Effects of ceramide C2 application on human laryngeal carcinoma cells: a cell culture study.

OBJECTIVE: In the present study, we investigated the effects of Ceramide C2 application on human laryngeal carcinoma cells. MATERIALS AND METHODS: Human larynx epidermoid carcinoma HEp-2 (ATCC® CCL-23™) cells were purchased from the American Type Culture Collection (ATCC, USA). Human larynx epidermoid carcinoma HEp-2 cells were cultured in complete Dulbecco's Modified Eagle's Medium (DMEM) supplemented with fetal bovine serum (FBS) (10%) and penicillin/streptomycin (1%) in a CO2 (5%) incubator under standard cell culture conditions. Ceramide C2 was prepared, and further dilutions ranging from 3.13 to 100 µM were prepared in a fresh culture medium. Cells on 96 well plates were exposed to the prepared concentrations of ceramide C2 for 24 and 48 hours. Cytotoxicity evaluation was performed by MTT. Apoptosis profiles of HEp-2 cells were detected by annexin-V analysis. The activated caspases 3/7 on HEp-2 cells after ceramide C2 exposure were evaluated with flow cytometric analysis. The morphological changes on HEp-2 cells caused by ceramide C2 were evaluated by staining with phalloidine and acridine orange via confocal microscopy. For the Wound Healing Assay, HEp-2 cells were cultured in 6 well-plates until they became confluent. RESULTS: MTT cytotoxicity test findings revealed that the viability of human laryngeal carcinoma cells decreased with the increased application of ceramide C2 for 24 hours compared to untreated (control) cells. The highest growth inhibition by ceramide C2 for short-term application for 24 hours was detected at the highest concentration of ceramide C2 (100 µM). Annexin-V findings showed that 98.97 of HEp-2 cells were alive, and 1.63% were detected as early apoptosis for the control group. The results showed that ceramide C2 triggered apoptosis on HEp-2 cells with a percentage of total apoptotic cells of 61,40 compared to untreated HEp-2 cells. Cysteine proteases (caspases) 3/7 activation percentages of HEp-2 cells exposed to ceramide C2 for 24 hours were compared to control cells, and the morphology of HEp-2 cells was changed with clear apoptotic signs that underlined the cytotoxicity and pro-apoptotic activity of ceramide C2. Scratch Assay assessed the migration capability of HEp-2 cells before and after the exposure to ceramide C2. It showed that ceramide C2 reduced human laryngeal carcinoma cells' migration capability and proliferation for 24 hours. CONCLUSIONS: Based on all study findings, it can be considered that short-chain ceramide C2 exerted cytotoxicity on human laryngeal carcinoma cells in a dose and time-dependent manner and reduced the viability via inducing caspase-dependent apoptosis. The overall effect might be derived from the elevated intracellular ceramide levels by the exogenous application of ceramide C2. Consequently, it was concluded that ceramide C2 has good potential to cause cytotoxicity and apoptosis in human laryngeal carcinoma cells and, after deeper in vitro and in vivo investigations, can be a good candidate for designing anti-cancer drugs with high efficiency.

Preventive effect of intravesical ozone supplementation on n-methyl-n-nitrosourea-induced non-muscle invasive bladder cancer in male rats.

Although non-muscle invasive bladder cancer (NMIBC) is widely seen in men, most laboratory studies of new intravesical therapies to prevent NMIBC have been conducted on female animals. In addition, ozone (O3) has been shown to be a beneficial agent as an intravesical application in the treatment of various disorders. In the current study, we evaluated the immunohistopathological and oxidative-antioxidative effects of intravesical O3 treatment on n-methyl-n-nitrosourea (MNU)-induced NMIBC. Male Wistar-Albino rats (n=51) were divided into four groups: sham (n=6), O3 only (n=15), MNU only (n=15), and MNU+O3 (n=15). The MNU-only and MNU+O3 groups received MNU, and the O3-only group received saline every other week for 10 weeks. The MNU-only group received 1 ml saline in place of O3 treatment, whereas the O3-only and MNU+O3 groups were treated with 1 ml 25 µg/ml O3 between the 7th and 12th weeks. Rat bladders were collected in the 15th week for immunohistopathology and oxidant-antioxidant quantitation. Oxidant-antioxidant parameters were determined by ELISA. Although all surviving rats in the MNU-only group had preneoplastic (4/11, 36.4%) or neoplastic changes (7/11, 63.6%), a completely normal urothelium was observed in 2 rats (2/12, 16.7%) in the MNU+O3-group (P=0.478). More high-grade lesions were observed in the MNU-only group (4/11, 36.4%) than in the MNU+O3 group (1/12, 8.3%) (P=0.120). All oxidant-antioxidant parameters significantly increased (P<0.05) in the O3-only group compared with the sham group. However, only antioxidant superoxide dismutase was remarkably higher (178.9%, P=0.060) in the MNU+O3 group compared with the MNU-only group. This is the first methodologically and pathologically well-described male rat orthotopic bladder carcinogenesis model with intravesical MNU and administration of O3 in NMIBC.