雷氏大疣蛛多肽毒素组分通过激活促凋亡通路和协同作用抑制癌细胞增殖

doi:10.12122/j.issn.1673-4254.2025.07.12

摘要/Abstract

摘要：

目的研究雷氏大疣蛛毒素对癌细胞增殖抑制的选择性和有效组分。方法采用CCK-8法检测共培养48 h后雷氏大疣蛛毒素对癌细胞增殖的影响；应用流式细胞术检测癌细胞凋亡率；采用caspase试剂盒检测癌细胞内caspase-8和9的表达水平；通过凝胶过滤层析和高效液相色谱（HPLC）将雷氏大疣蛛粗毒分为蛋白质组分、多肽组分、小分子化合物组分3个部分；通过蛋白质组学对蛋白质和多肽组分进行鉴定；采用核磁共振、质谱结合HPLC解析小分子化合物组分中各物质的结构。结果雷氏大疣蛛粗毒对乳腺癌MCF7和鼻咽癌（SUNE1、HONE1）细胞表现出很强的浓度依赖性增殖抑制作用，半抑制浓度（IC₅₀）分别为2.14 ±0.29、1.57±0.14、2.85±0.15 µg/mL；对多种胃癌和肠癌细胞系表现出较强的增殖抑制作用，对胃癌HGC27细胞的IC₅₀为3.02±0.27 µg/mL，对肠癌SW620细胞的IC₅₀为3.02±0.28 µg/mL。选用MCF7细胞来研究增殖抑制机制和筛选有效组分，发现粗毒可能通过激活caspase-8介导的信号通路诱导MCF7细胞凋亡。粗毒中蛋白质组分对MCF7细胞的增殖抑制作用很弱，10 µg/mL浓度仅抑制12.1%±1.9%。多肽组分抑制作用较强，IC₅₀为6.41±0.31 µg/mL。多肽组分相对分子质量集中在10 000左右，主要为与巨型上户蛛毒素同源的多肽；小分子组分未表现出增殖抑制作用，主要为核苷酸代谢物。多肽组分和小分子组分质量比4∶1混合抑制作用明显增强。结论雷氏大疣蛛毒素对不同癌细胞的抑制活性有很大差异，发挥对癌细胞增殖抑制作用的组分为多肽组分，可能与核苷酸代谢物存在协同作用。

关键词: 雷氏大疣蛛, 多肽毒素, 癌细胞, 抗增殖作用

Abstract:

Objective To evaluate the inhibitory effect of Macrothele raveni crude venom against proliferation of different cancer cells and identify the active components in the venom. Methods Different cancer cell lines were treated with different concentrations of Macrothele raveni venom for 48 h, and cell proliferation and the half-maximal inhibitory concentrations (IC₅₀) of the venom were assessed with CCK-8 assay. The apoptosis rate of breast cancer MCF7 cells following the treatment was analyzed with flow cytometry, and the changes in cellular caspase-8 and caspase-9 expressions were detected. The crude venom was separated into protein, peptide, and small-molecule compound fractions using gel filtration chromatography and high-performance liquid chromatography (HPLC). The protein and peptide components were identified using proteomics analysis, and small-molecule compounds were structurally characterized using nuclear magnetic resonance (NMR), mass spectrometry (MS), and HPLC. Results The crude venom exhibited strong concentration-dependent inhibitory effects on proliferation of MCF7 cells and nasopharyngeal carcinoma SUNE1 and HONE1 cells (IC₅₀ of 2.14±0.29, 1.57±0.14, and 2.85±0.15 µg/mL, respectively), with less potent inhibitory effects in gastric cancer HGC27 cells and colorectal cancer SW620 cells (IC₅₀ of 3.02±0.27 and 3.02±0.28 µg/mL, respectively). The crude venom significantly promoted MCF7 cell apoptosis likely via the caspase 8 signaling pathway. The protein fraction from the crude venom showed a weak inhibitory effect in MCF7 cells, whereas the peptide fraction exhibited a much stronger inhibitory effect (IC₅₀ of 6.41±0.31 µg/mL). The peptides in the peptide fraction, with relative molecular mass around 10 000, were homologous to those found in Macrothele gigas venom. The small-molecule fraction consisted mainly of nucleotide metabolites without obvious inhibitory effects in MCF7 cells, but its combination with the peptide fraction showed significantly enhanced inhibitory activity. Conclusion The inhibitory effects of Macrothele raveni venom, which vary significantly across different cancer cell lines, are attributed primarily to its peptide components, which may act synergistically with the nucleotide metabolites.

Key words: Macrothele raveni, peptide toxins, cancer cells, anti-proliferation

谢婷, 王云云, 郭婷, 袁春华. 雷氏大疣蛛多肽毒素组分通过激活促凋亡通路和协同作用抑制癌细胞增殖[J]. 南方医科大学学报, 2025, 45(7): 1460-1470.

Ting XIE, Yunyun WANG, Ting GUO, Chunhua YUAN. The peptide toxin components and nucleotide metabolites in Macrothele raveni venom synergistically inhibit cancer cell proliferation by activating the pro-apoptotic pathways[J]. Journal of Southern Medical University, 2025, 45(7): 1460-1470.

图/表 11

图1 雷氏大疣蛛毒素对癌细胞的增殖抑制作用

Fig.1 Inhibitory effects of Macrothele raven venom in different cancer cells assessed using CCK-8 assay at 48 h. A: The spider Macrothele raven. B: Inhibitory effects of the venom on proliferation of human breast cancer MCF7 cells, A2058 human Melanoma cells, and human oral epidermoid carcinoma KB cells. C-G: Inhibitory effects of the venom on proliferation of nasopharyngeal carcinoma cells (C), gastric cancer cells (D), colon cancer cells (E), hepatoma cells (F), and lung cancer cells (G). H: Inhibitory effects of the venom on human endometrial cancer Ishikawa cells, human cervical cancer HeLa cells, Siha cells, and human endometrial adenocarcinoma HEC-1B cells. I: Inhibitory effects of the venom on human glioma cells.

图2 雷氏大疣蛛毒素抑制癌细胞增殖的半抑制浓度

Fig.2 IC50 values of the venom of Macrothele raven in different cancer cell lines.

图3 雷氏大疣蛛粗毒诱导MCF7 细胞凋亡

Fig.3 Apoptosis of MCF7 cells following treatment with Macrothele raven venom. A: Flow cytometry of MCF7 cells treated with 5 and 10 µg/mL venom for 24 h. B: Analysis of caspase-8 and 9 activities in MCF7 cells treated with the venom at 5 and 10 µg/mL for 24 h (n=3). ***P<0.001 vs Control.

图4 雷氏大疣蛛毒素的分离纯化和SDS-PAGE分子量测定

Fig.4 Isolation, purification, and SDS-PAGE molecular weight determination of Macrothele raven venom. A: Separation of the crude venom by gel filtration (Sephadex G-75 column, 10 mm×100 mm). B: Analysis of the eluted fractions by SDS-PAGE with Coomassie Brilliant Blue staining. 1: Crude venom; 2: Fraction eluted at 5 min; 3, 4, 5: Fractions eluted at 10 min of different batches. C: Separation of the small molecule compounds from the crude venom by reversed-phase HPLC.

图5 雷氏大疣蛛毒素组分对乳腺癌MCF7细胞的增殖抑制作用

Fig.5 Inhibitory effects of the fractions from Macrothele raveni crude venom on proliferation of MCF-7 cells. A: Inhibitory effects of the 5-min and 10-min fractions separated by gel filtration and small compounds separated by HPLC on human breast cancer MCF-7 cells. B: Inhibitory effects of the fractions eluted at 10 min of different batches on MCF7 cells. C: Inhibitory effects of the 10 min-3 fraction on HEK-293 cells. D: Inhibitory effects of the combinations on human breast cancer MCF-7 cells. Data are presented as Mean±SD (n=3). *P<0.05, **P<0.01 vs 10-min peptide fraction.

图6 小分子化合物促进多肽诱导的MCF7 细胞凋亡

Fig.6 The small molecule compounds from Macrothele raveni venom promoted peptide-induced apoptosis of MCF7 cells. A: Apoptosis of MCF7 cells treated with 7.5 µg/mL venom fractions for 24 h analyzed by flow cytometry (10 min: 10 min peptide fraction). B: Analysis of the activities of caspase 8 and 9 in MCF7 cells treated with the venom fractions at 7.5 and 15 µg/mL for 24 h (n=3). ***P<0.001 vs Control, ##P<0.01, ###P<0.001 vs 10-min peptide fraction.

表1 凝胶过滤层析5 min组分中鉴定的蛋白质

Tab.1 Identified proteins in the 5-min component separated by gel filtration chromatography

Accession No.	Protein	#PSMs	MW	Score sequest HT
Q9NFL4	Hemocyanin G chain	8	71.766	17.8
P02241	Hemocyanin D chain	9	72.077	17.6
P02242	Hemocyanin E chain	8	71.63	15.3
P41340	Actin-3	6	41.781	11.7
Q9NFH9	Hemocyanin B chain	7	72.052	10.7
P14750	Hemocyanin A chain	4	72.273	6.0
P0C2V1	U15-hexatoxin-Mg1a	2	6.946	4.7
P80476	Hemocyanin AA6 chain	3	71.74	3.8
W4VS99	Neprilysin-1	1	82.004	2.4
B4GEL3	E3 UFM1-protein ligase 1 homolog	1	87.486	2.1
Q9NFL6	Hemocyanin C chain	2	72.521	2.1
Q9VL52	Putative ATP-dependent RNA helicase	1	168.374	2.0

表2 凝胶过滤层析10 min组分中鉴定的多肽和蛋白质

Tab.2 Identified peptides and proteins in the 10-min component separated by gel filtration chromatography

Accession No.	Protein	#PSMs	MW	Score sequest HT
P68423	U4-theraphotoxin-Hs1a	59	3.134	254.7
P0C2V2	U15-hexatoxin-Mg1b	32	6.904	101.0
P0C2V1	U15-hexatoxin-Mg1a	30	6.946	88.0
Q75WH4	U10-hexatoxin-Mg1a	9	8.876	35.2
Q75WH1	Mu-hexatoxin-Mg1c	14	11.64	34.2
P02241	Hemocyanin D chain	8	72.077	26.9
Q9NFL4	Hemocyanin G chain	8	71.766	25.2
P83558	Mu-hexatoxin-Mg1a	8	14.179	24.0
Q75WG7	U13-hexatoxin-Mg1a	8	13.18	21.2
P0DL75	RTX-VII	5	8.706	18.8
P83561	Beta-hexatoxin-Mg1a	5	8.785	17.5
P14750	Hemocyanin A chain	5	72.273	15.2
P02242	Hemocyanin E chain	4	71.63	13.4
P02572	Actin-42A	5	41.797	10.5
Q9NFH9	Hemocyanin B chain	6	72.052	10.2
P83559	Mu-hexatoxin-Mg2a	3	5.229	8.9
P56676	Mu/omega-theraphotoxin-Hs1a	2	9.313	5.5
P61233	U5-hexatoxin-Mr1a(Raventoxin-1)	1	4.845	4.3
C0JB02	Phospholipase D	1	30.78	4.0
Q9NFL6	Hemocyanin C chain	2	72.521	4.0
P82959	U1-theraphotoxin-Hs1a	1	9.423	3.5
Q86C49	U1-theraphotoxin-Hs1f	1	9.162	3.4

图7 雷氏大疣蛛毒素小分子化合物结构解析

Fig.7 Structural analysis of small molecule compounds isolated from Macrothele raveni venom. A: 600 MHz 13C NMR (left) and 31P NMR (right) spectrum of sample of peak a in Fig.4C. B: 600 MHz 13C NMR (left) and 31P NMR (right) spectrum of sample of peak b in Fig.4C. C: Mass spectrometry of the small molecule compounds isolated from Macrothele raveni venom.

表3 13C NMR 化学位移（ppm）：样本数据与文献数据或标准品数据比较

Tab.3 13C NMR chemical shifts （ppm）： comparison between sample and literature or standard data

ID	a peak	UMP	b peak	AMP
1	64.4	64.4	64.30	64.25
2	69.5	71	64.33	64.28
3	73.74	74.9	70.27	70.31
4	83.01	84.9	74.66	74.67
5	88.63	89.4	84.21	84.27
6	102.41	103.5	84.27	84.32
7	141.47	143	87.99	87.91
8	151.69	152.7	118.54	118.55
9	166.16	167.1	142.35	142.21
10			144.61	145.10
11			148.35	148.40
12			149.91	150.25

表4 雷氏大疣蛛毒素中小分子组分的分子量和洗脱保留时间与标准品比较。

Tab.4 Comparison relative molecular mass and retention time between small-molecule compounds from Macrothele raveni venom and standards

Sample	Small-molecule compounds		Standards
Sample	MW	Retention time (min)	Standards	MW	Retention time (min)
1	322.98 (-)	3.41	UMP	324.2	3.43
2	348.3 (-)	3.64	AMP	347.2	3.62
3	104.12 (+)	15.03	GABA	103.1	15.08
4	137.07 (+)	4.14	Hypoxanthine	136.1	4.11
5	244.01 (-)	4.91
6	267.02 (-)	4.45	Inosine	268.2	4.41
7	347.01 (-)	3.54	IMP	348.2	3.52
8	362.02 (-)	3.71	GMP	363.2	3.77
9	367.22 (-)	5.74

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