补骨脂素抑制膀胱癌T24细胞增殖和迁移的作用及机制研究

doi:10.3877/cma.j.issn.2095-1221.2022.04.001

目的

探讨补骨脂素对人膀胱癌T24细胞存活率、细胞周期、细胞凋亡和迁移的影响及其分子机制。

方法

分别用细胞培养液、3‰二甲基亚砜（DMSO）和不同浓度（10、30、50、100 μg/mL）补骨脂素处理膀胱癌细胞分成对照组、DMSO组和补骨脂素组，CCK-8检测细胞存活率。流式细胞术检测细胞周期和细胞凋亡。划痕实验检测划痕愈合率。RT-qPCR法检测磷脂酰肌醇3激酶（PI3K）和蛋白激酶B （AKT） mRNA表达水平、Western blot法检测PI3K和AKT蛋白的表达及磷酸化情况。多组间比较采用单因素方差分析，组间两两比较采用LSD-t检验。

结果

与DMSO组比较，除10 μg/mL补骨脂素作用24 h外，其余浓度补骨脂素作用不同时间的细胞存活率随着补骨脂素浓度增高、作用时间延长而逐渐降低（P < 0.05）。与DMSO组比较，30、100 μg/mL补骨脂素干预24 h后，G1期细胞比例增多，G2/M期比例减少，细胞凋亡率[（9.16±0.97）%、（15.45±1.57）%比（1.02±0.36）%]升高，划痕愈合率[24 h：（45.00±3.44）%、（27.60±2.21）%比（66.10±2.61）%，48 h：（70.00 ± 3.40）%、（45.17±2.44）%比（85.17±3.85）%]降低，PI3K、AKT mRNA表达以及PI3K、AKT蛋白表达水平和磷酸化水平均降低（P均< 0.05）。

结论

补骨脂素降低膀胱癌细胞存活率、阻滞细胞周期、诱导细胞凋亡和抑制细胞迁移，其机制可能与下调PI3K、AKT mRNA、蛋白表达及磷酸化水平有关。

关键词: 补骨脂素, 抗肿瘤, 膀胱癌, 磷脂酰肌醇3-激酶, 丝氨酸/苏氨酸激酶

Objective

To investigate the effects of psoralen on the viability, cell cycle, apoptosis, and migration of human bladder cancer T24 cells and its mechanisms.

Methods

Bladder cancer cells were treated with cell culture medium, 3‰ dimethyl sulfoxide (DMSO) and different concentrations of psoralen and divided into normal control group, DMSO group and psoralen groups (10、30、50、100 μg/mL) . CCK-8 was used to detect cell viabilities. FACS was performed to detect cell cycles and apoptosis. Scratch tests were used to detecte the scratch healing rates. The expression levels of phosphatidylinositol-3-kinase (PI3K) and protein kinase B (AKT) mRNA were detected by RT-qPCR, and the expression and phosphorylation levels of PI3K and AKT protein were detected by Western blot. One-way ANOVA was used for comparison among multiple groups, and LSD-t test was used for comparison between groups.

Results

Compared with DMSO groups, the cell viabilities were decreased gradually with the increase of psoralen concentration and action time (P < 0.05) , except the cells were treated with 10 μg/mL psoralen for 24 h. Compared with DMSO group, the proportion of cells in G1 phase was increased after treatment with 30, 100 μg/mL psoralen for 24 h. The proportion of cells in G2/M phase was decreased, the apoptosis rates were increased [ (9.16±0.97) %、(15.45±1.57) % vs (1.02±0.36) %], the scratch healing rates were decreased [24 h: (45.00±3.44) %, (27.60± 2.21) %vs (66.10±2.61) %, 48 h: (70.00± 3.40) %, (45.17±2.44) %vs (85.17± 3.85) %], PI3K and AKT mRNA expression levels, protein expression levels and phosphorylation levels were all decreased (all P < 0.05) .

Conclusion

Psoralen decreased cell viability, blocked cell cycle, induced apoptosis and inhibited cell migration. Its mechanism may be that psoralen down-regulates the expression levels of PI3K and AKT mRNA and the expression levels and phosphorylation levels of PI3K and AKT protein.

Key words: Psoralen, Antitumor, Bladder cancer, PI3K, AKT

表1 引物序列信息

基因	序列（5'- 3'）	预期扩增产物长度（bp）
PI3K	上游ACCACTACCGGAATGAATCTCT	207
	下游GGGATGTGCGGGTATATTCTTC
AKT	上游CACAGGTCGCTACTATGCC	152
	下游GGTCGTGGGTCTGGAATG
β-actin	上游CATGTACGTTGCTATCCAGGC	250
	下游CTCCTTAATGTCACGCACGAT

表1 引物序列信息

基因	序列（5'- 3'）	预期扩增产物长度（bp）
PI3K	上游ACCACTACCGGAATGAATCTCT	207
	下游GGGATGTGCGGGTATATTCTTC
AKT	上游CACAGGTCGCTACTATGCC	152
	下游GGTCGTGGGTCTGGAATG
β-actin	上游CATGTACGTTGCTATCCAGGC	250
	下游CTCCTTAATGTCACGCACGAT

表2 补骨脂素对膀胱癌T24细胞存活率的影响比较（%，±s）

分组	实验次数	24 h	48 h	72 h	96 h	F值	P值
正常对照	3	99.71±4.70	103.32±2.05	102.65±4.61	102.24±0.99
DMSO	3	100.00±0.00	100.00±0.00	100.00±0.00	100.00±0.00
10 µg/mL	3	96.50±2.70	94.53±1.68^a	90.34±3.07^aef	85.50±2.29^aefg	19.062	< 0.001
30 µg/mL	3	93.64±1.67^a	87.29±2.72^abe	83.67±5.76^abe	75.82±2.09^abefg	26.956	< 0.001
50 µg/mL	3	89.72±2.80^abc	83.47±2.12^abce	73.56±2.34^abcef	64.95±4.19^abcefg	79.297	< 0.001
100 µg/mL	3	85.45±2.21^abcd	77.60±2.45^abcde	64.24±3.00^abcdef	55.45±3.06^abcdefg	157.463	< 0.001
F值		21.192	56.974	51.392	124.302
P值		< 0.001	< 0.001	< 0.001	< 0.001

表2 补骨脂素对膀胱癌T24细胞存活率的影响比较（%，±s）

分组	实验次数	24 h	48 h	72 h	96 h	F值	P值
正常对照	3	99.71±4.70	103.32±2.05	102.65±4.61	102.24±0.99
DMSO	3	100.00±0.00	100.00±0.00	100.00±0.00	100.00±0.00
10 µg/mL	3	96.50±2.70	94.53±1.68^a	90.34±3.07^aef	85.50±2.29^aefg	19.062	< 0.001
30 µg/mL	3	93.64±1.67^a	87.29±2.72^abe	83.67±5.76^abe	75.82±2.09^abefg	26.956	< 0.001
50 µg/mL	3	89.72±2.80^abc	83.47±2.12^abce	73.56±2.34^abcef	64.95±4.19^abcefg	79.297	< 0.001
100 µg/mL	3	85.45±2.21^abcd	77.60±2.45^abcde	64.24±3.00^abcdef	55.45±3.06^abcdefg	157.463	< 0.001
F值		21.192	56.974	51.392	124.302
P值		< 0.001	< 0.001	< 0.001	< 0.001

表3 补骨脂素作用24 h时对膀胱癌T24细胞周期的影响比较（%，±s）

分组	实验次数	G1	S	G2/M
DMSO	3	57.91±0.84	25.73±0.17	16.37±1.00
30 µg/mL	3	62.34±1.13^a	32.77±1.35^a	4.89±1.38^a
100 µg/mL	3	72.08±2.75^ab	25.56±0.83^b	2.36±1.97^a
F值		49.678	59.844	73.895
P值		< 0.001	< 0.001	< 0.001

表3 补骨脂素作用24 h时对膀胱癌T24细胞周期的影响比较（%，±s）

分组	实验次数	G1	S	G2/M
DMSO	3	57.91±0.84	25.73±0.17	16.37±1.00
30 µg/mL	3	62.34±1.13^a	32.77±1.35^a	4.89±1.38^a
100 µg/mL	3	72.08±2.75^ab	25.56±0.83^b	2.36±1.97^a
F值		49.678	59.844	73.895
P值		< 0.001	< 0.001	< 0.001

图1 补骨脂素作用24 h时对T24细胞周期的影响

表4 补骨脂素对膀胱癌T24细胞凋亡和迁移率的影响比较（%，±s，n = 3）

分组	凋亡率（24 h）	划痕愈合率（24 h）	划痕愈合率（48 h）
DMSO	1.02±0.36	66.10±2.61	85.17±3.85
30 µg/mL	9.16±0.97^a	45.00±3.44^a	70.00±3.40^a
100 µg/mL	15.45±1.57^ab	27.60±2.21^ab	45.17±2.44^ab
F值	133.451	142.294	113.707
P值	< 0.001	< 0.001	< 0.001

表4 补骨脂素对膀胱癌T24细胞凋亡和迁移率的影响比较（%，±s，n = 3）

分组	凋亡率（24 h）	划痕愈合率（24 h）	划痕愈合率（48 h）
DMSO	1.02±0.36	66.10±2.61	85.17±3.85
30 µg/mL	9.16±0.97^a	45.00±3.44^a	70.00±3.40^a
100 µg/mL	15.45±1.57^ab	27.60±2.21^ab	45.17±2.44^ab
F值	133.451	142.294	113.707
P值	< 0.001	< 0.001	< 0.001

图2 补骨脂素对膀胱癌细胞凋亡的影响

图3 补骨脂素对膀胱癌细胞划痕愈合率的影响

表5 补骨脂素对膀胱癌细胞PI3K、AKT mRNA表达的影响比较（±s）

分组	实验次数	PI3K	AKT
DMSO	3	1.00±0.00	1.00±0.00
30 µg/mL	3	0.56±0.11^a	0.45±0.03^a
100 µg/mL	3	0.31±0.04^ab	0.26±0.06^a
F值		86.107	288.978
P值		< 0.001	< 0.001

表5 补骨脂素对膀胱癌细胞PI3K、AKT mRNA表达的影响比较（±s）

分组	实验次数	PI3K	AKT
DMSO	3	1.00±0.00	1.00±0.00
30 µg/mL	3	0.56±0.11^a	0.45±0.03^a
100 µg/mL	3	0.31±0.04^ab	0.26±0.06^a
F值		86.107	288.978
P值		< 0.001	< 0.001

表6 补骨脂素对膀胱癌T24细胞PI3K、AKT蛋白表达及磷酸化的影响比较（±s，n = 3）

分组	PI3K	AKT	p-PI3K	p-AKT
DMSO	1.83±0.17	1.75±0.12	3.59±0.42	2.82±0.25
30 µg/mL	1.35±0.07^a	1.34±0.09^a	1.98±0.07^a	1.60±0.20^a
100 µg/mL	1.00±0.10^ab	1.00±0.16^ab	1.00±0.27^ab	1.00±0.32^ab
F值	35.359	26.746	61.081	37.389
P值	< 0.001	0.001	< 0.001	< 0.001

表6 补骨脂素对膀胱癌T24细胞PI3K、AKT蛋白表达及磷酸化的影响比较（±s，n = 3）

分组	PI3K	AKT	p-PI3K	p-AKT
DMSO	1.83±0.17	1.75±0.12	3.59±0.42	2.82±0.25
30 µg/mL	1.35±0.07^a	1.34±0.09^a	1.98±0.07^a	1.60±0.20^a
100 µg/mL	1.00±0.10^ab	1.00±0.16^ab	1.00±0.27^ab	1.00±0.32^ab
F值	35.359	26.746	61.081	37.389
P值	< 0.001	0.001	< 0.001	< 0.001

图4 补骨脂素对膀胱癌T24细胞PI3K、AKT蛋白表达及磷酸化的影响

1	Pemov A, Wegman-Ostrosky T, Kim J, et al. Identification of genetic risk factors for familial urinary bladder cancer: An exome sequencing study[J]. JCO Precis Oncol, 2021,5:PO.21.00115. doi: 10.1200/PO.21.00115.
2	Liu X, Jiang J, Yu C, et al. Secular trends in incidence and mortality of bladder cancer in China, 1990-2017: A joinpoint and age-period-cohort analysis[J]. Cancer Epidemiol, 2019, 61:95-103.
3	Wang X, Xu C, Hua Y, et al. Psoralen induced cell cycle arrest by modulating Wnt/beta-catenin pathway in breast cancer cells[J]. Sci Rep, 2018, 8(1):14001.doi: 10.1038/s41598-018-32438-7.
4	Xiao W, Liu Y, Dai M, et al. Rotenone restrains colon cancer cell viability, motility and epithelialmesenchymal transition and tumorigenesis in nude mice via the PI3K/AKT pathway[J]. Int J Mol Med, 2020, 46(2):700-708.
5	Li F, Xie W, Fang Y, et al. HnRNP-F promotes the proliferation of bladder cancer cells mediated by PI3K/AKT/FOXO1[J]. J Cancer, 2021, 12(1):281-291.
6	Liu ST, Hui G, Mathis C, et al. The current status and future role of the phosphoinositide 3 Kinase/AKT signaling pathway in urothelial cancer: An old pathway in the new immunotherapy era[J]. Clin Genitourin Cancer, 2018, 16(2):e269-e276.
7	Rentsch CA, Muller DC, Ruiz C, et al. Comprehensive molecular characterization of urothelial bladder carcinoma: A step closer to clinical translation?[J]. Eur Urol, 2017, 72(6):960-961.
8	Jayaratna IS, Navai N, Dinney CP. Risk based neoadjuvant chemotherapy in muscle invasive bladder cancer[J]. Transl Androl Urol, 2015, 4(3):273-282.
9	Zhang HH, Huang B, Cao YH, et al. Role of 5-Aza-CdR in mitomycin-C chemosensitivity of T24 bladder cancer cells[J]. Oncol Lett, 2017, 14(5):5652-5656.
10	Yuan Y, Chiba P, Cai T, et al. Fabrication of psoralen-loaded lipid-polymer hybrid nanoparticles and their reversal effect on drug resistance of cancer cells[J]. Oncol Rep, 2018, 40(2):1055-1063.
11	杨阔，高茸，马亚中, 等. 补骨脂素药理作用及肝毒性机制的研究进展[J]. 中草药, 2021, 52(1):289-298.
12	Wang X, Xu C, Hua Y, et al. Exosomes play an important role in the process of psoralen reverse multidrug resistance of breast cancer[J]. J Exp Clin Cancer Res, 2016, 35(1):186.
13	Jiang J, Wang X, Cheng K, et al. Psoralen reverses the P-glycoprotein-mediated multidrug resistance in human breast cancer MCF-7/ADR cells[J]. Mol Med Rep, 2016, 13(6):4745-4750.
14	李少鹏，蔡建通，翁铭芳, 等. 补骨脂素对前列腺癌LNCaP-AI细胞增殖和周期调控及雌激素受体β表达的影响[J/CD]. 中华细胞与干细胞杂志（电子版）, 2018, 8(1):1-5.
15	Zhu S, Ma AH, Zhu Z, et al. Synergistic antitumor activity of pan-PI3K inhibition and immune checkpoint blockade in bladder cancer[J]. J Immunother Cancer, 2021, 9(11):e002917. doi: 10.1136/jitc-2021-002917.
16	吴琦. 三叶青黄酮调节PI3K/Akt-eNOS信号通路对膀胱癌细胞生物学行为的影响及其机制[J]. 中国现代医生, 2021, 59(18):31-34+193.
17	Lee JH, Parveen A, Do MH, et al. Lespedeza cuneata protects the endothelial dysfunction via eNOS phosphorylation of PI3K/Akt signaling pathway in HUVECs[J]. Phytomedicine, 2018, 48:1-9.
18	Zhang Q, Lyu W, Yu M, et al. Sulfur dioxide induces vascular relaxation through PI3K/Akt/eNOS and NO/cGMP signaling pathways in rats[J]. Hum Exp Toxicol, 2020, 39(8)1108-1117.
19	Xiao W, Zhang G, Chen B, et al. Mutational landscape of PI3K-AKT-mTOR pathway in breast cancer: implications for targeted therapeutics[J]. J Cancer, 2021, 12(14):4408-4417.
20	Nagai S, Kurebayashi Y, Koyasu S. Role of PI3K/Akt and mTOR complexes in Th17 cell differentiation[J]. Ann N Y Acad Sci, 2013, 1280:30-34.
21	Mao W, Wang K, Sun S, et al. ID2 inhibits bladder cancer progression and metastasis via PI3K/AKT signaling pathway[J]. Front Cell Dev Biol, 2021, 9:738364.doi: 10.3389/fcell.2021.738364.

[1]	张玉, 薛文瑞, 王鑫, 李旭瑜, 王旭东, 袁鹏飞, 梁雨润, 韩志兴, 张海建, 刘庆军, 纪世琪. 人类免疫缺陷病毒感染合并膀胱癌14例临床特点[J]. 中华实验和临床感染病杂志(电子版), 2023, 17(05): 354-358.
[2]	刘畅, 徐骋, 宁杨. 左颏下横纹肌肉瘤放化疗术后牙齿发育不良一例[J]. 中华口腔医学研究杂志(电子版), 2021, 15(05): 296-299.
[3]	顾志波, 郝林, 陆明, 陈建刚. 光动力纳米载体联合si-P3H4治疗膀胱癌的初步探索[J]. 中华腔镜泌尿外科杂志(电子版), 2023, 17(06): 633-641.
[4]	熊风, 林辉煌, 陈晓波. 铥激光在泌尿外科中的临床应用及研究进展[J]. 中华腔镜泌尿外科杂志(电子版), 2023, 17(05): 533-536.
[5]	彭龙飞, 汪鑫, 夏典, 颜松柏, 王进有, 毕良宽. 腹腔镜下腹膜外膀胱根治和原位新膀胱术的初步体会[J]. 中华腔镜泌尿外科杂志(电子版), 2023, 17(01): 7-10.
[6]	邰胜, 周骏, 施浩强, 杨诚, 尹水平, 牛迪, 郝宗耀, 傅强, 梁朝朝. 无线智能腔镜在腹腔镜膀胱癌根治术中的运用[J]. 中华腔镜泌尿外科杂志(电子版), 2023, 17(01): 22-25.
[7]	赵永录, 张兴盛, 宋广智, 黄斌, 张斌, 顾鹏守, 刘国淦. 经尿道二次电切在pT₁期非肌层浸润性膀胱癌的预后价值研究[J]. 中华腔镜泌尿外科杂志(电子版), 2022, 16(04): 316-319.
[8]	肖东明, 刘硕儒, 张斌, 李茂胤, 黄文涛, 张浩, 胡成. 腹腔镜膀胱癌根治术尿道改流留置外、内支架管与无管化的对比研究[J]. 中华腔镜泌尿外科杂志(电子版), 2022, 16(03): 198-203.
[9]	刘天琦, 张振辉, 刘豪圣, 方建雄, 赵超, 刘久敏, 蒲小勇. 腹腔镜下膀胱癌根治术中完全腹膜外经皮输尿管造口术[J]. 中华腔镜泌尿外科杂志(电子版), 2022, 16(02): 169-172.
[10]	朱金海, 左泽平, 宇洪涛, 钱俊杰. 术前外周血中淋巴细胞与单核细胞比值对高危非肌层浸润性膀胱癌预后的评估价值[J]. 中华腔镜泌尿外科杂志(电子版), 2022, 16(02): 124-127.
[11]	杨红霞, 龙喜花, 罗朝霞. 高原地区术中低体温对非肌层浸润性膀胱癌患者术后免疫功能和远期复发的影响[J]. 中华腔镜泌尿外科杂志(电子版), 2021, 15(05): 396-400.
[12]	陈令秋, 王鹏, 袁龙平, 乙从亮. 二次电切术在T2期膀胱癌保留膀胱综合治疗中的临床意义[J]. 中华腔镜泌尿外科杂志(电子版), 2021, 15(04): 325-329.
[13]	孙晶晶, 刘蓬杨, 张怡, 路君. STING信号通路介导抗肿瘤免疫的研究进展[J]. 中华细胞与干细胞杂志(电子版), 2022, 12(03): 186-190.
[14]	邓凌钢, 孙建明, 陈晓峰. FBXO6介导的ERO1L泛素化降解对膀胱癌细胞增殖、迁移和侵袭的作用研究[J]. 中华细胞与干细胞杂志(电子版), 2022, 12(03): 153-160.
[15]	邓世栋, 刘凌志, 郭大勇, 王超, 黄忠欣, 张晖辉. 沉默SNHG1基因对膀胱癌细胞增殖、凋亡、迁移和铁死亡的影响[J]. 中华临床医师杂志(电子版), 2023, 17(07): 804-811.

阅读次数

全文

摘要

选择文件类型/文献管理软件名称

选择包含的内容

Inhibitory effect and mechanisms of psoralen on proliferation and migration of bladder cancer T24 cells

模态框（Modal）标题

选择文件类型/文献管理软件名称

选择包含的内容

Inhibitory effect and mechanisms of psoralen on proliferation and migration of bladder cancer T24 cells