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.

Bromelain: a candidate to enhance wound healing after endonasal surgeries.

OBJECTIVE: In the present study, we investigated the topical bromelain's cytotoxic effects on mouse fibroblast NIH/3T3 cells via cell culture study. MATERIALS AND METHODS: In this cell culture study, Dulbecco's Modified Eagle Medium (DMEM) with fetal bovine serum (FBS, 10%) and penicillin/streptomycin (1%) was used as a cell growth medium for NIH/3T3 mouse fibroblast cells. MTT test was performed in 96-well plates seeded with NIH/3T3 cells 5x103/well and under standard cell culture conditions. Bromelain doses of 3.13 to 100 µM were administered to the wells and incubated for 24, 48, and 72 hours in the same cell culture conditions. For Confocal microscopic evaluation, NIH/3T3 cells were plated on cover slips in 6-well plates (105 cells/well) and treated with 100 µM concentration of bromelain for 24 h. Untreated cells were used as controls. RESULTS: MTT results showed that bromelain is not cytotoxic on mouse fibroblast NIH/3T3 cells. All three incubation times of 24, 48, and 72 hours bromelain initiated cell growth. A statistically significant rise in cell growth was detected in the only applied highest dose of 100 µM bromelain for all incubation times except for 24 hours. The nontoxic effect was further investigated by using confocal microscopy by applying the highest bromelain dose of 100 µM to NIH/3T3 mouse fibroblast cells. Confocal micrographs showed that bromelain did not change the morphology of mouse fibroblast cells at the incubation time of 24h. In untreated cells and bromelain-treated cells, the nucleus of NIH/3T3 cells was undamaged and compact, and the cytoskeleton was fusiform and non-fragmented. CONCLUSIONS: Bromelain is not cytotoxic on mouse fibroblast NIH/3T3 cells and enhances cell growth. If clinical trials will confirm this, it is possible that bromelain will be used topically in humans to enhance wound healing, in rhinosinusitis and chronic rhinosinusitis with nasal polyps and endonasal surgeries due to its anti-inflammatory effects.

Is G. cambogia a promising treatment? Effects on cultured nasal epithelial cells.

OBJECTIVE: The purpose of this study is to assess the effects of applying Garcinia cambogia to cultured human nasal epithelial cells. MATERIALS AND METHODS: A cell culture was set up consisting of human primary nasal epithelial cells harvested during septorhinoplasty from volunteers. The cells came from individuals with no history of rhinosinusitis. One assay for assessing cytotoxicity in cell culture utilizes MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). This method allows visualization of fragmented DNA, condensation of nuclei and changes to the external cellular membrane or cytoskeleton. Our study employed this method. Nasal epithelial cells at 37°C were exposed in culture to G. cambogia for a period of 24 hours. Afterwards an MTT assay was used in conjunction with confocal microscopy to assess evidence of toxicity. The proliferative capability of the nasal epithelial cells was also evaluated by inducing a scratch injury to cultured cells followed by light microscopic examination. RESULTS: Testing for cytotoxicity in this manner indicates that G. cambogia does not appear harmful to cultured nasal epithelial cells when applied directly. The cells exposed to this plant extract were still fully viable 24 hours afterwards. There was no increase in viability at the level of statistical significance. It was noted, however, that proliferation did increase slightly within the exposure period. The MTT assay and confocal microscopy confirm these findings. Under confocal microscopic examination, a compact morphology with unaltered nuclear and cytoskeletal appearances was observed. Thus, there is no evidence suggesting viability is impaired or that cytotoxicity occurs. Ordinary light microscopic examination showed the area denuded of cells had become re-covered completely within 24 hours in the cultures where G. cambogia had been applied. The result suggests that exposure to G. cambogia has no significant effect in terms of stimulating or inhibiting cellular proliferation. CONCLUSIONS: G. cambogia may offer clinical benefit as a supplementary topical treatment for inflammation of the nose and sinuses, as seen in chronic and acute rhinosinusitis, or nasal polyps. The plant appears to increase nasal epitheliocytic proliferation slightly, as revealed by the MTT assay. There were no indications of a cytotoxic effect on epithelial cells of the nose.