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. 2016 Dec 9;11(12):e0167672.
doi: 10.1371/journal.pone.0167672. eCollection 2016.

Ophiobolin A Induces Autophagy and Activates the Mitochondrial Pathway of Apoptosis in Human Melanoma Cells

Carlo Rodolfo  1 , Mariapina Rocco  2 , Lucia Cattaneo  1 , Maria Tartaglia  2 , Mauro Sassi  3 , Patrizia Aducci  1 , Andrea Scaloni  3 , Lorenzo Camoni  1 , Mauro Marra  1
Affiliations

Ophiobolin A Induces Autophagy and Activates the Mitochondrial Pathway of Apoptosis in Human Melanoma Cells

Carlo Rodolfo et al. PLoS One. .

Abstract

Ophiobolin A, a fungal toxin from Bipolaris species known to affect different cellular processes in plants, has recently been shown to have anti-cancer activity in mammalian cells. In the present study, we investigated the anti-proliferative effect of Ophiobolin A on human melanoma A375 and CHL-1 cell lines. This cellular model was chosen because of the incidence of melanoma malignant tumor on human population and its resistance to chemical treatments. Ophyobolin A strongly reduced cell viability of melanoma cells by affecting mitochondrial functionality. The toxin induced depolarization of mitochondrial membrane potential, reactive oxygen species production and mitochondrial network fragmentation, leading to autophagy induction and ultimately resulting in cell death by activation of the mitochondrial pathway of apoptosis. Finally, a comparative proteomic investigation on A375 cells allowed to identify several Ophiobolin A down-regulated proteins, which are involved in fundamental processes for cell homeostasis and viability.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. OP-A reduced A375 melanoma cells viability.
Cells were incubated with 0.3, 0.6 and 1.2 μM OP-A for 24 and 48 h. Panel A: metabolic activity assayed by MTS test. Data are expressed as % of cell survival with respect to control. ***, p<0.0001. Panel B: mitochondrial and nuclear morphology. Mitochondrial network imaging was performed by incubating untreated and treated cells for 20 min at 37°C, with 1 μM MitoTracker Red CMXRos reagent in RPMI medium and nuclei counterstained with 1 μM HOECHST 33342. Images were captured by means of a Floid instrument. Arrowheads indicated picnotic and fragmented nuclei.
Fig 2
Fig 2. OP-A induced autophagy and cell death by apoptosis.
A, flow cytometry analysis of cell death. HaCaT, CHL-1 and A375 cell lines were incubated with 0.3, 0.6 and 1.2 μM OP-A for 24 h and cell death analysis performed by propidium iodide staining and flow cytometry evaluation of the sub-G1 population. Where indicated, 20 μM necrostatin-1 was added to evaluate the occurrence of necrotic cell death. B, western blot of PARP, caspase-9 and -3, and LC3II proteins. C, D and E. Densitometric analysis of immunorecognized protein bands of caspase-3, caspase-9 and LC3II, respectively. Data were analysed with FlowJo software. *, p<0.05, **, p<0.01, ***, p<0.001.
Fig 3
Fig 3. OP-A effect on mitochondrial membrane potential, mitochondrial mass, lysosome content and mitochondrial ROS production.
HaCaT, CHL-1 and A375 cell lines incubated with 0.3, 0.6 and 1.2 μM OP-A for 24 h, were analysed to evaluate: A, mitochondrial membrane potential (MMP), (2 μM JC-1 staining); B, mitochondrial mass (100 nM MitoTracker Green staining); C, lysosome content (100 nM LysoTracker Red staining); D, mitochondrial ROS (5 μM MitoSox Red staining). Cells were incubated for 30 min at 37°C and then acquired by means of a FACSCalibur. Data, expressed as %, or fold increase with respect to controls, were analysed with FlowJo software. *, p<0.05, **, p<0.01, ***, p<0.001.
Fig 4
Fig 4. OP-A induced PINK1 accumulation, Bax and Bak translocation and cytochrome c release.
HaCaT, CHL-1 and A375 cell lines were incubated with 0.3, 0.6 and 1.2 μM OP-A for 24 h and whole cell extracts (WCE), mitochondrial-enriched and cytosolic fractions were analysed by western blot (A) with antibodies directed against PINK1, Bak, Bax, and cytochrome c. Panels B, C and D, densitometric analysis of PINK1, Bak and Bax levels present in the mitochondrial fraction. β-tubulin was used as loading control and standard for densitometry. Data, expressed as %, or fold variation with respect to β-tubulin, were analysed with FlowJo software. *, p<0.05, **, p<0.01, ***, p<0.001.
Fig 5
Fig 5. OP-A induced cell cycle alteration.
HaCaT, CHL-1 and A375 cell lines, incubated with 0.3, 0.6 and 1.2 μM OP-A for 24 h, were stained with propidium iodide and, after over-night incubation at 4°C, the percentage of cells in each phase of the cycle measured by flow cytometry. *, p<0.05, **, p<0.01, ***, p<0.001.
Fig 6
Fig 6. 2-DE map of whole cell protein extract of OP-A-treated A375 melanoma cells.
Proteins were extracted from cells treated with 0.6 μM OP-A for 24 h and from control cells. Proteins were electrophoretically separated in the non-linear pH range 3–10 and the 200–15 kDa molecular mass range, and visualized by colloidal Coomassie staining. Top panel: master gel. The encircled spots indicate the proteins affected by OP-A treatment, which were further subjected to nanoLC-ESI-LIT-MS/MS analysis. Data significance was evaluated by a Student’s t-test (p< 0.05). Bottom panel: representative gels of control (A) and OP-A treated (B) A375 melanoma cells.
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Grants and funding

This work was supported by funds from the Italian Ministry of Economy and Finance to the National Research Council for the project "Innovazione e sviluppo del Mezzogiorno - Conoscenze Integrate per Sostenibilità ed Innovazione del Made in Italy Agroalimentare" - Legge n.191/2009, and from Regione Campania for the Project BenTeN - Nuovi Processi e Prodotti per la Nutraceutica, la Cosmeceutica e la Nutrizione umana” (P.O.R. 2007/2013, objectives 2.1). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.