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.

The apoptotic effects of escin in the H-Ras transformed 5RP7 cell line.

Extracts of Aesculus hippocastanum L. (horse chestnut) seed have been used in the treatment of chronic venous insufficiency, edema and hemorrhoids. Most of the beneficial effects of horse chestnut are attributed to its principal component ß-escin or escin. We have evaluated the cytotoxic and apoptotic effects of escin in the H-Ras 5RP7 cell line by analyzing cell growth inhibition, apoptosis and caspase-3 dependent activity. We have also shown structural and ultrastructural changes in these cell using confocal and transmission electron microscopy. The results indicated that escin has significant inhibitory effects on cell growth and the percentage of apoptotic cells increased after treatment with escin, and the micrographs confirmed that escin damaged these cells and induced apoptosis.