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Modulation of Autophagy by a Thioxanthone Decreases the Viability of Melanoma Cells.

ABSTRACT: (1) 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; (2) 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; (3) Results: TXA1 induced autophagy of melanoma cells at the GI₅₀ concentration (3.6 ?M) 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; (4) 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.

SUBMITTER: Lima RT

PROVIDER: S-EPMC6274546 | biostudies-literature | 2016 Oct

REPOSITORIES: biostudies-literature

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Modulation of Autophagy by a Thioxanthone Decreases the Viability of Melanoma Cells.

Lima Raquel T RT Sousa Diana D Paiva Ana M AM Palmeira Andreia A Barbosa João J Pedro Madalena M Pinto Madalena M MM Sousa Emília E Vasconcelos M Helena MH

Molecules (Basel, Switzerland) 20161010 10

(1) 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 <i>N</i><sup>10</sup>-substituted phenoxazines (isosteres of thioxanthones, and autophagy inducers), this study aimed at further assessing its cytotoxic mechanism and evaluating its potential as an autophagy modul ...[more]

PMID: 27735867

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Project description:Glioblastoma multiforme is the most aggressive primary brain cancer in adults. Therefore, it is important to investigate the mechanisms associated with cell viability and invasion ability of the cells in glioblastoma multiforme. The opposite strand of the glial cell line-derived neurotrophic factor (GDNF) gene is used to transcribe the cis-antisense GDNF opposite strand (GDNFOS) gene, which belongs to the long noncoding RNAs. The current study assessed the effects of GDNFOS1 overexpression and interference on GDNF expression, cell viability and invasion ability in U87 and U251 MG glioblastoma cells. Overexpression and interference were performed using constructed lentiviral vectors, including long non-coding RNA GDNFOS1 overexpression vector, pL-short hairpin RNA (shRNA)-GDNFOS1-9, pL-shRNA-GDNFOS1-49, pL-shRNA-GDNFOS1-248, pL-shRNA-GDNFOS1-9+49, pL-shRNA-GDNFOS1-9+248 and pL-shRNA-GDNFOS1-49+248. Reverse transcription-quantitative PCR was used to determine the efficiency of interference and overexpression of GDNFOS1 in U87 and U251 MG cells. GDNF protein expression in U87 and U251 MG cells was detected using western blot analysis. In addition, cell viability was detected using a cell counting kit-8 assay at 24, 48 and 72 h after GDNFOS1 overexpression or interference. A transwell invasion assay was used to detect invasion ability. Different shRNA sequences were tested and the results revealed that a combination (pL-shRNA-GDNFOS1-49+248) was most effective in the knock-down GDNFOS1. Compared with the control group, GDNF expression in U87 MG cells was significantly increased in the GDNFOS1 overexpression group and decreased in the shRNA-GDNFOS1-248 group. U87 MG cell viability was significantly increased in the GDNFOS1 overexpression group at 24, 48 and 72 h compared with the negative control group. The viability of U87 MG cells was decreased in the GDNFOS1 interference group at 72 h when compared with the control group. The relative invasive ability was significantly increased in the GDNFOS1 overexpression group when compared with the negative control group. The invasive ability was significantly decreased in the GDNFOS1 interference group when compared with the negative control group. Similar results were exhibited by the U251 MG cells. Overall, GDNF expression, cell viability and invasion ability of glioblastoma cells significantly increased with GDNFOS1 overexpression and decreased with GDNFOS1 interference.

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Modulation of Autophagy by a Thioxanthone Decreases the Viability of Melanoma Cells.

Publications

Modulation of Autophagy by a Thioxanthone Decreases the Viability of Melanoma Cells.

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