[1]王荣坤,林简.3-甲基腺嘌呤增强ALA-PDT对皮肤鳞状细胞癌A431细胞的杀伤作用[J].中国皮肤性病学杂志,2020,(06):627-633.[doi:10.13735/j.cjdv.1001-7089.201908164]
 WANG Rongkun,LIN Jian.3-Methyladenine Enhances the Killing Effect of ALA-PDT on Cutaneous Squamous Cell Carcinoma A431 Cells[J].The Chinese Journal of Dermatovenereology,2020,(06):627-633.[doi:10.13735/j.cjdv.1001-7089.201908164]
点击复制

3-甲基腺嘌呤增强ALA-PDT对皮肤鳞状细胞癌A431细胞的杀伤作用
分享到:

《中国皮肤性病学杂志》[ISSN:1001-7089/CN:61-1197/R]

卷:
期数:
2020年06期
页码:
627-633
栏目:
论著
出版日期:
2020-06-01

文章信息/Info

Title:
3-Methyladenine Enhances the Killing Effect of ALA-PDT on Cutaneous Squamous Cell Carcinoma A431 Cells
文章编号:
1001-7089(2020)06-0627-07
作者:
王荣坤林简
三亚市人民医院皮肤性病科,海南 三亚 572000
Author(s):
WANG RongkunLIN Jian
(Dermatology & STD Department,Sanya People's Hospital,Sanya 572000,China)
关键词:
3-甲基腺嘌呤 ALA-PDT 皮肤鳞状细胞癌 凋亡 自噬
Keywords:
3-methyladenine ALA-PDT Cutaneous squamous cell carcinoma Apoptosis Autophagy
分类号:
R 739.5
DOI:
10.13735/j.cjdv.1001-7089.201908164
文献标志码:
A
摘要:
目的 探讨5-氨基酮戊酸光动力疗法(ALA-PDT)联合3-甲基腺嘌呤(3-MA)对皮肤鳞状细胞癌A431细胞的杀伤作用及其机制研究。方法 体外培养人皮肤鳞状细胞癌A431细胞,设置不同的ALA浓度(0、0.20、0.40、0.80、1.60 mmol/L)及不同的培养时间(12、24、48 h),对A431细胞进行ALA-PDT处理,采用MTT法检测细胞增殖活性并计算半抑制浓度(IC50)值。根据实验需要将A431细胞分为空白对照组、ALA-PDT组、3-MA组和ALA-PDT+3-MA组。分组处理后,MTT法检测细胞增殖; 流式细胞术分别检测细胞凋亡(Annexin V-FITC/PI双染法)以及细胞线粒体膜电位变化(JC-1染色法); 单丹磺酰尸胺(MDC)染色法观察细胞自噬情况; Western blot检测凋亡和自噬相关蛋白的表达水平。结果 ALA-PDT对A431细胞的增殖活性有明显的抑制作用,且呈剂量和时间依赖性。与对照组比较,ALA-PDT组细胞增殖活性、线粒体膜电位均显著降低(P<0.05),而细胞凋亡与自噬水平显著提高(P<0.05),Cleaved-caspase-3、Bax、胞质Cyt C、LC-3Ⅱ、Beclin1等蛋白表达显著上调(P<0.05),Bcl-2表达显著下调(P<0.05)。与ALA-PDT组比较,ALA-PDT+3-MA组细胞增殖活性、线粒体膜电位以及细胞自噬水平显著降低(P<0.05),细胞凋亡水平显著提高(P<0.05),Cleaved-caspase-3、Bax、胞质Cyt C等蛋白表达显著上调(P<0.05),而Bcl-2、LC-3Ⅱ、Beclin1等蛋白表达显著下调(P<0.05)。结论 3-MA可通过抑制细胞自噬增强ALA-PDT对A431细胞产生的杀伤作用。
Abstract:
Objective To investigate the killing effect of 5-aminolevulinic acid-mediated photodynamic therapy(ALA-PDT)combined with 3-methyladenine(3-MA)on cutaneous squamous cell carcinoma cell line A431,and to explore its possible mechanism.Methods A431 cells were cultured in vitro and treated with ALA-PDT at different concentrations of ALA(0,0.20,0.40,0.80,1.60 mmol/L)for different time(12,24,48 h).Cell viability was detected by MTT assay and the value of IC50 was calculated.A431 cells were divided into four groups:control group,ALA-PDT group,3-MA group and ALA-PDT+3-MA group.After treatment,cell proliferation was detected by MTT method.Apoptosis rates(Annexin V-FITC/PI double staining)and the change of mitochondrial membrane potential(JC-1 staining)were detected by flow cytometry.Autophagy was observed by monodansylcadaverin(MDC)staining.The expression levels of apoptosis and autophagy related proteins were detected by Western blot.Results ALA-PDT significantly inhibited the proliferation activity of A431 cells in a dose- and time-dependent manner.Compared with control group,ALA-PDT significantly decreased the cell proliferation activity and mitochondrial membrane potential of A431 cells(P<0.05),elevated the levels of apoptosis and autophagy(P<0.05),increased the protein expression levels of Cleaved-caspase-3,Bax,LC-3Ⅱ,Beclin1 and cytoplasmic Cyt C(P<0.05),and decreased the protein expression of Bcl-2(P<0.05).Compared with the ALA-PDT group,the combination of 3-MA and ALA-PDT significantly decreased the cell proliferation activity,mitochondrial membrane potential and autophagy(P<0.05),elevated the level of apoptosis(P<0.05),increased the protein expression levels of Cleaved-caspase-3,Bax,cytoplasmic Cyt C(P<0.05),while decreased the protein expression levels of Bcl-2,LC-3Ⅱ and Beclin1(P<0.05).Conclusion 3-MA enhances the killing effect of ALA-PDT on A431 cells by inhibiting autophagy.

参考文献/References:

[1] Brougham ND,Tan ST.The incidence and risk factors of metastasis for cutaneous squamous cell carcinoma--implications on the T-classification system[J].J Surg Oncol,2014,110(7):876-882.
[2] 李超,陈永锋.皮肤鳞状细胞癌的研究进展[J].皮肤性病诊疗学杂志,2019,26(1):49-52.
[3] 周萌,刘保国,顾静.5-氨基酮戊酸光动力疗法治疗皮肤恶性肿瘤及癌前病变的研究进展[J].中国激光医学杂志,2016,25(3):160-164.
[4] Bhatta AK,Wang P,Keyal U,et al.Therapeutic effect of Imiquimod enhanced ALA-PDT on cutaneous squamous cell carcinoma[J].Photodiagnosis Photodyn Ther,2018,23:273-280.
[5] Peng J,Feng W,Luo X,et al.A clinical trial using attrition combined with 5-aminolevulinic acids based photodynamic therapy in treating squamous cell carcinoma[J].Med Sci Monit,2017,23:1347-1354.
[6] Wu YT,Tan HL,Shui G,et al.Dual role of 3-methyladenine in modulation of autophagy via different temporal patterns of inhibition on class Ⅰ and Ⅲ phosphoinositide 3-kinase[J].J Biol Chem,2010,285(14):10850-10861.
[7] 雷思雨,陈曼曼,陈斌,等.细胞自噬与肿瘤微环境的关系研究进展[J].蚌埠医学院学报,2018,43(7):979-981.
[8] Zhang L,Zhang J,Chen L,et al.Autophagy in human skin squamous cell carcinoma:Inhibition by 3-MA enhances the effect of 5-FU-induced chemotherapy sensitivity[J].Oncol Rep,2015,34(6):3147-3155.
[9] Wang J,Wang C,Hu X,et al.Gefitinib-mediated apoptosis is enhanced via inhibition of autophagy by chloroquine diphosphate in cutaneous squamous cell carcinoma cells[J].Oncol Lett,2019,18(1):368-374.
[10] Zhou W,Zhang S,Li J,et al.lncRNA TINCR participates in ALA-PDT-induced apoptosis and autophagy in cutaneous squamous cell carcinoma[J].J Cell Biochem,2019,120(8):13893-13902.
[11] Hou H,Zhang Y,Huang Y,et al.Inhibitors of phosphatidylinositol 3'-kinases promote mitotic cell death in HeLa cells[J].PLoS One,2012,7(4):e35665.
[12] 余兵,高君,肖文.5-氨基酮戊酸光动力疗法在皮肤性病科的应用进展[J].中国皮肤性病学杂志,2016,30(10):1070-1072.
[13] Li B,Lin L,Lin H,et al.Photosensitized singlet oxygen generation and detection:Recent advances and future perspectives in cancer photodynamic therapy[J].J Biophotonics,2016,9(11):1314-1325.
[14] 姜彬,陈办成,于波,等.ALA-PDT对皮肤鳞状细胞癌SCL-1细胞体外活性的影响[J].中国皮肤性病学杂志,2018,32(12):1361-1366.
[15] Burke PJ.Mitochondria,bioenergetics and apoptosis in cancer[J].Trends Cancer,2017,3(12):857-870.
[16] Ola MS,Nawaz M,Ahsan H.Role of Bcl-2 family proteins and caspases in the regulation of apoptosis[J].Mol Cell Biochem,2011,351(1-2):41-58.
[17] Ravanan P,Srikumar IF,Talwar P.Autophagy:The spotlight for cellular stress responses[J].Life Sci,2017,188:53-67.

相似文献/References:

[1]朱世文.复方甘草酸苷联合ALA-PDT治疗中、重度痤疮44例临床观察[J].中国皮肤性病学杂志,2016,(10):1098.[doi:10.13735/j.cjdv.1001-7089.201601156]
 ZHU Shi-wen.Clinical Efficacy of 44 Cases of Compound Glycyrrhizin Combined with 5-aminolevulinic Acid Photodynamic Therapy in the Treatment of Moderate to Severe Acne[J].The Chinese Journal of Dermatovenereology,2016,(06):1098.[doi:10.13735/j.cjdv.1001-7089.201601156]

备注/Memo

备注/Memo:
[网络首发时间] 2020-4-15 10:49
[网络首发地址] http://kns.cnki.net/kcms/detail/61.1197.R.20200414.1458.009.html
更新日期/Last Update: 2020-06-05