山萘酚逆转肝癌耐药细胞Bel-7402/5-Fu的作用机制研究

doi:10.11958/20231828

摘要/Abstract

摘要：

目的探讨山萘酚（KAE）对肝癌耐药细胞Bel-7402/5-Fu功能的影响。方法采用KAE处理Bel-7402/5-Fu细胞，将细胞分为对照组和药物组（0.064、0.320、1.600、8、40、200 μmol/L KAE）；根据干扰DNA依赖性激酶催化亚基（DNA-PKcs）、加入蛋白酶体抑制剂MG132或加入自噬抑制剂CQ，将细胞分为si-NC组和DNA-PKcs干扰（siRNA-1664、siRNA-2142、siRNA-3785）组，对照组、KAE组和KAE+si-DNA-PKcs组，对照组、KAE+DMSO组、KAE+MG132组和KAE+CQ组。采用CCK-8检测细胞增殖，实时荧光定量PCR（RT-qPCR）和Western blot检测组蛋白H₂AX磷酸化（γ-H₂AX）、DNA-PKcs、DNA双链断裂修复/V（D）J重组蛋白（Artemis）和药泵基因（P-gp）mRNA和蛋白的表达，流式细胞术检测细胞周期和细胞凋亡，蛋白稳定性实验检测DNA-PKcs蛋白稳定性，免疫共沉淀检测DNA-PKcs蛋白泛素化。结果与对照组相比，8 μmol/L KAE处理细胞24 h可抑制约50%的细胞增殖能力，选择此时间和浓度进行后续研究。与对照组相比，KAE组γ-H₂AX mRNA和蛋白表达水平升高，DNA-PKcs、Artemis和P-gp mRNA和蛋白表达水平降低（P<0.05）；与对照组相比，KAE促进Bel-7402/5-Fu细胞周期阻滞于G2/M期，增加细胞凋亡；与si-NC组相比，siRNA-1664能显著下调DNA-PKcs mRNA和蛋白表达水平（P<0.05）；与KAE组相比，KAE+si-DNA-PKcs组进一步促进了KAE对细胞的效应；与对照组相比，KAE+DMSO组DNA-PKcs蛋白表达水平降低（P<0.05）；与KAE+DMSO组相比，KAE+MG132组DNA-PKcs蛋白表达水平升高（P<0.05），而KAE+CQ组DNA-PKcs蛋白表达水平无明显变化（P>0.05）；与对照组相比，KAE+DMSO组促进DNA-PKcs蛋白的泛素化，而KAE+MG132组则可抑制其泛素化（P<0.05）。结论 KAE能够诱导肝癌耐药细胞Bel-7402/5-Fu的细胞凋亡和细胞周期阻滞。

关键词: 癌，肝细胞, 山萘酚, 细胞凋亡, 细胞周期, Bel-7402/5-Fu, DNA-PKcs

Abstract:

Objective To investigate the effect of kaempferol (KAE) on the function of drug-resistant Bel-7402/5-Fu cells. Methods Bel-7402/5-Fu cells were treated with KAE, and cells were divided into the control group and the drug group (0.064, 0.320, 1.600, 8, 40, 200 μmol/L KAE). Cells were divided into the si-NC group and the DNA-PKcs interference group, or the control group, the KAE group, the KAE+si-DNA-PKcs group or the KAE+DMSO group, the KAE+MG132 group and the KAE+CQ group based on interfering DNA dependent kinase catalytic subunits (DNA-PKcs) or addition of proteasome inhibitor MG132 or autophagy inhibitor CQ. Cell proliferation was detected using CCK-8. The expression level of histone H₂AX phosphorylation (γ-H₂AX), DNA-PKcs, DNA double strand break repair/V(D)J recombinant protein (Artemis) and drug pump gene (P-gp) were analyzed using real-time fluorescence quantitative PCR (RT-qPCR) and Western blot assay. Cell cycle and apoptosis were detected by flow cytometry. The stability of DNA-PKcs proteins was analyzed by protein stability experiments. Ubiquitination of DNA-PKcs protein was evaluated by immunoprecipitation assay. Results Compared to the control group, treating cells with 8 μmol/L KAE for 24 h inhibited about 50% of cell proliferation ability. Therefore, this time and concentration were chosen for subsequent research. Compared to the control group, the expression level of γ-H₂AX mRNA and protein significantly increased, while expression levels of DNA-PKcs, Artemis and P-gp mRNA and proteins significantly decreased in the KAE group (P<0.05). Compared to the control group, KAE promoted cell cycle arrest in the G2/M phase of Bel-7402/5-Fu cells and increased cell apoptosis. Compared to the si-NC group, siRNA-1664 significantly downregulated the mRNA and protein expression levels of DNA-PKcs (P<0.05). Compared with the KAE group, the effect of KAE was further promoted in the KAE+si-DNA-PKcs group of Bel-7402/5-Fu cells. Compared with the control group, the protein expression level of DNA-PKcs decreased in the KAE+DMSO group (P<0.05). Compared with the KAE+DMSO group, the protein expression level of DNA-PKcs increased in the KAE+MG132 group (P<0.05), while there was no significant change in the protein expression level of DNA-PKcs in the KAE+CQ group (P>0.05). Compared to the control group, there was promoted ubiquitination of DNA-PKcs in the KAE+DMSO group, and the inhibited ubiquitination in the KAE+MG132 group (P<0.05). Conclusion KAE may induce cell apoptosis and cell cycle arrest in drug-resistant Bel-7402/5-Fu cells.

Key words: carcinoma, hepatocellular, kaempferol, apoptosis, cell cycle, Bel-7402/5-Fu, DNA-PKcs

中图分类号:

R735.7

图/表 19

参考文献 16

[1]	TANG Z, HE J, ZOU J, et al. Cisplatin-resistant HepG2 cell-derived exosomes transfer cisplatin resistance to cisplatin-sensitive cells in HCC[J]. PeerJ, 2021, 13(9):e11200. doi:10.7717/peerj.11200.
[2]	LEE K H. Primary cilia:a novel research approach to overcome anticancer drug resistance[J]. Front Mol Biosci, 2023, 2(10):1270639. doi:10.3389/fmolb.2023.1270639.
[3]	KRENNING L, BERG J, MEDEMA R H. Life or death after a break:what determines the choice?[J]. Mol Cell, 2019, 76(2):346-358. doi:10.1016/j.molcel.2019.08.023.
[4]	ZHAO Y, THOMAS H D, BATEY M A, et al. Preclinical evaluation of a potent novel DNA-dependent protein kinase inhibitor NU7441[J]. Cancer Res, 2006, 66(10):5354-5362. doi:10.1158/0008-5472.
[5]	FOK J H L, RAMOS M A, VAZQUEZ C M, et al. AZD7648 is a potent and selective DNA-PK inhibitor that enhances radiation,chemotherapy and olaparib activity[J]. Nat Commun, 2019, 10(1):5065. doi:10.1038/s41467-019-12836-9.
[6]	CHEN M, XIAO J, SEEDI H R, et al. Kaempferol and atherosclerosis:from mechanism to medicine[J]. Crit Rev Food Sci Nutr, 2024, 64(8):2157-2175. doi:10.1080/10408398.2022.2121261.
[7]	ZHANG Y, QU Y, CHEN Y Z. Influence of 6-shogaol potentiated on 5-fluorouracil treatment of liver cancer by promoting apoptosis and cell cycle arrest by regulating AKT/mTOR/MRP1 signaling[J]. Chin J Nat Med, 2022, 20(5):352-363. doi:10.1016/S1875-5364(22)60174-2.
[8]	BABY J, DEVAN A R, KUMAR A R, et al. Cogent role of flavonoids as key orchestrators of chemoprevention of hepatocellular carcinoma:a review[J]. J Food Biochem, 2021, 45(7):e13761. doi:10.1111/jfbc.13761.
[9]	WU H, DU J, LI C, et al. Kaempferol can reverse the 5-Fu resistance of colorectal cancer cells by inhibiting PKM2-mediated glycolysis[J]. Int J Mol Sci, 2022, 23(7):3544. doi:10.3390/ijms23073544.
[10]	RIAHI C I, SOUID S, OTHMAN H, et al. The Phenolic compound kaempferol overcomes 5-fluorouracil resistance in human resistant LS174 colon cancer cells[J]. Sci Rep, 2019, 9(1):195. doi:10.1038/s41598-018-36808-z.
[11]	DING J, LI X, KHAN S, et al. EGFR suppresses p53 function by promoting p53 binding to DNA-PKcs:a noncanonical regulatory axis between EGFR and wild-type p53 in glioblastoma[J]. Neuro Oncol, 2022, 24(10):1712-1725. doi:10.1093/neuonc/noac105.
[12]	SHABKHIZAN R, HAIATV S, MOSLEHIAN M S, et al. The beneficial and adverse effects of autophagic response to caloric restriction and fasting[J]. Adv Nutr, 2023, 14(5):1211-1225. doi:10.1016/j.advnut.2023.07.006.
[13]	KWON Y T, CIECHANOVER A. The ubiquitin code in the ubiquitin-proteasome system and autophagy[J]. Trends Biochem Sci, 2017, 42(11):873-886. doi:10.1016/j.tibs.2017.09.002.
[14]	FERGUSON B J, MANSUR D S, PETERS N E, et al. DNA-PK is a DNA sensor for IRF-3-dependent innate immunity[J]. Elife, 2012, 1:e00047. doi:10.7554/eLife.00047.
[15]	HO S R, MAHANIC C S, LEE Y J, et al. RNF144A,an E3 ubiquitin ligase for DNA-PKcs,promotes apoptosis during DNA damage[J]. Proc Natl Acad Sci U S A, 2014, 111(26):E2646-E2655. doi:10.1073/pnas.1323107111.
[16]	LI Y H, ZHONG M, ZANG H L, et al. MTA1 promotes hepatocellular carcinoma progression by downregulation of DNA-PK-mediated H1.2^T146 phosphorylation[J]. Front Oncol, 2020, 10:567. doi:10.3389/fonc.2020.00567.

干扰序列	序列（5'→3'）
siRNA-2142	正义：GCAAAGAGGUGGCAGUUAATT
siRNA-2142	反义：UUAACUGCCACCUCUUUGCTT
siRNA-3785	正义：GAACAUGGCAGGAGAGAAUTT
siRNA-3785	反义：AUUCUCUCCUGCCAUGUUCTT
siRNA-1664	正义：GUGAAUUCCUCCAGUGAAATT
siRNA-1664	反义：UUUCACUGGAGGAAUUCACTT
si-NC	正义：UUCUCCGAACGUGUCACGUTT
si-NC	反义：ACGUGACACGUUCGGAGAATT

干扰序列	序列（5'→3'）
siRNA-2142	正义：GCAAAGAGGUGGCAGUUAATT
siRNA-2142	反义：UUAACUGCCACCUCUUUGCTT
siRNA-3785	正义：GAACAUGGCAGGAGAGAAUTT
siRNA-3785	反义：AUUCUCUCCUGCCAUGUUCTT
siRNA-1664	正义：GUGAAUUCCUCCAGUGAAATT
siRNA-1664	反义：UUUCACUGGAGGAAUUCACTT
si-NC	正义：UUCUCCGAACGUGUCACGUTT
si-NC	反义：ACGUGACACGUUCGGAGAATT

基因名称	引物序列（5'→3'）	产物大小/bp
DNA-PKcs	上游：CCTGGCCGGTCATCAACTG	227
DNA-PKcs	下游：AGTAAGGTGCGATCTTCTGGC	227
γ-H₂AX	上游：CTACTCCGAACGAGTCGGG	125
γ-H₂AX	下游：GATGGTCACGCGACCTAGAAG	125
Artemis	上游：TATGCCGGTCTTCCCAAAGTA	164
Artemis	下游：GTGAAAAGTTTCCGGGTATGGA	164
P-gp	上游：GCACAGGAGAATTGATTAACCGC	118
P-gp	下游：ATACTGATGCCTACGGAAGCC	118
GAPDH	上游：TGTGGGCATCAATGGATTTGG	116
GAPDH	下游：ACACCATGTATTCCGGGTCAAT	116

基因名称	引物序列（5'→3'）	产物大小/bp
DNA-PKcs	上游：CCTGGCCGGTCATCAACTG	227
DNA-PKcs	下游：AGTAAGGTGCGATCTTCTGGC	227
γ-H₂AX	上游：CTACTCCGAACGAGTCGGG	125
γ-H₂AX	下游：GATGGTCACGCGACCTAGAAG	125
Artemis	上游：TATGCCGGTCTTCCCAAAGTA	164
Artemis	下游：GTGAAAAGTTTCCGGGTATGGA	164
P-gp	上游：GCACAGGAGAATTGATTAACCGC	118
P-gp	下游：ATACTGATGCCTACGGAAGCC	118
GAPDH	上游：TGTGGGCATCAATGGATTTGG	116
GAPDH	下游：ACACCATGTATTCCGGGTCAAT	116

组别	0 h	24 h	48 h	72 h	F
对照组	100.00±0.00	114.67±9.87	121.33±13.50	127.00±15.88	3.062
0.064 μmol/L组	102.00±1.00	107.33±11.02	115.67±9.61	116.67±10.63	1.175
0.320 μmol/L组	100.62±1.07	105.33±9.51	113.67±12.01	119.33±11.28	1.469
1.600 μmol/L组	98.50±1.80	103.00±7.55	112.67±8.62	116.86±16.01	2.107
8 μmol/L组	100.83±1.76	53.00±8.19^aA	41.00±6.56^aA	34.66±2.52^aA	90.160^＊
40 μmol/L组	99.80±1.26	40.65±7.50^aA	36.66±7.64^aA	29.67±3.22^aA	99.540^＊
200 μmol/L组	100.53±0.92	42.33±8.74^aA	31.33±6.51^aA	20.00±4.03^aA	113.700^＊
F	2.750	42.130^＊	60.040^＊	45.020^＊