抗菌肽LL-37对结肠癌小鼠肿瘤生长的抑制作用及其机制

doi:10.13481/j.1671-587x.20200324

摘要/Abstract

摘要： 目的：探讨抗菌肽LL-37对结肠癌小鼠肿瘤生长和细胞凋亡的影响，阐明其抗肿瘤作用的可能分子机制。方法：构建LL-37过表达结肠癌HT-29细胞，采用qRT-PCR和Western blotting法检测HT-29细胞中LL-37 mRNA和蛋白表达水平。30只BALB/c小鼠随机分为对照组（给予未经感染的HT-29细胞）、空载组（给予感染空载质粒的HT-29细胞）、LL-37过表达组（给予感染LL-37过表达载体的HT-29细胞）、AMPK抑制剂组[给予感染空载体的HT-29细胞后，立刻尾静脉注射2 mg·kg^-1（Dorsomorphin，Dor）]和联合组（给予感染LL-37过表达载体的HT-29细胞后，立刻尾静脉注射2 mg·kg^-1 Dor），每组6只。采用结肠癌HT-29细胞复制结肠癌小鼠模型，观察各组小鼠肿瘤体积并绘制生长曲线，感染24 d后剥离肿瘤测量质量并计算抑瘤率，TUNEL染色检测各组小鼠肿瘤细胞凋亡情况，Western blotting法检测各组小鼠肿瘤组织中自噬相关蛋白Beclin1、Atg5、LC3Ⅰ和LC3Ⅱ及AMPK/mTOR通路相关蛋白AMPK、p-AMPK、mTOR及p-mTOR表达水平。结果：与对照组和空载组比较，LL-37过表达组小鼠结肠癌HT-29细胞中LL-37 mRNA和蛋白表达水平明显升高（P<0.01），肿瘤质量和体积明显降低（P<0.05），抑瘤率升高（P<0.05），肿瘤细胞凋亡数减少，肿瘤组织中自噬相关蛋白Beclin1和Atg5表达水平、LC3Ⅱ/LC3Ⅰ比值及p-AMPK/AMPK比值均明显升高（P<0.05），p-mTOR/mTOR比值明显降低（P<0.05）；与空载组比较，AMPK抑制剂组小鼠肿瘤质量和体积明显升高（P<0.05）、抑瘤率降低（P<0.05），肿瘤细胞凋亡数减少，自噬相关蛋白Beclin1和Atg5表达水平、LC3Ⅱ/LC3Ⅰ比值及p-AMPK/AMPK比值明显降低（P<0.05），p-mTOR/mTOR比值明显升高（P<0.05）；与LL-37过表达组比较，联合组小鼠肿瘤组织中Beclin1和Atg5表达水平、LC3Ⅱ/LC3Ⅰ比值及p-AMPK/AMPK比值明显降低（P<0.05或P<0.01），p-mTOR/mTOR比值明显升高（P<0.01）。结论：抗菌肽LL-37能激活AMPK/mTOR信号通路促进细胞自噬，进而抑制结肠癌HT-29荷瘤小鼠的肿瘤生长。

关键词: 抗菌肽LL-37, AMPK/mTOR信号通路, 自噬, 结肠肿瘤

Abstract: Objective: To investigate the effects of antimicrobial peptide LL-37 on the tumor growth and apoptosis of the mice with colon cancer, and to elucidate the possible molecular mechanism of its anti-tumor effect. Methods: The LL-37 over-expression colon cancer HT-29 cells were constructed, and the expression levels of LL-37 mRNA and protein in the HT-29 cells were detected by qRT-PCR and Western blotting methods. A total of 30 BALB/c mice were randomly divided into control group (given the uninfected HT-29 cells), empty vector group (given the HT-29 cells infected with empty plasmid), LL-37 over-expression group (given the HT-29 cells infected with LL-37 over-expression vector), AMPK inhibitor group[given the HT-29 cells infected with empty vector, and then injected with 2 mg·kg^-1 Dorsomorphin (Dor) in the tail vein] and combination group (given the HT-29 cells infected with LL-37 over-expression vector, and then injected with 2 mg·kg^-1 Dor in the tail vein),and there were 6 mice in each group. The colon cancer HT-29 cells were used to replicate the colon cancer models of the mice. The tumor volumes of the mice in various groups were detected and the growth curve was drawn. After infected for 24 d, the weights of the tumor of the mice in various groups were measured and the inhibitory rates of the tumor were calculated. TUNEL staining was used to detect the apoptosis of tumor cells in various groups. Western blotting method was used to detect the expression levels of autophagy-related proteins Beclin1, ATG5, LC3Ⅰ, LC3Ⅱ and AMPK/mTOR pathway-related proteins AMPK, p-AMPK, mTOR and p-mTOR in tumor tissue of the mice in various groups. Results: Compared with control group and empty vector group, the expression levels of LL-37 mRNA and protein in the HT-29 cells of the mice in over-expression group were significantly increased(P<0.01),the weight and volume of the tumor were significantly decreased (P<0.05), the inhibitory rate of tumor was increased(P<0.05), the number of apoptotic cells was increased, the expression levels of autophage-related proteins Beclin1 and ATG5 in tumor tissue and the ratios of LC3Ⅱ/LC3Ⅰ and p-AMPK/AMPK were increased(P<0.05), while the ratio of p-mTOR/mTOR was significantly decreased (P<0.05).Compared with empty vector group,the weight and volume of the tumor of the mice in AMPK inhibitor group were increased (P<0.05), the inhibitory rate of the tumor was decreased (P<0.05), the number of apoptotic cells was decreased, the expression levels of autophagy-related proteins Beclin1 and Atg5,the ratios of LC3Ⅱ/LC3Ⅰ and p-AMPK/AMPK were decreased (P<0.05), and the ratio of p-mTOR/mTOR was increased (P<0.05).Compared with LL-37 over-expression group, the expression levels of Beclin1, ATG5,the ratios of LC3Ⅱ/LC3Ⅰ and p-AMPK/AMPK in tumor tissue of the mice in combination group were significantly decreased (P< 0.05 or P<0.01), and the ratio of p-mTOR/mTOR was significantly increased (P<0.01). Conclusion: The antimicrobial peptide LL-37 can activate the AMPK/mTOR signaling pathway and promote the autophagy of the cells, thereby inhibit the tumor growth in the colon cancer HT-29-bearing mice.

Key words: antimicrobial peptide LL-37, AMPK/mTOR signaling pathway, autophagy, colon neoplasms

中图分类号:

R735.35

闫圣玉, 谢亚锋, 许志杰, 刘英, 丁雅婷, 张侨, 刘菀莹, 刘丽兵. 抗菌肽LL-37对结肠癌小鼠肿瘤生长的抑制作用及其机制[J]. 吉林大学学报(医学版), 2020, 46(03): 575-581.

YAN Shengyu, XIE Yafeng, XU Zhijie, LIU Ying, DING Yating, ZHANG Qiao, LIU Wanying, LIU Libing. Inhibitory effect of antimicrobial peptide LL-37 on tumorgrowth of mice with colon cancer and its mechanism[J]. Journal of Jilin University(Medicine Edition), 2020, 46(03): 575-581.

参考文献

[1] ASHBY M,PETKOVA A,GANI J,et al. Use of peptide libraries for identification and optimization of novel antimicrobial peptides[J]. Curr Top Med Chem, 2017, 17(5):537-553.
[2] WANG S, ZENG XF, YANG Q,et al. Antimicrobial peptides as potential alternatives to antibiotics in food animal industry[J]. Int J Mol Sci, 2016, 17(5):E603.
[3] CIPE G,IDIZ U O,FIRAT D,et al. Relationship between intestinal microbiota and colorectal cancer[J]. World J Gastrointest Oncol, 2015, 7(10):233-240.
[4] PIKTEL E,NIEMIROWICZ K,WNOROWSKA U,et al. The role of cathelicidin LL-37 in cancer development[J]. Arch Immunol Ther Exp (Warsz), 2016, 64(1):33-46.
[5] OKAMOTO R, GERY S, KUWAYAMA Y,et al. Novel Gemini vitamin D3 analogs:large structure/function analysis and ability to induce antimicrobial peptide[J]. Int J Cancer, 2014, 134(1):207-217.
[6] YANG Y H,ZHENG G G,LI G,et al. Expression of LL-37/hCAP-18 gene in human leukemia cells[J]. Leuk Res, 2003, 27(10):947-950.
[7] REN S X,CHENG A S,TO K F,et al. Host immune defense peptide LL-37 activates caspase-independent apoptosis and suppresses colon cancer[J]. Cancer Res, 2012, 72(24):6512-6523.
[8] KIM K H,LEE I S,PARK J Y,et al. Cucurbitacin B induces hypoglycemic effect in diabetic mice by regulation of AMP-activated protein kinase alpha and glucagon-like peptide-1 via bitter taste receptor signaling[J]. Front Pharmacol, 2018, 9:1071. DOI:10.3389/fphar.2018.01071.
[9] KANG H K, KIM C, SEO C H,et al. The therapeutic applications of antimicrobial peptides (AMPs):a patent review[J]. J Microbiol, 2017, 55(1):1-12.
[10] JI P,ZHOU Y X,YANG Y B,et al. Myeloid cell-derived LL-37 promotes lung cancer growth by activating Wnt/β-catenin signaling[J]. Theranostics, 2019, 9(8):2209-2223.
[11] ISLAM D,BANDHOLTZ L,NILSSON J,et al. Downregulation of bactericidal peptides in enteric infections:a novel immune escape mechanism with bacterial DNA as a potential regulator[J]. Nat Med, 2001, 7(2):180-185.
[12] BERGMAN P,JOHANSSON L,ASP V,et al. Neisseria gonorrhoeae downregulates expression of the human antimicrobial peptide LL-37[J]. Cell Microbiol, 2005, 7(7):1009-1017.
[13] RAQIB R,SARKER P,BERGMAN P,et al. Improved outcome in shigellosis associated with butyrate induction of an endogenous peptide antibiotic[J]. Proc Natl Acad Sci U S A, 2006, 103(24):9178-9183.
[14] STEINMANN J,HALLDRSSON S,AGERBERTH B,et al. Phenylbutyrate induces antimicrobial peptide expression[J]. Antimicrob Agents Chemother, 2009, 53(12):5127-5133.
[15] MILY A,REKHA R S,KAMAL S M,et al. Oral intake of phenylbutyrate with or without vitamin D3 upregulates the cathelicidin LL-37 in human macrophages:a dose finding study for treatment of tuberculosis[J]. BMC Pulm Med, 2013, 13:23. DOI:10.1186/1471-2466-13-23.
[16] YUK J M,SHIN D M,LEE H M,et al. Vitamin D3 induces autophagy in human monocytes/macrophages via cathelicidin[J]. Cell Host Microbe, 2009, 6(3):231-243.
[17] REKHA R S, RAO MUVVA S S, WAN M, et al. Phenylbutyrate induces LL-37-dependent autophagy and intracellular killing of Mycobacterium tuberculosis in human macrophages[J]. Autophagy, 2015,11(9):1688-1699.
[18] 高旭峰,程先硕,周坦,等. 抗菌肽LL-37在结直肠癌中的研究进展[J]. 现代肿瘤医学, 2018, 26(9):1441-1444.
[19] PIERZYNOWSKA K, GAFFKE L, PODLACHA M, et al. Mucopolysaccharidosis and autophagy:controversies on the contribution of the process to the pathogenesis and possible therapeutic applications[J/OL]. Neuromolecular Med, 2019. https://doi.org/10.1007/s12017-019-08559-1.
[20] WANG Q, WEI S, ZHOU S,et al. Hyperglycemia aggravates acute liver injury by promoting liver-resident macrophage NLRP3 inflammasome activation via the inhibition of AMPK/mTOR-mediated autophagy induction[J/OL]. Immunol Cell Biol,2020,98(1):54-66.
[21] RANEK M J, KOKKONEN-SIMON K M, CHEN A N, et al. PKG1-modified TSC2 regulates mTORC1 activity to counter adverse cardiac stress[J]. Nature, 2019, 566(7743):264-269.
[22] DITE T A, LING N X Y, SCOTT J W, et al. The autophagy initiator ULK1 sensitizes AMPK to allosteric drugs[J]. Nat Commun, 2017, 8(1):571.
[23] 刘帛岩,李松岩,张红亮,等.肿瘤细胞减灭术联合腹腔热灌注化疗对结肠癌与直肠癌腹膜转移的远期疗效比较[J].解放军医学杂志,2020,45(1):79-83.