Study on the effect and mechanism of kaempferol in reversing drug-resistant Bel-7402/5-Fu cells

doi:10.11958/20231828

Abstract

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

CLC Number:

R735.7

Figures/Tables 19

References 16

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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^＊