Associação Portuguesa de Investigação em Cancro
Modulation of autophagy by a thioxanthone decreases the viability of melanoma cells
Modulation of autophagy by a thioxanthone decreases the viability of melanoma cells
Autophagy is a catabolic process which targets cellular organelles and cytoplasmic constituents to the lysosomes for degradation, allowing the cell to maintain homeostasis and being particularly relevant during nutrient deprivation and other stresses.
Alterations in autophagy are frequently found in diseases, such as cancer, and might be relevant for cancer response to therapy. Although autophagy is mainly considered a survival mechanism there is increasing evidence that it plays dual roles in cancer, acting also as a tumor suppressor mechanism, or even as a cell death mechanism. Excessive or sustained autophagy has the potential to induce tumor cell death and this may explain the antitumor effect of autophagy inducers. Thus, there is increasing interest in the development of compounds which modulate autophagy for anticancer therapy.
Our previous studies have unveiled a hit thioxanthone (TXA1) which presented tumor cell growth inhibitory activity towards several human tumor cell lines. In the present work, TXA1 was shown to modulate autophagy in melanoma cells, decreasing their viability. This work shows that TXA1 may serve as a lead compound for the development of autophagy modulators with antitumor activity. Moreover, it further supports the importance of autophagy modulation as an anticancer strategy.
Authors and Affiliations:
Raquel T. Lima 1,2,3,#, Diana Sousa 1,2,4,#, Ana M. Paiva 5, Andreia Palmeira 5, João Barbosa 6,
Madalena Pedro 6, Madalena M. Pinto 5,7, Emília Sousa 5,7 e M. Helena Vasconcelos 1,2,4
1 i3S-Instituto de Investigação e Inovação em Saúde, Universidade do Porto
2 Cancer Drug Resistance Group, IPATIMUP—Institute of Molecular Pathology and Immunology of the University of Porto
3 Department of Pathology, FMUP—Faculty of Medicine of the University of Porto
4 Laboratory of Microbiology, Department of Biological Sciences, FFUP—Faculty of Pharmacy, University of Porto
5 Laboratory of Organic and Pharmaceutical Chemistry, Department of Chemical Sciences, FFUP—Faculty of Pharmacy, University of Porto
6 CESPU, Instituto de Investigação e Formação Avançada em Ciências e Tecnologias da Saúde, IUCS— Instituto Universitário de Ciências da Saúde
7 CIIMAR/CIMAR— Centro Interdisciplinar de Investigação Marinha e Ambiental, Universidade do Porto
# Estes autores contribuíram igualmente para o trabalho.
Abstract:
Background: Our previous studies unveiled the hit thioxanthone TXA1 as an inhibitor of P-glycoprotein (drug efflux pump) and of human tumor cells growth, namely of melanoma cells. Since TXA1 is structurally similar to lucanthone (an autophagy inhibitor and apoptosis inducer) and to N10-substituted phenoxazines (isosteres of thioxanthones, and autophagy inducers), this study aimed at further assessing its cytotoxic mechanism and evaluating its potential as an autophagy modulator in A375-C5 melanoma cells;
Methods: Flow cytometry with propidium iodide (PI) for cell cycle profile analysis; Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, flow cytometry with Annexin V/PI labeling and Western blot for apoptosis analysis were conducted. A pharmacophore approach was used for mapping TXA1 onto pharmacophores for autophagy induction. Autophagy analyses included transmission electron microscopy for visualization of autophagic structures, fluorescence microscopy for observation of monodansylcadaverine (MDC) staining, pattern of LC3 expression in the cells and acridine orange staining, and Western blot for autophagic proteins expression.
Results: TXA1 induced autophagy of melanoma cells at the GI50 concentration (3.6 μM) and apoptosis at twice that concentration. Following treatment with TXA1, autophagic structures were observed, together with the accumulation of autophagosomes and the formation of autophagolysosomes. An increase in LC3-II levels was also observed, which was reverted by 3-methyladenine (3-MA) (an early stage autophagy-inhibitor) but further increased by E-64d/pepstatin (late-stage autophagy inhibitors). Finally, 3-MA also reverted the effect of TXA1 in cellular viability.
Conclusion: TXA1 decreases the viability of melanoma cells by modulation of autophagy and may, therefore, serve as a lead compound for the development of autophagy modulators with antitumor activity.
Journal: Molecules
