二甲双胍对弥漫大B细胞淋巴瘤细胞的抑制作用及机制探讨

doi:10.11958/20220684

天津医药 ›› 2023, Vol. 51 ›› Issue (4): 355-359.doi: 10.11958/20220684

二甲双胍对弥漫大B细胞淋巴瘤细胞的抑制作用及机制探讨

谭春莲¹(), 李晓红¹, 夏国栋¹, 张志红²^,^△(), 李晓明³

1 西南医科大学附属医院健康管理中心（邮编646000）
2 西南医科大学附属医院全科医学科（邮编646000）
3 西南医科大学附属医院血液内科（邮编646000）

收稿日期:2022-06-20 修回日期:2022-11-15 出版日期:2023-04-15 发布日期:2023-04-20
通讯作者: 张志红 E-mail:978174922@qq.com;zhihonglily@126.com
作者简介:谭春莲（1992），女，硕士，医师，主要从事血液疾病及慢病健康管理方面研究。E-mail：978174922@qq.com

Inhibitory effect and mechanism of metformin on diffuse large B-cell lymphoma cells

TAN Chunlian¹(), LI Xiaohong¹, XIA Guodong¹, ZHANG Zhihong²^,^△(), LI Xiaoming³

1 Health Management Center, the Affiliated Hospital of Southwest Medical University, Luzhou 646000, China
2 Department of General Medicine, the Affiliated Hospital of Southwest Medical University, Luzhou 646000, China
3 Department of Hematology, the Affiliated Hospital of Southwest Medical University, Luzhou 646000, China

Received:2022-06-20 Revised:2022-11-15 Published:2023-04-15 Online:2023-04-20
Contact: ZHANG Zhihong E-mail:978174922@qq.com;zhihonglily@126.com

谭春莲, 李晓红, 夏国栋, 张志红, 李晓明. 二甲双胍对弥漫大B细胞淋巴瘤细胞的抑制作用及机制探讨[J]. 天津医药, 2023, 51(4): 355-359.

TAN Chunlian, LI Xiaohong, XIA Guodong, ZHANG Zhihong, LI Xiaoming. Inhibitory effect and mechanism of metformin on diffuse large B-cell lymphoma cells[J]. Tianjin Medical Journal, 2023, 51(4): 355-359.

摘要/Abstract

摘要：

目的探讨二甲双胍对人弥漫大B细胞淋巴瘤（DLBCL）U2932细胞的抑制作用及其相关机制。方法采用不同浓度二甲双胍作用于体外培养的U2932细胞，分为对照组（0 mmol/L）和二甲双胍实验组（分别为5、10、20、40 mmol/L），分别培养24、48、72 h。通过CCK-8法检测细胞增殖活性改变；流式细胞术检测细胞周期分布及凋亡情况；Western blot检测AMP活化蛋白激酶（AMPK）、细胞周期蛋白D1（CyclinD1）、自噬及凋亡等通路相关蛋白的表达水平。结果经二甲双胍处理后的U2932细胞存活率较对照组明显下降（P<0.05）。5、10和20 mmol/L二甲双胍组G0/G1期细胞比例均较对照组增加（P<0.05）。20 mmol/L二甲双胍组24 h、48 h、72 h的细胞凋亡比例较对照组增加（P<0.05）。与对照组相比，5、10和20 mmol/L二甲双胍组磷酸化AMP活化蛋白激酶α亚基（p-AMPKα）、P53、B淋巴细胞瘤-2基因（Bcl-2）关联X蛋白（Bax）、活化的胱天蛋白酶3（Cleaved Caspase-3）、微管相关蛋白l轻链3B亚基（LC3B）蛋白表达水平增加，细胞自噬蛋白Beclin1表达水平仅部分增加，Bcl-2、磷酸化蛋白激酶B（p-AKT）、CyclinD1蛋白表达水平降低（P<0.05）。结论二甲双胍能够激活AMPK、凋亡及自噬通路，抑制U2932细胞增殖，促进其凋亡。

关键词: 二甲双胍, 淋巴瘤，大B细胞，弥漫性, AMP活化蛋白激酶类, 细胞凋亡, 细胞增殖, 自噬, 信号通路

Abstract:

Objective To investigate the inhibitory effect and related mechanism of metformin on human diffuse large B-cell lymphoma (DLBCL) U2932 cells. Methods U2932 cells cultured in vitro were treated with different concentrations of metformin, and cells were divided into the control group (0 mmol/L) and the metformin experimental groups (5, 10, 20 and 40 mmol/L). Cells were cultured for 24 h, 48 h and 72 h, respectively. Changes of cell proliferation activity were detected by CCK-8 assay. Cell cycle distribution and apoptosis were detected by flow cytometry. Expression levels of AMPK, CyclinD1, autophagy and apoptosis pathway-related proteins were detected by Western blot assay. Results The proliferation activity of U2932 cells treated with metformin was significantly lower than that of the control group (P<0.05). Compared with the control group, the percentage of cells in G0/G1 phase treated with 5, 10, 20 mmol/L metformin and the apoptosis rate of U2932 cells treated with 20 mmol/L metformin for 24 h, 48 h and 72 h were significantly increased (P<0.05). Western blot assay showed that 5, 10, 20 mmol/L metformin could up-regulate the expression of p-AMPKα, P53, Bax, Cleaved Caspase-3, LC3B proteins, but Beclin1 only partially increased, and down-regulate the expression levels of Bcl-2, p-AKT, CyclinD1 proteins in U2932 cells (P<0.05). Conclusion Metformin can activate AMPK, apoptosis and autophagy pathways, inhibit the proliferation of U2932 cells, and promote their apoptosis.

Key words: metformin, lymphoma, large B-cell, diffuse, AMP-activated protein kinases, apoptosis, cell proliferation, autophagy, signaling pathway

中图分类号:

R733.41

图/表 8

表1 各组U2932细胞经二甲双胍处理后72 h内细胞存活率比较（n=3，%，$\bar{x} \pm s$）

Tab.1 Comparison of cell viability of U2932 cells treated with different concentrations of metformin within 72 h in each group

组别	24 h	48 h	72 h	F
对照组	123.77±2.40	204.57±16.26	253.37±7.54	117.969^**
5 mmol/L组	96.63±2.40^a	81.67±1.65^a	61.33±5.15^a	80.585^**
10 mmol/L组	86.87±1.76^ab	75.10±4.78^a	51.37±1.67^ab	102.305^**
20 mmol/L组	69.33±8.00^abc	50.23±1.19^abc	38.97±3.40^abc	27.543^**
40 mmol/L组	61.67±0.97^abcd	39.83±1.10^abc	22.77±0.85^abcd	1 188.112^**
F	112.884^**	224.552^**	1 372.132^**

表2 各组U2932细胞经二甲双胍处理48 h后对其周期的影响（n=3，%，$\bar{x} \pm s$）

Tab.2 Effects of different concentrations of metformin on the cell cycle of U2932

组别	G0/G1期	S期	G2/M期
对照组	37.11±1.57	30.65±1.56	30.46±1.46
5 mmol/L组	47.24±1.85^a	27.68±0.77^a	24.63±0.78^a
10 mmol/L组	57.92±1.49^ab	24.42±0.92^ab	17.54±0.81^ab
20 mmol/L组	64.11±9.62^ab	17.97±1.02^abc	12.67±0.79^abc
F	16.906^**	72.239^**	189.181^**

图1 流式细胞术检测各组细胞周期变化

Fig.1 The cell cycle detected by flow cytometry

图2 二甲双胍对U2932细胞凋亡的影响

Fig.2 The effect of metformin on the apoptosis of U2932 cells

图3 U2932细胞经不同浓度的二甲双胍处理后AMPK、AKT、CyclinD1及P53通路蛋白水平的变化

Fig.3 Western blot results of p-AMPKα, p-AKT, CyclinD1 and P53 proteins in U2932 cells treated with different concentrations of metformin

表3 各组p-AMPKα、p-AKT、CyclinD1及P53蛋白表达水平比较（n=3，$\bar{x} \pm s$）

Tab.3 Comparison of expression levels of p-AMPKα, p-AKT, CyclinD1 and P53 proteins between the four groups of cells

组别	p-AMPKα	p-AKT	CyclinD1	P53
对照组	1.00±0.00	1.00±0.00	1.00±0.00	1.00±0.00
5 mmol/L组	1.06±0.01^a	0.61±0.01^a	0.18±0.00^a	3.36±0.52^a
10 mmol/L组	1.33±0.01^ab	0.51±0.01^ab	0.17±0.00^ab	8.91±0.35^ab
20 mmol/L组	1.60±0.01^abc	0.21±0.01^abc	0.16±0.00^abc	9.29±0.16^ab
F	5 145.734^**	8 145.469^**	175 735.307^**	484.500^**

图4 U2932细胞经不同浓度的二甲双胍处理后凋亡及自噬相关蛋白水平的变化

Fig.4 Western blot results of apoptosis and autophagy proteins in U2932 cells treated with different concentrations of metformin

表4 各组Cleaved Caspase-3、Bcl-2、Bax、LC3B及Beclin1蛋白表达水平比较（n=3，$\bar{x} \pm s$）

Tab.4 Comparison of expression levels of Cleaved Caspase-3, Bcl-2, Bax, LC3B and Beclin1 proteins between the four groups of cells

组别		Cleaved Caspase-3		Bcl-2
对照组		1.00±0.00		1.00±0.00
5 mmol/L组		3.65±0.01^a		0.54±0.17^a
10 mmol/L组		3.78±0.05^ab		0.47±0.14^ab
20 mmol/L组		3.88±0.08^abc		0.16±0.00^abc
F		2 740.077^**		2 988.028^**
组别	Bax		LC3B		Beclin1
对照组	1.00±0.00		1.00±0.00		1.00±0.00
5 mmol/L组	1.23±0.03^a		1.55±0.02^a		1.01±0.01
10 mmol/L组	1.40±0.01^ab		1.69±0.03^ab		1.66±0.03^ab
20 mmol/L组	1.53±0.03^abc		2.98±0.13^abc		2.70±0.32^abc
F	286.269^**		414.542^**		74.086^**

参考文献 17

[1]	COCCARO N, ANELLI L, ZAGARIA A, et al. Molecular complexity of diffuse large B-cell lymphoma:Can it be a roadmap for precision medicine?[J]. Cancers, 2020, 12(1):185. doi:10.3390/cancers12010185.
[2]	LIU M K, SUN X J, GAO X D, et al. Methylation alterations and advance of treatment in lymphoma[J]. Front Biosci(Landmark Ed), 2021, 26(9):602-613. doi:10.52586/4970.
[3]	MA T, TIAN X, ZHANG B, et al. Low-dose metformin targets the lysosomal AMPK pathway through PEN2[J]. Nature, 2022, 603(7899):159-165. doi:10.1038/s41586-022-04431-8.
[4]	SAENGBOONMEE C, SANLUNG T, WONGKHAM S. Repurposing metformin for cancer treatment:A great challenge of a promising drug[J]. Anticancer Res, 2021, 41(12):5913-5918. doi:10.21873/anticanres.15410.
[5]	YANG J, ZHOU Y, XIE S, et al. Metformin induces ferroptosis by inhibiting UFMylation of SLC7A11 in breast cancer[J]. J Exp Clin Cancer Res, 2021, 40(1):206. doi:10.1186/s13046-021-02012-7.
[6]	CADEDDU G, HERVÁS-MORÓN A, MARTÍN-MARTÍN M, et al. Metformin and statins:A possible role in high-risk prostate cancer[J]. Rep Pract Oncol Radiother, 2020, 25(2):163-167. doi:10.1016/j.rpor.2019.12.027.
[7]	EIBL G, ROZENGURT E. Metformin:Review of epidemiology and mechanisms of action in pancreatic cancer[J]. Cancer Metastasis Rev, 2021, 40(3):865-878. doi:10.1007/s10555-021-09977-z.
[8]	TSENG C H. Metformin is associated with a lower risk of non-Hodgkin lymphoma in patients with type 2 diabetes[J]. Diabetes Metab, 2019, 45(5):458-464. doi:10.1016/j.diabet.2019.05.002.
[9]	WANG Y, MAURER M J, LARSON M C, et al. Impact of metformin use on the outcomes of newly diagnosed diffuse large B-cell lymphoma and follicular lymphoma[J]. Br J Haematol, 2019, 186(6):820-828. doi:10.1111/bjh.15997.
[10]	陈惠丽, 马平, 陈艳丽, 等. 二甲双胍对K562细胞增殖、凋亡及糖酵解的影响[J]. 中国实验血液学杂志, 2019, 27(5):1387-1394.
	CHEN H L, MA P, CHEN Y L, et al. Effect of metformin on proliferation capacity,apoptosis and glycolysis in K562 cells[J]. Journal of Experimental Hematology, 2019, 27(5):1387-1394. doi:10.19746/j.cnki.issn.1009-2137.2019.05.006.
[11]	VISNJIC D, DEMBITZ V, LALIC H. The role of AMPK/mTOR modulators in the therapy of acute myeloid leukemia[J]. Curr Med Chem, 2019, 26(12):2208-2229. doi:10.2174/0929867325666180117105522.
[12]	XIONG Z S, GONG S F, SI W, et al. Effect of metformin on cell proliferation,apoptosis,migration and invasion in A172 glioma cells and its mechanisms[J]. Mol Med Rep, 2019, 20(2):887-894. doi:10.3892/mmr.2019.10369.
[13]	ZHAO Y, SUN H, FENG M, et al. Metformin is associated with reduced cell proliferation in human endometrial cancer by inbibiting PI3K/AKT/mTOR signaling[J]. Gynecol Endocrinol, 2018, 34(5):428-432. doi:10.1080/09513590.2017.1409714.
[14]	NOZHAT Z, MOHAMMADI-YEGANEH S, AZIZI F, et al. Effects of metformin on the PI3K/AKT/FOXO1 pathway in anaplastic thyroid cancer cell lines[J]. Daru, 2018, 26(2):93-103. doi:10.1007/s40199-018-0208-2.
[15]	ZHANG C, HU J, SHENG L, et al. Metformin delays AKT/c-Met-driven hepatocarcinogenesis by regulating signaling pathways for de novo lipogenesis and ATP generation[J]. Toxicol Appl Pharmacol, 2019, 365:51-60. doi:10.1016/j.taap.2019.01.004.
[16]	HONG Y, REN T, WANG X, et al. APR-246 triggers ferritinophagy and ferroptosis of diffuse large B-cell lymphoma cells with distinct TP53 mutations[J]. Leukemia, 2022, 36(9):2269-2280. doi:10.1038/s41375-022-01634-w.
[17]	CHUKKAPALLI V, GORDON L I, VENUGOPAL P, et al. Metabolic changes associated with metformin potentiates Bcl-2 inhibitor,Venetoclax,and CDK9 inhibitor,BAY1143572 and reduces viability of lymphoma cells[J]. Oncotarget, 2018, 9(30):21166-21181. doi:10.18632/oncotarget.24989.

二甲双胍对弥漫大B细胞淋巴瘤细胞的抑制作用及机制探讨

Inhibitory effect and mechanism of metformin on diffuse large B-cell lymphoma cells

引用本文

RichHTML

PDF (PC)

可视化

摘要/Abstract

使用本文

图/表 8

参考文献 17

相关文章 15

编辑推荐

Metrics

本文评价

[1]	钟玉梅, 周海燕, 张敏. ASIC1a介导类风湿关节炎软骨细胞损伤机制的研究进展[J]. 天津医药, 2024, 52(9): 1004-1008.
[2]	张晋玮, 王燕, 王通. miR-107对口腔鳞癌细胞系CAL27增殖、侵袭及迁移的影响[J]. 天津医药, 2024, 52(9): 897-899.
[3]	梁大敏, 杨正久, 张子萍, 钱静, 毛朝坤. 山萘酚逆转肝癌耐药细胞Bel-7402/5-Fu的作用机制研究[J]. 天津医药, 2024, 52(9): 900-906.
[4]	方杰, 黄芮, 郑红慧, 贾倩倩, 鲍静. miR-9-5p靶向TIMP2诱导多发性骨髓瘤细胞自噬和凋亡的机制[J]. 天津医药, 2024, 52(8): 785-790.
[5]	李大强, 李坚, 陆哲明, 曹阳. 毛蕊异黄酮对脊髓损伤大鼠神经元自噬及凋亡的影响[J]. 天津医药, 2024, 52(8): 798-803.
[6]	黄愿, 王刚, 李燕玲, 谢萍. 放射性心脏损伤相关信号通路及药物干预的研究进展[J]. 天津医药, 2024, 52(8): 888-892.
[7]	侯维玲, 乔云阳, 吴小芸, 施会敏, 曲高婷, 张爱青. 锌指蛋白281抑制高糖诱导的肾小管上皮细胞上皮间质转化和细胞外基质合成[J]. 天津医药, 2024, 52(7): 720-726.
[8]	徐琼芳, 钟斐, 李子帅. 淫羊藿苷调节SDF-1/CXCR4信号通路对多囊卵巢综合征大鼠卵巢颗粒细胞凋亡的影响[J]. 天津医药, 2024, 52(7): 727-732.
[9]	钟敏, 施震, 周劲松, 李晋杰. GABA信号通路对脓毒症大鼠急性肺损伤内质网应激和线粒体自噬的影响[J]. 天津医药, 2024, 52(7): 733-737.
[10]	陈芋洁, 黄霞, 邓铂林, 贾文文. 金合欢素调节Hippo信号通路对糖尿病视网膜病变大鼠血管生成的影响[J]. 天津医药, 2024, 52(6): 578-583.
[11]	刘丹阳, 李永涛, 张海燕, 李林, 刘洋, 沈雷. 乳腺癌细胞条件培养基对骨髓间充质干细胞生物学行为的影响[J]. 天津医药, 2024, 52(5): 454-458.
[12]	王柯, 叶寒露. 隐丹参酮调节HIF-1α/BNIP3信号通路对兔膝骨关节炎模型软骨细胞自噬和凋亡的影响[J]. 天津医药, 2024, 52(4): 372-378.
[13]	何亚男, 蔡翔, 邱百怡, 孙邦梅, 李伶华. 穿心莲内酯调节cGAS-STING信号通路对银屑病小鼠的治疗作用[J]. 天津医药, 2024, 52(4): 379-386.
[14]	何颖, 张广华, 田立东, 于泳浩. 富氢液通过增加自噬治疗大鼠神经病理性疼痛[J]. 天津医药, 2024, 52(3): 261-265.
[15]	王扶凝, 代会博, 单云, 俞曼殊, 盛梅笑. 骨髓间充质干细胞对腹膜间皮细胞凋亡的影响[J]. 天津医药, 2024, 52(2): 113-118.