Medet Gulsah, Inal Ahmet
Department of Pharmacology, Institute of Health Sciences, Erciyes University, Kayseri 38039, Turkey.
Department of Pharmacology, School of Medicine, Erciyes University, Kayseri 38039, Turkey.
Int J Mol Sci. 2025 Aug 22;26(17):8151. doi: 10.3390/ijms26178151.
This study aimed to evaluate the effects of dextromethorphan (DX), alone and in combination with gemcitabine (GEM), on cell viability, apoptosis, and epithelial-mesenchymal transition (EMT) markers in PANC-1 human pancreatic cancer cells. PANC-1 human pancreatic cancer cells were cultured and treated with varying concentrations of dextromethorphan (DX), gemcitabine (GEM), and 5-fluorouracil (5-FU), both as monotherapies and in combination. Cytotoxic effects were assessed using the MTT assay, and IC values were calculated at 24, 48, and 72 h. Apoptotic responses were evaluated using Annexin V-FITC/PI staining followed by flow cytometry. Protein expression levels of Bax, Bcl-2, and Vimentin were determined via immunocytochemistry, while EMT markers (E-cadherin, N-cadherin, Vimentin) were analyzed using flow cytometry. Relative mRNA expression of apoptotic and EMT-related genes was quantified by qRT-PCR. DX exhibited time- and dose-dependent cytotoxicity in PANC-1 cells, with IC values of 280.4 µM at 24 h, 163.2 µM at 48 h, and 105.6 µM at 72 h. For GEM, the 72 h IC was 57.53 µM. The combination of DX 50 µM + GEM 12.5 µM resulted in significantly lower cell viability (24.93 ± 3.12%) compared to GEM 25 µM (35.33 ± 5.22%) and DX 100 µM (51.40 ± 3.10%) ( < 0.001). Flow cytometry revealed significant increases in early (21.83 ± 1.32%) and late apoptotic cells (32.20 ± 0.84%) in the combination group, with a corresponding reduction in viable cells compared to control (24.93 ± 3.12% vs. 89.53 ± 0.97%, < 0.001). Immunocytochemical analysis showed increased Bax-positive cell count (62.0 cells/unit area), and decreased Bcl-2 (19.0) and Vimentin (28.0) levels in the combination group compared to control (Bax: 15.0, Bcl-2: 60.0, Vimentin: 70.0) ( < 0.001). Flow cytometry for EMT markers demonstrated increased E-cadherin (83.84 ± 0.65%) and decreased Vimentin (71.04 ± 1.17%) and N-cadherin (30.47 ± 0.72%) expression in the DX + GEM group compared to EMT control (E-cadherin: 68.97 ± 1.43%, Vimentin: 91.00 ± 0.75%, N-cadherin: 62.47 ± 1.13%) ( < 0.001). qRT-PCR supported these findings with increased Bax (2.1-fold), E-cadherin (2.0-fold), and reduced Bcl-2 (0.3-fold) and XIAP (0.6-fold) in the combination group ( < 0.05). Dextromethorphan, particularly in combination with gemcitabine, appears to enhance apoptosis and suppress EMT-associated marker expression in PANC-1 cells, supporting its potential as an adjuvant agent in pancreatic cancer therapy.
本研究旨在评估右美沙芬（DX）单独及与吉西他滨（GEM）联合使用对PANC - 1人胰腺癌细胞的细胞活力、凋亡及上皮 - 间质转化（EMT）标志物的影响。培养PANC - 1人胰腺癌细胞，并用不同浓度的右美沙芬（DX）、吉西他滨（GEM）和5 - 氟尿嘧啶（5 - FU）进行单药治疗及联合治疗。使用MTT法评估细胞毒性作用，并在24、48和72小时计算IC值。使用Annexin V - FITC/PI染色后通过流式细胞术评估凋亡反应。通过免疫细胞化学测定Bax、Bcl - 2和波形蛋白的蛋白表达水平，同时使用流式细胞术分析EMT标志物（E - 钙黏蛋白、N - 钙黏蛋白、波形蛋白）。通过qRT - PCR定量凋亡和EMT相关基因的相对mRNA表达。DX在PANC - 1细胞中表现出时间和剂量依赖性细胞毒性，24小时的IC值为280.4 µM，48小时为163.2 µM，72小时为105.6 µM。对于GEM，72小时的IC为57.53 µM。与GEM 25 µM（35.33 ± 5.22%）和DX 100 µM（51.40 ± 3.10%）相比，DX 50 µM + GEM 12.5 µM的联合用药导致细胞活力显著降低（24.93 ± 3.12%）（<0.001）。流式细胞术显示联合治疗组早期凋亡细胞（21.83 ± 1.32%）和晚期凋亡细胞显著增加（32.20 ± 0.84%），与对照组相比活细胞相应减少（24.93 ± 3.12%对89.53 ± 0.97%，<0.001）。免疫细胞化学分析显示联合治疗组中Bax阳性细胞计数增加（62.0个细胞/单位面积），与对照组相比Bcl - 2（19.0）和波形蛋白（28.0）水平降低（Bax：15.0，Bcl - 2：60.0，波形蛋白：70.0）（<0.001）。EMT标志物的流式细胞术显示，与EMT对照组相比，DX + GEM组中E - 钙黏蛋白表达增加（83.84 ± 0.65%），波形蛋白（71.04 ± 1.17%）和N - 钙黏蛋白（30.47 ± 0.72%）表达降低（E - 钙黏蛋白：68.97 ± 1.43%，波形蛋白：91.00 ± 0.75%，N - 钙黏蛋白：62.47 ± 1.13%）（<0.001）。qRT - PCR支持这些发现，联合治疗组中Bax（2.1倍）、E - 钙黏蛋白（2.0倍）增加，Bcl - 2（0.3倍）和XIAP（0.6倍）降低（<0.05）。右美沙芬，特别是与吉西他滨联合使用时，似乎可增强PANC - 1细胞的凋亡并抑制EMT相关标志物的表达，支持其作为胰腺癌治疗辅助药物的潜力。
