Type of cell death induced by alpha-trifluoromethyl acyloins in oral squamous cell carcinoma.

We previously reported that alpha-trifluoromethyl acyloins (TFs) induced various types of cell death, depending on the target cancer cell line. We investigated here what type of cell death is induced by a-trifluoromethyl acyloins in two human oral squamous cell carcinoma cell lines (HSC-2, HSC-4). TFs produced few TUNEL-positive cells. TFs induced annexin V/PI-double positive HSC-2 cells and annexin V-positive/PI-negative HSC-4 cells, respectively, but failed to activate caspase-3, capase-8 and caspase-9 in both HSC-2 and HSC-4 cells. On the other hand, TFs induced the formation of acidic organelles (detected by acridine orange staining) in both HSC-2 and HSC-4 cells. When HSC-2 and HSC-4 cells that had been transfected with expression vector encording the microtubule-associated protein 1 light chain 3 (LC3) gene fused to green fluorescent protein (GFP) were treated with TFs, LC3-GFP fusion protein was accumulated as granular dots in autophagosomes. Pretreatment with 3-methyladenine, an inhibitor of autophagy, partially inhibited the cytotoxicity of TFs, the formation of acidic organelles and LC3 accumulation in the autophagosome. These data suggest that alpha-trifluoromethyl acyloins may induce autophagic cell death in HSC-2 and HSC-4 cells following the early stage of necrosis or apoptosis, respectively.

Re-evaluation of tumor-specific cytotoxicity of mitomycin C, bleomycin and peplomycin.

Three antitumor antibiotics, mitomycin C, bleomycin sulfate and peplomycin sulfate, were compared for their tumor-specific cytotoxicity, using human oral squamous cell lines (HSC-2, HSC-3, HSC-4, Ca9-22 and NA), human promyelocytic leukemic cell line HL-60 and human normal oral cell types (gingival fibroblast HGF, pulp cell HPC and periodontal ligament fibroblast HPLF). Among these three compounds, mitomycin C showed the highest tumor-specificity, due to its higher cytotoxic activity against human oral tumor cell lines than bleomycin and peplomycin. However, there was considerable variation of drug sensitivity among the six tumor cell lines. Mitomycin C induced internucleosomal DNA fragmentation and caspase-3, -8 and -9 activation in HL-60 cells only after 24 h. On the other hand, mitomycin C induced no clear-cut DNA fragmentation in HCS-2 cells, although it activated caspase-3, -8 and -9 to a slightly higher extent. Western blot analysis demonstrated that mitomycin C did not induce any apparent change in the intracellular concentration of anti-apoptotic protein (Bcl-2) and pro-apoptotic proteins (Bax, Bad). Electron microscopy of mitomycin C-treated HL-60 cells showed intact mitochondria (as regards to integrity and size) and cell surface microvilli, without production of an apoptotic body or autophagosome, at an early stage after treatment. The present study suggests the incomplete induction of apoptosis or the induction of another type of cell death by mitomycin C treatment.

Cytotoxic activity of selected trifluoromethyl ketones against oral tumor cells.

Several trifluoromethyl ketones (TF1-4) and related non-fluorinated ketones (TF5 and 6) were tested for their relative cytotoxicity on four human tumor cell lines (oral squamous cell carcinoma HSC-2, HSC-3, HSC-4 and promyelocytic leukemia HL-60) and three normal human cells [gingival fibroblasts (HGF), pulp cells (HPC) and periodontal ligament fibroblasts (HPLF)]. Trifluoromethylated a-diketone (TF1, CF3COCOPh) and alpha-hydroxy ketones (TF2, CF3CH(OH)COPh; TF3, CF3CH(OH)COCH2Ph) showed higher tumor-specific cytotoxic activity than the corresponding non-fluorinated analogs (TF5, CH3COCOPh; TF6, CH3CH(OH)COPh), while the anti-tumor potency of trifluoromethyl ketone (TF4, CF3COCH2Ph) was lower. Among four tumor cell lines, HL-60 cells were the most sensitive to TF1-4, followed by HSC-2 and HSC-3 cells. HSC-4 cells were the most resistant in most cases. Agarose gel electrophoresis showed that TF1-3 did not induce intemucleosomal DNA fragmentation nor activated caspase-3. The cytotoxic activities of TF1-3 were not significantly affected by FeCl3. Electron microscopy of TF2- or 3-treated HL-60 cells showed the development of autophagosomes in HL-60 cells, without the production of an apoptotic body, or affecting the mitochondria and cell surface microvilli. The autophagy inhibitor, 3-methyladenine (3-MA), partially inhibited the TF2- or 3-induced cytotoxicity. These data suggest the induction of non-apoptotic cell death by TF2 or 3.

3-(3,4,5-Trimethoxyphenyl)-1-oxo-2-propene: a novel pharmacophore displaying potent multidrug resistance reversal and selective cytotoxicity.

This study revealed that various alicyclic and acyclic compounds containing the 3-(3,4,5-trimethoxyphenyl)-2-propenoyl group displayed potent MDR reversal properties. In particular, a concentration of 4 microg/ml of 2,5-bis(3,4,5-trimethoxyphenylmethylene)cyclopentanone was 31 times more potent than verapamil as a MDR revertant. In general, they were selectively toxic to malignant rather than normal cells. Two representative compounds induced apoptosis in human HL-60 cells and markedly activated caspase-3.