Modulation of autophagy by a thioxanthone decreases the viability of melanoma cells

(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. SinceTXA1 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 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; (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.This work was financed by FEDER—Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020—Operacional Programme for Competitiveness and Internationalisation (POCI), Portugal 2020, and by Portuguese funds through FCT—Fundação para a Ciência e a Tecnologia/ Ministério da Ciência, Tecnologia e Inovação in the framework of the project “Institute for Research and Innovation in Health Sciences (POCI-01-0145-FEDER-007274)”. The work was also funded by ERDF, COMPETE, and FCT under the projects PTDC/SAU-OSM/101437/2008, PTDC/MAR-BIO/4694/2014, and INNOVMAR—reference NORTE-01-0145-FEDER-000035, Research Line NOVELMAR. The authors also thank: FCT for D. Sousa and R.T. Lima grants (PTDC/SAU-FCT/100930/2008 and SFRH/BPD/68787/2010, respectively), QREN for D. Sousa grant (NORTE-07-0124-FEDER-000023)

The antitumor activity of a lead thioxanthone is associated with alterations in cholesterol localization

The search for novel anticancer small molecules and strategies remains a challenge. Our previous studies have identified TXA1 (1-{[2-(diethylamino)ethyl]amino}-4-propoxy-9H-thioxanthen-9-one) as a hit compound, with in vitro antitumor potential by modulating autophagy and apoptosis in human tumor cell lines. In the present study, the mechanism of action and antitumor potential of the soluble salt of this molecule (TXA1.HCl) was further investigated using in vitro and mouse xenograft tumor models of NSCLC. Our results showed that TXA1.HCl affected steroid biosynthesis, increased RagD expression, and caused abnormal cellular cholesterol localization. In addition, TXA1.HCl treatment presented no toxicity to nude mice and significantly reduced the growth of human NSCLC cells xenografts in mice. Overall, this work provides new insights into the mechanism of action of TXA1, which may be relevant for the development of anticancer therapeutic strategies, which target cholesterol transport. © 2018 by the authors.Acknowledgments: FCT for D.S. and R.T.L. grants (PTDC/SAU-FCT/100930/2008 and SFRH/BPD/68787/2010, respectively) and FCT PhD Programme BiotechHealth grant of A.S.G. (PD/BD/114046/2015); QREN for D. Sousa grant (NORTE-07-0124-FEDER-000023); P. Castro, R. Barros, L. Pereira and S. Ricardo, who provided technical assistance. Funding: IPATIMUP integrates the i3S Research Unit, which is partially supported by FCT, the Portuguese Foundation for Science and Technology. This work is funded by FEDER funds through the Operational Programme for Competitiveness Factors-COMPETE and National Funds through the FCT-Foundation for Science and Technology, under the projects “PEst-C/SAU/LA0003/2013”, ERDF, programme PT2020—Contributos para o reforço da capacidade do IPATIMUP enquanto actor do sistema regional de inovação” and NORTE-07-0162-FEDER-000067—Reforço e consolidação da capacidade infraestrutural do IPATIMUP para o sistema regional de inovação”, both supported by Programa Operacional Regional do Norte (ON.2—O Novo Norte), through FEDER funds under the Quadro de Referência Estratégico Nacional (QREN). This work was also financed by FEDER—Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020—Operacional Programme for Competitiveness and Internationalisation (POCI), Portugal 2020, and by Portuguese funds through FCT—Fundação para a Ciência e a Tecnologia/ Ministério da Ciência, Tecnologia e Inovação in the framework of the project “Institute for Research and Innovation in Health Sciences” (POCI-01-0145-FEDER-007274). This work was also supported through national funds provided by FCT/MCTES—Foundation for Science and Technology from the Minister of Science, Technology and Higher Education (PIDDAC) and European Regional Development Fund (ERDF) through the COMPETE—Programa Operacional Factores de Competitividade (POFC) programme, under the Strategic Funding UID/Multi/04423/2013, in the framework of the programme PT2020

In Vitro Structural and Functional Evaluation of Gold Nanoparticles Conjugated Antibiotics

Bactericidal efficacy of gold nanoparticles conjugated with ampicillin, streptomycin and kanamycin were evaluated. Gold nanoparticles (Gnps) were conjugated with the antibiotics during the synthesis of nanoparticles utilizing the combined reducing property of antibiotics and sodium borohydride. The conjugation of nanoparticles was confirmed by dynamic light scattering (DLS) and electron microscopic (EM) studies. Such Gnps conjugated antibiotics showed greater bactericidal activity in standard agar well diffusion assay. The minimal inhibitory concentration (MIC) values of all the three antibiotics along with their Gnps conjugated forms were determined in three bacterial strains,Escherichia coli DH5α,Micrococcus luteusandStaphylococcus aureus. Among them, streptomycin and kanamycin showed significant reduction in MIC values in their Gnps conjugated form whereas; Gnps conjugated ampicillin showed slight decrement in the MIC value compared to its free form. On the other hand, all of them showed more heat stability in their Gnps conjugated forms. Thus, our findings indicated that Gnps conjugated antibiotics are more efficient and might have significant therapeutic implications

‘A sword of Damocles’ : patient and caregiver beliefs, attitudes and perspectives on presymptomatic testing for autosomal dominant polycystic kidney disease: a focus group study

Background and objectives: Presymptomatic testing is available for early diagnosis of hereditary autosomal dominant polycystic kidney disease (ADPKD). However, the complex ethical and psychosocial implications can make decision-making challenging and require an understanding of patients’ values, goals and priorities. This study aims to describe patient and caregiver beliefs and expectations regarding presymptomatic testing for ADPKD. Design, setting and participants: 154 participants (120 patients and 34 caregivers) aged 18 years and over from eight centres in Australia, France and Korea participated in 17 focus groups. Transcripts were analysed thematically. Results: We identified five themes: avoiding financial disadvantage (insecurity in the inability to obtain life insurance, limited work opportunities, financial burden); futility in uncertainty (erratic and diverse manifestations of disease limiting utility, taking preventive actions in vain, daunted by perplexity of results, unaware of risk of inheriting ADPKD); lacking autonomy and support in decisions (overwhelmed by ambiguous information, medicalising family planning, family pressures); seizing control of well-being (gaining confidence in early detection, allowing preparation for the future, reassurance in family resilience); and anticipating impact on quality of life (reassured by lack of symptoms, judging value of life with ADPKD). Conclusions: For patients with ADPKD, presymptomatic testing provides an opportunity to take ownership of their health through family planning and preventive measures. However, these decisions can be wrought with tensions and uncertainty about prognostic implications, and the psychosocial and financial burden of testing. Healthcare professionals should focus on genetic counselling, mental health and providing education to patients’ families to support informed decision-making. Policymakers should consider the cost burden and risk of discrimination when informing government policies. Finally, patients are recommended to focus on self-care from an early age

Horizontal Branch Stars: The Interplay between Observations and Theory, and Insights into the Formation of the Galaxy

We review HB stars in a broad astrophysical context, including both variable and non-variable stars. A reassessment of the Oosterhoff dichotomy is presented, which provides unprecedented detail regarding its origin and systematics. We show that the Oosterhoff dichotomy and the distribution of globular clusters (GCs) in the HB morphology-metallicity plane both exclude, with high statistical significance, the possibility that the Galactic halo may have formed from the accretion of dwarf galaxies resembling present-day Milky Way satellites such as Fornax, Sagittarius, and the LMC. A rediscussion of the second-parameter problem is presented. A technique is proposed to estimate the HB types of extragalactic GCs on the basis of integrated far-UV photometry. The relationship between the absolute V magnitude of the HB at the RR Lyrae level and metallicity, as obtained on the basis of trigonometric parallax measurements for the star RR Lyrae, is also revisited, giving a distance modulus to the LMC of (m-M)_0 = 18.44+/-0.11. RR Lyrae period change rates are studied. Finally, the conductive opacities used in evolutionary calculations of low-mass stars are investigated. [ABRIDGED]Comment: 56 pages, 22 figures. Invited review, to appear in Astrophysics and Space Scienc