wallis test. **p < 0.01, ***p < 0.001, ****p < 0.0001, Dunn’s test, compared to perinecrotic zone, ++++p < 0.0001, Dunn’s test, compared to pseudopalisading cells around necrotic areas. (I) CD8a+ cells in the tumor microenvironment in control- and lucanthone-treated tumors. *p < 0.05, Mann-Whitney test Bars are mean +/- SEM, N=4 animals per group.
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