Propranolol Sensitizes Vascular Sarcoma Cells to Doxorubicin by Altering Lysosomal Drug Sequestration and Drug Efflux

Angiosarcoma is a rare cancer of blood vessel–forming cells with a high patient mortality and few treatment options. Although chemotherapy often produces initial clinical responses, outcomes remain poor, largely due to the development of drug resistance. We previously identified a subset of doxorubicin-resistant cells in human angiosarcoma and canine hemangiosarcoma cell lines that exhibit high lysosomal accumulation of doxorubicin. Hydrophobic, weak base chemotherapeutics, like doxorubicin, are known to sequester within lysosomes, promoting resistance by limiting drug accessibility to cellular targets. Drug synergy between the beta adrenergic receptor (β-AR) antagonist, propranolol, and multiple chemotherapeutics has been documented in vitro, and clinical data have corroborated the increased therapeutic potential of propranolol with chemotherapy in angiosarcoma patients. Because propranolol is also a weak base and accumulates in lysosomes, we sought to determine whether propranolol enhanced doxorubicin cytotoxicity via antagonism of β-ARs or by preventing the lysosomal accumulation of doxorubicin. β-AR-like immunoreactivities were confirmed in primary tumor tissues and cell lines; receptor function was verified by monitoring downstream signaling pathways of β-ARs in response to receptor agonists and antagonists. Mechanistically, propranolol increased cytoplasmic doxorubicin concentrations in sarcoma cells by decreasing the lysosomal accumulation and cellular efflux of this chemotherapeutic agent. Equivalent concentrations of the receptor-active S-(−) and -inactive R-(+) enantiomers of propranolol produced similar effects, supporting a β-AR-independent mechanism. Long-term exposure of hemangiosarcoma cells to propranolol expanded both lysosomal size and number, yet cells remained sensitive to doxorubicin in the presence of propranolol. In contrast, removal of propranolol increased cellular resistance to doxorubicin, underscoring lysosomal doxorubicin sequestration as a key mechanism of resistance. Our results support the repurposing of the R-(+) enantiomer of propranolol with weak base chemotherapeutics to increase cytotoxicity and reduce the development of drug-resistant cell populations without the cardiovascular and other side effects associated with antagonism of β-ARs

Discutindo a educação ambiental no cotidiano escolar: desenvolvimento de projetos na escola formação inicial e continuada de professores

A presente pesquisa buscou discutir como a Educação Ambiental (EA) vem sendo trabalhada, no Ensino Fundamental e como os docentes desta escola compreendem e vem inserindo a EA no cotidiano escolar., em uma escola estadual do município de Tangará da Serra/MT, Brasil. Para tanto, realizou-se entrevistas com os professores que fazem parte de um projeto interdisciplinar de EA na escola pesquisada. Verificou-se que o projeto da escola não vem conseguindo alcançar os objetivos propostos por: desconhecimento do mesmo, pelos professores; formação deficiente dos professores, não entendimento da EA como processo de ensino-aprendizagem, falta de recursos didáticos, planejamento inadequado das atividades. A partir dessa constatação, procurou-se debater a impossibilidade de tratar do tema fora do trabalho interdisciplinar, bem como, e principalmente, a importância de um estudo mais aprofundado de EA, vinculando teoria e prática, tanto na formação docente, como em projetos escolares, a fim de fugir do tradicional vínculo “EA e ecologia, lixo e horta”.Facultad de Humanidades y Ciencias de la Educació

Combinatorial Treatment of DNA and Chromatin- Modifying Drugs Cause Cell Death in Human and Canine

Downregulation of microRNAs (miRNAs) at the 14q32 locus stabilizes the expression of cMYC, thus significantly contributing to osteosarcoma (OS) pathobiology. Here, we show that downregulation of 14q32 miRNAs is epigenetically regulated. The predicted promoter regions of miRNA clusters at 14q32 locus showed no recurrent patterns of differential methylation, but Saos2 cells showed elevated histone deacetylase (HDAC) activity. Treatment with 4-phenylbutyrate increased acetylation of histones associated with 14q32 miRNAs, but interestingly, robust restoration of 14q32 miRNA expression, attenuation of cMYC expression, and induction of apoptosis required concomitant treatment with 5-Azacytidine, an inhibitor of DNA methylation. These events were associated with genome-wide gene expression changes including induction of pro-apoptotic genes and downregulation of cell cycle genes. Comparable effects were achieved in human and canine OS cells using the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA/Vorinostat) and the DNA methylation inhibitor Zebularine (Zeb), with significantly more pronounced cytotoxicity in cells whose molecular phenotypes were indicative of aggressive biological behavior. These results suggested that the combination of these chromatin-modifying drugs may be a usefu

Combinatorial Treatment of DNA and Chromatin-Modifying Drugs Cause Cell Death in Human and Canine Osteosarcoma Cell Lines

<div><p>Downregulation of microRNAs (miRNAs) at the 14q32 locus stabilizes the expression of cMYC, thus significantly contributing to osteosarcoma (OS) pathobiology. Here, we show that downregulation of 14q32 miRNAs is epigenetically regulated. The predicted promoter regions of miRNA clusters at 14q32 locus showed no recurrent patterns of differential methylation, but Saos2 cells showed elevated histone deacetylase (HDAC) activity. Treatment with 4-phenylbutyrate increased acetylation of histones associated with 14q32 miRNAs, but interestingly, robust restoration of 14q32 miRNA expression, attenuation of cMYC expression, and induction of apoptosis required concomitant treatment with 5-Azacytidine, an inhibitor of DNA methylation. These events were associated with genome-wide gene expression changes including induction of pro-apoptotic genes and downregulation of cell cycle genes. Comparable effects were achieved in human and canine OS cells using the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA/Vorinostat) and the DNA methylation inhibitor Zebularine (Zeb), with significantly more pronounced cytotoxicity in cells whose molecular phenotypes were indicative of aggressive biological behavior. These results suggested that the combination of these chromatin-modifying drugs may be a useful adjuvant in the treatment of rapidly progressive OS.</p> </div

SAHA and Zeb show additive cytotoxicity in human and in canine OS cells with aggressive biological behavior.

<p><b>A</b>) Human Saos2 and U2OS cells were cultured in the presence of SAHA and Zeb at the indicated concentrations at 48 hr. Cell viability was measured at 48 hrs using the MTS assay. Data are normalized hence100% viability reflects the number of cells present in control cultures (treated with vehicle only). <b>B</b>) Canine OSCA-40, OSCA-78, and OSCA-32 cells were cultured in the presence of SAHA and Zeb at the indicated concentrations and cell viability was measured as in panel A. Data show means ± SD for three independent experiments, performed in triplicates. Data for each condition were normally distributed with <15% intra- and inter-experimental variation from the mean. Conditions that were significantly different from control by paired Student's t-test are denoted by asterisks (* p≤0.05; ** p≤0.01).</p

5-AzadC and 4-PBA activates 14q32 miRNAs expression and destabilize cMYC in Saos2 cells.

<p><b>A</b>) 5-AzadC and 4-PBA treated Saos2 cells were subjected to western blot analysis with Anti-acetylated histone-H3 antibody; and GAPDH is shown as control. Note an increase in the level of acetylated Histone-H3 in the treated cells. <b>B</b>) 5-AzadC and 4-PBA significantly increased the acetylation of histone-H3 of representative 14q32 miRNA upstream regions (−200 bp); *p values <0.005. <b>C</b>) Quantitative RT-PCR analysis of 14q32 miRNAs following the treatments with 3 µM 5-AzadC and 3 mM 4-PBA. Saos2 cells were treated with 3.0 µM 5-AzadC and 3.0 mM 4-PBA for 6 days, unless otherwise mentioned.</p

5-AzadC and 4-PBA induce apoptosis in Saos2 cells.

<p><b>A</b>) Cells treated with 5-AzadC and 4-PBA alone or in combination along with sham and untreated cells were subjected to western blot analysis using anti-cMYC antibody. The blot was stripped and re-probed with anti-GAPDH antibody. Signal intensity was measured by densitometry analysis using image J software. cMYC/GAPDH ratio of sham treated cells was considered as unity and relative cMYC/GAPDH ratios were calculated (plotted on the top of the blot). The results show that treatment of Saos2 cells with 5-AzadC and 4-PBA decreased endogenous cMYC levels. <b>B</b>) Cells were treated with 5-AzadC and 4-PBA at concentrations of i) 0 µM & 0 mM; ii) 0 µM & 3 mM; iii) 1.5 µM & 1.5 mM; iv) 1.5 µM & 3 mM; v) 3 µM & 1.5 mM; vi) 3 µM & 0 mM; and vii) 3 µM & 3 mM, respectively. Green and red color staining represents live and dead cells, respectively. <b>C</b>) FACS analysis of cells treated with 5-AzadC and 4-PBA at various dose combinations led to apoptosis (% given in parenthesis). FACS analysis of cells treated with 5-AzadC and 4-PBA together with Z-VAD-fmk, which led to a significant reduction in the apoptosis of treated cells shown on the right panel. <b>D</b>) Cytotoxicty assay in 5-Aza and 4-PBA treated Saos2 cells with untreated (control) and sham treated (0/0) controls. Maximum LDH release from equal number of sham treated cells grown under identical condition is shown as positive control.</p