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- Author or Editor: Sridhar Murahari x
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Objective—To determine whether exposure of canine cancer cells to histone deacetylase (HDAC) inhibitors S(+)-N-hydroxy-4-(3-methyl-2-phenyl-butyrylamino)benzamide (OSU-HDAC42) or suberoylanilide hydroxamic acid (SAHA) results in increased histone acetylation and decreased cell viability and whether any changes in viability involve induction of apoptosis or alterations in progression of the cell cycle.
Sample Population—9 canine cancer cell lines.
Procedures—Cells from 9 canine cancer cell lines were treated with dimethyl sulfoxide vehicle, OSU-HDAC42, or SAHA, then assays of cell viability were performed. Histone acetylation was assessed by use of western blot analysis. Apoptosis was assessed via ELISA to detect fragmentation of cytoplasmic nucleosomal DNA and western blot analysis to detect cleavage of caspase 3. Cell cycle analysis was performed by use of propidium iodide staining and flow cytometry.
Results—Concentrations of OSU-HDAC42 and SAHA required to achieve 50% inhibition of cell viability (IC50) were reached in cells of 6 and 4 canine cancer cell lines, respectively, and ranged from approximately 0.4 to 1.3μM for OSU-HDAC42 and 0.6 to 4.8μM for SAHA. Cells from T-cell lymphoma, mast cell tumor, osteosarcoma, and histiocytic sarcoma lines were most sensitive to HDAC inhibition, with IC50s of < 1μM for OSU-HDAC42 and < 5μM for SAHA. Induction of apoptosis was indicated via cleavage of caspase 3 and increases in cytoplasmic nucleosomes and the subG1 cell population.
Conclusions and Clinical Relevance—Micromolar concentrations of HDAC inhibitors OSU-HDAC42 and SAHA induced histone acetylation, cytotoxicity, and apoptosis in canine cancer cells. In general, OSU-HDAC42 was more potent than SAHA.
Objective—To evaluate the biological activity of dihydroartemisinin on canine osteosarcoma cell lines in vitro.
Sample Population—4 canine osteosarcoma cell lines.
Procedures—Cell viability assays were performed on canine osteosarcoma cell lines OSCA2, OSCA16, OSCA50, and D17 after 24, 48, and 72 hours of treatment with dihydroartemisinin at concentrations of 0.1 to 100μM. Apoptosis was assessed by use of an ELISA for free nuclosomal DNA fragmentation and by western blot analysis for cleavage of caspase 3. Cell cycle analysis was performed by use of staining with propidium iodide and flow cytometry. Detection of reactive oxygen species (ROS) was conducted in the D17 cell line by use of 6-carboxy-2′,7′-dihydrofluorescein diacetate and flow cytometry.
Results—The concentration of dihydroartemisinin required for 50% inhibition of cell viability (IC50) was achieved in all 4 canine osteosarcoma cell lines and ranged from 8.7 to 43.6μM. Induction of apoptosis was evident as an increase in nucleosomal DNA fragmentation, cleavage of caspase 3, and an increase in the population in the sub G0/G1 phase of the cell cycle detected by flow cytometry. Exposure to dihydroartemisinin also resulted in a decrease in the G0/G1 population. Iron-dependent generation of ROS was detected in dihydroartemisinin-treated D17 cells; ROS generation increased in a dose-dependent manner.
Conclusions and Clinical Relevance—Incubation with dihydroartemisinin resulted in biological activity against canine osteosarcoma cell lines, which included induction of apoptosis and arrest of the cell cycle. Clinical trials of dihydroartemisinin in dogs with osteosarcoma should be conducted.
Objective—To evaluate in vitro effects of gemcitabine alone and in combination with carboplatin on canine transitional cell carcinoma (TCC) cell lines.
Sample—In vitro cultures of 5 canine TCC cell lines.
Procedures—Cells were treated with gemcitabine, carboplatin, or a combination of both at various concentrations. Cell proliferation was assessed via a fluorescence-based microplate cell proliferation assay. Cell cycle was evaluated via propidium iodide staining, and apoptosis was assessed by measurement of caspase 3 and 7 enzymatic activity. Synergy between gemcitabine and carboplatin was quantified via combination index analyses.
Results—Treatment of 5 canine TCC cell lines with gemcitabine or carboplatin decreased cell proliferation, increased apoptosis, and induced cell cycle arrest. Cell cycle arrest and apoptosis were markedly increased when cell lines were treated with both gemcitabine and carboplatin simultaneously or sequentially. Order of administration during sequential treatment did not consistently affect cell proliferation results in TCC cell lines. When TCC cell lines were treated with gemcitabine and carboplatin in combination at therapeutically relevant concentrations (gemcitabine concentration, < 10μM; carboplatin concentration, < 250μM), a significant decrease in cell proliferation was observed, compared with cell proliferation following treatment with gemcitabine or carboplatin alone. In combination, the effects of gemcitabine and carboplatin were synergistic in 3 of 5 cell lines and additive in the other 2.
Conclusions and Clinical Relevance—Gemcitabine had antitumor effects on canine TCC cells in vitro, and the combination of gemcitabine and carboplatin had synergistic activity at biologically achievable concentrations.