Oğuz O, Manole F, Bayar Muluk N, Vejselova Sezer C, Kutlu H M, Cingi C
Department of Audiology, Istanbul Nişantaşı University, Health Services Vocational School, Istanbul, Turkey.
Eur Rev Med Pharmacol Sci. 2023 Oct;27(5 Suppl):109-120. doi: 10.26355/eurrev_202310_34079.
In the present study, we investigated the effects of Ceramide C2 application on human laryngeal carcinoma cells.
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.
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.
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.
在本研究中，我们探究了应用神经酰胺C2对人喉癌细胞的影响。
人喉表皮样癌HEp-2（ATCC® CCL-23™）细胞购自美国典型培养物保藏中心（美国ATCC）。人喉表皮样癌HEp-2细胞在添加有胎牛血清（FBS）（10%）和青霉素/链霉素（1%）的完全杜氏改良 Eagle 培养基（DMEM）中，于二氧化碳（5%）培养箱中在标准细胞培养条件下培养。制备神经酰胺C2，并在新鲜培养基中进一步稀释至3.13至100 μM的浓度范围。将96孔板中的细胞暴露于制备好的不同浓度神经酰胺C2中24小时和48小时。通过MTT法进行细胞毒性评估。通过膜联蛋白-V分析检测HEp-2细胞的凋亡情况。通过流式细胞术分析评估神经酰胺C2处理后HEp-细胞中活化的半胱天冬酶3/7。通过共聚焦显微镜用鬼笔环肽和吖啶橙染色评估神经酰胺C2对HEp-2细胞造成的形态变化。对于伤口愈合实验，将HEp-2细胞接种于6孔板中，直至细胞汇合。
MTT细胞毒性测试结果显示，与未处理（对照）细胞相比，随着神经酰胺C2作用时间延长至24小时，人喉癌细胞的活力降低。在最高浓度的神经酰胺C2（100 μM）下，检测到其对短期作用24小时的人喉癌细胞生长抑制作用最强。膜联蛋白-V检测结果显示，对照组中98.97%的HEp-2细胞存活，1.63%的细胞被检测为早期凋亡。结果表明，与未处理的HEp-2细胞相比，神经酰胺C2可诱导HEp-2细胞凋亡，总凋亡细胞百分比为61.40%。将暴露于神经酰胺C2中24小时后的HEp-细胞的半胱氨酸蛋白酶（半胱天冬酶）3/7活化百分比与对照细胞进行比较，HEp-2细胞的形态发生改变，出现明显的凋亡迹象，突出了神经酰胺C2的细胞毒性和促凋亡活性。划痕实验评估了暴露于神经酰胺C2前后HEp-2细胞的迁移能力。结果表明，神经酰胺C2在24小时内降低了人喉癌细胞的迁移能力和增殖能力。
基于所有研究结果，可以认为短链神经酰胺C2对人喉癌细胞具有剂量和时间依赖性的细胞毒性，并通过诱导半胱天冬酶依赖性凋亡降低细胞活力。总体效应可能源于外源性应用神经酰胺C使细胞内神经酰胺水平升高。因此，得出结论，神经酰胺C2在人喉癌细胞中具有良好的诱导细胞毒性和凋亡的潜力，经过更深入的体外和体内研究后，有望成为高效抗癌药物设计的良好候选物。
