The peptide toxin components and nucleotide metabolites in Macrothele raveni venom synergistically inhibit cancer cell proliferation by activating the pro-apoptotic pathways

doi:10.12122/j.issn.1673-4254.2025.07.12

Abstract

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

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.

Figures/Tables 11

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.

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

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.

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.

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.

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.

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

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

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.

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

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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